LIBRO DE RESUMENES - Congreso SEBBM Salamanca 2016

Entidades colaboradoras:
Expositores:
SEBBM Salamanca 2016
Organiza:
SEBBM
Salamanca 2016
XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular
Del 5 al 8 de septiembre
Colaboradores:
Patrocinadores de Reuniones de Grupos y Cursos:
Patrocinador general del Congreso:
Socios Protectores:
XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular
Patrocinadores de Conferencias y Premios:
LIBRO DE
RESÚMENES
www.sebbm.es/xxxixcongreso
XXXIX Congreso
de la Sociedad Española
de Bioquímica
y Biología Molecular
SEBBM
Salamanca 2016
Del 5 al 8 de septiembre
Primera edición: Septiembre 2016
© los autores, 2016
XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular
Publica: Sociedad Española de Bioquímica y Biología Molecular (SEBBM)
Producción editorial: Rubes Editorial, S.L.
Sicilia, 253 – 6º 4ª. 08025 Barcelona
Índice
Invitación. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Agradecimientos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
Comité organizador . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
Junta directiva de la SEBBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
Socios protectores de la SEBBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
Calendario del Congreso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Conferencias plenarios / Plenary Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Simposios / Symposia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
S1.
S2.
S3.
Biomedicina molecular / Molecular biomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulación génica y comunicación celular / Gene regulation and cellular communication . . . . . . . . . . . . . . . . .
Estructura y función de biomoléculas / Structure and function of biomolecules . . . . . . . . . . . . . . . . . . . . . . . .
17
23
29
Pósters / Posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35
P01.
P02.
P03.
P04.
P05.
P06.
P07.
P08.
P09.
P10.
P11.
P12.
P13.
P14.
P15.
P16.
Apoptosis / Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bases moleculares de la patología / Molecular bases of pathology . . . . . . . . . . . . . . . . . . .
Biología del desarrollo / Developmental biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biología molecular computacional / Computational molecular biology . . . . . . . . . . . . . . . .
Biomembranas y bioenergética / Biomembranes and bioenergetics . . . . . . . . . . . . . . . . . .
Bioquímica de la nutrición / Nutritional biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bioquímica perinatal / Perinatal biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bioquímica y biología molecular de plantas / Biochemistry and molecular biology of plants .
Biotecnología molecular / Molecular biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estructura y función de proteínas / Protein structure and function . . . . . . . . . . . . . . . . . .
Genómica y proteómica / Genomics and proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Metabolismo del nitrógeno / Nitrogen metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Neurobiología molecular / Molecular neurobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Parasitología molecular / Molecular parasitology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Radicales libres y estrés oxidativo / Free radicals and oxidative stress . . . . . . . . . . . . . . . .
Regulación de la expresión génica y dinámica del genoma / Regulation of gene expression
and genome dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P17. Regulación metabólica / Metabolic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P18. Señalización celular / Cellular signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P19. Transgénesis en mamíferos / Transgenesis in mammals . . . . . . . . . . . . . . . . . . . . . . . . . .
P20. Transportadores de membrana / Membrane transporters . . . . . . . . . . . . . . . . . . . . . . . . . .
PW1. Enseñanza de la bioquímica / Teaching biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . .
PW2. Workshop: Investigador emergente / Workshop emergent investigator . . . . . . . . . . . . . . . .
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37
42
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120
127
140
152
154
159
162
Índice de autores / Authors index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
Invitación
Querid@ amig@,
En nombre del comité organizador te doy la bienvenida al
XXXIX Congreso de la SEBBM. La sede, el Palacio de Congresos de Castilla y León, está situada en el mismo centro
de Salamanca, a pocos metros del Edificio Histórico de la
Universidad o de la plaza Mayor. El acto inaugural (5 de septiembre a las 19:00 h) contará con la presencia del Prof. Sir
Paul Nurse, premio Nobel de Fisiología y Medicina en 2001,
quien impartirá una conferencia. Seguidamente, iremos paseando al Colegio Arzobispo Fonseca para disfrutar del cóctel
de bienvenida en su bello claustro.
Durante los dos días y medio siguientes ofreceremos el marco que nos permita disfrutar de las discusiones científicas.
Para facilitarlo, hemos dotado de tiempo suficiente entre
las sesiones de simposios y las conferencias plenarias con
la pretensión de que ningún póster quede sin ser visitado ni
discutido. Además, hemos recuperado el Poster Party de la
tarde, con el cual pretendemos catalizar el intercambio de
ideas. Animo también a los congresistas a interesarse por los
nuevos y excelentes productos que las empresas patrocinadoras nos muestran desde sus stands, colocados junto a los
pósters. Desde aquí me gustaría agradecer a las empresas
patrocinadoras su constante apoyo, sin el cual este Congreso
no podría celebrarse.
Me gustaría destacar dos novedades en este Congreso. La primera, facilitar que sean los propios socios de la SEBBM los
que tomen la iniciativa de elaborar propuestas de simposios
mediante concurrencia competitiva. Y, la segunda, destacar
la figura del investigador emergente en un simposio plenario
que nos permita conocer mejor nuestro potencial investigador.
Naturalmente, con respecto a los congresos anteriores mantendremos las ya clásicas conferencias plenarias y premios
(Alberto Sols-Fundación BBVA, Fundación Ramón Areces,
L’Oréal-UNESCO, Leloir, Niemeyer, PABMB, Fischer-Scientific, Joven Investigador-Biotools, Margarita Lorenzo-Fundación Lilly), así como las reuniones de grupo, esencia del
Congreso de la SEBBM. Por otro lado, continuaremos con las
actividades satélite, tales como el Curso de Iniciación a la
Investigación en Bioquímica y Biología Molecular, el Foro del
Emprendedor, la Reunión de Coordinadores de Másteres
del Área de Bioquímica, la Bioquímica en la Ciudad, y los
Talleres, de enorme interés entre los más jóvenes, y entre
los no tan jóvenes.
Con la nueva conexión ferroviaria de vía rápida, la ciudad de
Salamanca está a tan solo una hora y media de Madrid. La
coincidencia del Congreso con las fiestas patronales de la
ciudad nos permitirán además disfrutar de los conciertos en
la plaza Mayor y otras distracciones de índole gastronómica.
Con sus universidades, claustros y palacios, Salamanca es de
incuestionable interés cultural. Por todos estos motivos, en
nombre del Comité Organizador es un placer darte la bienvenida y animarte a participar activamente en el XXXIX Congreso de la SEBBM.
JUAN PEDRO BOLAÑOS
Presidente del Comité Organizador
4
Agradecimientos
Nuestro agradecimiento a las entidades públicas y privadas que han colaborado económicamente en la realización del XXXIX
Congreso de la SEBBM.
Ministerio de Economía y Competitividad
Consejo Superior de investigaciones científicas (CSIC)
Junta de Castilla y León
Ayuntamiento de Salamanca
Universidad de Salamanca
Instituto de Biología Fundacional y Genómica (IBFG)
Instituto de Investigación Biomédica de Salamanca (IBSAL)
Instituto de Neurociencias Castilla y León
Centro de Investigación del Cáncer (CIC)
Fundación BBVA
Fundación Ramón Areces
L’Oréal – For Women in Science
Fundación lilly
FEBS
PABMB
Eppendorf
Bruker
Fisher Scientific
Biotools
Roche
Geirli
Bio-Rad
Biogen
Conda – Pronadisa
Controltécnica
Cultek
Ecogen
Frontexbiomed
GE Healthcare
Gilson
Marie Curie Alumni Association
Merck
Nucleus
Panreac AppliChem
Stab Vida
Vitro
VWR International
Werfen
5
Comité organizador
COMITÉ HONORÍFICO
Excmo. Sr. Juan Vicente Herrera, Presidente
de la Comunidad Autónoma de Castilla y León
Crisanto Gutiérrez (Tesorero de la SEBBM)
Joaquim Ros (Coordinador de la Comisión Asesora
de Congresos de la SEBBM)
Ilma. Sra. Dra. Carmen Vela, Secretaria de Estado
de Universidades e Investigación
Excmo. Sr. Daniel Hernández Ruipérez, Rector Magnífico
de la Universidad de Salamanca
COMITÉ CIENTÍFICO
Ángeles Almeida (Instituto de Investigación
Biomédica de Salamanca, Hospital Universitario
de Salamanca-Universidad de Salamanca)
COMITÉ EJECUTIVO
Juan Pedro Bolaños (Presidente, Instituto de Biología
Funcional y Genómica, Universidad de Salamanca)
Isabel Fariñas (Universidad de Valencia)
Ángeles Almeida (Secretaría Científica, Instituto de
Investigación Biomédica de Salamanca, Hospital
Universitario de Salamanca-Universidad de Salamanca)
Arantxa Tabernero (Coordinación Actividades Satélite,
Instituto de Neurociencias de Castilla y León, Universidad
de Salamanca)
Emilio Fernández (Tesorero, Instituto de Biología Funcional
y Genómica)
Carlos López-Otín (Universidad de Oviedo)
Salvador Moncada (Universidad de Manchester, Reino
Unido)
OTROS COMITÉS
Curso de Iniciación a la Investigación
Arantxa Tabernero (Instituto de Neurociencias de Castilla
y León, Universidad de Salamanca)
Reunión de coordinadores de grados y posgrados
Carmen Guerrero (Coordinación Logística, Centro
de Investigación del Cáncer)
Isabel Muñoz-Barroso (Departamento de Bioquímica
y Biología Molecular, Universidad de Salamanca)
Ángel Hernández (Coordinación Logística, Departamento de
Bioquímica y Biología Molecular, Universidad
de Salamanca)
Foro del emprendedor
César Roncero (Coordinación Logística, Instituto de Biología
Funcional y Genómica)
Juan Carlos Arévalo (Instituto de Neurociencias de Castilla
y León, Universidad de Salamanca)
Actividades Bioquímica en la Ciudad
Federico Mayor (Presidente de la SEBBM)
Concepción Lillo (Instituto de Neurociencias de Castilla
y León, Universidad de Salamanca)
María Ángeles Ros (Vocal de Congresos de la Junta Directiva
de la SEBBM)
Ana Velasco (Instituto de Neurociencias de Castilla y León,
Universidad de Salamanca)
Itziar Alkorta (Vocal de Empresas y Cónsules de la Junta
Directiva de la SEBBM)
David Díaz (Instituto de Neurociencias de Castilla y León,
Universidad de Salamanca)
6
Junta directiva de la SEBBM
PRESIDENTE
Federico Mayor Menéndez
VICEPRESIDENTE
Manuel Palacín Prieto
VOCALES
Juan Pedro Bolaños Hernández
Carmen Caelles Franch
Irene Díaz Moreno
Iztiar Alkorta Calvo
SECRETARIA
Almudena Porras Gallo
Fernado Moreno Herrero
TESORERO
Crisanto Gutiérrez Armenta
PRESIDENTE ELECTO
Felix María Goñi Urcelay
Marian Ros Lasierra
Socios protectores de la SEBBM
Los socios protectores de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM) contribuyen al progreso de la ciencia.
7
Calendario del Congreso
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Conferencias plenarios
Plenary Lectures
9
10
Salamanca 2016
Conferencia de apertura / Opening Lecture
Alberto Sols - Fundación BBVA
CP01-1
Controlling the Cell Cycle
Matthew Swaffer, Andrew Jones, Helen Flynn, Bram Snijders,
Paul Nurse
The Francis Crick Institute, Londres, UK
Like other eukaryotes wild type fission yeast cells control their cell cycle
through multiple cyclin dependent kinases (CDKs) with roles at different
cell cycle stages. However, all these CDKs can be replaced by a single
CDK, suggesting that the order of cell cycle events can be controlled
by quantitative changes in CDK activity rather than by qualitatively
different CDKs. Using phosphoproteomics combined with a novel in
vivo protein kinase assay, we have shown that activity increases as cells
proceed through the cell cycle and that different substrates have differing
sensitivities to absolute kinase activity. Substrates required early in the
cell cycle for S-phase become phosphorylated at low CDK activity whilst
substrates require later for mitosis need higher CDK activity to become
phosphorylated. We propose that this is the core organising principle
ordering events in the cell cycle which helps explain the plasticity and
redundancy observed between CDKs in Metazoan eukaryotes.
Conferencia Premio Fisher Scientific:
mejor artículo científico de joven
científico/a / Fisher Scientific Award
Lecture: best scientific paper of a young
scientist
CP02-1
AMPK and PFKFB3 mediate glycolysis and survival
in response to mitophagy during mitotic arrest
Elena Doménech Cruz1, Carolina Maestre1, Lorena Esteban2, David
Partida1, Gonzalo Fernández-Miranda3, Ramón Campos-Olivas1,
Manuel Pérez1, Diego Megías1, Katherine Allen4, Miguel López5,
Asish Saha6, Guillermo Velasco7, Eduardo Rial2, Raúl Méndez3,
Patricia Boya2, María Salazar-Roa1, Marcos Malumbres1
1
Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ES,
2
CIB, Madrid, ES, 3IRB Barcelona, Barcelona, ES, 4Boston Universisty
School of Medicine, Massachusetts, US, 5CIMUS, University of
Santiago de Compostela-Instituto de Investigación Sanitaria,
Santiago de Compostela, ES, 6Division of Endocrinology, Diabetes
& Nutrition, Boston University School of Medicine, Massachusetts, US,
7
Universidad Complutense de Madrid, Madrid, ES
Blocking mitotic progression has been proposed as an attractive therapeutic
strategy to impair proliferation of tumour cells. Inhibition of mitotic kinases
such as Aurora A or Plk1, or treatment of cells with microtubule poisons
that disrupt the proper dynamics of the spindle, results in mitotic arrest, a
feature that may be used to prevent tumour cell proliferation. However,
how cells survive during prolonged mitotic arrest is not well understood.
At this stage, transcription is strongly suppressed, translation is limited
to a subset of messenger RNAs, and the absence of the nuclear envelope
temporally maintains on hold multiple regulatory mechanisms that are
based on the separation of the nuclear and cytoplasmic compartments.
Although death in mitosis is at least partially mediated by apoptosis, the
molecular pathways that control survival or death in mitosis are not well
understood. Here we show that survival during mitotic arrest is affected
by the special energetic requirements of mitotic cells. Prolonged mitotic
Conferencias plenarios / Plenary Lectures
arrest results in mitophagy-dependent loss of mitochondria, accompanied
by reduced ATP levels and the activation of AMPK. Oxidative respiration
is replaced by glycolysis owing to AMPK-dependent phosphorylation
of PFKFB3 and increased production of this protein as a consequence
of mitotic-specific translational activation of its mRNA. Induction of
autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death
in mitosis and improves the anti-tumoral efficiency of microtubule poisons
in breast cancer cells. Thus, survival of mitotic-arrested cells is limited
by their metabolic requirements, a feature with potential implications in
cancer therapies aimed to impair mitosis or metabolism in tumour cells.
Conferencia plenaria Leloir /
Leloir Plenary Lecture
CP03-1
Más que un inhibidor de quinasas dependientes
de ciclinas: una nueva función de p21 en el
replisoma
Sabrina Mansilla1, Agustina P Bertolin1, Valerie Bergoglio2, Marina
Gonzalez Besteiro1, María Belén de la Vega1, Marie-Jeanne Pillaire2,
Nicolás Calzetta1, Natalia Paviolo1, Paola Campodónico1, María
Belén Federico1, Jean-Sebastien Hoffmann2, Vanesa Gottifredi1
1
Fundación Instituto Leloir-Instituto de Investigaciones Bioquímicas
de Buenos Aires. Consejo de Investigaciones Científicas y Técnicas,
C.A.B.A, AR, 2Cancer Research Center of Toulouse, Inserm U1037,
CNRS ERL5294 ; University Paul Sabatier, Toulouse, FR
Los niveles del inhibidor p21 quinasa dependiente de ciclina (CDK) son bajos
en la fase S e insuficiente para inhibir CDK. Hemos demostrado que la p21
endógena, en lugar de ser residual es funcional y necesaria para preservar la
estabilidad genómica de las células no estresadas. La pérdida de expresión
basal de p21 retrasa la elongación del ADN naciente, provocando defectos
permanentes en la finalización de la replicación y promoviendo la inestabilidad
de regiones genómicas que son difíciles de replicar, es decir, sitios frágiles
comunes (SFC). Encontramos que la región de p21 necesaria para promover la
replicación es su dominio de interacción con PCNA (PIR ) y no su dominio de
unión CDK. Por otro lado, identificamos a la polimerasa especializada kappa
como la responsable de los defectos replicativos que aumentan cuando los
niveles de p21 se eliminan, ya que no se detectaron alteraciones en el proceso
de replicación ni aumento de la inestabilidad genómica en células donde p21
y la polimerasa kappa fueron eliminadas. Por lo tanto, con un mecanismo
que es completamente independiente de su capacidad de regular CDK, p21
endógeno impide un tipo de inestabilidad genómica que no se desencadena por
las lesiones del ADN endógenas sino por una desregulación en la elección de
la polimerasa de ADN durante la síntesis de ADN genómico.
Conferencia plenaria / Plenary Lecture
L’Oréal-UNESCO For Women in Science
CP04-1
Modulation of neural plasticity in Alzheimer’s
disease
Nancy Ip
Division of Life Science and State Key Laboratory of Molecular
Neuroscience, The Hong Kong University of Science and
Technology, Hong-Kong, CN
Proper brain function depends on the intricate interplay of well
co-ordinated signaling pathways among different types of brain cells,
deregulation of which can lead to neural dysfunctions that contribute
11
Conferencias plenarios / Plenary Lectures
to various brain disorders. Our research interest is focused on
unravelling the molecular and cellular mechanisms underlying these
pathways to enhance our understanding of brain functions as well as
to identify specific molecular targets associated with brain disorders.
In this seminar, I will talk about our recent work, which has led to the
identification of new targets for Alzheimer’s disease (AD). Increasing
evidence suggests that synaptic dysfunction plays a significant role in
the onset and progression of AD. We have recently identified distinct
receptor-mediated signaling pathways, including receptor tyrosine
kinase EphA4, which can regulate hippocampal synaptic strength
and plasticity. Specifically, we have demonstrated the role of EphA4
as a negative regulator of synaptic plasticity and its importance in
mediating synaptic dysfunctions during progression of AD, thus
identifying this receptor as a promising target for drug development.
We have also identified a small molecule EphA4 inhibitor from an
in-house traditional Chinese medicine database and validated its ability
to alleviate impaired synaptic plasticity in an AD transgenic mouse
model. In addition to synaptic plasticity, innate immunity has emerged
to be an important mediator of the pathogenesis of AD. Our recent work
has suggested that impairment of interleukin-33 (IL-33) signaling is
associated with early progression of the disease. Importantly, injection
of IL-33 rescues synaptic dysfunctions and contextual memory deficits
in an AD transgenic mouse model. The beneficial action of IL-33 is
mediated by regulating the activation state of microglia: enhancing
their β-amyloid phagocytic capacity and reducing proinflammatory
response in the brain. Together, our work highlights the identification
of novel molecular targets for neurodegenerative diseases, and opens up
new opportunities in AD drug discovery and development
Conferencia Premio Joven Investigador
SEBBM-BIOTOOLS: a la actividad
científica en España /
SEBBM-BIOTOOLS Young Award
Lecture: for scientific work in Spain
CP05-1
The emerging role of stress kinases in tissue
homeostasis and pathophysiology
Guadalupe Sabio
Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid, ES
Obesity is currently a major health problems that has reached epidemic
proportions. Obesity is associated with other disorders collectively
known as “metabolic syndrome”, characterized by impaired glucose
tolerance, dyslipidaemia and hypertension. Moreover, obesity may be a
predisposing factor for the development of some types of cancer, such
as hepatocellular carcinoma. Inflammation plays an important role in
obesity and its complications. Stress activated protein kinases (SAPKs),
an important family of kinases implicated in the transduction of stress
signals into the cell, could be key regulators of the development of
diabetes and cancer. While the role of JNK in metabolic diseases
has been studied for a long time, less is known about the role of the
other stress kinase family, p38MAPK. We will discuss about the role
of p38MAPK pathway in the pathophysiology of obesity and the
development of related complications, such as cancer, diabetes and
cardiovascular diseases.
12
XXXIX Congreso SEBBM
Conferencia Premio Científico Margarita
Lorenzo (Fundación Lilly) / Award Lecture
Margarita Lorenzo (Fundación Lilly)
CP06-1
La disminución de la expresión de s-resistina
en el hipotálamo mejora la respuesta central
y periférica a la insulina en ratas Wistar
María Rodríguez Pérez
Área de Bioquímica, Facultad de Ciencias Ambientales y
Bioquímica; Centro Regional de Investigaciones Biomédicas,
UCLM, Toledo
La s-resistina es una variante no secretable de resistina, generada por
splicing alternativo, que se expresa principalmente en tejido adiposo
blanco e hipotálamo de ratas Wistar. Resultados previos confirmaron que
células 3T3-L1 que expresaban s-resistina mostraron un incremento en la
respuesta inflamatoria, así como un descenso en el transporte de glucosa
estimulado por insulina. Además, la s-resistina, al igual que la resistina,
bloqueó la vía de señalización de la insulina inhibiendo la fosforilación de
IR, IRS-1 y Akt, e incrementó los niveles de SOCS-3. Por tanto, s-resistina
podría actuar como un factor no secretado que regula la sensibilidad del
adipocito a la insulina. En este trabajo se analiza el papel de s-resistina en el
hipotalámo. Para ello se ha disminuido la expresión central de esta isoforma
corta de resistina, inyectando, de forma i.c.v, un lentivirus que contiene un
RNAi frente a dicha isoforma (LV-RNAi-s-res). La diminución central de
s-resistina incrementa los niveles basales de fosforilación en Tyr, tanto en
IR como en IRS-1, mientras que disminuye los niveles de fosforilación
en Ser307 en IRS-1 en el hipotálamo. También se observa un incremento
de los niveles de mRNA y de fosforilación en STAT-3, mientras que éstos
disminuyen en el caso de PTP1b. Además, en los animales tratados con
el LV-RNAi-s-res se promueven los efectos anorexigénicos, reduciendo
la expresión de NPY e incrementando la de POMC, lo que conlleva un
descenso de la ingesta. Estos datos muestran, por tanto, una mejora en la vía
de señalización de insulina en aquellos animales que presentan disminuidos
los niveles de expresión de s-resistina en el hipotálamo. Asimismo, se
observa un incremento en la sensibilidad periférica a la insulina, como lo
demuestran las curvas de tolerancia a la glucosa obtenidas en los animales
tratados. Todos estos resultados sugieren que s-resistina podría actuar
como un sensor neuronal intracrino, que actuaría regulando la sensibilidad
a la insulina en todo el organismo.
Conferencia plenaria Niemeyer /
Niemeyer Plenary Lecture
CP07-1
Mitochondrial non-coding RNAs: novel targets
for cancer therapy?
Luis Burzio
Andes Biotechnologies Sp.A, Fundación Ciencia & Vida, and
Facultad de Ciencias Biológicas, Universidad Andrés Bello,
Santiago de Chile, CL
A family of mitochondrial lncRNAs (ncmtRNAs) arising from the
mitochondrial 16S rRNA gene will be described. They comprise a
Sense (SncmtRNA) and Antisense (ASncmtRNA) members, containing
long inverted repeats (IR) and stem. These transcripts are differentially
expressed according to proliferative status; both transcript types are
expressed in normal proliferating cells, but not in resting cells. Tumor
cells (human and mouse) express the SncmtRNA but down regulate the
Salamanca 2016
ASncmtRNAs. We proposed that the ASncmtRNAs is tumor suppressor
and a generalized hallmark of cancer. Knock-down of ASncmtRNA with
antisense oligonucleotides (ASK for short) induces selective death of
tumor cells. Tumor cell death displays characteristics of apoptosis and is
mediated by down-regulation of Survivin and XIAP. Cell death is preceded
by a drastic reduction in proliferative index, mediated through a marked
decrease only of Cyclin D1 and B1. In addition, ASK induces drastic
inhibition of sphere formation together with down regulation of N-cadherin
and β-caveolin, pro-metastasic factors.The reduction in survivin, both
cyclins, N-cadherin and β-caveolin seems to be mediated by microRNAs
arising upon ASncmtRNA knock down. Indeed, ASK induces several
microRNAs derived from the ASncmtRNAs. Among them, we found strong
increase of hsa-miR-1973 and hsa-miR-4485 derived from the stem of the
ASncmtRNAs. Mimics assay demonstrate that hsa-miR-4485 expression
inhibition of cyclin D1 and B1 plus N-cadherin. Translation studies
demonstrate that the ASncmtRNAs are potent target for cancer therapy in
vivo. This results will include examples of syngeneic (melanoma cells and
renal carcinoma cells) as well as xenograph assays with human prostate
carcinoma (PC3 cells) and breast carcinoma (MDA-MB-231 cells).
Conferencia plenaria SEBBM /
SEBBM Plenary Lecture
CP08-1
Epithelial plasticity in health and disease
Ángela Nieto
Instituto de Neurociencias, CSIC-UMH, Alicante, ES
Epithelial homeostasis is crucial to maintain tissue architecture, and
therefore, it needs to be tightly regulated in the adult. By contrast, embryonic
cells show a high degree of epithelial plasticity required for proper
morphogenesis and, in particular, for the implementation of massive cell
movements that occur at gastrulation and neural crest delamination among
other processes. We have been interested in the analysis of cell movements,
plasticity and epithelial to mesenchymal transitions (EMT) for many years
and found that the reactivation of developmental EMT-like programs in
adult cells leads to several pathologies including tumor progression and
organ degeneration. While the epithelial and mesenchymal cells are
usually considered as extreme phenotypes, intermediate EMT states also
exist. Under those circumstances cells depict an intermediate phenotype
expressing both epithelial and mesenchymal markers and from which they
can reverse to the original state or move towards a more mesenchymal
phenotype. This hybrid transitory state can favor coordinated cell migration
or wound healing and it can also enable the formation of clusters of
migratory cancer cells than can better survive in the blood-stream and have
increased tumor initiating potential. In contrast to the hybrid transitory
state, there are particular contexts in which an intermediate phenotype
Conferencias plenarios / Plenary Lectures
can be defined as a final state, as we have recently shown to occur during
renal fibrosis. I will present new data to suggest that evolution has favored
the maintenance of the epithelial phenotype by attenuating the function of
some EMT inducers. I will discuss the implications that this mechanism
might have in embryonic development and in tumor progression toward
the metastatic state.
Conferencia de clausura / Closing Lecture
Fundación Ramón Areces
CP09-1
Angiogenesis revisited: role and (therapeutic)
implications of endothelial metabolism
Peter Carmeliet
Laboratory of Angiogenesis and Vascular Metabolism, Vesalius
Research Centre (VRC), Department of Oncology, KU Leuven,
Leuven, BE
The past 40 years of research in the angiogenesis field have focused on
identifying genetic signals such as VEGF and Notch, which determine
vessel sprouting. However, the role and therapeutic potential of targeting
endothelial cell (EC) metabolism have been largely overlooked.
We have recently reported that ECs are glycolysis addicted and that
glycolysis importantly co-determine vessel sprouting downstream of
VEGF and other pro-angiogenic signals. In addition, we documented
that ECs are rather unique in utilizing fatty acid-derived carbons for the
de novo synthesis of deoxyribonucleotides for DNA synthesis during
EC proliferation when vessels sprout. Moreover, targeting (blocking)
glycolysis and fatty acid oxidation inhibit pathological angiogenesis
and induce tumor vessel normalization (thereby reducing metastasis and
improving chemotherapy), suggesting that these metabolic pathways
are new targets for anti-angiogenic drug development without evoking
systemic side effects. Furthermore, lymphatic ECs differ from other EC
subtypes in their metabolic requirements for lymphangiogenesis. Since
many of these metabolic targets are pharmacologically druggable, these
metabolic pathways represent a new promising target for therapeutic
anti-angiogenesis.
References
[1] S. Schoors, et al. & P. CARMELIET. Fatty acid carbon is essential for
dNTP synthesis in endothelial cells. Nature 520, 192-197 (2015).
[2] S. Schoors, et al. & P. CARMELIET. Partial and transient reduction of
glycolysis by PFKFB3-blockade reduces pathological ocular angiogenesis.
Cell Metab 2014; 19(1):37-48.
[3] K. De Bock, et al. & P. CARMELIET. Role of PFKFB3-driven glycolysis
in vessel sprouting.Cell 154, 651-663 (2013).
13
14
Simposios
Symposia
15
16
Simposio 1.
Biomedicina molecular / Molecular biomedicine
17
18
Salamanca 2016
S1.1. Señalizacion por quinasas
de estrés en la salud y en la
enfermedad / Stress kinase
signaling in heart and disease
Simposios / Symposia
to the effects of feeding a high fat diet, including protection against insulin
resistance and failure of obesity development. We have used tissue-specific
JNK-deficient mice to probe the mechanism of JNK regulation of insulin
resistance and obesity. We show that JNK plays different roles in multiple
tissues and that the phenotype of whole body JNK-deficient mice reflects
the interactions between these different JNK-dependent processes. The
molecular mechanisms of JNK function will be discussed.
S1.1-1
News from alternative p38 in inflammation
Ana Cuenda
Centro Nacional de Biotecnología, CSIC, Madrid, ES
p38MAPK pathways are central to inflammatory processes. The p38MAPK
group has four members encoded by different genes, p38α, p38β, p38γ and
p38δ. While the roles of the p38 isoform have been widely studied in the
context of inflammation and tumour development, the knowledge of the
in vivo role of p38γ and p38δ in these processes is still very limited. Our
laboratory is studying essential functions of these two less studied alternative
p38MAPKs, in vivo and in cultured cells. This strategy has lead to the
discovery of unexpected cross talk between p38γ/p38δ and ERK1/2 pathway
controlling cytokine production in cells during inflammation. The regulation
of inflammatory processes by p38γ/p38δ has an impact in the development
of different diseases such as colon and skin inflammation, arthritis, or colon
cancer associated to colitis. We will discuss how, in specific settings, the
p38MAPK components, p38γ and p38δ, operate in inflammatory processes,
and the mechanisms underlying these unexpected functions.
S1.1-2
Regulation of tumorigenesis by p38 MAPK
signaling
Ángel R. Nebreda
Institute for Research in Biomedicine (IRB Barcelona) and ICREA,
Barcelona, ES
The p38a MAPK pathway plays key roles in the cellular responses to stress
but can also integrate signals that affect many other cellular processes in
a cell context- and cell type-specific manner. Studies using genetically
modified mice have elucidated in vivo functions for p38a, and provided
insights into how this pathway can suppress tumor initiation. There is
evidence that normal cells rely on p38a signaling to engage various tumor
suppressor mechanisms. However, p38a does not seem to be usually mutated
in human tumors, and it appears that thissignaling pathway sometimes can
acquire new roles facilitating tumor development. Using mouse models of
cancer, we have obtained genetic evidence that transformed epithelial cells
rely on the p38a pathway for survival and proliferation. Experiments using
chemical inhibitors support a role for p38a signaling in tumor development
in vivo. We are also investigating how the p38a pathway in stromal cells
contributes to tumorigenesis. I will present results showing that myeloid cells
rely on p38a signaling to support tumor progression by several mechanisms.
S1.1-3
CONFERENCIA PABMB
Metabolic stress signaling by the JNK signaling
pathway
S1.2. Versatilidad de las células
mieloides. Aspectos básicos en
polarización y reprogramación
funcional de las mismas y su
potencial terapéutico / Versatility
of myeloid cells: basic aspects
aspects on polarization and
functional reprogramming and
therapeutic potential
S1.2-1
Polarización de macrófagos: aspectos básicos
y nuevos reguladores
Sonsoles Hortelano
Unidad de Terapias Farmacológicas, ISCIII, Madrid, ES
Las células mieloides constituyen un grupo heterogéneo esencial en
inmunidad. En los últimos años, los macrófagos han adquirido especial
relevancia, no solo porque desempeñan un papel clave en la defensa
contra patógenos y contribuyen a la homeostasis del tejido y la reparación;
sino porque además se encuentran implicados en un amplio espectro de
patologías como el cáncer, la obesidad, enfermedades respiratorias o en
enfermedades inflamatorias crónicas como la enfermedad de Crohn.
Esta diversidad en sus funciones viene determinada por la capacidad que
presentan estas células para adaptarse al medio ambiente que les rodea,
pudiendo adoptar distintos estados de activación. Simplificando la gran
heterogeneidad de estas células, podemos distinguir dos tipos de macrófagos,
identificados como M1 (macrófagos clásicos o pro-inflamatorios) y M2
(macrófagos alternativos o anti-inflamatorios), que difieren en la expresión
de receptores, funciones y producción de citoquinas y quimioquinas. El
delicado balance entre ambas poblaciones viene marcado por un conjunto
de señales, incluyendo factores de transcripción, cambios epigenéticos,
reguladores post-transcripcionales,…, que modifican sus estados de
activación. A pesar de los recientes avances en el estudio de la biología de
los macrófagos, todavía queda mucho camino por recorrer. Sin duda, una
mejor comprensión de las bases moleculares y los mecanismos implicados
en la plasticidad de estas células permitirá el diseño de estrategias
terapéuticas más eficaces.
S1.2-2
Los receptores Notch en el control de la
activación proinflamatoria de los macrófagos
Roger J. Davis
Howard Hughes Medical Institute (HHMI), University
of Massachusetts, Chevy Chase, US
María José Martínez Díaz-Guerra
Departamento de Química Inorgánica, Orgánica y Bioquímica,
Facultad de Medicina, Universidad de Castilla-La Mancha,
Albacete, ES
The cJun NH2-terminal kinase (JNK) signaling pathway is implicated
in the pathogenesis of diabetes.High fat diet-induced obesity causes
activation of JNK in insulin target tissues. JNK-deficient mice are resistant
Los receptores Notch constituyen una ruta de señalización muy conservada
que es esencial en procesos de desarrollo y diferenciación celular. En el
sistema inmune está bien documentada su participación en el desarrollo
19
Simposios / Symposia
y la activación de las células T y B. Múltiples trabajos han asociado la
activación de la ruta de Notch con procesos inflamatorios, sin embargo
los mecanismos por los que la infección y la inflamación modulan la
señal de Notch, y cómo esta ruta puede condicionar la respuesta de las
células mieloides, no están completamente establecidos. Nuestro grupo
estudia la función que los receptores Notch desarrollan en la respuesta
de los macrófagos a la activación por los receptores Toll. Estos inducen
la expresión de todos los receptores Notch, especialmente Notch1, y
de ligandos de las familias Jagged y Delta, así como la de genes diana
clásicos de la vía como Hes1 y Hey1 y 2. La activación de los receptores
Toll incrementa también la expresión y/o activación de diferentes proteasas
implicadas en el procesamiento de los receptores Notch como la furina,
ADAM10 y ADAM17, y la presenilina1 que forma parte del complejo
γ-secretasa, así como el de otras moléculas que favorecen la activación
de estas proteasas como la tetraspanina TSPAN33. La señalización a
través de los receptores Notch a su vez favorece la expresión de diversas
citoquinas proinflamatorias tras la activación de los receptores Toll como
IL-6, IL-12 o TNFα, o enzimas como la Óxido Nítrico Sintasa inducible,
o COX-2. Este efecto está mediado por la unión directa de Notch/RBP-J
a los promotores de algunos de estos genes, o de forma indirecta por un
incremento en la actividad de factores de transcripción como NFκB e IRF8.
Nuestros resultados y los de otros grupos muestran que los receptores
Notch actúan favoreciendo la activación de los macrófagos en la respuesta
inmune e inflamatoria. Este proceso parece condicionar el desarrollo
de algunas patologías inflamatorias, lo que sitúa a estos receptores, sus
moduladores y sus dianas como posibles puntos de acción farmacológica.
S1.2-3
Control de la polarización antiinflamatoria
de macrófagos humanos y sus implicaciones
patológicas
Ángel L. Corbí
Grupo de Biología de las Células mieloides, Centro
de Investigaciones Biológicas (CIB), CSIC, Madrid, ES
Macrophages display a huge functional heterogeneity and acquire proor anti-inflammatory functions depending on the surrounding tissue
microenvironment. In response to Th1 cytokines, macrophages acquire
pro-inflammatory, bactericidal, tumoricidal and immunogenic activities
(“M1 polarization”), whose hallmark is the production of TNFalpha
and IL-12/IL-23. Conversely, other factors (IL-4, TGFbeta) promote
“M2 polarization”, characterized by enhanced anti-inflammatory,
scavenging, tumor-promoting, tissue repair and pro-angiogenic functions.
The pathophysiological relevance of the balance between macrophage
polarization states is best exemplified during inflammation. M1
macrophages predominate at the initial stages of an inflammatory response,
whereas M2 macrophages drive resolution of inflammation, and their
sequential occurrence is required for termination of inflammatory responses,
and for tissue repair after injury. In fact, deregulation of the macrophage
polarization balance leads to chronic inflammatory pathologies (e.g.,
rheumatoid arthritis, fibrosis, obesity), and its subversion is used by tumors
to escape from immune-surveillance and to foster their own proliferation
and dissemination. Thus, determination of the mechanisms underlying the
differences between polarization states should lead to the identification of
molecules whose targeting might allow modulating macrophage effector
functions under homeostatic or pathological conditions.
We have previously defined the transcriptomic profiles of macrophages
generated in the presence of either GM-CSF (pro-inflammatory GM-MØ)
or M-CSF (anti-inflammatory M-MØ), and identified genes/proteins whose
expression distinguishes pro-inflammatory macrophages or homeostatic
tissue-resident macrophages in vivo. With this background, the presentation
will focus on the existence of an anti-inflammatory gene signature in human
macrophages, whose acquisition is dependent on the MAFB transcription
factor. The importance of MAFB in the anti-inflammatory polarization of
human macrophages will be illustrated with data generated on samples from
20
XXXIX Congreso SEBBM
a rare osteolytic disease (MCTO). Besides, the presentation will address
the molecular basis of the serotonin ability to inhibit pro-inflammatory
cytokine production, describing the receptors involved and identifying the
serotonin-regulated genes in human macrophages.
S1.3. Hipoxia: de los mecanismos
moleculares a las aplicaciones
clínicas/ Hypoxia: from molecular
mechanisms to clinical applications
S1.3-1
Acute oxygen sensing mechanisms
José López-Barneo
Departamento de Fisiología Médica y Biofísica, Universidad
de Sevilla, Sevilla, ES
Acute O2-sensing is essential for adaptation of mammals to changing
environments and pathophysiological conditions. Upon exposure to
hypoxia, arterial chemoreceptors, in particular the carotid body (CB),
mediate fast (seconds) cardiorespiratory responses (hyperventilation and
sympathetic activation) that compensate the lack of O2. These life-saving
reflexes are necessary for survival at high altitude and at sea level
contribute to the eupneic drive to breathe as well as to increase ventilation
in hypoxemic patients with chronic obstructive pulmonary diseases. In
addition to the CB, other organs, such as the adrenal medulla (AM) and
the pulmonary arteries, are also responsive to acute hypoxia. Therefore, the
understanding of the acute O2 sensing mechanisms has a major biomedical
interest. Responsiveness to hypoxia depends on specialized chemoreceptors
cells, which contain O2-sensitive K+ channels. In CB cells, closure of these
channels during hypoxia leads to cell depolarization and the release of
transmitters that activate excitatory fibers terminating at the brain stem
respiratory center. We have shown that ablation of the Ndufs2 gene, which
encodes a 49 kD protein at the ubiquinone binding site of mitochondrial
complex Iresults in mice with selective abolition of the hypoxic ventilatory
response. Ndufs2-deficient chemoreceptor cells from the CB and the AM
are insensitive to hypoxia although show normal responses to other stimuli
such as hypercapnia or hypoglycemia. Our current model of acute O2
sensing proposes that the slow down of mitochondrial electron transport
during hypoxia causes a succinate-dependent accumulation of reduced
quinone (QH2), which results in a burst of NADH and ROS. These, in turn,
modulate ion channels in O2-sensing membrane microdomains.
S1.3-2
Hypoxic regulation of the innate immune response
Sarah Walmsley
MRC Centre for Inflammation Research, University of Edinburg,
Edinburg, UK
The treatment of both sepsis and the adult respiratory distress syndrome
represent huge clinical challenges and areas of unmet therapeutic need.
In acute settings, hypoxemia is associated with increased morbidity and
mortality of bacterial infections, particularly in the context of development
of ARDS. We questioned whether more prolonged hypoxic preconditioning
could modify the clinical outcome to infection. In both a local subcutaneous
S. aureus skin infection model and a systemic S. pneumoniae model of
bacteraemic pneumonia pathophysiological responses to infection are
pre-determined by prior exposure to hypoxia. We observe these outcomes
to be a consequence of the transcriptional regulation of leukocyte HIF-1a
pathways, with myeloid cell responses to altered oxygen and nutrient
availability a critical determinant of overall morbidity and mortality. This
work provides evidence of imprinted changes in the functional memory of
Salamanca 2016
myeloid cell populations, and supports the concept that strategies to target
the host response in addition to direct anti-microbial targeting will be
required to improve outcomes where HIF-1 hypoxia and infection co-exist.
S1.3-3
Identification of non-coding genetic variants
in samples from respiratory disease patients that
affect the transcriptional response to hypoxia
Luis del Peso
Departamento de Bioquímica, Universidad Autónoma de Madrid,
Madrid, ES
A wide range of diseases course with an unbalance between the
consumption of oxygen by tissues and its supply. This situation triggers
a transcriptional response, mediated by the hypoxia inducible factors
Simposios / Symposia
(HIFs), that aims to restore oxygen homeostasis. Little is known about the
inter-individual variation in this response and its role in the progression
of disease. Herein, we sought to identify common genetic variants
affecting the hypoxia response elements (HREs) and characterize their
effect on transcription. To this end, we constructed a list of genome-wide
HIF-binding regions from publicly available experimental datasets and
studied the genetic variability in these regions by targeted re-sequencing
of genomic samples from 96 chronic obstructive pulmonary disease
patients and 144 obstructive sleep apnea patients. This study identified
13 frequent variants in potential HREs, which is significantly lower than
expected by chance. Analysis of the loci containing these variants by
means of reporter assays showed that these regions drove the transcription
of reporter genes under low oxygen that the alternate alleles blunted this
response. Finally, using genome editing we confirmed the functional role
of these variants in a physiological context. Altogether our results suggest
that common polymorphisms contribute to determine the efficiency of
transcriptional activation of target genes by hypoxia.
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22
Simposio 2.
Regulación génica y comunicación celular /
Gene regulation and cellular communication
23
24
Salamanca 2016
S2.1. Fronteras en biología de plantas:
de las moléculas al organismo /
Frontiers in plant biology: from
molecules to the organism
(Simposio Hispano-Mexicano)
Simposios / Symposia
demostrado que la señal responsable de los fenotipos de interés es un
apocarotenoide generado por el rompimiento de carotenos lineales por la
digoxigenasa CCD4. Estudios recientes han identificado genes alterados
por esta señal con funciones en el desarrollo de la hoja, así como secuencias
en cis importantes para esta regulación.
S2.1-3
La reparación por escisión de bases
como mecanismo de control epigenético
S2.1-1
Abordajes sistemáticos para el estudio
de la división de las células madre en plantas
Ana I. Caño Delgado
Centre de Recerca en Agrigenòmica (CRAG), Bellaterra (Cerdanyola
del Vallés), ES
El mantenimiento de los nichos de células madre en una pregunta central de
la biología fundamental. Nuestro laboratorio ha identificado recientemente
una vía de señalización necesaria para la quiescencia celular del nicho
de células madre en la raíz primaria de la planta modelo Arabidopsis.
BRAVO (BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING
CENTRE) es un factor de transcripción de tipo R2R3-MYB que funciona
como un represor específico de la división del centro quiescente en el nicho
de células madre de la raíz. El análisis molecular de la vía BRAVO ha
revelado un mecanismo que controla las divisiones de células específicas
de esteroides mediada por Brasinoesteroides. Estos resultados abren una
nueva línea para el estudio de las células madre de la planta. Actualmente,
estamos llevando a cabo la identificación de las redes de señalización
que operan durante la división de las células madre basándonos en la
integración de los datos genómicos con resolución celular, en combinación
con imágenes de alta resolución espaciotemporal y modelado matemático.
En Salamanca, presentaremos una visión actualizada de la señalización en
el Centro Quiescente (QC) y en las células madre vasculares adyacentes, así
como nuestro avances hacia la identificación de las firmas transcripciones
de células madre basadas en el análisis genómico de una sola célula.
S2.1-2
La participación de carotenos lineales
en el desarrollo de la hoja
Patricia León, Julio Sierra, Elizabeth Cordoba, Ernesto Llamas
Instituto de Biotecnología UNAM MX, Cuernavaca, MX
Los cloroplastos son esenciales para la fotosíntesis, sin embargo, estos
organelos también funcionan como fabricas en la síntesis de múltiples
compuestos esenciales de las plantas. La diferenciación del cloroplasto
depende de señales nucleares coordinadas a la diferenciación del tejido
fotosintético. Sin embargo, los plastidos también generan señales
retrógradas con que transmiten su estado metabólico y de diferenciación
alterando la expresión de nuclear y regulando el desarrollo y las respuestas
de la planta. Se reconocen múltiples vías de señalización retrógrada, pero
sólo pocas de las señales responsables. Estudios recientes han mostrado que
los carotenoides en adición a su función capatadora de luz y fotoprotectiva,
también sirven como señales regulatorias. La caracterización de la mutante
clb5 de Arabidopsis ha proporcionado evidencias que intermediarios
lineales de carotenoides son capaces de modular la expresión nuclear y
plastídica, así como el desarrollo de la hoja. Nuestro trabajo demuestra
que los fenotipos asociados asociados a clb5 son una consecuencia de
la mutación en la enzima zeta coroteno desaturasa (ZDS), que provoca
la acumulación de los intermediarios fitoflueno y -carotenos, y no se
observan en otras mutantes deficientes de carotenos. El patrón de expresión
de ZDS en la planta apoya a que esta enzima juega un papel importante
durante el desarrollo de la planta. Análisis genéticos y moleculares han
Dolores Córdoba-Cañero, María Isabel Martínez-Macías, Jara
Teresa Parrilla-Doblas, María Isabel Ponferrada-Marín, Teresa
Morales-Ruiz, María Victoria García-Ortiz, Casimiro Barbado,
Iván Devesa, Raphael Ricon, Rafael R. Ariza,Teresa Roldán-Arjona
Departamento de Genética, Universidad de Córdoba (UCO)/Instituto
Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/
Hospital Universitario Reina Sofía, Córdoba, ES
La ruta de Reparación por Escisión de Bases (Base Excision Repair, BER)
es esencial para eliminar lesiones frecuentes del ADN y se ha estudiado
con detalle en modelos animales y microbianos, pero no en plantas. Este
mecanismo de reparación consta de varias etapas y se inicia por la acción
de enzimas denominadas ADN glicosilasas, que eliminan bases dañadas.
El trabajo llevado a cabo en Arabidopsis por nuestro grupo y por otros
laboratorios ha permitido identificar una familia de ADN glicosilasas
específicas de plantas (familia ROS1/DME) que eliminan 5-meticitosina
(5-meC). La 5-meC no es una lesión, sino una marca epigenética que
suprime la expresión génica y desempeña un papel clave en el desarrollo y la
diferenciación celular. Las enzimas de la familia ROS1/DME inician una ruta
de escisión para borrar la metilación del ADN y no tienen equivalentes en
células animales. Son ADN glicosilasas atípicas, anormalmente voluminosas
y con un dominio catalítico discontinuo con residuos que intervienen en
el reconocimiento de la base metilada y su escisión. Tras liberar la 5-meC
estas enzimas rompen el esqueleto azúcar-fosfato, generando un hueco
mono-nucleotídico con un extremo 3´ bloqueado por un fosfato o por
un aldehído insaturado. Una ADN fosfatasa (ZDP) o una endonucleasa
(APE1L) convierten estos extremos en un grupo 3´-OH, permitiendo
así que una ADN polimerasa aún por identificar inserte una citosina no
metilada y una ADN ligasa (LIG1) selle la cadena procesada. En esta ruta de
desmetilación también intervienen proteínas adicionales. En conjunto, estos
resultados demuestran que las plantas usan la ruta BER no solo para proteger
su genoma, sino también para reprogramar su epigenoma.
S2.2. Más allá de los genes: análisis
funcional del resto del genoma /
Beyond the genes functional
analysis of the rest of the genome
S2.2-1
Regulation of genome function: genes versus
structure
Miguel Manzanares
Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid, ES
Recent studies have demonstrated an unexpected link between
chromatin structure and gene activity. The generation of high-resolution
chromatin interaction maps has revealed that the genome folds into
distinct megabase-scale modules termed topologically associated
domains (TADs) that show a direct relationship to gene expression and
regulation within them. CTCF is a ubiquitously expressed and highly
conserved chromatin architectural protein enriched at the boundaries
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of these domains. Most strikingly, TAD organization is mostly stable
among different cell-types and developmental stages independent
of the gene activity status within them, and also shows a surprising
evolutionary conservation in structure. Furthermore, depletion of
CTCF or other chromatin structural proteins does not cause a dramatic
disassembling of the organization of the genome. Altogether, these
observations pose the conundrum of why is the genome organized in
such way, and how does this occur? We are exploring these issues in
different developmental systems to explore how the structuring of the
genome can regulate gene expression.
S2.2-2
Transcriptional regulation of CD69 gene
through the enhancer located in the conserved
non-coding sequence (CNS)2
Pilar Lauzurica, Laura Notario, Teresa Laguna, Miguel G. Fontela,
Isabel Cervera, Jennifer Redondo, Berta Vázquez
Instituto de Salud Carlos III, Majadahonda, Madrid, ES
The CD69 type II C-type lectin is one of the earliest indicators of
leukocyte activation acting in lymphocyte migration and cytokine
secretion. CD69 expression in hematopoietic lineage undergoes rapid
changes depending on the cell-lineage, the activation state or the
localization of the cell where it is expressed, suggesting a complex
and tightly controlled regulation. We have examined the molecular
mechanism underlying mouse CD69 gene transcription in silico, in
vitro and in vivo. Analysis of the CD69 gene revealed evolutionary
conservation at the promoter and at four noncoding sequences (CNS)
that were called CNS1, CNS2, CNS3, and CNS4. These regions were
found to be hypersensitive sites in DNase I digestion experiments, and
chromatin immunoprecipitation assays showed specific epigenetic
modifications. Through in silico studies, we analyzed several regulatory
features of the 4 CNSs, confirming a major function of CNS2 in the
transcriptional regulation of CD69. In addition, multiple transcription
binding sites are identified in the CNS2 region by DNA cross-species
conservation analysis. CNS2 displayed constitutive and inducible
enhancer activity in transient transfection assays in T cells. By functional
approaches we defined a core region of 226bp located within CNS2 as
the main enhancer element of CD69 transcription in the hematopoietic
cells analyzed. However, using a transgenic approach to test CNS
function, we found that the CD69 promoter conferred developmentally
regulated expression during positive selection of thymocytes but could
not support regulated expression in mature lymphocytes. Inclusion of
CNS1 and CNS2 caused suppression of CD69 expression, whereas
further addition of CNS3 and CNS4 supported developmental-stage and
lineage-specific regulation in T cells but not in B cells.
S2.2-3
Using CRISPR tools to functionally analyze
the non-coding genome
Davide Seruggia1, Santiago Josa1, Almudena Fernández López2,
Lluís Montoliu3
1
CNB-CSIC, Madrid, ES, 2CIBERER-ISCIII y CNB-CSIC, Madrid, ES,
3
Centro Nacional de Biotecnología (CNB-CSIC), Madrid, ES
Most part of mammalian genomes is non-coding and contains DNA
regulatory elements that are essential for proper gene expression.
However, these key genetic elements are surrounded by many
repetitive elements, such as retrotransposons, which interfere with
homologous recombination-mediated approaches and, hence, usually
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XXXIX Congreso SEBBM
prevent any direct functional assessment. The recent development
and implementation of genome-editing technologies, notably
CRISPR-Cas, has enabled, for the first time, targeting these small
regulatory elements and allowed their functional evaluation to assess
their relevance in overall gene expression regulation. For many years
we were traditionally using large-genomic size transgenes (i.e. yeast
artificial chromosomes) to assess the function of regulatory elements
of certain experimental gene models. However, this approach had
many limitations, mostly related with the random nature of the
genetic modification, prone to chromosomal position effects. We have
applied CRISPR-Cas9 tools to functionally analyze several key DNA
regulatory elements that we have been characterizing in the mouse
tyrosinase locus. We tested the gene-editing tools in cells first and
we next produced the corresponding genome-edited animals. In this
presentation we will discuss the variety of mutated alleles generated,
the mosaicism detected in these experiments and how we have
been able to segregate most of these alleles in order to analyze the
phenotype of any of them, independently.
S2.3. Papel de los exosomas y carga
exosómica en sinapsis inmune,
cáncer y células neuronales /
Role of exosomes and exosomic
cargo in the immune synapse,
cáncer and neural cells
S2.3-1
Immune Cell-to-Cell communication:
Mechanisms of microRNA and proteins
sorting into exosomes
Francisco Sánchez-Madrid
Intercellular Communication Laboratory, National Center
Cardiovascular Research and Instituto Investigación Sanitaria
Princesa, Universidad Autónoma de Madrid, Madrid, ES
Exosomes are released by most cells to the extracellular environment
and are involved in cell-to-cell communication, mediating transfer of
proteins and RNAs through structures as the Immunological synapse.
Exosomes arising from different cell types show enrichment for
a shared set of specific proteins, including tetraspanins, adhesion
molecules, endosomal proteins, and heat-shock proteins, among others.
They are also very much enriched in RNAs, including miRNAs and
other non-coding RNAs. Exosome composition is not a mere copy
of cytosolic content; rather, specific proteins and nucleic acids are
selectively sorted into exosomes. The amount and content of exosomes
can moreover change in response to stimuli such as cell activation,
hypoxia or cell stress. Such changes in exosome composition determine
the final outcome of exosome-mediated communication. However,
the mechanisms that control these processes are not well understood.
First, we will discuss on the mechanisms of miRNA and protein
sorting into EVs. In this regard, we have identified sequence motifs
present in miRNAs that control their localization into exosomes. The
protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1)
specifically binds exosomal miRNAs through the recognition of these
motifs and controls their loading into exosomes. Moreover, hnRNPA2B1
in exosomes is sumoylated, and sumoylation controls the binding of
hnRNPA2B1 to miRNAs. Second, we will discuss post-translational
modifications of proteins that control exosomal sorting. Our recent data
demonstrate that ISGylation, an ubiquitin-like modification, controls
exosome secretion.
Salamanca 2016
Simposios / Symposia
S2.3-2
S2.3-3
Exosomes and metastasis: the dangerous alliance
Extracellular vesicles as messengers between
neural cells and beyond
Héctor Peinado
Microenvironment and Metastasis Group, Molecular Oncology Program,
Spanish National Cancer Research Centre (CNIO), Madrid, ES
Cancer is a systemic disease, while most of the research effort has been
focused on analyzing the primary tumor, there is a lack of information
on how the tumor microenvironment influences metastasis. Prominent
roles for stromal cells, such as fibroblasts, endothelial cells, lymphatic
endothelial cells, bone marrow-derived cells, soluble factors and secreted
vesicles have been established. Exosomes are secreted vesicles carrying
lipids, proteins, RNA and DNA molecules. By carrying these molecules,
and facilitating their cell-to-cell transfer, exosomes can modulate the
behavior of resident cells that can impact disease progression. Exosomes
can serve as vehicles for horizontal transfer of proteins, RNA and DNA
to the surrounding cells, thus promoting additional modifications in the
tumor and metastatic microenvironments. Our studies in demonstrated
that tumor-secreted exosomes are a major tumor-derived factor that acts
systemically to promote metástasis in a process that we have termed
“education”. Tumor exosomes fuse specifically to stromal cells within
the metastatic microenvironments favoring organotropic metastasis.
Our most recent data demonstrate that exosomes secreted by metastatic
models promote vascular leakiness in the sentinel lymph node favoring
metastatic spread. We found that lymphatic endothelial cells and
subscapular sinus macrophages are the main cell types uptaking exosomes
in the lymph nodes promoting cellular and molecular alterations in the
lymph node microenvironment fostering metastasis. The analysis of
human lymphatic fluid suggest that proteomic cargo and particles are
increased in melanoma patients opening the possibility of the use of
circulating vesicles in lymphatic fluid as biomarkers and as a source of
novel markers in pre-metastatic sentinel lymph nodes that predict relapse
and metastatic potential.
Eva-Maria Krämer-Albers
Department of Molecular Cell Biology, Johannes Gutenberg
University (JGU), Mainz, DE
Exosomes are universally secreted vesicles that mediate cell communication
in multiple tissues, including the CNS. Exosomes deliver a specific set
of biomolecules including proteins, lipids and small non-coding RNAs
modulating the phenotypic behavior of recipient cells.Our recent work revealed
that exosomes, are involved in bidirectional interaction between CNS glia cells
and neurons. Myelinating oligodendrocytes release exosomes in response
to neurotransmitter signalling and activation of glial ionotropic glutamate
receptors. These exosomes are internalized by neurons via endocytosis and the
exosome cargo is functionally recovered by the recipient neurons. Neurons
appear to benefit from exosome internalization by increased resistance to
stress conditions such as oxidative stress, starvation and ischemia indicating
a role of glia to neuron exosome transfer in glial support and neuroprotection.
Oligodendroglial exosomes have the ability to modulate a broad spectrum of
neuronal functions. Treatment of cultured neurons with isolated glial exosomes
affects action potential firing and axonal transport, activates signal transduction
pathways, and regulates neuronal gene expression. We further studied exosome
release from oligodendrocytes derived from PLP- and CNP-deficient mice,
which are characterized by secondary axonal degeneration, and found that
exosome secretion in these mouse models is impaired. Intriguingly, exosomes
derived from knock-out oligodendrocytes lack neuroprotective activity
and the ability to promote axonal transport. In summary, we propose that
oligodendroglial exosomes function as vehicles for the transfer of biomolecules
from oligodendrocytes to neurons and are implicated in neuroprotection and
glial maintenance of axonal integrity.
Supported by DFG.
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Simposio 3.
Estructura y función de biomoléculas /
Structure and function of biomolecules
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Salamanca 2016
S3.1. Dinámica estructural del genoma/
Genome structural dynamics
S3.1-1
Mammalian sperm exhibit priming of embryonic
and adult regulatory landscapes
Víctor Corcés
Department of Biology, O. Wayne Rollings Research Center Emory
University, Atlanta, US
Environmental factors increase the frequency of metabolic disorders.
The mechanisms by which these epiphenotypes are transmitted between
the exposed and subsequent generations through the paternal germline
remains poorly understood. The nuclei of mammalian sperm are highly
condensed, the DNA is mostly covered by protamines with only a few
retained nucleosomes, and epigenetic information stored in the form
of DNA methylation is quickly erased from paternal chromosomes
shortly after fertilization. Experiments carried out in our lab suggest
a more complex picture of mouse sperm, suggesting the presence
of multiple histone modifications, nucleosomes positioned around
transcription start sites and transcription factor binding sites, and the
presence of multiple transcription factors. Most promoters in mouse
sperm contain the elongating form of RNA polymerase II, and are
flanked by several positioned nucleosomes marked by a variety of
active histone modifications. The sperm genome is bound by several
transcription factors, including Nanog, Oct 4, Mediator, condensin,
and the pioneer factor FoxA1. These proteins are found at promoters,
enhancers, and super-enhancers, many of which are active in mESCs
or adult tissues. CTCF and cohesin are also present in sperm DNA,
where they mediate interactions that organize the sperm genome into
domains and compartments that overlap extensively with those found
in mESCs. This information suggests that mammalian sperm contain a
rich and complex palette of epigenetic information that could be altered
by environmental factors to paint novel phenotypic outcomes in the
next generation.
Simposios / Symposia
S3.1-3
Structural dynamics of the breast cancer genome
in response to hormones
Miguel Beato
Centro de Regulación Genómica (CRG), Barcelona Science and
Technology Institute (BIST), Barcelona, ES
Eukaryotic cells decode environmental information via receptors and
signalling networks that converge in the cell nucleus to adjust an integrated
gene expression response. We study the response of breast cancer cells to
progestins (Pg) acting via the progesterone receptor (PR) to decipher the
underlying molecular mechanisms.
The organization in nucleosomes of the DNA sequences recognized
by PR is a requisite for the initiation of chromatin remodelling leading
to displacement of histones H1 and H2A/H2B. Remodelling depends
on PR-associated enzymes, including histone modifying enzymes and
ATP-dependent remodelers, as well as activated PARP1 that leads to
accumulation of Poly-ADP-Ribose (PAR) in he cell nucleus. These
epigenetic processes are required for the rapid regulation of thousand of
genes and ultimately for cell proliferation in response to hormone.
In addition to nucleosomes, higher levels of genome organization also
participates in hormone action. The conserved partition of the genome
in consecutive topological associating domains (TADs) contributes to
coordination of the hormonal response. Hormone regulated genes tend
to segregate into TADs that respond as a whole with either activation
or repression of transcription. Thus, TADs behave as “regulons” in the
response of cells to external signals. High-resolution Hi-C data reveal
the role of dominant enhancers in organizing the differential hormonal
response of the TADs.
The extensive chromatin remodelling in response to hormones requires
large amounts of ATP, which is mainly generated in the cell nucleus by
NUDIX5 that uses ADP-Ribose (ADPR) derived from PARG-mediated
hydrolysis of PAR. NUDIX5 is an homodimer that catalyses hydrolysis
of ADPR to AMP and R-5-P. In response to hormone NUDIX5 is
dephosphorylated at T45, leading to a conformational change that
enables the reaction of ADPR with PPi to generate ATP and R-5-P.
NUDIX5 is overexpressed in breast cancers and is a marker for
poor prognosis. Thus, it represents a novel target for breast cancer
management.
S3.1-2
Genome architecture mapping of multi-enhancer
contacts in embryonic stem cells
Ana Pombo
Berlin Institute for Medical Systems Biology, Max Delbrück Center
for Molecular Medicine (MDC), Berlín, DE
S3.2. Formación y funcionamiento
de los ribosomas / The synthesis
and function of ribosomes
S3.2-1
The organization of the genome in the nucleus and the interactions of
genes with their regulatory elements are key features of transcriptional
control and their disruption causes disease. Technologies based on
chromosome conformation capture (3C) have profoundly expanded
our understanding of the role of genome architecture in gene
regulation. We introduce Genome Architecture Mapping (GAM),
a novel genome-wide method for measuring three-dimensional
chromatin topology. Exploration of the most prominent chromatin
contacts detected in mouse ES cells using GAM identifies most
specific chromatin contacts between active genes and enhancers
across very large genomic distances. GAM also reveals abundant
three-way contacts genome-wide, especially between the enhancers
most highly occupied by pluripotency transcription factors and
highly transcribed genomic regions.Our results highlight a previously
inaccessible complexity in genome architecture and a major role for
contacts related with gene expression in the structural organisation of
the genome in mammalian nuclei.
Ribosome assembly and functions of ribosomal
proteins
Jesús de la Cruz
Instituto de Biomedicina de Sevilla, Universidad de Sevilla,
Sevilla, ES
Ribosomes are highly complex ubiquitous macromolecular assemblies
that are responsible for the synthesis of all cellular proteins from mRNA
templates. In all cells, ribosomes are composed of two ribosomal subunits,
the large one about twice the size of the small one. In eukaryotes, biogenesis
of cytoplasmic ribosomes is a complex process that takes successively place
in the nucleolus, nucleoplasm and cytoplasm. The speed, directionality and
accuracy of this process depend on a myriad of small nucleolar RNAs and
protein trans-acting factors. Functional analyses have also demonstrated
that many ribosomal proteins directly contribute to the maturation and
nucleo-cytoplasmic transport of pre-ribosomal particles. The principles
governing the assembly of ribosomal proteins and how these molecules
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Pósters / Posters
assemble together remain challenging questions, especially in eukaryotes.
Our laboratory is interested in understanding the role of ribosomal
proteins, mainly from the large subunit, in ribosome biogenesis and
function. In this presentation, I provide details on how selected ribosomal
proteins contribute to these processes. Moreover, many evolutionarily
conserved ribosomal proteins possess extra eukaryote-specific aminoor carboxy-terminal extensions and/or internal loops. I will also
discuss on an emerging interesting question in our field, the functional
significance of these extensions for the proper assembly and function
of ribosomes.
S3.2-2
The activity of the ribosome synthesis pathway
during proliferation. Causes and consequences of
nucleolar stress
Mercedes Dosil
Departamento de Bioquímica y Biología Molecular y Centro de
Investigación del Cáncer, Universidad de Salamanca, Salamanca, ES
Ribosome synthesis takes place within the most prominent nuclear
substructure, the nucleolus. It is a multistep process that entails
pre-rRNA transcription, pre-rRNA processing, and assembly of
ribosomal proteins imported from the cytoplasm. These activities
require the participation of more than 200 trans-acting factors. The
majority of studies on ribosome formation have been performed in
yeast, and it has long been assumed that most features are similar in
humans. However, although the basic aspects are conserved, there are
many maturation events, and links to other processes, that are unique
of higher eukaryotes. One such link is the nucleolar stress response,
which is mediated by p53, and is triggered by alterations of ribosome
synthesis. This response is receiving intense attention because it
is the cause of various human diseases known as ribosomopathies.
Despite this growing interest, progress in understanding ribosome
synthesis in humans is greatly hampered by technical limitations.
Powerful approaches used in yeast, such as genetic screens to
identify trans-acting factors or purification of pre-ribosomes by
tandem-affinity chromatography, are not possible in human cells. In
our laboratory we have optimized a series of techniques to monitor
the formation of different pre-ribosomal complexes in human cells.
We are studying normally-growing cells and cells blocked at specific
steps of ribosome maturation. Data will be shown on the defects in
ribosome synthesis that occur in Diamond-Blackfan anemia and on
the mechanisms underlying p53 activation in this ribosomopathy.
S3.2-3
The function of ribosomes at the structural level.
Cryo-electron microscopy analysis of translating
ribosomes
Mikel Valle
Unidad de Homeostasis de Proteínas, CIC BioGUNE, Derio, Bizkaia, ES
The ribosome is the large macromolecular complex where translation
takes place. This bio-synthesis of proteins involves numerous steps
and the functioning of ribosomes is modulated by the interaction
with several translation factors. The design of ribosomes into two
subunits is related to their specialized work in reading the mRNA
(small ribosomal subunit) and writing the coded polypeptide (large
ribosomal subunit). The interplay between subunits takes care of the
selection of cognate aminoacyl-tRNAs, and the maintenance of the
reading frame in the mRNA. The presence of structured mRNAs,
the nature of the nascent polypeptide chain, and the involvement of
cellular factors during stress conditions, are some of the elements
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XXXIX Congreso SEBBM
that regulate translation by ribosomes. The highly dynamic ribosomal
structure has been a subject of deep investigation. Cryo-electron
microscopy (cryoEM) has contributed to unveil the structure/function
relationship of the translating ribosome. The latest developments in
cryoEM technique have improved the resolution of the attainable 3D
maps, and there is a large number of ribosomal cryoEM structures
at atomic detail. These advances will be discussed in the context of
ribosomal functioning.
S3.3. Dinámica de la cromatina y
estabilidad genómica / Chromatin
dynamics and genome stability
S3.3-1
Nucleosome positioning in vivo driven
by the DNA sequence
Francisco Antequera
Instituto de Biología Funcional y Genómica, CSIC, Universidad
de Salamanca, Salamanca, ES
Nucleosomes facilitate the packaging of the genome inside the nucleus
and modulate the access of regulators to the DNA molecule. Most of
the yeast genome is organized in a pattern of positioned nucleosomes
that is stably maintained under different genetic backgrounds and
physiological conditions. While the contribution of chromatin
remodelers and DNA binding proteins to maintain this organization
is well established, the relevance of the DNA sequence to nucleosome
positioning in the genomic context remains controversial. We have
searched for sequence determinants associated with positioned
nucleosomes in several species of fission and budding yeasts. We
have found that the distribution of the four mononucleotides along
mononucleosomal DNA follows a well-defined pattern. Despite the
high degree of histone conservation, these nucleosomal signatures are
species-specific and are present in coding and non-coding regions. In
the case of open reading frames, they correctly predict the relative
distribution of codons on mononucleosomal DNA and establish a direct
connection between the position of each codon around the histone
octamer and protein composition. High-resolution mutagenenesis
of mononucleosomal DNA reveals that sequence changes alter the
nucleosomal pattern at the level of individual nucleosomes suggesting
the existence of sequence elements contributing to positioning.
Functional analyses of these elements show that it is possible to
target nucleosomes to specific positions on natural or synthetic DNA
molecules in the genomic context.
S3.3-2
The importance of the mitotic spindle integrity
in DNA repair
Facundo Ramos, María Teresa Villoria, Encarnación Dueñas,
Andrés Clemente-Blanco
Instituto de Biología Funcional y Genómica, CSIC, Universidad
de Salamanca, Salamanca, ES
Maintenance of genome integrity is a vital aspect of our cellular
physiology. Our hereditary information encoded in the DNA is
constantly threatened by both endogenous and environmental
genotoxic stresses that could alter our genomic material. To combat
this threat, eukaryotic cells have evolved a series of mechanisms,
collectively known as DDR, to survey several features of the cellular
response, including the detection of the lesion, a transient cell cycle
Salamanca 2016
arrest and the repair of the broken DNA. During the last years it
has becoming clearer the biochemical mechanisms operating at
each stage of the DDR. However, little is known about the spatial
regulation of their components during the DNA damage response
and how the relocation of the DNA lesion itself can influence in the
repair process. Interestingly, previous studies have indicated that
DNA breaks are re-localized from the nucleoplasm to the nuclear
periphery, suggesting that nuclear compartmentalization of DNA
lesions could comprise another layer in the regulation of the DNA
repair pathway. Nevertheless, whether the nuclear periphery harbours
an environment that is permissive for DNA repair and its implications
in maintaining genome integrity is a subject that still under debate.
Remarkably, new data coming from our group indicate that the cell
cycle phosphatase Cdc14 is involved in DNA repair by controlling
the tethering of a DNA lesion into the spindle pole body (SPB) region
of the nuclear envelope. This function is attained by preserving the
integrity of the metaphase spindle, a vital requirement to stimulate
DSB-SPB interaction and thus DNA repair. Accordingly, disruption
of spindle stability impairs both DSB-SPB interaction and DNA
repair by homologous recombination. These observations directly
connect spindle integrity with DNA repair and reveal that DSBs are
preferentially tethered to the SPBs to be restored. Importantly, this
new function of Cdc14 could provide a physiological mechanism that
spatially regulates the DNA damage response and therefore the fate of
the repair process.
Simposios / Symposia
S3.3-3
Cohesin functions in chromosome architecture
and inheritance
Ana Losada
Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ES
Cohesin is a ring-shaped complex that entraps the sister chromatids from
the time they emerge from the replication fork until their separation in
anaphase, thereby ensuring both faithful chromosome segregation and
accurate DNA repair by homologous recombination. Cohesin plays also an
important role in genome compartmentalization. It promotes long-range
DNA looping which contributes to transcriptional regulation, organization
of DNA replication factories and locus rearrangement by recombination.
Recently, mutations in cohesin genes have been identified in a number of
human cancers. Understanding the contribution of cohesin mutations to
tumourigenesis requires a deep knowledge of the multiple functions of the
complex. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21
and either SA1 or SA2. Cohesion factors Pds5, Wapl and Sororin bind cohesin
and modulate its interaction with chromatin. There are also two versions of
Pds5 in vertebrates, Pds5A and Pds5B, and both interact with cohesin-SA1
and cohesin-SA2. In order to investigate the functional specificity of all these
different cohesin complexes both at the cellular level and in the context of an
organism, we have generated mouse models carrying knock out alleles for the
variant subunits SA1, SA2, Pds5A and Pds5B. I will discuss what we have
learnt from the characterization of these mice.
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Posters
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Salamanca 2016
Pósters / Posters
Las comunicaciones libres aceptadas al XXXIX Congreso SEBBM se muestran por grupos científicos. El doble código R indica que
el póster ha sido seleccionado como comunicación oral en una reunión de grupo científico. Las letras “r” y “m” tras el código
de la comunicación indican, respectivamente, que optan al Premio Roche al mejor panel y al Premio Lilly–Margarita Lorenzo.
El Libro de resúmenes on-line, en formato buscador, está disponible en la red y para smartphone y Tablet en:
http://www.sebbm.com/xxxixcongreso/
Además de este listado, existe el Libro de resúmenes impreso que se entrega a los congresistas que lo solicitaron en el
momento de formalizar su inscripción.
P01. Apoptosis / Apoptosis
P01-1
Lipid raft platforms as major scaffolds
in regulating apoptotic signaling
Faustino Mollinedo, Consuelo Gajate
Laboratory of Cell Death and Cancer Therapy, Department of Cellular
and Molecular Medicine, Centro de Investigaciones Biológicas,
Consejo Superior de Investigaciones Científicas (CSIC), Madrid, ES
Membrane lipid rafts are highly ordered membrane domains enriched
in cholesterol, sphingolipids and gangliosides, which form specialized
domains with distinct composition and physical properties. Rafts provide
an additional level of compartmentalization, serving as sorting platforms
and hubs for recruitment of distinct signaling processes. We found
that the ether phospholipid edelfosine, considered as the prototype of a
family of compounds collectively known as alkylphospholipid analogs
with interesting biomedical activities, showed affinity for cholesterol
and accumulated in lipid rafts in a number of malignant hematological
cells. This led to an efficient in vitro and in vivo antitumor activity by
inducing translocation of Fas/CD95 death receptor as well as downstream
signaling molecules to rafts, as assessed by different approaches,
including microscopy techniques. Then, additional antitumor drugs were
shown to act, at least in part, by recruiting death receptors in lipid rafts.
The partition of death receptors together with downstream apoptotic
signaling molecules in membrane rafts led us to postulate the concept
of a special liquid-ordered membrane platform coined as “cluster of
apoptotic signaling molecule-enriched rafts” (CASMER). CASMERs act
as scaffolds for apoptosis signaling compartmentalization, facilitating
and stabilizing protein-protein interactions by local assembly of
cross-interacting molecules, thus leading to apoptosis. This process also
occurs under physiological death receptor activation. The fact that raft/
death receptor-mediated apoptotic activation can be pharmacologically
promoted, exacerbating a physiological process, opens up a new way to
modulate apoptosis with interesting biomedical applications.
P01-2
Killing cancer cells safely. The new antitumoral
drug ABTL0812 induces autophagy-mediated
cancer cell death by a novel and drugable
mechanism of action
José Miguel Lizcano de Vega
Protein Kinases & Signal Transduction Lab Unitat de Medicina,
Dept. Bioquímica i Biologia Molecular & Institut de Neurociències.
Universitat Autònoma de Barcelona, Bellaterra, ES
ABTL0812 is a novel first-in-class, small molecule which showed
antiproliferative effects on tumor cells in phenotypic assays. Here we
described the mechanism of action of this antitumoral drug, which is
currently in clinical development. We used two human cancer paradigms,
lung and pancreatic adenocarcinoma (A549 and MiaPaCa-2 cells,
respectively). ABTL0812 impairs cancer cell proliferation and tumor growth
in animal models (tumor xenografts), resulting in autophagy-mediated
cancer cell death, without affecting non-tumoral cells. No signs of
apoptosis were observed, and autophagy-deficient cells (Atg5-/-) were
resistant to ABTL cytotoxicity. Importantly, ABTL0812 synergizes with
standard fist-line chemotherapy (docetaxel and gemcitabine) to induce
cancer cell death and tumor growth reduction in animal models.
We identified the primary target of ABTL0812 by in silico high-throughput
screening, comparing ABTL0812 structure against ChEMBL15 database.
ABTL0812 binds and activates Peroxisome Proliferator-Activators
Receptors (PPAR) alpha and gamma, resulting in upregulation of Tribbles-3
pseudokinase (TRIB3) gene expression. Upregulated TRIB3 binds cellular
Akt, which induces suppression of the Akt/mTORC1 axis. Pharmacological
inhibition of PPARs or TRIB3 silencing prevents ABTL0812 cytotoxicity.
ABTL0812 induces Akt inhibition in cancer cells, human tumor xenografts,
and peripheral blood mononuclear cells from patients enrolled in phase
I/Ib fist-in-human clinical trial. Thus, ABTL0812 has a unique and novel
mechanism of action, that induces autophagic cancer cell death and
defines a new and drugable cellular route: PPARs-TRIB3-Akt-mTORC1.
Furthermore, the low toxicity and high tolerability support further clinical
development of ABTL0812.
P01r-3
Temozolomide does not improve
γ-irradiation-triggered caspase-dependent cell
death in glioblastoma-derived human cells
Laura Martínez-Escardó1, María Sánchez-Osuna1, Pilar Baldominos1,
Jordi Bruna2, Victor J. Yuste1
1
Cell Death, Senescence & Survival Research Group, Dept.
Bioquímica y Biología Molecular - Instituto de Neurociencias,
Facultad de Medicina, Universitat Autònoma de Barcelona
(UAB), Cerdanyola del Vallès, ES, 2Neuro-Oncology Unit, Hospital
Universitari de Bellvitge - Institut Català d´Oncologia (ICO),
Duran i Reynals, L’Hospitalet de Llobregat, ES
Despite treatment with the most rigorous surgical interventions, along with
radio-chemotherapy regimens, glioblastoma (GBM) continues to rank
amongthe most aggressive human cancers. Although radio-chemotherapy
has improved the overall survival of GBM-affected patients up to ~14
months, almost all of them suffer recurrences. Previous results from
our group demonstrated that after a broad panel of cytotoxic stimuli,
GBM cells undergo caspase-dependent cell death without showing both
oligonucleosomal DNA degradation and apoptotic nuclear morphology.
Therefore we hypothesize that the refractoriness of this kind of tumors
towards the current treatment could be due to an impairment of GBM cells
to undergo canonical apoptotic cell death. With this aim we decided to
characterize the biochemical and morphological changes that GBM cells
suffer when exposed to the standard combination employed in the clinics,
which combines γ-irradiation and the alkylating agent temozolomide. In
37
Pósters / Posters
this sense, γ-irradiation triggered caspase-dependent cell death and partially
provoked the appearance of apoptotic nuclear morphologies. On the other
hand, GBM-derived cells cultured in the presence of high concentrations
of temozolomide were unable to undergo cell death. Unexpectedly, the
cytotoxic effect by γ-irradiation was not enhanced when temozolomide
was added to the culture media. Although preliminary, our results suggest
that the cytotoxic effect of the current standard treatment could be mainly
due to radiotherapy instead of temozolomide or their combination.
L.M.-E. holds a predoctoral contract (Personal Investigador en Formació)
from Universitat Autònoma de Barcelona. P.B. is a M.Sc. student in
Advanced Immunology from Universitat de Barcelona and Universitat
Autònoma de Barcelona. V.J.Y. is a Doctor Distinguished Researcher
under a “Retention of Research Talent” contract from “Programa Banco
de Santander”.
This work has been supported by MINECO/FEDER (SAF2012-31485),
Generalitat de Catalunya, AGAUR, Grups de Recerca Consolidats
(2014SGR-1609) and Centro de Investigación Biomédica en Red sobre
Enfermedades Neurodegenerativas (CIBERNED).
P01-4
Cytochrome P450 induction in SH-SY5Y
cells and its effect on neurotoxicity promoted
by xenobiotics
Jesús Fernández Abascal, Massimo Valoti
University of Siena, Siena, IT
Background: Cytochrome P-450 (CYP) is involved in the oxidative
metabolism of both endogenous and exogenous compounds such as
drugs, pesticides, or hormones. Some CYP isoforms are inducible by
β-naphthoflavone (β-N), phenobarbital and ethanol in the rat (Raza and
Avadhani 1988); and they can be found in brain mitochondria, leading to
a potential toxicity generated by several apoptotic mechanisms (Boopathi,
Anandatheerthavarada et al. 2000). An important question is whether their
induction would promote neurotoxicity due to activation of endogenous
substrates, or whether induction would lead to a more sensitive system to
exogenous toxins. This metabolic activity may be related with promotion
of Parkinson Disease (PD). In fact, some in vitro studies indicate that CYP
is able to protect cells against MPP+ toxicity (Mann and Tyndale 2010).
This is an important regard of the neurodegenerative process as the
mechanisms involved in PD are thought to be multifactorial in nature.
Hence, the study of how CYP-induction may predispose the brain to
subsequent damage caused by exogenous toxins is significant. In addition,
knowledge of CYP metabolism of neuroprotective compounds in the
dopaminergic neurons is important for treatment of PD patients.
Aim: The main goal of this project is to elucidate any involvement of CYP
in mitochondrial impairment and any subsequent damage. The induction
of CYP isoforms in SH-SY5Y cells and their potential effect upon
neurotoxicity of several xenobiotics has been studied.
Methods: Undifferentiated neuroblastoma cells has been treated with
various CYP inducers: β-N, cyclophosphamide (CPA), ethanol and
acetaminophen. Western blot and qRT-PCR were used to identify the
expression of different CYP isoforms. Cell viability was recorded by
colorimetric assay: pre-incubation for 24 hours with each inducer alone,
then either co-incubation of the same inducer and a toxin (Rotenone,
Paraquat or MPP+), or incubation just with the toxin alone. A study of
apoptotic population was carried out by fluorescent-activated cell sorting
(FACS); and the mitochondrial membrane potential was measured to
identify the early apoptotic cells.
Results: Cyclophosphamide (CPA) showed to induce the isoforms 2D6,
2E1, and 1A1. β-Naphtoflavone (β-N) increased the expression of CYP 2E1
and 1A1. Ethanol increased the expression of CYP 2E1. Acetaminophen
only increased the 2E1 isoform. The MTT assay showed β-N to partially
neuroprotect cells against MPP+ toxicity only when the induction of
38
XXXIX Congreso SEBBM
CYP was maintained during the treatment with the toxin. The analysis of
apoptotic population by FACS supported this results, showing a decrease
of late apoptotic cells. However, preliminary results from mitochondrial
membrane potential measurement indicate that early apoptotic cell
populations remain equal.
Conclusions: CYP isozymes are inducible by different compounds in
the SH-SY5Y cells, and are involved in cell survival against toxicity of
inhibitors of Complex I of electron transport chain. These results could
provide a new insights in the development of PD and the metabolism of
dopaminergic cells. Other experiments will be done in order to study the
relationship between neurotoxins and CYP, either in undifferentiated or
RA-differentiated SH-SY5Y cells.
Acknowledgment: The presented research is granted by TINTIN - a Marie
Curie ITN funded project (2013-17). GA: 608381.
References
[1] Boopathi, E., H. K. Anandatheerthavarada, S. V. Bhagwat, G. Biswas,
J. K. Fang and N. G. Avadhani (2000). “Accumulation of mitochondrial
P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat
brain during chronic treatment with beta-naphthoflavone. A role in the
metabolism of neuroactive drugs.” J Biol Chem 275(44): 34415-34423.
[2] Mann, A. and R. F. Tyndale (2010). “Cytochrome P450 2D6 enzyme
neuroprotects against 1-methyl-4-phenylpyridinium toxicity in SH-SY5Y
neuronal cells.” Eur J Neurosci 31(7): 1185-1193.
[3] Raza, H. and N. G. Avadhani (1988). “Hepatic mitochondrial cytochrome
P-450 system. Purification and characterization of two distinct forms of
mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat
liver.” J Biol Chem 263(19): 9533-9541.
P01-5
Estudio de la correlación entre la expresión
de la enzima α(1,6)fucosiltransferasa y la
proteína DR4 de la vía de proapoptosis celular
TRAIL/DR4 en las líneas de cáncer colorrectal
SW480/SW620 y validación en una cohorte
de pacientes
Rubén López-Cortés1, Elsa Núñez Rodríguez1, Laura Muinelo
Romay2, Emilio Gil Martín1, Almudena Fernández Briera1
1
Grupo BB1, Dpto. Bioquímica, Genética e Inmunología,
Universidad de Vigo, Vigo, ES, 2Grupo de Oncología Médica
Traslacional, CHUS, Santiago de Compostela, ES
DR4 (Death Receptor 4) es una glicoproteína proapoptótica de la que
se sospecha que está core-fucosilada. Estudios previos de nuestro grupo
investigador han encontrado indicios de correlación inversa entre los niveles
de expresión de la alfa(1,6)fucosiltransferasa (FucT-8), enzima responsable
de la core-fucosilación, y la expresión de DR4. Nuestro trabajo actual
pretende profundizar en esta observación inicial. Usando como modelo
celular las líneas isogénicas SW480 y SW620, junto con sus respectivos
clones shRNA atenuados para la expresión de FucT-8, hemos logrado
demostrar la up-regulation de DR4 en los clones knocked-down de ambas
líneas celulares. La inducción de apoptosis con TRAIL (Tumor Necrosis
Factor-Related Apoptosis Inducing Ligand), por otra parte, condujo a una
mayor expresión de FucT-8, mientras, sin embargo, la expresión de DR4
se mantuvo inalterada. En SW480 todos los clones (silvestre, control y
knocked-down) mostraron fragmentación de las caspasas iniciadoras Casp8
y Casp9, pero la mayor transducción de la señal apoptótica se halló a través
de las caspasas efectoras Casp3, Casp6 y Casp7 en los clones atenuados. El
perfil de expresión de las caspasas en SW620 reflejó el bloqueo de la vía
mitocondrial descrito en esta línea. Otro foco de nuestra atención ha sido la
vía de supervivencia celular MAPK/ERK, que se activa por fosforilación
del transductor ERK1/2 (pERK1/2). Los clones knocked-down de SW480
mostraron un nivel de expresión de pERK1/2 mayor que los control pero
Salamanca 2016
insensible a la estimulación con TRAIL. Esta diferencia no se observó en
SW620. Para validar la correlación entre FucT-8 y DR4 se recurrió al análisis
de fracciones de membrana procedentes de una cohorte de pacientes con
cáncer colorrectal. Los especímenes de tejido tumoral mostraron mayores
niveles de expresión de FucT-8, y menores de DR4, que los de tejido sano,
lo que refuerza los indicios de partida sobre la evolución inversa de la
expresión de ambas proteínas en el cáncer colorrectal.
Estudio financiado mediante el Contrato-Programa CN2011/024, Xunta de
Galicia. RLC es contratado FPU12/03662 del MECD.
P01-6
Las caspasas 3 y 6 participan en la proteólisis
de Tau y en la apoptosis inducidas por estrés
hiperosmótico en células de neuroblastoma
humano SH-SY5Y
Marta Olivera Santa-Catalina1, Montaña Caballero Bermejo1,
Ricardo Argent1, Juan Carlos Alonso1, Francisco Centeno2, María
Jesús Lorenzo1
1
Departamento de Bioquímica y Biología Molecular y Genética.
Facultad de Veterinaria. Universidad de Extremadura, Cáceres, ES,
2
Departamento de Bioquímica y Biología Molecular y Genética.
Facultad de Ciencias. Universidad de Extremadura, Badajoz, ES
La proteína asociada a microtúbulos Tau tiene como función principal
la regulación de la dinámica de los microtúbulos. Este proceso está a
su vez regulado por las modificaciones postraduccionales que sufre la
proteína Tau, como la proteólisis. Esta modificación aberrante promueve
la formación de filamentos y agregados de la proteína (NFT, del inglés,
neurofibrilary tangles), que llevan a la muerte de las neuronas. Entre las
proteasas responsables de la proteólisis de Tau destacan las caspasas, que
también juegan un papel fundamental en la apoptosis celular. El objetivo
del presente trabajo fue estudiar el efecto del estrés hiperosmótico inducido
por sorbitol sobre la proteólisis de Tau. Además, se analizó la posible
relación entre la activación de caspasas y apoptosis observada bajo estas
condiciones. Células de neuroblastoma humano SH-SY5Y se incubaron con
sorbitol durante diferentes periodos de tiempo y la fragmentación de Tau y
la activación de las caspasas 3 y 6 fueron analizadas mediante Western blot.
El estrés hiperosmótico promovió la proteólisis de Tau y la activación de
ambas caspasas. Usando inhibidores farmacológicos específicos, se detectó
que la proteólisis de Tau tenía lugar en el extremo amino-terminal y que las
caspasas 3 y 6 participaron en dicha fragmentación. De forma paralela, el
sorbitol indujo apoptosis en las células tratadas, proceso en el que también
están involucradas las caspasas. Nuestros resultados sugieren una posible
relación entre proteólisis de Tau y apoptosis celular en condiciones de
estrés hiperosmótico.
Trabajo financiado por los proyectos: BFU2007-67577-C02-02/BMC,
PRIS10018, GRU10046 y GR15164 FEDER-Junta de Extremadura.
P01-7
Células madre amnióticas epiteliales: cambios
en su proliferación y apoptosis durante su
diferenciación hepática
Rodrigo Riedel1, Antonio Pérez Pérez2, Bernardo Maskin3, Mariana
Jaime3, Ornella Parolini4, Víctor Sánchez Margalet2, Cecilia Varone1,
Julieta Maymó1
1
IQUIBICEN-CONICET-Depto. de Química Biológica, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Buenos Aires, AR, 2Depto. de Bioquímica Médica y Biología
Molecular, Universidad de Sevilla, Sevilla, ES, 3Hospital Nacional
Alejandro Posadas, Buenos Aires, AR, 4Centro di Ricerca E. Menni
-Fondazione Poliambulanza- Istituto Ospedaliero, Brescia, IT
Pósters / Posters
Las células madre aisladas de la placenta y sus membranas fetales, representan
una atractiva e importante alternativa para la medicina regenerativa, dadas
las ventajas que poseen respecto a otras células madre: el fácil acceso a
los tejidos, un uso que no genera debate ético y un potencial ilimitado para
proveer células. Las células madre amnióticas epiteliales (hAEC), aisladas
del amnios de la placenta humana a término, son pluripotentes, expresan
marcadores embrionarios, no son tumorigénicas y poseen propiedades
inmunomoduladoras. Estas características posicionan a las hAEC como
ideales candidatas para su utilización en la medicina regenerativa. La
falla hepática es una de las mayores causas de morbilidad y mortalidad
en el mundo; y si bien el trasplante representa la mejor manera de tratar la
enfermedad hepática crónica y aguda, existen varios obstáculos al respecto.
Las hAEC han sido propuestas como fuente alternativa de hepatocitos, dado
su capacidad de diferenciación hepática. El objetivo del presente trabajo
fue estudiar algunos aspectos de la proliferación y apoptosis de las hAEC,
durante su diferenciación hepática temprana y tardía. La diferenciación se
llevo a cabo durante 30 días con factores específicos (EGF + dexametasona)
o con medio condicionado de la línea celular hepática HepG2 (MC).
Previamente hemos determinado que las hAEC adquieren morfología
hepática y expresan marcadores específicos hepáticos (α-fetoproteína,
α1-AT, albúmina, CYP3A4, CYP7A1), luego del tratamiento con factores.
Determinamos que el EGF incrementó significativamente la proliferación
y la viabilidad de las hAEC medidas por incorporación de 3H-T y ensayo
de MTT, respectivamente. Se produjo un aumento en la expresión de Ki-67
y una disminución de Caspasa-3 clivada y PARP clivada, determinadas por
IF y WB. El tratamiento con MC produjo el efecto contrario. Los resultados
obtenidos contribuyen a la comprensión de los mecanismos celulares y
moleculares que ocurren durante la diferenciación hepática de las hAEC.
P01r-8
Impaired function of the WD40 domain of
ATG16L1 caused by the T300A Crohn’s disease
risk polymorphism
Inmaculada Serramito Gómez, Felipe Xosé Pimentel Muiños
Instituto de Biología Molecular y Celular del Cáncer (IBMCC),
Salamanca, ES
A coding allele of human ATG16L1 (rs2241880; T300A) increases the risk
of Crohn’s disease, but the underlying molecular defects introduced by this
mutation are not fully understood. We show that T300A alters the ability of the
C-terminal WD40-repeat domain of ATG16L1 to interact with an amino acid
motif that recognizes this region. This alteration impairs the unconventional
autophagic activity of TMEM59, a transmembrane protein that contains the
WD40 domain-binding motif, and disrupts its normal intracellular trafficking
and ability to engage ATG16L1 in response to bacterial infection. Notably,
these defects are independent of ATG16L1 T300A caspase cleavage. In
addition, TMEM59-induced autophagy is blunted in cells expressing
the fragments generated by caspase 3 cleavage of the risk allele, whereas
canonical autophagy remains unaffected. These results suggest that the
T300A polymorphism alters the function of motif-containing molecules
that engage ATG16L1 through the WD40 domain, either by influencing this
interaction under non-stressful conditions or by inhibiting their downstream
autophagic signalling after caspase-mediated cleavage.
P01r-9
Efecto protector de la leptina en explantos
placentarios sometidos a un pH ácido
Antonio Pérez Pérez1, Ayelen Rayen Toro2, Teresa Vilariño-García3,
Julieta Maymó2, Cecilia Varone2, Víctor Sánchez-Margalet3
1
Hospital Universitario Virgen Macarena, Sevilla, ES, 2Departamento
de Química Biológica, FCEN-UBA. IQUIBICEN, CONICET, Buenos
Aires, AR, 3Departamento de Bioquímica Médica, Biología
Molecular e Inmunología, Universidad de Sevilla, Hospital
Universitario Virgen Macarena, Sevilla, ES
39
Pósters / Posters
La disminución del intercambio gaseoso a nivel de la placenta es una
complicación grave durante la gestación. La perturbación del pH del medio
interno puede alterar funciones fetales, maternas y también placentarias.
En particular, la disminución del pH puede alterar la homeostasis fetal
provocando daños tisulares irreparables o hasta la muerte fetal por acidosis
metabólica. En este contexto decidimos estudiar el efecto de la disminución
del pH sobre el proceso de apoptosis en explantos placentarios y la acción
de la leptina bajo dichas condiciones. Basándonos en antecedentes previos
de nuestro grupo de investigación y en el rol central de p53 en la regulación
de la apoptosis, hipotetizamos que los cambios de pH en las células
placentarias aumentan la apoptosis alterando la expresión de p53 así como
también la de sus genes diana. En este contexto nos planteamos estudiar
un posible efecto antiapoptótico de la leptina en células trofoblásticas
sometidas a diferentes pH.
Explantos de placenta humana a término fueron incubados en medio
DMEM F-12 a diferentes pH (7,4, 7 y 6,8) en presencia o ausencia de
leptina 10 nM. Se analizaron por Western blot y qRT-PCR los niveles
de p53 y sus efectores p21, BAX y BCL-2, así como también el
fragmento derivado por rotura de PARP-1 y la forma activa de caspasa-3.
Encontramos un aumento en la fosforilación (residuo Ser 46) de p53,
así como de la expresión de p21, PARP-1 y la activación de caspasa-3
en los explantos incubados a pH 7 y 6,8. Por el contrario, estos efectos
fueron atenuados en presencia de leptina 10 nM tanto a pH 7 como a pH
6,8. La relación BCL-2/BAX disminuyó a pH 7 y 6,8 en comparación
con explantos incubados a pH 7,4, efecto que se contrarrestó por el
tratamiento con leptina. Estos datos demuestran un potencial efecto
antiapoptótico de la leptina que involucra el eje de regulación de p53 en
explantos placentarios cultivados a pH ácidos, sugiriendo que la leptina
placentaria posee un efecto protector contra el daño generado por el pH
acido en células trofoblásticas.
P01-10
Characterization of the release of histones
in glioblastoma-derived cells after cytotoxic
insult and its relationship with the apoptotic
endonuclease DFF40/CAD
Pilar Baldominos, Laura Martínez-Escardó, Víctor J. Yuste
Cell Death, Senescence & Survival Research Group, Dept.
Bioquímica y Biología Molecular - Instituto de Neurociencias,
Facultad de Medicina, Universitat Autònoma de Barcelona (UAB),
Cerdanyola del Vallès, Bellaterra, ES
Among primary brain tumours, glioblastoma (GBM) is the most lethal
and the one with highest prevalence. Conventional therapy involves
a combinatorial strategy of surgical resection, chemotherapy with
temozolomide and irradiation. However, the benefits of the current
approach are negligible and the overall survival of patients is prolonged,
at beast, for no more than 16 months. Our group has previously
described the resistance of glioblastoma-derived cells to chemotherapy
and their inability to complete the apoptotic cell programme. Histones
are DNA binding proteins that have been described to be released to
the extracellular milieu during apoptosis. In these conditions, histones
are recognized by receptors of the toll-like receptor family from innate
immune system cells, so they are characterized as DAMPs (damage
associated molecular patterns) molecules. Here, we have studied the
ability of glioblastoma-derived cells to liberate histones after challenged
with different cytotoxic stimuli, with the immunological implications it
might have. Moreover we have characterized the relationship between
the release of histones and the endonuclease DFF40/CAD. Previous
studies of our group showed DFF40/CAD is essential for finishing the
apoptotic programme and it is down-regulated in glioblastoma-derived
cells.
40
XXXIX Congreso SEBBM
Work supported by “Ministerio de Economía y Competitividad (MINECO)/
Fondo Europeo de Desarrollo Regional (FEDER)” Grant SAF2012-31485,
and by “Generalitat de Catalunya” Grant SGR2009-346.
P.B. performed this work under the Final Master Thesis Work of the
interuniversitary MSc in Advance Immunology from “Universitat de
Barcelona” and “Universidad Autónoma de Barcelona”.
P01r-11
The new antitumor drug ABTL0812 induces
autophagy-mediated cell death in human
glioblastoma cell lines
Pau Muñoz-Guardiola1, Valeria Rizzuto2, Tatiana Erazo2, Nora
Diéguez2, Rubén Melgarejo2, Elisabet Megías2, Carles Domènech3,
José Miguel Lizcano2
1
Protein Kinases & Signal Transduction Lab. Unitat de Medicina,
Dept. Bioquímica i Biologia Molecular & Institut de Neurociències.
Universitat Autònoma de Barcelona (UAB); Ability Pharmaceuticals
SL. Campus UAB, Tiana, ES, 2Protein Kinases & Signal
Transduction Lab. Unitat de Medicina, Dept. Bioquímica i Biologia
Molecular & Institut de Neurociències. Universitat Autonoma de
Barcelona (UAB), Bellaterra, ES, 3Ability Pharmaceuticals SL.
Campus UAB, Bellaterra, ES
ABTL0812 is a first-in-class molecule with antitumor activity which has
successfully passed phase I/Ib clinical trials in patients with advanced solid
tumors. ABTL0812 is a polyunsaturated fatty acid derivativewhich induces
cell death in a broad panel of cancer cell lines, without affecting cell viability
of non-tumoral cells. ABTL0812 inhibits Akt by upregulating expression
of the pseudokinase TRIB3, leading to suppression of the Akt-mTORC1
axis and autophagy-mediated cancer cell death. ABTL0812 do not activate
apoptosis in any of the cancer cell lines tested. ABTL0812 also inhibits
tumor growth of lung and pancreatic xenograft models, with a similar
efficacy to that of gold-standards-of-care docetaxel and gemcitabine.
Here, we present preliminary results on the effect of ABTL0812 treatment
in two human glioblastoma cell lines, LN18 and U87-MG. Glioblastoma
multiforme is the most common and lethal primary malignant brain
tumor, and the therapeutic strategies are few and poor. Aggressiveness
of glioblastoma is mostly due to diffuse infiltration, angiogenesis and
resistance to apoptosis. To overcome apoptotic resistance, autophagy has
emerged as an alternative to kill cancer cells. Here we describe by first
time that ABTL0812 induces autophagy-mediated cell death of human
glioblastoma cell lines LN18 and U87-MG, with an IC50 value similar
to that showed in other human cancer cell lines (pancreas, lung, prostatic,
neuroblastoma, breast or endometrial cancers). We also characterize the
precise mechanism of action by which ABTL0812 exerts a cytotoxic effect
in glioblastoma.
Acknowledgements: ABTL0812 is owned by Ability Pharmaceuticals SL,
a Biotech Company of the UAB Parc de Recerca. Work supported by
MINECO, Grant INNPACTO Ref. IPT-2012-0614-010000, and Fondo
Europeo de Desarrollo Regional (FEDER).
P01-12
Desarrollo de ensayos citómicos de alto
contenido para la predicción y clasificación
del riesgo tóxico de compuestos químicos
José Enrique O`Connor1, Susana Balaguer1, Guadalupe Herrera2
Universidad de Valencia, Valencia, ES, 2Fundación Incliva,
Valencia, ES
1
El desarrollo de métodos in vitro de análisis de la toxicidad bioquímica de
compuestos químicos es una prioridad y ha conducido a la disponibilidad
Salamanca 2016
de técnicas relativamente sencillas con aceptable capacidad de detección
de efectos in vitro y de predicción de toxicidad aguda in vivo. La citómica
se está convirtiendo en una herramienta esencial para el estudio de
toxicidad celular in vitro. En nuestro laboratorio, los ensayos citómicos
de toxicidad sobre líneas celulares humanas clasifican razonablemente
los compuestos según su riesgo de toxicidad en una escala de toxicidad
in vivo en humanos. Sin embargo, la comparación de los datos con el
sistema internacional de clasificación (Global Harmonizing System,
GHS), basado en datos de toxicidad in vivo en rata, resultaba en una
escasa capacidad de clasificación correcta.Hemos puesto a punto y
validado una plataforma de ensayos citómicos miniaturizados y de alto
contenido (integridad de membrana, toxicidad mitocondrial y estrés
oxidativo/nitrosativo) para la determinación de parámetros generales de
citotoxicidad y específicos de estrés oxidativo en tres líneas celulares
de rata. Aplicando técnicas estadísticas y bioinformáticas hemos
determinado la capacidad la plataforma de ensayos para predicción
de toxicidad in vivo y clasificación de compuestos según el GHS. Los
resultados muestran que los ensayos citómicos miniaturizados y de
alto contenido clasifican correctamente la toxicidad de un gran número
de compuestos químicos de acuerdo al criterio GHS. La capacidad de
clasificación mejora respecto al método in vitro de referencia, basado en
los valores de IC50 en el test de captación de Rojo Neutro por 3T3.
Financiado por Universitat de València, Convocatòria Accions Especials
2015 (UV-INV-AE15-349700).
P01r-13
El bloqueo farmacológico de Mcl-1 sensibiliza
a las células de cáncer de vejiga al tratamiento
con Paclitaxel
Rocío Jiménez Guerrero1, Jessica Gasca Bellido1, Mª Luz Flores
de Mera1, María Tortolero García2, Francisco Romero Portillo2,
Miguel Ángel Japón Rodríguez3, Carmen Sáez Torres3
1
Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario
Virgen del Rocío/CSIC, Universidad de Sevilla, Jerez de la Frontera,
ES, 2Departamento de Microbiología, Facultad de Biología,
Universidad de Sevilla, Sevilla, ES,3Instituto de Biomedicina
de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC,
Universidad de Sevilla. Departamento de Anatomía Patológica,
Hospital Universitario Virgen del Rocío, Sevilla, ES
Los taxanos como Paclitaxel y Docetaxel son agentes antimitóticos
que se utilizan en diferentes tipos de cáncer incluyendo el de vejiga, y
constituyen una alternativa terapéutica para el tratamiento en segunda
línea del cáncer avanzado de vejiga músculo-invasivo. Sin embargo,
las células tumorales acaban desarrollando mecanismos de resistencia
que hacen necesaria la búsqueda de nuevas alternativas y/o de terapias
sensibilizadoras. En este sentido aparece Obatoclax (GX15-070) como
inhibidor específico del dominio BH3 de Mcl-1, una proteína clave en
la muerte celular por apoptosis tras parada en mitosis, y que está muy
relacionada con la resistencia al tratamiento con taxanos en diversos tipos
tumorales. Actualmente están en marcha varios ensayos clínicos en fase I
y II con Obatoclax en algunas enfermedades hematológicas, pero aún se
desconoce su papel en tumores sólidos y su mecanismo de acción sigue
siendo motivo de controversia. Nosotros hemos observado que en células
de cáncer de vejiga Obatoclax induce muerte celular y que en células
resistentes a Paclitaxel es capaz de sensibilizar a dicho tratamiento.
Además, hemos estudiado la influencia de Obatoclax en la progresión del
ciclo celular.Hemos observado también que este fármaco induce autofagia
en líneas celulares de cáncer de vejiga y que dicha autofagia puede ser
protectora o inductora de muerte celular. En este trabajo discutimos los
posibles mecanismos por los que Obatoclax sensibiliza al tratamiento
con taxanos relacionados con el papel de la autofagia citoprotectora o
citotóxica dependiendo del tipo celular.
Pósters / Posters
P01r-14
Histone H2AZ is involved in transmissible
cytotoxicity mechanisms leading to multiple
myeloma cell death
Beatriz Martín-Antonio1, Guillermo Suñe1, Lorena Pérez1,
Amer Najjar2, Álvaro Urbano-Ispizua1
1
IDIBAPS-Hospital Clinic Josep Carreras Leukaemia Research
Institute, Barcelona, ES, 2The University of Texas M.D. Anderson
Cancer Center. Department of Cancer Systems Imaging, Houston,
Texas, US
Cord blood derived natural killer cells (CB-NK) are a feasible source
of NK cells for immune cell therapy in hematological malignancies.
We have shown that CB-NK exert a transmissible cytotoxicity towards
multiple myeloma (MM) cells which involves protein vesicle transfer
between MM cells exposed to CB-NK (1ºMM) and MM cells exposed
to 1ºMM cells (2ºMM cells). However, this transmission between MM
cells could have a diluent effect of the CB-NK cytotoxicity which
could be a tumor cell survival mechanism. To gain insights into this
mechanism, we performed TRANS-SILAC proteomics to identify
which proteins were transferred between CB-NK and MM cells. 1ºMM
cells acquired 9.5% of CB-NK proteins, these proteins were transferred
to 2ºMM cells. As a consequence, 1ºMM cells diluted their CB-NK
protein content. Furthermore, while in resting conditions, the proteome
transfer between MM cells was minimal, under the presence of CB-NK,
1ºMM cells transferred a higher amount of their proteome to CB-NK
and to 2ºMM cells.
Among the transferred proteins, we focused on the Histone H2AZ
variant 1. In vivo confocalmicroscopy showed direct and secondary
transfer of H2AZ between MM cells associated to cell death. H2AZ
overexpression in CB-NK increased CB-NK cytotoxicity vs MM cells.
H2AZ overexpression in MM cells caused Caspase independent MM cell
death with features of DNA damage. H2AZ-mediated cytotoxicity was
transmissible from H2AZ-overexpressing MM cells to neighboring MM
cells causing DNA damage in the 2ºMM population. While the transfer of
H2AZ from CB-NK to MM cells occurred through vesicles, between MM
cells it was through intercellular nanotubes connecting MM cells. These
findings show the relevance of cell-cell communication in mechanisms
leading to MM cell death.
P01m-15
Apoptosis in adventitial layer of hydatid cysts
Edgar Mauricio Jiménez Vargas, Rodolfo Paredes, Felipe Correa,
Carlos González, Ivan Contreras, Caroll Stoore
Escuela de Medicina Veterinaria, Facultad de Ecología y Recursos
Naturales, Universidad Andrés Bello, Santiago, CL
Cystic Echinococcosis (CE) is a zoonotic infection with high prevalence
in part of Eurasia, Africa, Australia, and South America that represents a
major public health and economic burden in many countries. Fertile cysts
are capable to generate protoscolex, while for unknown reason, some cysts
are unable to produce (infertile hydatid cysts). Previous reports showed
that apoptosis could be involved in a negative regulation of protoscoleces
generation, leading to hydatid cyst infertility but no report are available
about cell apoptosis in adventitial layer and the relationship with other
parasitic disease like fascioliasis.
Materials and Methods: Animals were evaluated to characterize the
presence of CE and fasioliasis (FS) in cattle slaughtered at abattoir
in Santiago, Chile. Samples were processed for routine histology and
stained with heamatoxylin/eosin. Samples from animal with both fertile
and infertile cyst from CE and with or without DS was included in this
study, using 5 samples per condition. Histological samples were evaluated
by immunofluorescence and TUNEL Assay; digital images were obtained
41
Pósters / Posters
XXXIX Congreso SEBBM
using an Olympus BX 41 Microscope and analyzed with software for
morphometric analysis (Image Pro-Plus, Media Cybernetics, USA). The
adventitial layer apoptotic index was calculated as follows: apoptotic
nuclear area in adventitial layer x 100/total nuclear area in adventitial layer.
Kruskal-Wallis test was performed using IBM SPSS Statistics 22 (IBM
Corporation) software, p <0.05.
Results: The apoptotic index in infertile cysts (0.18 ± 0.35%) was
significantly higher (p = 0.032) compared with fertile cysts (0.045 ±
0.068%), as the presence of co-infection with liver fluke, both fertile cysts
as infertile has a lower apoptotic index without significant difference
(p > 0.05).
Conclusion: The highest index of apoptosis cells in the adventitia layer
of hydatid cysts is related to infertility cysts. The presence of distomatosis
decreases cell apoptosis.
Acknoledgments: Fondecyt /Chile No. 1161475, Proyecto interno UNAB
DI-1388-16/I.
P02. Bases moleculares de la patología /
Molecular bases of pathology
P02-1
Sensibilización de las células de cáncer de
mama a la radioterapia mediante modulación
epigenética
José Neptuno Rodríguez López1, Rebeca González Guerrero1,
Luis Sánchez del Campo1, Ana Moreno1, Juan Cabezas Herrera2,
María Fernanda Montenegro1
1
Departamento de Bioquímica y Biología Molecular A, Universidad
de Murcia, Murcia, ES, 2Instituto Murciano de Investigación
Biosanitaria (IMIB), Murcia, ES
El cáncer, además de considerarse una enfermedad genética, puede
también considerarse una enfermedad epigenética. Las modificaciones
epigenéticas además de ser causantes de los cambios en la expresión
de los genes, participan activamente en la respuesta de las células al
daño del ADN. Como los factores epigenéticos están regulados por
múltiples elementos, las terapias dirigidas a componentes específicos
de la maquinaria epigenética pueden no ser eficaces. A diferencia de
estas, las terapias dirigidas a la inhibición del ciclo de la metionina
pueden inhibir indirectamente la metilación tanto de proteínas como
del ADN afectando a una gran cantidad de genes y vías de señalización.
Siguiendo esta misma línea, hemos desarrollado una terapia adyuvante
dirigida a la epigenética de la respuesta al daño del ADN en las células
de cáncer de mama que consigue aumentar la apoptosis y reducir, in
vivo, la metástasis a distancia. En nuestro laboratorio hemos observado
que una terapia combinada dirigida a desacoplar el metabolismo de
la adenosina usando dipiridamol en presencia de un nuevo antifolato
sintético, 3-O-(3,4,5-trimetoxibenzoil)-(-)–catequina, bloquea eficiente
y simultáneamente el ciclo del ácido fólico y de la metionina en células
de cáncer de mama sensibilizándolas a la radioterapia. Esta combinación
impide, entre otros, la movilización de 53BP1 y BRCA1 a las zonas
de la cromatina que flanquean las roturas de doble cadena del ADN
evitando así la reparación del daño del ADN en las células de cáncer
de mama expuestas a la radiación ionizante. Así, la terapia combinada
que proponemos puede definirse como una estrategia global dirigida a la
maquinaria epigenética de las células tumorales.
Financiado por un proyecto del
(SAF2013-48375-C2-1R) y la FAECC.
42
MINECO-Fondos
FEDER
P02-2
Extracellular heat shock protein 90 binding to
TGFβ receptor I participates in TGFβ-mediated
collagen production in myocardial fibroblasts
Jenny Milagros Gómez1, Francisco Javier Ruano Calvo2, Antonio
Aires Trapote3, Iñigo Casafont Parra4, Ana María Palanca Cuñado4,
Aitziber López Cortajarena3, Ana Victoria Villar Ramos4
1
Hospital de Laredo, Cantabria, ES, 2Hospital Universitario Marqués
de Valdecilla, Cantabria, ES, 3CIC-BiomaGUNE, Gipuzcoa, ES,
4
Universidad de Cantabria, Cantabria, ES
The pathological remodeling heart shows an increase in left ventricular
mass and an excess of extracellular matrix deposition that can over time
cause heart failure. Transforming growth factor β (TGFβ) is the main
cytokine controlling this process. The molecular chaperone heat shock
protein 90 (Hsp90) has been shown to play a critical role in TGFβ signaling
by stabilizing the TGFβ signaling cascade. We detected extracellular
Hsp90 in complex with TGFβ receptor I (TGFβRI) in fibroblasts and
determined a close proximity between both proteins suggesting a potential
physical interaction between the two at the plasma membrane. It was
supported by in silico studies predicting Hsp90 and TGFβRI extracellular
domain interaction. Extracellular Hsp90 inhibition lessened the yield of
collagen production as well as the canonical TGFβ signaling cascade.
These observations together with the significant increased activity of
Hsp90 at the plasma membrane of TGFβ-treated myocardial fibroblasts
pointed to a functional cooperative partnership between the extracellular
Hsp90 and TGFβRI in the fibrotic process. We propose that an extracellular
population of Hsp90 bound to TGFβRI behaves as an active participant in
collagen production in TGFβ-activated fibroblasts. We also offer an in vivo
insight into the role of Hsp90 during cardiac remodeling in murine aortic
banding model suffering from pathological cardiac remodeling and detect
circulating Hsp90 overexpressed in remodeling mice.
P02r-3
Identification of MicroRNAs involved
in the metabolic basis of renal fibrogenesis
Verónica Miguel Herranz1, Marta Fierro Fernández1, Ricardo
Ramos2, Santiago Lamas1
1
Centro de Biología Molecular Severo Ochoa CSIC-UAM, Madrid,
ES, 2Genomic Facility, Parque Científico de Madrid, Madrid, ES
Excessive accumulation of extracellular matrix (ECM) proteins is the
hallmark of fibrotic diseases. In the kidney, it is the final common pathway
of prevalent clinical conditions as type 2 diabetes mellitus and arterial
hypertension, leading to chronic renal failure. This process is dependent on
the activation of ECM synthesis in phenotypically transformed fibroblasts
known as myofibroblasts. While archetypal cytokine mediators such as
TGF-β play a fundamental role in this transformation, recent work has shown
that lipid metabolic alterations, specifically a reduction in fatty acid oxidation
(FAO) are important pathogenetic coadjuvators. MicroRNAs are small
non-coding RNAs that regulate gene expression at the post-transcriptional
level and controlling multiple biological processes, including fibrogenesis.
To identify miRNAs involved in the metabolic regulation of renal fibrosis, we
performed studies in in the renal fibrosis mouse models of unilateral ureteral
obstruction (UUO) and folic acid nephropathy (FAN). From a total of 175
array-analyzed microRNAs in the UUO model 74 showed altered expression
in the fibrotic samples. In silico analysis of the FAO-related enzyme
carnitin-palmitoyl transferase 1 (CPT1A) 3´UTR identified miR-33-5p
and miR-124-3p as potential candidates. We found downregulation of
miR-33-5p in the UUO and FAN models by 40 and 50%, respectively, while
miR-124-3p levels were unchanged. TGF-β1-induced fibrogenesis in the
human renal cell line HKC-8 as well as in primary human renal epithelial
cells (RECs) and primary human tubular renal epithelial cells (HTECs). We
found 60% downregulation of miR-124-3p levels in TGF-β1-treated HTEC.
Salamanca 2016
A 60% decrease in the luciferase reporter activity of the CPT1A 3´UTR after
transfection with miR-124-3p confirmed its target specificity. Consistently,
miR-124 overexpression downregulated CPT1A mRNA and protein levels
by 60% in HTECs and resulted in a 3-fold increase of TGF-β1-induced
α-SMA expression. These results support the role of miRNAS in a novel and
potentially complex metabolic regulatory pathway related to renal fibrosis.
P02m-4
La disminución de la expresión de s-resistina
en el hipotálamo mejora la respuesta central
y periférica a la insulina en ratas Wistar
María Rodríguez Pérez1, C. Pintado1, V. López-Gómez Carreño2,
N. Gallardo2, E. Moltó1, C. Arribas1, A. Andrés2
1
Área de Bioquímica, Facultad de Ciencias Ambientales y
Bioquímica; Centro Regional de Investigaciones Biomédicas,
UCLM, Toledo, ES, 2Facultad de Ciencias y Tecnologías Químicas,
Ciudad Real, ES; Centro Regional de Investigaciones Biomédicas,
UCLM, Toledo, ES
La s-resistina es una variante no secretable de resistina, generada por
splicing alternativo, que se expresa principalmente en tejido adiposo blanco
e hipotálamo de ratas Wistar. Resultados previos confirmaron que células
3T3-L1 que expresaban s-resistina mostraron un incremento en la respuesta
inflamatoria, así como un descenso en el transporte de glucosa estimulado
por insulina. Además, la s-resistina, al igual que la resistina, bloqueó la vía de
señalización de la insulina inhibiendo la fosforilación de IR, IRS-1 y Akt, e
incrementó los niveles de SOCS-3. Por tanto, s-resistina podría actuar como
un factor no secretado que regula la sensibilidad del adipocito a la insulina.
En este trabajo se analiza el papel de s-resistina en el hipotalámo. Para ello
se ha disminuido la expresión central de esta isoforma corta de resistina,
inyectando, de forma i.c.v, un lentivirus que contiene un RNAi frente a dicha
isoforma (LV-RNAi-s-res). La diminución central de s-resistina incrementa
los niveles basales de fosforilación en Tyr, tanto en IR como en IRS-1,
mientras que disminuye los niveles de fosforilación en Ser307 en IRS-1 en el
hipotálamo. También se observa un incremento de los niveles de mRNA y de
fosforilación en STAT-3, mientras que éstos disminuyen en el caso de PTP1b.
Además, en los animales tratados con el LV-RNAi-s-res se promueven los
efectos anorexigénicos, reduciendo la expresión de NPY e incrementando la
de POMC, lo que conlleva un descenso de la ingesta. Estos datos muestran,
por tanto, una mejora en la vía de señalización de insulina en aquellos
animales que presentan disminuidos los niveles de expresión de s-resistina
en el hipotálamo. Asimismo, se observa un incremento en la sensibilidad
periférica a la insulina, como lo demuestran las curvas de tolerancia a la
glucosa obtenidas en los animales tratados. Todos estos resultados sugieren
que s-resistina podría actuar como un sensor neuronal intracrino, que actuaría
regulando la sensibilidad a la insulina en todo el organismo.
Financiación: PI-2007/60 (JCCM) FISCAM y BFU2012-39705-C03-01
Mineco, España.
P02-5
Identification of new mechanisms involved
in the postnatal regeneration of endocrine
pancreas: role of the autophagy in beta-cells
and its relationship with IGF-2
Elisa Fernández-Millán1, David Álvarez-Cilleros2, Juan de
Toro-Martín3, Esther Lizárraga-Mollinedo4, Fernando Escrivá2,
Carmen Álvarez Escolá2
1
CIBER de Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM, ISCIII), Madrid, ES, 2Dpto. Bioquímica y Biología
Molecular II, Facultad de Farmacia, UCM, Madrid, ES, 3Institute
of Nutrition and Functional Foods (INAF), Laval University,
Pósters / Posters
Quebec, CA, 44ULB Center for Diabetes Research, Medical Faculty,
Université Libre de Bruxelles (ULB), Bruxelles, BE
Aim: The developing endocrine pancreas undergoes substantial remodeling
during the postnatal period, which triggers a transformation from fetal to
adult phenotype of beta-cells. This beta-cell turnover is achieved by a
transient wave of apoptosis that is temporally associated with a lack of
expression of IGF-2 within islets. Because IGF-1R pathway is upstream
of mTORC1, a negative regulator of autophagy, we expected autophagy
to be activated in beta-cells during neonatal period. Thus, our study aimed
to characterize the basal autophagic activity during endocrine pancreas
remodeling and its contribution to neonatal beta-cell apoptosis.
Methods: Autophagy markers were measured by Western blot in the
pancreas of rats on postnatal (PN) day 4, 14 and 23. Autophagosome
formation was analyzed by TEM. Neonates were treated with rapamycin
(3 mg/kg, 5 days) and beta-cell apoptosis was quantified by TUNEL assay.
In order to determine the effect of IGF-2 on autophagy, in vitro studies
were performed in INS-1E cells and neonatal islets.
Results: Under basal conditions, a significant decrease of LC3II levels was
observed in the pancreas of neonates on PN14 and PN23 compared with
PN4. This decrease was accompanied by an accumulation of p62 (>2-fold
vs. PN4). Similarly, TEM revealed a reduction in the number of autophagic
vacuoles per beta cell in PN14 (49.7%, p<0.05) and PN23 rats (64.24%,
p<0.05) compared with PN4 rats. Sub-chronic treatment with rapamycin,
induced a significant reduction (p<0.01) in the beta-cell apoptosis observed
on PN14. Finally, kinetic experiments showed that long- but not short-term
supplementation of beta-cells with IGF-2 resulted in a significant increase
in LC3-II formation.
Conclusion: The blockade of autophagic activity in the endocrine pancreas
seems to be required during postnatal remodeling in order to ensure the
correct beta-cell turnover by apoptosis. This event is probably associated
to disappearance of islet IGF-2 expression.
Acknowledgments: MINECO (BFU2011/25420), CAM (S2010/BMD-2423)
and CIBERDEM (ISCIII), Spain.
P02-6
Development of new cell genetic tracing tools
for the study of intratumour heterogeneity
Laura Quevedo1, Laura González-Silva1, Thaidy Moreno1,
Carlos Revilla1, Dieter Saur2, Roland Rad2, Ignacio Varela1
1
Instituto de Biomedicina y Biotecnología de Cantabria
(CSIC-UC-Sodercan), Departamento de Biología Molecular,
Universidad de Cantabria, Santander, ES, 2Department of Internal
Medicine II, Klinikum rechts der Isar, Technische Universität
München, Munich, DE; German Cancer Consortium (DKTK),
German Cancer Research Center (DKFZ), Heidelberg, DE
Intratumour heterogeneity has been observed in multiple cancers and
has been postulated as a critical aspect for tumour metastasis and
treatment resistance. Therefore, a further characterization of its role in
cancer progression and metastasis has become essential to increase our
understanding of cancer biology and to improve the treatment of cancer
patients1. The use of cell lineage tracing systems, combined with the use
of genetically modified mouse models, could be applied in this context.
Several genetic tracing systems, based in a random CRE-mediated
recombination event in an allele with multiple fluorescent markers
surrounded by incompatible lox sites have been created2. Once this
recombination occurs, the cell and its genetic descendants are permanently
labelled with the same fluorescent marker. Nevertheless, these systems
present several limitations like a reduced number of potential colour
combinations or problems in the unique identification of the markers.
Here we have identified new incompatible lox sites that, together with an
efficient selection of fluorescent markers, have allowed us to design a new
system that will be able to produce up to 15 different colour combinations
43
Pósters / Posters
that can be uniquely identified by confocal microscopy and FACS. This
system will be combined with cancer mouse models to study the role and
dynamics of intratumour heterogeneity in cancer progression.
References
[1] Gerlinger, M. et al. “Intratumor heterogeneity and branched evolution
revealed by multiregion sequencing”. N. Engl. J. Med. 366, 883–892 (2012).
[2] Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W. & Sanes, J. R. “Improved
tools for the Brainbow toolbox”. Nat. Methods 10, 540–547 (2013).
P02r-7
Obesity and type 2 diabetes alters the immune
properties of human adipose derived stem cells
Noelia Keiran, Carolina Serena, Victoria Ceperuelo-Mallafre, Miriam
Ejarque, Kelly Roche, Catalina Núñez-Roa, Joan Vendrell, Sonia
Fernández-Veledo
Hospital Universitari de Tarragona Joan XXIII. Institut d´Investigació
Sanitària Pere Virgili, Universitat Rovira i Virgili, Tarragona, ES;
CIBER de Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM), Instituto de Salud Carlos III, Madrid, ES
Adipose tissue-derived stem cells (ASCs) are proposed as an alternative
stem cell source to bone marrow-derived cells for immune cell therapy.
However, microenvironmental factors may impact the functionality of this
population in human adipose tissue (AT). We hypothesized that the fat
depot in addition to the donor phenotype controls the immunomodulatory
capacity of ASCs. Focusing on obesity and type 2 diabetes (T2D) as
metabolic disorders that might affect the immune response of ASCs, we
compared the inflammatory response of ASCs from subcutaneous and
visceral AT of age-matched donors (lean n=4, body mass index [BMI]
21.981.9; obese n=4 BMI 33.12.1 and T2D n=4 BMI 35.31.5). Obese
and particularly T2D-derived ASCs showed increased expression of
inflammatory markers, activation of NLRP3 inflammasome and higher
migration, invasion and phagocytosis capacities than those derived from
lean donors. Remarkably, ASCs derived from obese and T2D subjects
exhibited a reduction in typical immunosuppressive activities attributed
to stem cells. Accordingly, obese and T2D-ASCs were less effective in
suppressing lymphocyte proliferation, activating the M2 macrophage
phenotype, and in increasing TGF-b1 secretion, than lean-derived ASCs.
Treatment of lean hASCs with IL-1b mimicked thedysfunctional immune
behaviour of obese and T2D hASCs. Conversely, combined treatment with
IL1RA and TGF-b1 reverted the phenotype of obese- and T2D-ASCs.
These data indicate that the donor metabolic phenotype compromises the
immunomodulatory properties of ASCs. These results are relevant not only
for understanding the physiology of ASCs in terms of cell-based therapies
but also for their role as key regulators of the immune response.
P02-8
Papel de la Kleisina NCAPH en el desarrollo y
evolución del cáncer de mama HER2 positivo
Sonia Castillo-Luva1, Adrián Blanco Gómez2, Jesús Pérez-Losada2
Universidad Complutense de Madrid, Madrid, ES, 2Instituto de
Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de
Salamanca/CSIC. Salamanca, España, Salamanca, ES
1
El cáncer de mama es la neoplasia más frecuente en mujeres y causa de
gran morbimortalidad siendo un problema importante de salud pública. Es
una enfermedad compleja y heterogénea, con un rango de manifestaciones
variable tanto a nivel clínico, como histopatológico, molecular y de la
respuesta al tratamiento. (1) Los estudios de expresión génica en carcinoma
ductal invasivo (IDC), obtenidos mediante análisis de microarrays en
bases de datos, nos han permitido identificar a un miembro del complejo
Condensina I como un transcrito sobreexpresado en IDC versus tejido de
44
XXXIX Congreso SEBBM
mama normal. El complejo Condensina I participa en la correcta segregación
cromosómica y su alteración podría dar lugar a aneuploidías. (2) Hemos
encontrado asociación significativa entre el pronóstico de la enfermedad
y la expresión de este complejo en la base de datos pública TCGA que
contiene información molecular y clínica de más de 825 pacientes con
cáncer de mama. (3) Mujeres con niveles altos de expresión del complejo en
el tumor presentaron una menor supervivencia que aquellas que presentaron
niveles bajos. Es más, niveles elevados de expresión de Condensina I se
asociaron con una reducción de supervivencia libre de metástasis. Para
estudiar el posible papel que desempeña Condensina I en la evolución
del cáncer de mama se ha generado un modelo de ratón transgénico que
sobre-expresa el gen del componente Kleisina I del complejo Condensina I
y el proto-oncogén Erbb2/Neu. (4) Nuestros datos preliminares indican que
niveles altos de expresión de KLEISINA I modifican las características
de desarrollo tumoral como son el número de tumores y la capacidad de
diseminación en animales expuestos a un estímulo hormonal, como es
el embarazo. Sin embargo, no afecta a características temporales de la
enfermedad como son la latencia o la duración de la enfermedad.
Financiación: Este proyecto está financiado por el Ministerio de Ciencia e
Innovación (MINECO/FEDER) SAF2015-64499-R
Referencias
[1] Polyak K. Heterogeneity in breast cancer. J Clin Invest. 2011
Oct;121(10):3786-8.
[2] Wood AJ, Severson AF, Meyer BJ. Condensin and cohesin
complexity: the expanding repertoire of functions. Nat Rev Genet. 2010
Jun;11(6):391-404.
[3] Cancer Genome Atlas Network. Comprehensive molecular portraits of
human breast tumours. Nature, 2012 Oct 4;490(7418):61-70.
[4] Guy CT, Webster MA, Schaller M, Parsons TJ, CardiffRD, Muller
WJ. Expression of the neu protooncogene in the mammary epithelium
of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A.
1992 Nov 15;89(22):10578-82.
P02-9
Tumor suppressor ARF regulates tissue
microenvironment and tumor growth through
modulation of macrophage polarization
Lidia Jiménez, Sandra Herranz, María Ángeles Higueras, Alfonso
Luque, Sonsoles Hortelano
Unidad de Terapias Farmacológicas. Instituto de Investigaciones
de Enfermedades Raras (IIER), Instituto de Salud Carlos III,
Majadahonda, ES
Tumor microenvironment has been described to play a key role in tumor
growth, progression, and metastasis. Macrophages are a major cellular
constituent of the tumor stroma, and particularly tumor associated
macrophages (TAMs or M2-like macrophages) exert important
immunosuppressive activity and a pro-tumoral role within the tumor
microenvironment. Alternatively-reading frame (ARF) gene is widely
inactivated in human cancer. We have previously demonstrated that ARF
deficiency severely impairs inflammatory response establishing a new
role for ARF in the regulation of innate immunity. On the basis of these
observations, we hypothesized that ARF may also regulates tumor growth
through recruitment and modulation of the macrophage phenotype in the
tumor microenvironment. Xenograft assays of B16F10 melanoma cells into
ARF-deficient mice resulted in increased tumor growth compared to those
implanted in WT control mice. Tumors from ARF-deficient mice exhibited
significantly increased number of TAMs as well as microvascular density.
Transwell assays showed crosstalk between tumor cells and macrophages.
On the one hand, ARF-deficient macrophages modulate migratory ability
of the tumor cells. And on the other, tumor cells promote the skewing of
ARF-/- macrophages toward a M2-type polarization. In conclusion, these
results demonstrate that ARF deficiency facilitates the infiltration of
Salamanca 2016
macrophages into the tumor mass and favors their polarization towards
a M2 phenotype, thus promoting tumor angiogenesis and tumor growth.
This work provides novel information about the critical role of ARF in the
modulation of tumor microenvironment.
P02-10
8,9-dehydrohispanolone-15,16-lactol diterpene
prevents LPS-triggered inflammatory responses
by inhibiting endothelial activation
Lidia Jiménez-García1, Paqui G. Través2, Raquel López-Fontal3,
Sandra Herranz1, Sandra Amarilla-Quintana1, Beatriz de las Heras4,
Sonsoles Hortelano1, Alfonso Luque1
1
ISCIII, Majadahonda, ES, 2The Salk Institute, La Jolla, California,
US, 3Universidad Europea de Madrid, Villaviciosa de Odón, ES,
4
Universidad Complutense de Madrid, Madrid, ES
Endothelial activation contributes to lung inflammatory disorders by
inducing leukocyte recruitment to pulmonary parenchyma. Consequently,
vascular-targeted therapies constitute promising strategies for the
treatment of inflammatory pathologies. Here we evaluated the effect of
8,9-dehydrohispanolone-15,16-lactol diterpene (DT), previously described
as a regulator of LPS-treated macrophages, on lung endothelium during
inflammation. Pulmonary lung endothelial cells pre-treated with DT
and activated with LPS or TNF- exhibited reduced expression of the
pro-inflammatory cytokines Cxcl10, Ccl5 and Cxcl1 compared with
non-treated cells, whereas the anti-inflammatory molecules IL1r2 and
IL-10 were induced. Consistent with this result, DT pre-treatment inhibited
NF-B nuclear translocation in endothelial cells activated by LPS or
TNF-. Besides, conditioned medium from these cells failed to stimulate
leukocytes as measured by a reduction in adhesive ability of the leukocyte
cell line J774 to fibronectin. Additionally, DT reduced the expression of
the endothelial adhesion molecules E-selectin, VCAM-1 and ICAM-1 after
activation. Similarly, expression of VCAM-1 and ICAM-1 molecules on the
lung endothelial layer of C57/BL6 mice pretreated with DT and challenged
with LPS were unchanged. Finally, inhibition of vascular adhesion
molecule expression by DT decreased the interaction of J774 cells with
lung endothelial cells in an inflammatory environment. Thus, DT exhibits
an efficient anti-inflammatory activity on lung vasculature by inhibiting
the expression of endothelial adhesion molecules and chemotactic factors.
Our findings establish DT as a novel endothelial inhibitor for the treatment
of inflammatory-related diseases triggered by gram-negative bacteria or by
the associated cytokine TNF-.
P02-11
Telomere status and telomerase activity: clinical
usefulness in non-small cell lung and colorectal
cancer
Tamara Fernández-Marcelo1, Carmen De Juan Chocano1, Ana
Gómez2, Andrés Sánchez Pernaute3, Florentino Hernando2,
José-Ramón Jarabo2, Marina Zorita4, Antonio-José Torres García3,
Pilar Iniesta Serrano1
1
Departamento de Bioquímica y Biología Molecular II. Facultad
de Farmacia. Universidad Complutense; Instituto de Investigación
Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, ES,
2
Servicio de Cirugía Torácica. Hospital Clínico San Carlos. Instituto
de Investigación Sanitaria Hospital Clínico San Carlos (IdISSC),
Madrid, ES, 3Servicio de Cirugía General y del Aparato Digestivo.
Hospital Clínico San Carlos. Instituto de Investigación Sanitaria
Hospital Clínico San Carlos (IdISSC), Madrid, ES, 4Departamento
de Bioquímica y Biología Molecular II. Facultad de Farmacia.
Universidad Complutense, Madrid, ES
Pósters / Posters
Lung cancer remains the most common cancer in men and colorectal cancer
(CRC) is the 3rd most common in men and the 2nd in women1. For both, the
identification of new biomarkers is still needed to predict the course of the
disease. Telomere dysfunction and increase in telomerase activity (TA) are
frequent events in cancer2,3; however, their role as prognostic factors remains
unconfirmed. We evaluated TA and telomere length (TL) in 142 non-small
cell lung cancers (NSCLCs) and 132 CRCs, and their corresponding control
tissues, from patients submitted to potentially curative surgery at S. Carlos
Hospital, Madrid. Group oriented curves for disease-free survival were
calculated according to the Kaplan-Meier method considering TA, TL and
T/N ratio (TL in tumour vs. control) and differences were evaluated using
the Log-Rank test (only patients submitted to curative surgery & median
follow-up period: 5 years). Software: SPSS 19 & Cutoff Finder4. Positive
TA was found in 86.6% of NSCLCs. The mean TL (MTL) was 6.56±0.26
Kb in NSCLCs and 7±0.19 Kb for controls (P=0.027). The mean T/N ratio
was 0.93±0.03. Positive TA was detected in 82.5% of CRCs. The MTL was
5.49±0.23 Kb in CRCs and 7.32±0.33 Kb in controls (P<0.001). The mean
T/N ratio was 0.79±0.02. In NSCLCs, the patients whose tumours showed a
TL<7.29 Kb or T/N ratio<0.97 had a significantly worse clinical evolution
(P=0.034 & 0.040, respectively; and both were independent prognostic
markers of the stage). The absence of TA in the tumour conferred a better
clinical evolution (P=0.039). In CRCs, the most favorable prognosis was
found in the patients with a MTL in tumours <6.35 Kb or T/N ratio<0.665
(P<0.001 & P=0.043, respectively). The MTL in CRCs was shown to be
an independent prognostic parameter. No recurrences were detected in the
group of patients who had TA negative tumours (P=0.184)5,6. Telomere
attrition seems to confer a different prognosis according to the tumour
type: negative prognosis in NSCLCs and positive in CRCs. Exploring the
molecular basis which underlies telomere shortening, as we previously
hypothesized7, could be useful to establish personalized therapies.
References
[1] http://globocan.iarc.fr/
[2] J Cell Mol Med. 2011;15(6):1227-38.
[3] Cancer Treat Rev. 2013;39(5):444-56.
[4] PLoS One. 2012;7(12):e51862.
[5] J Exp Clin Cancer Res. 2015;34:78.
[6] PLoS One. 2016;11(2):e0149626.
[7] Oncology. 2012;82(3):153-64.
P02r-12
Relevance of BMP9-Mediated signaling in oval
cell function during chronic liver injury. Crosstalk
with the HGF/c-MET pathway
Annalisa Addante1, María García-Álvaro2, César Roncero1, Margarita
Fernández1, Laura Almalé1, Nerea Lazcanoiturburu1, Julian Sanz3,
Patricia Saperas3, Se-Jin Lee4, Isabel Fabregat5, Steven Dooley6,
Peter ten Dijke2, Blanca Herrera1, Aránzazu Sánchez1
1
Department of Biochemistry and Molecular Biology, Faculty of
Pharmacy, Complutense University of Madrid (UCM), Instituto de
Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC),
Madrid, ES, 2Department of Molecular Cell Biology and Cancer
Genomics Centre Netherlands, Leiden University Medical Centre,
RC Leiden, Leiden, NL, 3Department of Pathology. Hospital Clínico
San Carlos, Madrid, ES, 4Department of Molecular Biology and
Genetics, Johns Hopkins University School of Medicine, Baltimore,
US, 5Bellvitge Biomedical Research Institute - IDIBELL, L’Hospitalet
de Llobregat, Barcelona, ES, 6Department of Medicine II, Medical
Faculty Mannheim, Heidelberg University, Mannheim, DE
Oval cells (OCs) constitute a bi-potential progenitor cell population from
adult liver. Under chronic liver disease (CLD), they become activated and
proliferate and differentiate into cholangiocytes and/or hepatocytes to
compensate for the cellular loss contributing to maintain liver homeostasis.
It is well recognized the critical role of the TGF-β in CLD, but the role
45
Pósters / Posters
of other members of the TGF-β superfamily, the BMPs, is only partly
understood. We focused our attention in BMP9, a member of the BMP
family that has recently emerged as a critical regulator of liver pathology;
and explored its potential role in the regulation of OC. For this, WT and
BMP9 KO mice were submitted to 0.1% DDC-supplemented diet for 2,
4 or 6 weeks, as a model of liver damage associated with OC expansion.
We found a greater expansion of the oval cell population in BMP9 KO
mice respect to WT in response to a DDC diet, which suggests that BMP9
plays a negative role in OC-mediated liver regeneration. BMP9 suppressor
effects were confirmed in vitro, as BMP9 decreases oval cell number and
induces apoptosis. Using a model of OC lines harboring a genetically
inactivated HGF receptor (c-Met) tyrosine kinase (Met-/- cells) and its
control (Metflx/flx cells) we found that HGF/c-Met signaling inhibits BMP9
suppressor effects. Furthermore, our results indicate that BMP9 activates
the Smad pathway in OCs, being this activation diminished in Met-/- OC.
Consistently, Smad activation by BMP9 in Metflx/flx oval cells is increased
after co-treatment with HGF. Searching for the mechanisms mediating the
BMP9/HGF crosstalk, we found that the expression of ALK1, the BMP9
high affinity type I receptor, is up-regulated when OC are treated with
BMP9 and HGF. More importantly, loss of ALK1 expression abolishes
both the HGF-mediated amplification of BMP9-triggered Smad signaling
in OC and HGF protective effects. In conclusion, BMP9 emerges as a novel
regulator of OC population exerting suppressor effects. We also provide
novel evidence of an interesting signaling crosstalk between BMP9 and
HGF/c-Met pathways in OC regulation during CLD.
P02-13
Ifg1r media en la regeneración del epitelio
bronquiolar murino regulando la cinética
de recuperación tras la ablación selectiva
de las células club
Icíar Paula López García1, Sergio Piñeiro-Hermida1, Rosete Sofia
Pais1, Raquel Torrens1, Andreas Hoeflich2, José G. Pichel1
1
Centro de Investigación Biomédica de La Rioja (CIBIR) /
Fundación Rioja Salud, Logroño, ES, 2Institute for Genome Biology,
Leibniz-Institute for Farm Animal Biology (FBN), Dummerstorf, DE
La regeneración y reparación del epitelio pulmonar son vitales para
mantener la función e integridad de las vías aéreas. El desequilibrio
entre el daño epitelial y la recuperación de éste es la causa de numerosas
enfermedades crónicas pulmonares como asma, EPOC, fibrosis
pulmonar y cáncer de pulmón. La señalización mediada por IGFs
(Insulin-like Growth Factors) se ha relacionado con estas patologías,
pero sus mecanismos de acción en este órgano son poco conocidos.
La inmuno-localización de su receptor IGF1R revela altos niveles de
expresión en las células del epitelio pulmonar. Para determinar la función
de IGF1R en el epitelio del pulmón murino se generaron mutantes
condicionales Nkx2.1-Cre; Igf1r fl/fl que expresan la recombinasa Cre en
el epitelio respiratorio. La falta de IGF1R altera la diferenciación del
epitelio bronquiolar en ratones adultos ocasionando un incremento de
la proliferación y cambios en la morfología de las células club en los
bronquiolos distales, pero sin ocasionar impacto morfológico aparente
en el parénquima alveolar. Durante la recuperación del daño bronquiolar
inducido por la ablación selectiva de las células club con naftaleno, la
deficiencia de IGF1R mantiene los defectos histológicos, aumenta la
proliferación y retrasa la diferenciación de las células club y ciliadas.
Además, los pulmones de los ratones mutantes también revelan un
aumento de expresión de Igf1, Insr, Igfbp3 y de marcadores de precursores
epiteliales, un descenso en la proteína Scgb1a1, y alteraciones de los
mediadores de la señalización de IGFs. Estos resultados demuestran que
IGF1R está implicado en el control de la proliferación y diferenciación
celular del epitelio bronquiolar durante su regeneración, y fundamentan
su posible participación en patologías respiratorias.
46
XXXIX Congreso SEBBM
P02m-14
Effects of a novel synthethic
microneurotrophin DHEA-derivative BNN27
in the neurodegenerative and inflammatory
component of diabetic retinopathy
Silvia Lisa Ferrer1, Ruth Iban-Arias2, Niki Mastrodimou2,
Despina Kokona3, Panagiota Iordanidou2, Mara Boumpouli2,
Achilleas Gravanis2, Ioannis Charalampopoulos2, Kyriaki Thermos2
1
Instituto de Neurociencias de Castilla y Leon, Salamanca, ES,
2
University of Crete, Heraklion, GR, 3Inselspital, Universitätsspital
Bern, Bern, CH
Diabetes may induce diabetic retinopathy (DR), a chronic eye disease that
lead to retinal cell death and blindness. DR is caused by three components:
neurodegeneration, inflammation and neovascularization, but available
treatments target only the neovascularization component. Therefore,
new therapeutics must be developed to treat the neurodegeneration
and inflammatory components. DHEA, a neurosteroid hormone that
binds to TrkA receptor with high specificity (Lazaridis, 2011), has
neuroprotective and anti-inflammatory effects in the retina (Kokona,
2012; Straub, 1998). Novel DHEA derivatives, such as the spiro-epoxy
derivative BNN27, have been synthesizedthat lacking the endocrine side
effects of DHEA (Calogeropoulou, 2009). The main objective of this
study was to investigate the neuroprotective and anti-inflammatory effect
of BNN27 in the rat streptozotocin (STZ) model of DR. We have shown
that 4 weeks after STZ injection, the number of ganglion and amacrine
cells decreased (Mastrodimou, 2015). Treatment of the diabetic rat
with BNN27 (10 mg/kg, ip) for one week, protected amacrine cells that
express nitric oxide synthetase and tyrosine hydroxylase and ganglion cell
axons. This neuroprotection was shown to be due to BNN27 activation/
phosphorylation of the TrkA receptor and its prosurvival signaling
pathway (ERK1/2 kinases) as well as the reduction of the phosphorylation
of pro-apoptotic kinases SAPK/JNK. In addition, the transcription factor
related to the activation of the inflammatory response, NFκΒ, increased
in diabetic retina. An increase in the phosphorylation of Akt, the kinase
implicated in the upstream signaling of pro-inflammatory cytokines, was
observed in DR. BNN27 reversed Akt activation and the expresion of the
pro-inflammatory cytokines TNFα and IL-1β, and increased the expression
of the anti-inflammatory cytokines, IL-10 and IL-4 in DR.In conclusion,
our results suggest that the DHEA-derivative BNN27 has neuroprotective
and anti-inflammatory effects in the diabetic retina and is a putative
therapeutic candidate for the treatment of DR.
P02-15
Inflammatory up-regulation in the pathogenesis
of calcific aortic valve stenosis
Iván Parra Izquierdo1, Irene Castaños-Mollor1, Javier López2,
Cristina Gómez1, Sergio Ayuso1, Alberto San Román2,
Mariano Sánchez Crespo1, Carmen García-Rodríguez1
1
Instituto de Biología y Genética Molecular, CSIC-Universidad
de Valladolid, Valladolid, ES, 2Instituto de Ciencias del Corazón,
Hospital Clínico Universitario, Valladolid, ES
Background and aim: Calcific aortic valve stenosis (CAVS), the most
prevalent valvulopathy in Western countries, is characterized by valve
thickening. Many evidences point to inflammation as a key event for the
development of fibrosis and valve calcification. Since interferons (IFN)
coordinate a diverse array of cellular programs through transcriptional
regulation of immunologically relevant genes, the aim of this study was
to elucidate their role in the inflammatory process in human valve cells.
Materials and methods: Endothelial and interstitial valve cells were
isolated from stenotic aortic valve (obtained from valve replacement), and
non-stenotic aortic and pulmonary valves (obtained from heart transplant
recipients). After exposure to recombinant type I and II IFN, the expression
Salamanca 2016
of pro-inflammatory molecules was addressed by Western blot and flow
cytometry, and monocyte adhesion assayed.
Results: Experiments showed a blatant array of cell-specific responses
to IFN, including the expression of COX-2, ICAM-1 and E-selectin in
endothelial valve cells. Notably, the response was higher in aortic than
in pulmonary valve cells. IFN also promoted monocyte adhesion to
endothelial monolayers. In control/stenotic interstitial cells, IFNγstrongly
induced ICAM-1; in addition, stenotic cells were responsive to IFNα.
Strikingly, IFN strongly cooperated with LPS (TLR4 ligand) and
Pam2CSK4 (TLR2/6 ligand) to induce COX-2 and ICAM-1 expression,
being the synergistic effect higher in aortic than in pulmonary valve cells.
Conclusions: IFN are more potent inducers of inflammation in aortic
than pulmonary valve cells, thus correlating with clinical differences since
pulmonary valve rarely suffer CAVS. Data open the way to explore IFN as
a potential therapeutic target for CAVS.
P02m-16
La expresión de la ciclooxigenasa-2 en
hepatocitos atenúa la esteatohepatitis no
alcohólica y la fibrosis hepática en ratones
Omar Motiño García-Miguel1, Noelia Agra Andrieu1, Rocio Brea
Contreras1, Carmelo García-Monzón2, Javier Vargas-Castrillón2,
Lisardo Boscá Gomar3, Marta Casado Pinna4, Rafael Mayoral
Moñibas5, Ángela María Martínez Valverde6, Daniel Frances7,
Paloma Martín-Sanz3
1
Instituto de Investigaciones Biomédicas Alberto Sols-UAM-CSIC,
Madrid, ES, 2Instituto de Investigación Sanitaria Princesa-Hospital
Universitario Santa Cristina, Madrid, ES, 3Instituto de
Investigaciones Biomédicas Alberto Sols-UAM-CSIC y Centro
de Investigación Biomédica en Red de Enfermedades Hepáticas y
Digestivas (CIBERrhd), Madrid, ES, 4Instituto de Biomedicina de
Valencia-CSIC, Valencia, ES, 5Centro de Investigación Biomédica
en Red de Enfermedades Hepáticas y Digestivas (CIBERrhd) y
Universidad de California-La Jolla, San Diego, US, 6Instituto de
Investigaciones Biomédicas Alberto Sols-UAM-CSIC y Centro
de Investigación Biomédica en Red de Diabetes y Enfermedades
Metabólicas asociadas (CIBERdem), Madrid, ES, 7Instituto de
Fisiología Experimental-CONICET, Rosario, AR
La ciclooxigenasa 2 (COX-2) participa en diferentes enfermedades
hepáticas, pero se conoce muy poco sobre la importancia de COX-2 en el
desarrollo y progresión de la esteatohepatitis no alcohólica. Este estudio se
diseñó con el objetivo de esclarecer el papel de la expresión de COX-2 en
hepatocitos en la patogénesis de la esteatohepatitis y la fibrosis hepática.
En el presente trabajo, ratones transgénicos para COX-2 específica del
hepatocito (hCOX-2-Tg) y sus correspondientes controles wild-type (Wt)
fueron alimentados con una dieta deficiente en metionina y colina (MCD)
para establecer un modelo experimental de esteatohepatitis no alcohólica,
o inyectados con tetracloruro de carbono (CCl4) para inducirles fibrosis
hepática. En nuestro modelo animal, los ratones hCOX-2-Tg alimentados
con la dieta MCD presentaron menor grado de esteatosis, “ballooning”
e inflamación que los ratones Wt, en parte debido a una disminución
del reclutamiento e infiltración de los macrófagos hepáticos, con la
correspondiente disminución en los niveles de citoquinas pro-inflamatorias
en suero. Además, los ratones hCOX-2-Tg mostraron una atenuación
significativa del incremento del estrés oxidativo y la apoptosis hepática
inducida por la dieta MCD observada en los ratones Wt. Aún más, los
ratones hCOX-2-Tg tratados con CCl4 presentaban, de forma significativa,
estadíos menores de fibrosis y un menor contenido hepático de colágeno,
hidroxiprolina y marcadores pro-fibrogénicos que los controles Wt.
De forma conjunta, nuestros datos indican que la expresión constitutiva
de COX-2 en el hepatocito atenúa el desarrollo de la esteatohepatitis no
alcohólica y la fibrosis hepática en ratones, mediante una reducción de la
inflamación, el estrés oxidativo y la apoptosis, y mediante una modulación
de la activación de las células estrelladas hepáticas, respectivamente,
Pósters / Posters
sugiriendo un posible papel protector de la inducción de COX-2 en la
progresión de la enfermedad del hígado graso no alcohólico, EHGNA.
P02-17
Study of genetic factors determining the
heterogeneous activation of signaling pathways
associated with cardiac pathophysiology and their
contribution to the individual susceptibility to
cardiotoxicity caused by chemotherapy
Aurora Gómez Vecino1, Roberto Corchado Cobos2, Susana Fraile
Martín3, Carmen García Macías3, María Isidoro García4
1
Instituto de Biología Molecular y Celular del Cáncer (IBMCC)Centro de Investigación del Cáncer (CIC), Zamora, ES, 2Instituto de
Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad
de Salamanca/CSIC, Salamanca, ES, 3Servicio de Patología
Molecular Comparada. Instituto de Biología Molecular y Celular
del Cáncer (IBMCC-CIC). Universidad de Salamanca, Salamanca,
ES, 4Instituto de Investigación Biosanitaria de Salamanca
(IBSAL); Servicio de Bioquímica Clínica. Hospital Universitario de
Salamanca, Salamanca, ES
The cardiotoxicity of anthracyclines is a complex trait. The degree of
cardiac damage is in part mediated by an imbalance between different
intracellular signaling pathways such as p38MAPK or PI3K / AKT which
at the same time, have been described as being important in other processes
of cardiac tissue.
Working hypothesis: (i) Interindividual differences in cardiac levels of
signaling pathways may contribute to the different susceptibility among
patients to cardiac damage produced by anthracyclines (ii) Identification of
genomic regions associated with different levels of these signaling proteins
is a strategy to identify part of the genetic component of cardiotoxicity.
Material and methods: We studied a cohort of mice with ERBB2 + breast
cancer generated by a backcross between two syngeneic strains, C56BL/6 and
FVB (carrier of the transgene MMTV-ErbB2 / Neu). The animals were treated
with doxorubicin alone or in combination with docetaxel. Histopathological
parameters of cardiac damage were quantified using the Ariol automated
system. Levels of the following proteins were quantified by Luminex: pCREB
(Ser133), pAKT (Ser473), pSTAT5A / B (Tyr694 / Tyr699), pSTAT3 (Ser707),
p70S6K (Thr412), p38 MAPK (Thr180 / Tyr182), pJNK (Thr183 / Tyr185),
NFKB (Ser536) and pERK1 / 2 (Thr185 / Tyr187).
Results: (i) The genetic background influences the activation of heart
intracellular signaling pathways. (ii) The levels of activation of these
pathways are correlated with histopathological parameters of heart damage.
(iii) There are specific and common genetic regions of susceptibility of
complex trait (QTL) associated with both processes.
Conclusion: We used the levels of different intramyocardial signaling
pathways as intermediate phenotypes for the identification of part of
the genetic component of susceptibility to heart damage caused by
chemotherapy. These results will require further validation to be later
transferred to the human population.
P02-18
Effects of Tributyltin Chloride on Cybrids with
or without an ATP Synthase Pathologic Mutation
Ester López Gallardo1, Laura Llobet Sese2, Sonia Emperador Ortiz1,
Julio Montoya2, Eduardo Ruiz Pesini3
1
Universidad de Zaragoza- CIBER de Enfermedades Raras,
Zaragoza, ES, 2Universidad de Zaragoza, Zaragoza, ES, 3Universidad
de Zaragoza-Fundación ARAID, Zaragoza, ES
Background: The oxidative phosphorylation system (OXPHOS) includes
nuclear chromosome (nDNA)- and mitochondrial DNA (mtDNA)-encoded
47
Pósters / Posters
polypeptides. Many rare OXPHOS disorders, such as striatal necrosis
syndromes, are due to genetic mutations. Despite important advances in
sequencing procedures, causative mutations remain undetected in some
patients. It is possible that etiologic factors, such as environmental toxins,
are the cause of these cases. Indeed, the inhibition of a particular enzyme by
a poison could imitate the biochemical effects of pathological mutations in
that enzyme. Moreover, environmental factors can modify the penetrance
or expressivity of pathological mutations.
Objectives: To study the interaction between p.MT-ATP6 and an
environmental exposure that may contribute phenotypic differences
between healthy individuals and patients suffering from striatal necrosis
syndromes or other mitochondriopathies.
Methods: We analyzed the effects of the ATP synthase inhibitor
tributyltin chloride (TBTC), a widely distributed environmental factor that
contaminates human food and water, on transmitochondrial cell lines with
or without an ATP synthase mutation that causes striatal necrosis syndrome.
Doses were selected based on TBTC concentrations previously reported in
human whole blood samples.
Results: TBTC modifies the phenotypic effects caused by a pathological
mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic
show similar bioenergetics when compared with the untreated mutated
cells.
Conclusions: In addition to the known genetic causes, our findings suggest
that environmental exposure to TBTC might contribute to the etiology of
striatal necrosis syndromes.
P02-19
Puesta a punto de un modelo celular para el
estudio de la función OXPHOS en la enfermedad
de Alzheimer
Alba Pesini, Eldris Iglesias, Pilar Bayona-Bafaluy, Nuria Garrido,
Carmen Hernández-Ainsa, Ester López-Gallardo, Julio Montoya,
Eduardo Ruiz-Pesini
Universidad de Zaragoza, Zaragoza, ES
La enfermedad de Alzheimer (AD) es un proceso neurodegenerativo
del sistema nervioso central. Esta patología se caracteriza por pérdida
de memoria, cambios de comportamiento y demencia. Estos procesos
mentales dependen del correcto funcionamiento de las redes neuronales,
que a su vez dependen de las sinapsis entre las neuritas. La generación de
neuritas requiere producción de membrana plasmática.
Nosotros proponemos que el sistema OXPHOS, por su implicación en la
síntesis de novo de nucleótidos de pirimidina que participan en la síntesis
de fosfolípidos, juega un papel muy importante en la elaboración de estas
neuritas.
Para probar nuestra hipótesis, usamos una línea celular, neuroblastoma
SH-SY5Y, con capacidad de diferenciación a neurona. Dado que las
neuronas colinérgicas son una de las células más afectadas en la AD, vamos
a poner a punto un modelo de neurona colinérgica para el estudio de la
función OXPHOS. Lo primero que llevamos a cabo es la caracterización
genética y celular. Posteriormente estudiamos mediante citometría de
flujo diferentes parámetros de diferenciación neuronal y de neurona
colinérgica. También analizamos por ELISA los niveles de acetilcolina
celular. Finalmente, hemos estudiado los cambios que operan en el sistema
OXPHOS (actividad y cantidad del complejo respiratorio IV y consumo
de oxígeno) durante este tipo de diferenciación celular y los efectos que
provocan diferentes compuestos que actúan sobre el sistema OXPHOS en
la diferenciación.
Esta caracterización basal nos va a permitir analizar la función OXPHOS
en la síntesis de novo de nucleótidos de pirimidina, en la generación de
membrana y, finalmente, en la diferenciación neuronal.
48
XXXIX Congreso SEBBM
P02-20
Analysis of genetic and phenotypic interactions
between DNA damage / genotoxicity pathways
in heart tissue and heart damage caused by
anthracyclines and taxanes
Roberto Corchado Cobos(#)1, Aurora Gomez Vecino(#)2, Carmen García
Macías3, Telmo Rodrigues Teixeira3, María Isidoro García4, María
Asunción García Sánchez5, Julie Milena Galvis Jiménez6, Isabel
Ramos Fernández1, Adrián Blanco Gómez(*)7, Pedro Luis Sánchez
Fernández(*)8, Jesús Pérez Losada(*)7
1
Instituto de Biología Molecular y Celular del Cáncer
(IBMCC-CIC). Universidad de Salamanca/CSIC, Plasencia, ES,
2
Instituto de Investigación Biosanitaria de Salamanca (IBSAL),
Salamanca, ES, 3Servicio de Patología Molecular Comparada.
Instituto de Biología Molecular y Celular del Cáncer (IBMCCCIC). Universidad de Salamanca, Salamanca, ES, 4Instituto
de Investigación Biosanitaria de Salamanca (IBSAL); Servicio
de Bioquímica Clínica. Hospital Universitario de Salamanca,
Salamanca, ES, 5Departamento de Ciencias Biomédicas y del
Diagnóstico. Facultad de Medicina. Universidad de Salamanca,
Salamanca, ES, 6Instituto de Biología Molecular y Celular del
Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC, Salamanca.
Instituto de Investigación Biosanitaria de Salamanca (IBSAL)
Salamanca. Instituto Nacional de Cancerología de Colombia,
Bogotá, CO, 7Instituto de Biología Molecular y Celular del Cáncer
(IBMCC-CIC). Universidad de Salamanca/CSIC. Instituto de
Investigación Biosanitaria de Salamanca (IBSAL), Salamanca, ES,
8
Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC).
Universidad de Salamanca/CSIC. Servicio de Cardiología Hospital
Universitario de Salamanca, Salamanca, ES
Introduction: Anthracyclines are among the most widely used
chemotherapeutic agents in the treatment of a variety of tumors. The
identification of genetic and molecular factors responsible for the increased
risk of CDA (cardiotoxicity due to anthracyclines) will contribute to a
better understanding of their pathophysiology, which could lead to new
approaches to predict, prevent and treat this serious complication of
chemotherapy.
Working hypothesis: Based on two premises: (i) anthracyclines have a
pro-genotoxicity effect. Differences in anti-genotoxicity pathways and
genetic variants could contribute to different susceptibility to CDA. (ii) The
usefulness of a simplified model system to identify genetic determinants
involved in the quantitative inheritance of complex traits.
Materials and methods: We treated a cohort of mice carrying ERBB2
breast cancer with doxorubicin alone (N = 85) or in combination with
docetaxel (N = 77). The cohort was generated by a backcross between
two genetically homogeneous strains, C57BL/6 and FVB, with the latter
carrying the MMTV- ErbB2 / Neu transgene. Histopathologic heart
damage was assessed by quantification of histologic parameters using
Ariol automated system. Cardiac level of some key anti-genotoxicity
proteins: ATR total, pp53 (Ser15), P21 Total, Total MDM2, pHistone
H2AX (Ser139), pCHK1 (Ser345) and pCHK2 (Thr68) were quantified.
Results: We identified: (i) differences dependent on the genetic background
in both cardiotoxicity and the levels of proteins implicated in the pathways
protecting against genotoxicity; (ii) activation of anti-genotoxicity
pathways were associated with chemotherapy cardiotoxicity; (iii)
quantitative trait loci (QTLs) specific and common to cardiotoxicity and
the levels of the pathways studied.
Conclusion: We identified genetic determinants associated with
anthracycline cardiotoxicity using components of the anti-genotoxic
pathways as subphenotypes. Crosses of syngeneic mouse strains are useful
in these studies, but require further validation in the human population.
(#) Igual contribución como primeros autores.
(*) Igual contribución como autores senior.
Salamanca 2016
P02r-21
Xenobiotics that affect oxidative phosphorylation
alter differentiation of human adipose-derived
stem cells at concentrations that are found in
human blood
Laura Llobet, Janne M. Toivonen, Julio Montoya, Eduardo
Ruiz-Pesini, Ester López-Gallardo
Universidad de Zaragoza, Zaragoza, ES
Adipogenesis is accompanied by differentiation of adipose
tissue-derived stem cells to adipocytes. As part of this differentiation,
biogenesis of the oxidative phosphorylation system occurs. Many
chemical compounds used in medicine, agriculture or other human
activities affect oxidative phosphorylation function. Therefore, these
xenobiotics could alter adipogenesis. We have analyzed the effects
on adipocyte differentiation of some xenobiotics that act on the
oxidative phosphorylation system. The tested concentrations have
been previously reported in human blood. Our results show that
pharmaceutical drugs that decrease mitochondrial DNA replication,
such as nucleoside reverse transcriptase inhibitors, or inhibitors
of mitochondrial protein synthesis, such as ribosomal antibiotics,
diminish adipocyte differentiation and leptin secretion. By contrast, the
environmental chemical pollutant tributyltin chloride, which inhibits
the ATP synthase of the oxidative phosphorylation system, can promote
adipocyte differentiation and leptin secretion, leading to obesity and
metabolic syndrome as postulated by the obesogen hypothesis.
P02r-22
Una mutación en TSFM causa ataxia de inicio
infantil y cardiomiopatía no obstructiva
Sonia Emperador Ortiz1, M. Pilar Bayona-Bafaluy1, Ana
Fernández-Marmiesse2, Carmen Hernández-Ainsa1, Mercedes
Pineda3, Blanca Felgueroso4, Ester López-Gallardo1, Rafael Artuch3,
Iria Roca2, Eduardo Ruiz-Pesini1, María Luz Couce2, Julio Montoya1
1
Universidad de Zaragoza, Zaragoza, ES, 2Hospital Clínico
Universitario de Santiago de Compostela. Instituto de Investigación
Sanitaria de Santiago de Compostela (IDIS), Santiago de
Compostela, ES, 3Hospital Sant Joan de Deu. Institut de Recerca
Pediàtrica (IRP-HSJD), Barcelona, ES, 4Hospital Materno Infantil
Teresa Herrera, A Coruña, ES
El sistema de fosforilación oxidativa (OXPHOS) es una ruta
bioquímica implicada en muchos procesos celulares clave. Este
sistema está constituido por la cadena de transporte de electrones
(ETC) formada por los complejos I al IV (CI-CIV) y la ATP sintasa
(CIV). Estos complejos que forman el sistema OXPHOS incluyen
13 polipéptidos codificados en el DNA mitocondrial (mtDNA), pero
este mtDNA también codifica para 22 tRNA y 2 rRNA necesarios
para la expresión de dichos polipéptidos. El sistema de traducción
mitocondrial depende también de muchas proteínas codificadas en el
DNA nuclear (nDNA) de manera que mutaciones en cualquiera de
los componentes del sistema de traducción mitocondrial pueden ser
responsables de diferentes patologías implicadas en la disfunción
mitocondrial. En este trabajo describimos un paciente que sufre
ataxia progresiva de inicio infantil y cardiomiopatía hipertrófica,
portador de una mutación en homocigosis en el gen que codifica para
el factor de elongación de la traducción mitocondrial Ts (TSFM).
Mediante estudios bioquímicos, moleculares y celulares confirmamos
la patogenicidad de esta mutación, al rescatar el fenotipo normal en
fibroblastos del paciente tras la transfección de la proteína wild-type.
Recientemente se han descrito varios casos de diferentes mutaciones
en este mismo gen, en pacientes con fenotipos similares, y hay un
número creciente de publicaciones que describen mutaciones en genes
nucleares relacionados con la maquinaria de traducción mitocondrial.
Pósters / Posters
Por ello consideramos necesario el análisis de estos genes en pacientes
con una clínica de enfermedad mitocondrial tras descartar alteraciones
en el mtDNA.
Financiación: ISCIII; FIS (PI14/00005); CIBERER.
P02-23
The Fibronectin Synergy Site is Essential
for Platelet Function
María Benito-Jardón1, Mercedes Costell Rosselló2
Universitat de València ERI en Biotecnología y Biomedicina
Campus de Burjassot, Burjassot, ES, 2Universitat de València,
Burjassot, ES
1
The large extracellular matrix (ECM) protein Fibronectin (FN) is a
structural component of tissues but it is also found as a soluble plasma
protein. FN regulates cell activity by binding cellular receptors, like
integrins. The major binding site for integrins in FN is the RGD
motif located in the 10 th FN type III repeat. Additionally, the α5β1
and αIIb3 integrins can also bind the synergy site (DRVPPSRN) in
the FN type III9. The synergy site has been described as important
to mediate either the initial cell adhesion or the reinforcement of the
integrin-FN bond. The arginines within the synergy sequence were
mutated to generate a mouse with impaired synergy site (FNsyn).
The homozygous synergy mutant mice were viable and fertile, but
tail bleeding times were 1.6-fold increased compared to wild-type
littermates. Study of the thrombus formation after inducing an injury
in the microvasculature by intravital microscopy showed that the
occlusion of arterioles in FNsyn mice occurred significantly slower.
Platelets mediate this process and contain the FN ligands α5β1 and
αIIb3 integrins. Platelets of the FNsyn mice accumulated less FN,
pointing that their ability of binding FN is reduced. Since αIIb3
can also bind fibrinogen (Fg), to abolish any compensation by Fg we
generated the double mutant mice FNsynβ3-/-. Contrary to the β3 -/-, the
FN synβ3-/-mice died at E16.5 and from E12.5 display multiple skin
bleedings and oedema due to a defect in the separation of the lymphatic
system from the blood vessels. This process is platelet-dependent. To
deepen in the platelet function on a FNsyn background we performed
platelet spreading and flow chamber adhesion assays on FNsyn and
FN wt, which showed that β3-/- platelets are able to spread on FNwt but
not on FN syn and both β3 -/- and β3 +/+ were unable to adhere to FN syn
under physiological flow conditions while they could adhere to the
FN wt. These results confirmed the in vivo observations that the FN
synergy site plays an important role in haemostasis and in absence of
β3 integrin becomes essential for platelet function.
P02-24
Role of interferons and pro-inflammatory
mediators on macrophages in mechanical stress
in asthma
María Ester Quesada del Bosque, Hitasha Rupani, Arjana A.
Sivaloganathan, Peter H. Howarth, Tilman Sanchez-Elsner
Clinical and Experimental Sciences, Faculty of Medicine, University
of Southampton, Southampton, UK
Asthma is a chronic inflammatory disease that is characterised by airway
narrowing due to inflammation and remodeling. This pathology makes
airways hyperresponsive to a variety of environmental factors allergens,
viruses, among others.
Our group has studied the role of the alveolar macrophages (AMs)
from asthmatics in response to stress produced by respiratory viruses,
mainly rhinovirus (RV), which have shown a reduction in RV-induced
interferons (INFs) in severe asthmatics compared to healthy. Airways
49
Pósters / Posters
epithelium also plays an active role in immunity and inflammation and
has been shown to be impaired in asthmatics, being it more permeable
to environmental factors and overexpresses proinflammatory cytokines.
Part of our team (Grainge et al., N Engl J Med 2011;364:2006-15) showed
how bronchoconstriction affects epithelium remodeling stimulating
collagen synthesis and an increase in the number of glandular cells.
Numerous environmental factors interact to influence susceptibility and
trigger bronchoconstriction.
Our goal is to evaluate the effect of stress suffered from asthmatics
when exposed to triggers environments on the immune system, alveolar
macrophages, on their response to virus. We have recruited 24 mild
asthmatics that underwent methacholine challenge (bronchoconstriction)
or saline challenge (control group, no bronchoconstriction). Our
preliminary data show significant differences in the immune
response after the asthma attack, with macrophages expressing less
inflammatory cytokines after bronchoconstriction than before, when
exposed to RV. These results suggest that macrophages may become
less pro-inflammatory after bronchoconstriction, thus enhancing
inflammation in the lungs.
P02-25
PSA-NCAM en el cáncer colorrectal: expresión
en pacientes, líneas tumorales y estabilidad
de la cola de PSA
Almudena Fernández Briera1, Lucía Patiño Álvarez1, Almudena
Fernández Briera2, Carlos Villaverde Taboada3, Emilio Gil Martín1
1
Dpto. Bioquímica, Genética e Inmunología. Universidad de Vigo,
Vigo, ES, 2Universidad de Vigo, Vigo, ES, 3Dpto. Estadística e
Investigación Operativa. Universidad de Vigo, Vigo, ES
La pérdida de la adhesión entre células tumorales es uno de los factores
que fomenta su desagregación y posterior diseminación y metástasis.
Nuestro grupo viene trabajando en una de las moléculas de adhesión,
la NCAM (o molécula de adhesión de células neurales) en el cáncer
colorrectal (CCR). La NCAM es una glicoproteína de membrana
involucrada en la adhesión célula-célula y célula-matriz. Presenta tres
isoformas principales, conocidas por sus respectivas Mr, NCAM-120,
140 y 180. Estas isoformas pueden unir un homopolímero dealfa(2,8)
siálico (ácido polisiálico o PSA) en 2 de sus 6 sitios de N-glicosilación.
La retención de agua y fuerte repulsión electrostática del PSA se cree
que favorecen la diseminación de algunos tipos tumorales. La puesta
a punto de un protocolo para la inmunodetección de NCAM y PSA
en una misma muestra (patente Ref. ES2571302 A1) nos ha permitido
valorar la expresión de la proteína y de su grado de polisialilación en
la fracción citosólica y de membrana de 21 especímenes pareados de
tejido sano y tumoral de 21 pacientes con CCR, así como en 7 líneas
tumorales de colon y en cerebro de ratón. Los resultados demuestran
la expresión de NCAM-140 en 20 de los 21 pacientes analizados y
una aparente tendencia a sobreexpresarse en el tejido tumoral. Por su
parte, el PSA rinde una señal intensa y continuada desde Mr ~180 hasta
>260, que sugiere la presencia también de NCAM-180 fuertemente
polisialilada. Esta polisialilación (en su mayoría presumiblemente
NCAM-180) disminuye en gran parte en el tejido tumoral (p=0,008, de
acuerdo con el test de Wilcoxon). El estudio de la estabilidad térmica
y frente al pH del PSA, tanto en el tejido sano y tumoral de CCR como
en cerebro postnatal de ratón, confirman la presencia de NCAM-140
y 180 en ambos tejidos. Asimismo, se ha comprobado la expresión de
NCAM-140 en las líneas tumorales de colon HT-29, Caco-2, SW480
(no así en las SW620), DLD-1, HTC116 y Colo-205 en sus estados
adhesivo y en suspensión.
50
XXXIX Congreso SEBBM
P02r-26
Mitochondrial dynamics and mitochondrial DNA
Aida Rodríguez Nuevo, Eduard Noguera, Àngels Díaz Ramos,
Antonio Zorzano
Institute for Research in Biomedicine (IRB Barcelona), Barcelona,
ES; CIBER de Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM), Instituto de Salud Carlos III, Barcelona, ES;
Departament de Bioquímica i Biomedicina Molecular, Facultat de
Biologia, Universitat de Barcelona, Barcelona, ES
Mitochondria are dynamic organelles, which undergo fission and fusion
events. Fission is the process by which mitochondria are divided. The
opposite process is fusion: Mitofusin 1 and 2 are responsible for the outer
membrane fusion, whereas Opa1 is for the inner membrane fusion. The
flexibility entailed by these events is crucial to ensure mitochondrial
quality and functionality. Moreover, mitochondrial DNA (mtDNA)
integrity is tightly related to these proteins. Several authors purpose
Opa1 as determining for mtDNA stability. The absence of diffusion of
photo-activatable mitochondrial GFP protein in Opa1 deficient cell showed
complete fragmentation of the mitochondrial network. Using a C2C12
muscle cells we observed that ablation of Opa1 induced mtDNA instability.
Confocal microscopy analysis revealed a reduction of mtDNA labelling
and a misplacement of the mtDNA extra-mitochondrially. We plan to
investigate the effects of these observations, which are expected to be
crucial to understand the importance of both Opa1 and mtDNA regulation
in the skeletal muscle cell context.
P02-27
Non-invasive monitoring of hypoxia-inducible
factor activation by optical imaging during
antiangiogenic treatment in a xenograft model
of ovarian carcinoma
Beatriz Martínez-Poveda1, Valentí Gómez2, Marisa Alcaide-Germán2,
Sara Perruca2, Silvia Vázquez2, Emilio Alba3, Oriol Casanovas4,
María Laura García-Bermejo5, Luis del Peso2, Benilde Jiménez2
1
Universidad de Málaga, Andalucía Tech, Departamento de
Biología Molecular y Bioquímica, Facultad de Ciencias, Málaga,
ES, 2Departamento de Bioquímica, Instituto de Investigaciones
Biomédicas (CSIC-UAM), Madrid, ES,3Servicio de Oncología
Médica, Hospital Virgen de la Victoria, Málaga, ES, 4Laboratorio
de Angiogénesis Tumoral, IDIBELL, L’Hospitalet de Llobregat, ES,
5
Departamento de Patología, Hospital Ramón y Cajal, Madrid, ES
Targeting the hypoxia response pathway and angiogenesis are two
promising therapeutic strategies for cancer treatment. Their use as single
strategies has important limitations. Thus, development of combined
regimens has become an important step toward improving therapeutic
efficacy. Also, non-invasive monitoring of the response to targeted
biological therapies, as well as determination of the optimal schedule for
combination regimens has become an active field of research over the
last five years, with relevance for both preclinical and clinical settings.
Here, we used an optical imaging method to non-invasively monitor the
functional changes in HIF activity in response to antiangiogenic treatment
in a xenograft model of human ovarian carcinoma. A bioluminescent
reporter construct containing nine copies of the hypoxia response element
upstream of the luciferase gene (9xHRE-luciferase) was characterized in
vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft
model of ovarian carcinoma by means of optical imaging. We showed
that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the
HIF functional reporter occurs before the onset of massive tumor growth.
However, this system failed to detect hypoxia induced upon antiangiogenic
treatment due to the compensating effects of increased hypoxia and
decreased tumor cell viability caused by imbalanced neovascularization vs.
tumor expansion. Therefore, the readout based on HIF functional reporter
Salamanca 2016
could be conditioned by the dynamics of tumor growth and angiogenesis,
which is highly variable depending on the tumor type, tumor model and
stage of progression
P02r-28
Unraveling new roles for macrophages in cardiac
repair upon myocardial infarction
Laura Alonso Herranz1, Pilar Gonzalo1, Marta Cedenilla1, Vanessa
Nuñez1, Luis Jesús Jiménez-Borreguero1, Carlos López-Otín2, Alicia
G. Arroyo1, Mercedes Ricote1
1
Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid, ES, 2Universidad de Oviedo, Oviedo, ES
After myocardial infarction (MI), monocyte infiltration and subsequent
differentiation into macrophages is essential for cardiac recovery,
but mechanisms remain not well defined. To understand the role of
macrophages, we performed two different models of injury in C57BL/6
mice: permanent left anterior coronary artery ligation (LAD-ligation) and
cryoinjury. LAD-ligation resulted in more pronounced worsening of the LV
function and higher fibrotic deposition compared to cryoinjury.
In order to analyze the possible role of macrophages in the different
functional outcome, we compared the genetic signature of isolated
cardiac macrophages upon injury and found mainly differences in
matrix-remodeling genes. Interestingly persistent elevated levels of
MT1-MMP were found in post-LAD versus post-CRYO macrophages.
This result prompted us to investigate the particular functions of this
protease in macrophages after cardiac injury. Consequently, LAD-ligation
was performed in a transgenic mouse line with macrophage specific
deletion of the MT1-MMP(MT1-MMPDLysM mice). Histological and
echocardiographic analysis in MT1-MMPDLysM mice upon MI showed a
decrease in LV dilatation and fibrosis, better preserved healthy myocardium
and higher density of arterioles that finally lead to amelioration of cardiac
dysfunction compared to their control littermates.
This study unravel that macrophage MT1-MMP absence attenuates cardiac
dysfunction by increasing arteriogenesis, and suggest new treatment
options for cardiac ischaemic disease based on metalloprotease modulation
in cardiac macrophages.
Funding: TV3 Marató Foundation (MTV/PIC1203) and “Obra Social La
Caixa” (International PhD “la Caixa” - Severo Ochoa 2014).
P02-29
Lack of LOXL2 (lysil oxidase-like 2) reduces
mouse renal fibrosis after surgical unilateral
ureteral obstruction
Pósters / Posters
roles in organ development, cicatrization, and fibrosis. Irrespective
of its catalytic activity, LOXL2 has been shown to be an inductor of
epithelial-mesenchymal transition (EMT). In this work, unilateral ureteral
obstruction (UUO), an experimental model of obstructive nephropathy
characterized by EMT and renal fibrosis (Grande et al., Nat Med. 2015;
21:989-97) was performed in LOXL2 gain and loss of function mice
(Martin et al., EMBO J. 2015; 34:1090-109), and its effects in ECM proteins
expression and renal fibrosis were analyzed as previously described
(Muñoz-Felix et al.; KidneyInt. 2014; 85:319-32). Lack of expression
of LOXL2 resulted in lowered ECM proteins (collagen-I and alfa-SMA)
expression and reduced interstitial renal fibrosis compared with WT mice
after 15 days of UUO. These results uncover a role of LOXL2 in kidney
fibrosis, thus being a potential target to pharmacological intervention in
human chronic kidney disease.
P02m-30
Vochysia rufa stem bark extract protects
endothelial cells against high glucose damage
Neire Moura de Gouveia1, Sonia Ramos2, M. Ángeles Martín2,
Luis Goya Suárez2, Olga Palomino3
1
Department of Biofunctional Science, Faculty Mineirense,
Mineiros, Goias, Brasil, BR, 2Department of Metabolism and
Nutrition, Institute of Science and Food Technology and Nutrition
(ICTAN – CSIC), Madrid, ES, 3Department of Pharmacology, Faculty
of Pharmacy, Universidad Complutense de Madrid, Madrid, ES
Increased oxidative stress by persistent hyperglycemia is a widely accepted
factor in vascular damage responsible for type 2 diabetes complications.
The plant Vochysia rufa (Vr) has been used in folk medicine in Brazil for
the treatment of type 1 and 2 diabetes. In this study, the protective effect of
a Vr stem bark extract against a challenge by a high glucose concentration
on EA.hy926 (EA) endothelial cells was evaluated. The Vr stem bark was
extracted with water by maceration and then evaporated until dryness
under vacuum. Treatment with 0.5-100 μg/mL of Vr extract for 24h
did not affect cell viability. The extract was diluted at concentrations of
5, 10 and 25 μg/mL and maintained for 24 hours along with 30 mM of
glucose to evaluate the protective effect of the extract on the EA cells.
The treatment of EA cells with 30 mM of glucose for 24h significantly
increased the cell damage. Furthermore, EA cells treated with 30 mM of
glucose showed a decrease of reduced glutathione (GSH) concentration
and increased protein carbonyl levels and activity of antioxidant enzymes,
compared to control. Vr concentrations significantly reduced cell damage
evoked by glucose. While 5 and 10 μg/mL Vr evoked a partial protection
against the glucose insult, 25 μg/mL Vr fully recovered GSH, antioxidant
enzymes and carbonyls to baseline levels. This study demonstrates that
phytochemicals from V. rufa stem bark may help to protect endothelial cells
against oxidative damage by modulating GSH concentration, antioxidant
enzyme activity and protein carbonyl levels.
Carmen Arizmendi López1, María Teresa Grande2, María Auxiliadora
Aparicio2, Alberto Martín Martín3, Annette G. Düvet2, Luis
Gamello-Pozuelo2, Miguel Ángel Arévalo4, Amparo Cano3,
José Miguel López Novoa2
1
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca, Salamanca, ES, 2Departamento de Fisiología
y Farmacología, Universidad de Salamanca, Salamanca, ES,
3
Departamento de Bioquímica, UAM, Instituto de Investigaciones
Biomédicas Alberto Sols, CSIC-UAM, Madrid, ES, 4Departamento
de Histología y Anatomía Humana, Universidad de Salamanca,
Salamanca, ES
M. Rodríguez Quiroga1, L. Vázquez Iglesias1, L. Barcia Castro1,
M. Páez de la Cadena1, F. J. Rodríguez Berrocal1, O. J. Cordero2
1
Departamento de Bioquímica, Genética e Inmunología.
Universidad de Vigo, Vigo, ES, 2Departamento de Bioquímica
y Biología Molecular. Universidad de Santiago de Compostela,
Santiago de Compostela, ES
LOXL2 is the member of the lysil oxidase family most abundant in the
kidney. The family classical catalytic activity is the formation of aldehydes
from lysine residues in collagen and elastin precursors. These aldehydes
undergo spontaneous chemical reactions with other lysyl oxidase-derived
residues that results in cross-linking of proteins of the extracellular matrix
(ECM) resulting in its stabilization. Lox enzymes have shown important
Cancer Stem Cells (CSCs) include a subpopulation of cells responsible for
tumour survival, resistance to chemotherapy, recurrence and metastases,
called Metastatic Stem Cells (MetSCs). Recently, DPP-IV/CD26 was
proposed as a new CSC and MetSCs biomarker in colorectal cancer
(CRC). In a panel of CRC cell lines from patients at different stage:
SW1116 (stage A), HT-29, Caco-2, SW480 (stage B), SW620 (lymph node
P02-31
CD26: a colorectal cancer stem cell marker
51
Pósters / Posters
metastasis), DLD -1 (stage C), COLO205 (peritoneal metastasis) and T84
(lung metastasis), we had evaluated by flow cytometry the presence of the
most favourite biomarkers candidates for CRC CSCs and MetSCs: CD133,
CD44, EpCAM, LGR5, riboflavin-dependent autofluorescence, and CD26.
Here we studied them in the spheroids originated from those lines in vitro.
All cell lines could form spheres in the first generation, confirming the
presence of CSCs in all cell lines. SW1116 showed the smallest spheres
and the lower number of cells disaggregated from the spheres. All cell
lines, except SW1116, did form subspheres in serial passages. Efficiency
was essentially maintained through passages in these cell lines with
self-renewal, except SW620 (loss of efficiency) and T84 (enhanced
efficiency). E-cadherin expression among sphere-forming cells showed
quite equivalent levels and higher than in the cell lines, as expected for
proliferating cells in epithelial state, although not all (particularly Colo205
cells) are E-cadherin+. Sphere-forming cells also showed enhanced
levels of LGR5 expression (2-3-fold in general). Still, the heterogeneity
remains, as DLD-1 and Colo205 showed similar frequencies (11%) in
comparison to the HT-29 (52%) and Caco-2 (88%) cell lines with much
higher expression. To note, the cell lines with higher frequencies of LGR5+
also show a high frequency of the LGR5+ EpCAMhigh subset. The other
biomarkers showed an important heterogeneity where CD26 and CD133
mark different CSCs subsets.
Financiación: Plan Nacional I+D+I (PI15/02007), Fundación Científica
AECC (GCB13131592CAST) y Xunta de Galicia (GRC2014/019 y
R2014/039).
P02-32
Relación entre arginina, poliaminas, obesidad
y resistencia insulínica: estudio en ratones ob/ob,
un modelo de obesidad y riesgo cardiovascular
Sandra Tavárez Alonso, Giovanna Pulido Núñez, Encarna Morant
Peiró, Lidia López López, Eulalia Alonso Iglesias
Departamento de Bioquímica y Biología Molecular, Facultad de
Medicina, Universidad de Valencia, Valencia, ES
Numerosas evidencias experimentales sustentan beneficios de la arginina
en las patologías de riesgo cardiovascular y, concretamente, en la obesidad
y la resistencia insulínica (RI). Los mecanismos moleculares propuestos
para la acción de la arginina se han relacionado con su papel como sustrato
precursor del óxido nítrico. Sin embargo la implicación en estos efectos de
las poliaminas, otros metabolitos derivados de la arginina relacionados tanto
con la obesidad como con la liberación y acción de la insulina, permanece
sin ser evaluada. Con este objetivo hemos analizado en nuestro estudio
los niveles de poliaminas (putrescina, espermidina y espermina; HPLC)
en páncreas y músculo de ratones obesos (ob/ob) y control, los efectos
sobre ellos de la suplementación de arginina (1% en el agua de bebida), así
como su relación con la RI (índice HOMA), grado de adiposidad y otros
parámetros de deterioro metabólico. En nuestro modelo, la suplementación
de arginina redujo significativamente la ganancia ponderal, el peso de
los paquetes grasos, el estrés oxidativo (MDA), la inflamación (IL-6) y,
particularmente, la RI (disminución de glucemia e insulinemia). Estos
efectos se relacionan con cambios en los niveles de poliaminas que
incluyen disminución de espermina en páncreas y elevación muscular de
putrescina. De acuerdo con estos resultados, los efectos metabólicos de la
arginina parecen implicar diferentes procesos y vías, algunas de las cuales
podrían suponer cambios sustanciales en los niveles de poliaminas. Estos
policationes, según se ha propuesto, podrían ejercer papeles selectivos
estimuladores o permisivos en relación a la síntesis y liberación de insulina
y en la sensibilidad a la misma.
Financiado con las ayudas CONSOLIDER-INGENIO CSD2007-00063 y
AGL2014-58205-REDC.
52
XXXIX Congreso SEBBM
P02-33
La citoquina proinflamatoria TNF-α promueve
la fosforilación y la deslocalización de la proteína
asociada a microtúbulos Tau en células de
neuroblastoma humano SH-SY5Y diferenciadas
Montaña Caballero Bermejo, Marta Olivera Santa-Catalina, Juan
Carlos Alonso, Ricardo Argent, Francisco Centeno, Mª Jesús Lorenzo
Benayas
Universidad de Extremadura, Cáceres, ES
La enfermedad de Alzheimer (EA) es un desorden neurodegenerativo asociado
a la edad. Uno de los marcadores neuropatológicos de esta enfermedad es la
presencia de ovillos neurofibrilares (NFT) compuestos por la proteína Tau
hiperfosforilada y proteolizada. Se ha descrito que TNF-α colocaliza con los
NFT en las zonas del cerebro afectadas por la EA, lo que sugiere que esta
citoquina puede regular la formación, la agregación y la deposición de los
NFT. Además, la fosforilación de Tau aumenta en un entorno inflamatorio,
aunque de momento se desconoce la importancia de las citoquinas sobre
la fosforilación de residuos específicos de Tau. El objetivo de este trabajo
fue estudiar el efecto de la citoquina TNF-α sobre la fosforilación residuo
específica y la localización intracelular de la proteína Tau en células SH-SY5Y
diferenciadas. El tratamiento de las células con TNF-α produjo un aumento
de los niveles y de la fosforilación de Tau en los residuos Thr-50 y Thr-69 de
manera dependiente a la concentración utilizada. El aumento en los niveles
de Tau inducidos por TNF-a se previno en presencia de actinomicina D y de
cicloheximida lo que sugiere que esta citoquina induce la expresión genética
de Tau en nuestras condiciones experimentales. Además, TNF-a promovió
cambios en la localización de las formas de Tau fosforiladas, que pasaron de
encontrarse principalmente en las neuritas a concentrarse en el soma celular y
en núcleo. Nuestros resultados sugieren que la inflamación puede contribuir a
la patología neurofibrilar de la proteína Tau.
Trabajo financiado por los proyectos: GRU10046, GRU15164 (FEDERJunta de Extremadura) y FIS PS/1330.
P02-34
Different expression levels of DLK1 inversely
modulate proliferation and oncogenic potential of
human MDA-MB-231 breast cancer cells through
inhibition of NOTCH signaling
María Julia González Gómez1, Ana Isabel Naranjo Pastor2, Victoriano
Baladrón García2, Jorge Laborda Fernández1, María Luisa Nueda
Sanz1
1
Biochemistry and Molecular Biology Branch, Department of
Inorganic, Organic Chemistry and Biochemistry. School of
Pharmacy. University of Castilla-La Mancha/CSIC, Albacete, ES,
2
Biochemistry and Molecular Biology Branch, Department of
Inorganic, Organic Chemistry and Biochemistry. Medical School;
CRIB/Biomedicine Unit. University of Castilla-La Mancha/CSIC,
Albacete, ES
NOTCH receptors participate in cell proliferation and survival of several
types of cancer cells. Evidence accumulated indicates that these receptors
can function as oncogenes or as tumor suppressors depending on the cellular
context. The EGF-like protein DLK1 acts as a NOTCH signaling inhibitor
and it is also involved in the regulation of cell growth and cancer. In this
work, we focused on the role of the protein DLK1 in the control of breast
cancer cell growth, where NOTCH receptors have been shown to play both
antagonist roles. We found that human DLK1 inhibit NOTCH1 signaling
in MDA-MB-231 breast cancer cells. The proliferation rate and migration
capabilities of these cells depended upon the level of NOTCH1 activation
and signaling as regulated by DLK1. In particular, high levels of DLK1
expression leaded to a great decrease in NOTCH1 signaling, which associated
Salamanca 2016
to a decrease of breast cancer cell proliferation and migration. On the contrary,
lower levels of NOTCH1 signaling inhibition caused by lower levels of DLK1
overexpression led to an increase of in vitro MDA-MB-231 cell migration,
and both in vitro and in vivo cell proliferation. The data presented in this work
suggest that a fine regulation of NOTCH signaling plays an important role in
the control of breast cancer cell proliferation and migration.
Financial Support: This work was supported by funds from the Spanish
Cancer Association (AECC) and the Health Council of the Regional
Government of Castilla-La Mancha, Spain (PI-2008/20), and from the
Ministry of Economy and Competitiveness (BFU2010-16433).
P02-35
Relación entre la calpaína-2 y la actividad
nucleolar en células humanas de cáncer de colon
Marcelino Telechea-Fernández1, Rosa Zaragozá2, Concha García3,
Juan R. Viña3, Andrés Cervantes1, Elena R. García-Trevijano3
1
Dpto. Hematología y Oncología Médica. Facultad Medicina. Univ.
Valencia. IIS INCLIVA, Valencia, ES, 2Dpto. Anatomía y Embriología
Humana. Facultad Medicina. Univ. Valencia. IIS INCLIVA, Valencia,
ES, 3Dpto. Bioquímica y Biología Molecular. Facultad Medicina.
Univ. Valencia. IIS INCLIVA, Valencia, ES
La desregulación de la calpaína-2 (CAPN2), una Cys-proteasa que participa
en el procesamiento limitado de múltiples proteínas, se han asociado a
una menor supervivencia de pacientes con cáncer colorectal (CCR). Sin
embargo, aún no se ha establecido el papel de la CAPN2 en la progresión
tumoral y su posible valor pronóstico. El tamaño/número de nucléolos son
factores de mal pronóstico en la progresión tumoral. Se analizó la posible
relación entre la CAPN2 y la actividad nucleolar en células humanas de
CCR, DLD-1. No se observó una correlación entre la expresión de CAPN2
y el tamaño/número de nucléolos como indicador de malignidad tumoral.
Sin embargo, mediante inmunotinción, se identificó a la CAPN2 como
una CAPN de localización preferentemente nucleolar. Se ha sugerido la
autoproteólisis como forma de limitar la actividad CAPN sobre sus sustratos.
Se estudiaron la autoproteólisis de la CAPN2 y su papel en el nucléolo.
Mientras que la CAPN2 se encontraba en su forma completa en el citosol, en
el nucleolo estaba proteolizada. Es más, observamos una correlación entre el
procesamiento de la CAPN2 nucleolar y la malignidad tumoral en células de
CCR con mutaciones KRASG13D frente a KRASWT. Las medidas de actividad
enzimática en el nucléolo confirman que la CAPN2 nucleolar se encontraba
plenamente activa. El extremo Nt de la histona H3 aparecía proteolizado en
nucleolos donde se observaba una colocalización de CAPN2 y el extremo
Ct de la histona H3. El tratamiento de las células con calpeptina bloquea
dicha proteólisis, lo que sugiere que la CAPN2 puede ser un mediador del
procesamiento de la histona H3 nucleolar. Nuestros datos sugieren que el
papel de la CAPN2 nucleolar en células tumorales podría ser el de estimular
la biogénesis ribosomal a través del procesamiento de la histona H3 y
consecuentemente, la estimulación de la expresión de genes ribosomales.
Financiado: BFU-2013-46434-P a J.R.V y R.Z; PI12/02394 a E.R.GT
ambos proyectos cofinanciados por FEDER; GVPROMETEOII/2014-055
a J.R.V. y GVPROMETEO/2013-005 a AC.
P02-36
Regulators of metal homeostasis normalize the
FRDA phenotypes in a Drosophila melanogaster
model of the disease
Pablo Calap Quintana1, Sirena Soriano2, José Vicente LLorens1,
María José Martínez-Sebastián1, María Dolores Moltó3
1
University of Valencia, Burjassot, ES, 2University of Valencia,
Burjassot, ES; Baylor College of Medicine, Houston, US, 3University
of Valencia, Burjassot, ES, CIBERSAM, INCLIVA, Valencia, ES
Pósters / Posters
The neurodegenerative disease Friedreich’s ataxia (FRDA) is caused by a
reduction in the synthesis of the mitochondrial protein frataxin. Frataxin
deficiency results in several biochemical disturbances including impaired
iron-sulphur cluster biogenesis and accumulation of mitochondrial
iron coupled to cytosolic iron depletion. Besides the alteration in the
iron homeostasis, it has been recently shown that other metals such as
Cu or Zn could play a role in the pathogenesis of the disease. Using an
RNAi-based model of FRDA in Drosophila, we previously found that
the levels of iron, zinc, copper, manganese and aluminum were also
increased in the FRDA flies. We therefore set out to test whether genetic
modification of key pathways regulating metal content and distribution
would improve FRDA phenotypes by restoring metal homeostasis in
fly models of the disease. For this purpose, two frataxin knockdown
lines were used showing a neurodegenerative phenotype (rough eye) or
a decreased motor performance phenotype, respectively. Changes in the
expression of different homeostasis regulators, transporters or chaperones
of Fe, Zn and Cu were able of suppressing the rough eye and the motor
performance phenotype of the FRDA model flies. Overexpression of the
Metal-responsive Transcription Factor-1 and knockdown of the metal
sequestering metallothioneins (MT) also had a beneficial effect on both
phenotypes. According to our results, the Fe related modifiers, some of the
zinc transporters and the overexpression of MTF-1 could be exerting their
beneficial effect by restoring the normal iron level. The knockdown of the
MT as well as the MTF-1 overexpression also restored the increased level
of lipid peroxidation found in the FRDA flies, highlighting the critical role
of metal dysregulation in the oxidative stress alterations of FRDA. Different
chelators for Cu and Zn were able of improving the motor impairment of
frataxin depleted flies, showing that the imbalance of other metals besides
Fe must be taken into account to fully understand the pathology of FRDA.
P02-37
Análisis de la metilación del gen DPPIV en ADN
sérico de pacientes con cáncer colorrectal como
posible mecanismo regulador
Loretta De Chiara1, Mar Ferreira Carrera1, Olalla Otero Estévez1,
Oscar Cordero2, María Páez de la Cadena1, José Ignacio Rodríguez
Prada3, Pamela Estévez Boullosa3, Lucía Cid Gómez3, David
Remedios Espino4, Francisco Javier Rodríguez Berrocal1,
Vicenta Soledad Martínez Zorzano1
1
Dpto. de Bioquímica, Genética e Inmunología. Universidade
de Vigo, Vigo, ES, 2Dpto. Bioquímica y Biología Molecular,
Universidade de Santiago de Compostela, Santiago de Compostela,
ES, 3Servicio de Aparato Digestivo. Hospital Álvaro Cunqueiro
EOXI Vigo, Vigo, ES, 4Servicio de Aparato Digestivo. Complexo
Hospitalario Universitario de Ourense, Ourense, ES
Previamente hemos descrito el descenso de los niveles séricos de la
proteína CD26 soluble (CD26s) en pacientes con cáncer colorrectal (CCR)
y adenomas avanzados (AA). En este estudio se analiza la metilación
del promotor del gen codificante de CD26, gen DPPIV, en ADN sérico
de pacientes con neoplasia avanzada (CCR o AA) y de individuos sanos,
para determinar si la disminución de CD26s en el suero de los pacientes
se debe al silenciamiento de DPPIV por hipermetilación de la isla CpG
analizada. Se cuantificó el porcentaje de metilación en las muestras (21 con
neoplasia avanzada y 27 sanos) mediante qPCR específica de metilación
con sondas TaqMan. Para facilitar el estudio, la isla CpG de 317 pb descrita
en el promotor de DPPIV, se subdividió en 3 subislas CpG (1, 2 y 3). No
se encontraron diferencias estadísticamente significativas en el porcentaje
medio de metilación para ninguna de las subislas analizadas entre los
individuos sanos y los pacientes con neoplasia avanzada. Se observó
una correlación positiva y estadísticamente significativa en la metilación
entre las 3 subislas en los individuos sanos. Sin embargo, en los pacientes
no encontramos correlación entre las subislas 1-2 y 1-3, pero sí entre
las subislas 2-3. Tampoco se observó correlación entre el porcentaje de
metilación y los niveles séricos de CD26s. Estos resultados sugieren que
53
Pósters / Posters
la metilación del promotor de DPPIV, al menos en la isla CpG estudiada,
no está relacionada con la reducción de CD26s en suero de pacientes con
neoplasia avanzada.
Financiación: Plan Nacional I+D+I 2014-2020 (AES) ISCIII-FEDER
(PI15/02007), Fundación Científica AECC (GCB13131592CAST),
“Axudas consolidación e estructuración de unidades de investigación
competitiva” (GRC2014/019) y REGICC (R2014/039) de Xunta de Galicia.
P02-38
Effects of Cyclin D-CDK4/6 axis inhibition
on PTEN-deficient neoplasias
Maria Alba Dosil1, Cristina Mirantes1, Nuria Eritja1, Isidre Felip1,
Raul Navaridas1, Sonia Gatius1, Maria Santacana1, Eloi Garí2, Xavier
Matias-Guiu1, Xavier Dolcet1
1
Oncologic Pathology Group. Dept. Ciències Mèdiques Bàsiques,
Universitat de Lleida. Hospital Universitari Arnau de Vilanova.
Institut de Recerca Biomèdica de Lleida, IRBLleida, Lleida, ES,
2
Cell Cycle Group. Dept. Ciències Mèdiques Bàsiques, Universitat
de Lleida. Hospital Universitari Arnau de Vilanova. Institut de
Recerca Biomèdica de Lleida, IRBLleida, Lleida, ES
PTEN is one of the most frequently mutated genes in human cancers. A
significant proportion of human malignancies present PTEN deficiencies.
Among all of them, the frequency of alterations in PTEN is particularly
high in endometrial carcinomas. Absence of PTEN leads to an abnormal
cell division, which is one of the most important hallmarks in cancer. CDKs,
their cognate Cyclins and CDKs inhibitors have been found mutated in a
significant fraction of human neoplasias and are presented as key targets in
the treatment of the pathogenesis of cancer. For that reason, several CDK
inhibitors have been developed as a strategy to generate new anticancer
drugs. Palbociclib or PD-332991 inhibits specifically CDK4/CDK6 and
it has been approved by the US Food and Drug Administration for use
in metastatic breast cancer. First of all, we have studied PTEN-driven
tumorigenesis in the context of Cyclin D1 deficiency in the endometrium,
thyroid and prostate. Histological examination by pathologists revealed
that Cyclin D1 deficiency did not impairs PTEN tumorogenesis neither in
thyroid nor in prostate. Interestingly, Cyclin D1 absence displayed a slight
but not statistically significant reduction in the incidence and progression
of endometrial lesions. Next, we have used a Tamoxifen-inducible PTEN
knock-out mouse model to assess the anti-tumoral effects of Palbociclib
on endometrial and prostate tumors, and thyroid hyperplasias. Our results
demonstrate that Palbociclib treatment triggers shrinkage of endometrial
lesions, but show poors beneficial effect on thyroid hyperplasias and
prostate carcinomas. To date, this is the first pre-clinical study evaluating the
response to Palbociclib in endometrial, thyroid and prostate malignancies
driven by PTEN deficiency.
P02-39
Estudios de preferencia metabólica en células
del “microambiente angiogénico”
Mª Carmen Ocaña1, Beatriz Martínez-Poveda1, Ana R. Quesada2,
Miguel Ángel Medina2
1
Universidad de Málaga, Andalucía Tech, Departamento de Biología
Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto
de Biomedicina de Málaga), Málaga, ES, 2Universidad de Málaga,
Andalucía Tech, Departamento de Biología Molecular y Bioquímica,
Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga);
Unidad 741, CIBER de Enfermedades Raras (CIBERER), Málaga, ES
El “redescubrimiento” del efecto Warburg a principios de este siglo XXI y
de la elevada glutaminolisis tumoral han contribuido a un renovado interés
por el metabolismo en oncología. Además, estudios recientes han dado
54
XXXIX Congreso SEBBM
importancia al metabolismo de células endoteliales. Resulta interesante
indagar más en el metabolismo de este tipo celular así como de otras
células de su microentorno (células tumorales e inflamatorias, entre otras).
Mediante distintas aproximaciones metodológicas y utilizando distintos
tipos celulares del “microambiente angiogénico” se pretende conocer
más sobre el papel de la glucosa y la glutamina, así como la posible
utilización de ácidos grasos como el palmitato, en estos tipos celulares.
La adquisición de estos conocimientos podría suponer el comienzo de
una posible vía para “atacar” a la angiogénesis patológica no solo con
compuestos anti-angiogénicos, sino utilizando simultáneamente como
diana el metabolismo endotelial y/o tumoral.
Our experimental work is supported by grants BIO2014-56092-R
(MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and
FEDER) and funds from group BIO-267 (Andalusian Government). The
“CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain)].
This communicaction has the support of a travel grant “Universidad de
Málaga. Campus de Excelencia Internacional Andalucía Tech”.
P02-40
Generación de una proteína quimera de
endoglina soluble y proteína fluorescente verde
(GFP). Posibles aplicaciones en el estudio
de la función de endoglina soluble
Lidia Ruiz Llorente, Carmen Langa, Carmelo Bernabeu
Centro de Investigaciones Biológicas (CIB) y Centro de Investigación
Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, ES
Endoglina (Eng; CD105) es una glicoproteína de membrana de tipo I que
funciona en células endoteliales como receptor auxiliar de TGF-βs y BMPs,
y que modula la fisiopatología vascular. Además de las dos isoformas de
membrana, L-endoglina (long endoglin) y S-endoglina (short endoglin), existe
una forma de endoglina soluble (sol-Eng) que se genera por la acción de la
metaloproteasa-14 (MMP-14) sobre la región extracelular de la proteína de
membrana (López-Novoa y Bernabeu, 2010). Se han descrito niveles elevados
de esta forma soluble en pacientes con preeclampsia, hipercolesterolemia,
aterosclerosis y cáncer (Valbuena-Díez et al., 2012). Además, sol-Eng es un
marcador de daño cardiovascular en pacientes con hipertensión y diabetes
y tiene una acción patogénica en preeclampsia. Endoglina soluble inhibe la
angiogénesis, así como la proliferación, migración e invasión celular (del
Castillo et al., 2015 y Gallardo-Vara et al., 2016). Sin embargo, los mecanismos
de acción de sol-Eng no han sido elucidados todavía y son necesarias nuevas
herramientas y aproximaciones experimentales en este campo. En nuestro
laboratorio hemos clonado el dominio extracelular de endoglina en el plásmido
pEGFP-N1 para generar una proteína de fusión con GFP dando lugar al vector
pEGFP-N1/ENG.EC. Este vector ha sido secuenciado y la proteína de fusión
ha sido caracterizada mediante transfecciones transitorias y estables en células
CHO-K1 por técnicas de citometría, SDS-PAGE, inmunodetección, ELISA y
microscopio de fluorescencia. La construcción aquí descrita podría utilizarse
como herramienta en el estudio de la funcionalidad de sol-Eng tanto en
situaciones control como patológicas.
P02r-41
Diseño y caracterización de modelos celulares
para el estudio del síndrome de Pearson
Carmen Hernández Ainsa, Pilar Bayona-Bafaluy, Julia Barrios,
Eduardo Ruiz Pesini, Julio Montoya, Sonia Emperador
Universidad de Zaragoza, Zaragoza, ES
El síndrome de Pearson (PS, OMIM-557000) es una enfermedad rara,
caracterizada principalmente por anemia sideroblástica y disfunción
pancreática exocrina, aunque se pueden ver implicados otros sistemas como
el renal. Se debe a la presencia de una única deleción grande en el mtDNA,
Salamanca 2016
Pósters / Posters
o SLSMD (single large-scale mtDNA deletion), en un alto porcentaje de
heteroplasmia, que suele ser más abundante en sangre que en otros tejidos.
El tratamiento es sintomático y se basa en transfusiones y reemplazamiento
de enzimas pancreáticos, pero a pesar de ello los pacientes suelen morir en la
infancia. Los individuos que sobreviven desarrollan síndrome de Kearns-Sayre
(KSS), por lo que se intenta retrasar la aparición de los síntomas neurológicos
mediante el tratamiento con ácido ascórbico y ácido folínico. Para estudiar si
la dinámica de propagación de las SLSMD se podría ver afectada por factores
como su tamaño y localización, porcentaje de heteroplasmia, fondo genético
nuclear o mitocondrial, estado celular proliferativo o no proliferativo, el tipo
celular maduro o las condiciones de cultivo; se están generando y estudiando
distintos modelos celulares. En primer lugar, se están realizando estudios
genéticos y bioquímicos con fibroblastos en cultivo obtenidos a partir de
biopsias de piel de dos pacientes PS, con deleciones de distinto tamaño, y de
un paciente control. También se han obtenido híbridos transmitocondriales o
cíbridos mediante la fusión fibroblastos enucleados y plaquetas de pacientes,
con la línea celular de osteosarcoma 143b rho0. En todos los casos se han
conseguido diferentes porcentajes de heteroplasmia, lo que permitirá hacer
estudios comparativos. Actualmente, también se está trabajando en el
desarrollo de células pluripotentes inducidas (iPSC) a partir de fibroblastos de
pacientes PS. Estos modelos celulares permitirán aumentar el conocimiento
sobre esta enfermedad.
(ALP) and generated by hydrolysis of ATP via eNPP1. For the first time,
the present study investigated extracellular pyrophosphate metabolism
during hemodialysis sessions (including its synthesis via eNPP1 and its
degradation via ALP) in physiological conditions.
Methods and Findings: 45 patients in hemodialysis were studied.
Physiological ALP activity represents only 4–6% of clinical activity.
ALP activity increased post-hemodialysis by 2% under ideal conditions
(87.4 ± 3.3 IU/L vs. 89.3 ± 3.6 IU/L) and 48% under physiological
conditions (3.5 ± 0.2 IU/L vs. 5.2 ± 0.2 IU/L). Pyrophosphate
synthesis by ATP hydrolysis remained unaltered post-hemodialysis.
Post-hemodialysis plasma pH (7.45 ± 0.02) significantly increased
compared with the pre-dialysis pH (7.26 ± 0.02). The slight variation
in pH (~0.2 units) induced a significant increase in ALP activity (9%).
Addition of phosphate in post-hemodialysis plasma significantly
decreased ALP activity, although this effect was not observed with the
addition of urea. Reduction in phosphate levels and increment in pH
were significantly associated with an increase in physiological ALP
activity post-hemodialysis. A decrease in plasma pyrophosphate levels
(3.3 ± 0.3 μmol/L vs. 1.9 ± 0.1 μmol/L) and pyrophosphate/ATP ratio
(1.9 ± 0.2 vs. 1.4 ± 0.1) post-hemodialysis was also observed.
Conclusion. Extraction of uremic toxins, primarily phosphate and
hydrogen ions, dramatically increases the ALP activity under physiological
conditions. This hitherto unknown consequence of hemodialysis suggests a
reinterpretation of the clinical value of this parameter
P02-42
Evaluation of the kinase inhibitory and
anti-angiogenic effects of damnacanthal
1
2
P02-44
2
Javier A. García-Vilas , Ana R. Quesada , Miguel Ángel Medina Torres
Universidad de Málaga, Andalucía Tech, Departamento de Biología
Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto
de Biomedicina de Málaga), Málaga, ES, 2Universidad de Málaga,
Andalucía Tech, Departamento de Biología Molecular y Bioquímica,
Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga),
Unidad 741, CIBER de Enfermedades Raras (CIBERER), Malaga, ES
1
Damnacanthal is a natural anthraquinone initially isolated from roots of
Morinda citrifolia L., a small evergreen tropical tree usually known as noni.
Damanacanthal is the most potent known inhibitor of p56Ick tyrosine kinase.
Herein we show that damnacanthal is a broad spectrum kinase inhibitor.
We use docking analysis and molecular dynamics simulations to get further
insight on the inhibition of FAK, VEGFR-2 and c-Met by damnacanthal.
Since these three and other kinases inhibited by damnacanthal are involved
in angiogenesis, we suspected that this natural compound could also have
anti-angiogenic effects. This was confirmed by a combination of in vitro,
ex vivo and in vivo assays.
[Our experimental work is supported by grants BIO2014-56092-R
(MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and
FEDER) and funds from group BIO-267 (Andalusian Government). The
“CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain)].
This communication has the support of a travel grant “Universidad de
Málaga. Campus de Excelencia Internacional Andalucía Tech”.
P02-43
Alkalosis and dialytic clearance of phosphate
increases phosphatase activity: a hidden
consequence of hemodialysis
El análogo de las estrigolactonas GR-24 inhibe
la angiogénesis in vitro e in vivo
Paloma Carrillo1, Beatriz Martínez-Poveda1, Miguel Ángel Medina2,
Ana R. Quesada2
1
Universidad de Málaga, Andalucía Tech, Departamento de Biología
Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto
de Biomedicina de Málaga), Málaga, ES, 2Universidad de Málaga,
Andalucía Tech, Departamento de Biología Molecular y Bioquímica,
Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga);
Unidad 741, CIBER de Enfermedades Raras (CIBERER), Málaga, ES
Muchas fitohormonas han mostrado un gran potencial en la prevención
y terapia contra el cáncer. Las estrigolactonas son hormonas vegetales
derivadas de los caroteinoides que están implicadas en la inhibición de
la ramificación de la raíz y el brote, promover la germinación de plantas
parásitas e intervenir en el establecimiento de simbiosis con micorrizas
arbusculares. Se ha descrito la capacidad antitumoral de diferentes
análogos de estrigolactonas, entre ellos GR-24, frente a diferentes líneas
celulares tumorales in vitro y en modelos xenográficos. En este estudio se
ha evaluado la capacidad citotóxica y anti-angiogénica de GR-24, tanto in
vitro como in vivo. In vitro, GR-24 presenta una IC50 entre 50 y 90 μM en
diversas líneas celulares tumorales y endoteliales. Además, afecta a pasos
clave del proceso angiogénico, como son la proliferación, diferenciación,
migración y capacidad de degradación de la matriz extracelular de células
endoteliales, a concentraciones menores que su IC50. En los ensayos in
vivo, GR-24 muestra un gran efecto inhibidor sobre la formación de vasos
sanguíneos en la membrana corioalantoidea de pollo y sobre la formación
de vasos intersegmentales en embriones de Danio rerio. En conjunto, estos
resultados sugieren que GR-24 puede ser un nuevo compuesto prometedor
en la terapia anti-angiogénica y otras enfermedades dependientes de
angiogénesis.
Ricardo Villa Bellosta1, Emilio González Parra2, Jesús Egido2
1
Instituto de Investigacion Sanitaria, Fundación Jiménez Díaz,
Madrid, ES, 2Fundación Jiménez Díaz, Madrid, ES
[Our experimental work is supported by grants BIO2014-56092-R
(MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and
FEDER) and funds from group BIO-267 (Andalusian Government). The
“CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain)].
Background: Extracellular pyrophosphate is a potent endogenous inhibitor
of vascular calcification, which is degraded by alkaline phosphatase
This communicaction has the support of a travel grant “Universidad de
Málaga. Campus de Excelencia Internacional Andalucía Tech”.
55
Pósters / Posters
P02-45
Statins upregulate microRNA-122
and microRNA-27n in HepG2 cells
A. Benito-Vicente, A. Etxebarria, X. Moreno-Monreal, U.
Galicia-García, S. Jebari, E. Aizquibel, A Larrea-Sebal, A. Etxaniz,
D. González-Bullón, K. B.Uribe, R. Alonso-Estrada, H. Ostolaza,
C. Martín
Instituto Biofísika (UPV/EHU, CSIC) and Dpt. Biochemistry and
Molecular Biology, UPV/EHU, Portugalete, ES
Statin administration decreases cardiovascular risk and, cholesterol
lowering with statins has become a cornerstone of cardiovascular disease
prevention for a wide range of patients. Despite this, muscle problems,
increased incidence of diabetes, increased body weight, reduced exercise
capacity and other adverse symptoms are associated with statin use. Besides
of their major positive health benefits by inhibiting hydroxymethylglutarylCoAReductase (HMGCR), the rate-limiting enzyme of cholesterol
synthesis, it has been proposed that statinsinteract directly with the
epigenome. The epigenetic mechanisms postulated to be responsible for
these changes included DNA methylation and histone modification. To
date, there is limited evidence available on the effects of statins on a third,
recently described arm ofepigenetics: microRNA (miRNA) expression.
It has been established that miRNAs coordinate metabolism by directly
affecting the transcriptome, modulate multiple target transcripts and thus
heavily influence gene expression patterns. miR-33, may be key to control
HDL levels via repression of sterol transporters in the liver. In addition,
miRNAs are involved in molecular mechanisms that regulate the lipid
homeostasis and can contribute importantly to cardiovascular disease
development. Generally, miRNAs act intracellularly, but it has been
described that they can be released to the extracellular medium and be
detected circulating in plasma where they are specially stable and resistant
to RNAse degradation. miRNA profiles have been detected extracellularly
in inflammation processes, cardiovascular disease, atherosclerosis, diabetes,
obesity and aging. Different studies show that miRNAs in the medium
can be protected in lipid vesicles or by conjugation onto proteins. It has
been demonstrated that HDL can bind and transport endogenous miRNAs,
particularly miR-223 to hepatocytes and that the inflammatory properties of
HDL are due in part to the transference of miR-223 to the endothelial cells
where the expression of adhesion molecules is suppressed. Very recently, it
has been proposed that atorvastatin induces energy depletion and inhibition
of creatine synthesis in liver cells. Understanding how the statin treatment
cause adverse effects on triglyceride and fatty acid synthesis, lipid
infiltration in the liver and peripheral tissues, or the mechanism that leads
to enhanced risk of statin- induced type 2 diabetes mellitus would help the
management of these diseases. In this work we examined the effects of four
statins (atorvastatin, pravastatin, rosuvastatin and simvastatin; 5 μM for 24
and 48 h) on miR-122, mir-33a and mir-27b levels in HepG2 cells and in
the culture medium.
XXXIX Congreso SEBBM
these circumstances, we did not observe a direct correlation between ROS
production and cell death caused by the Aß because both Aß 40 and Aß
42 increased neuronal death while Aß 40 did not change ROS production.
Conversely, we observed that those Aß that are able to cross neuron
membrane (Aß 25-35 and Aß 42) increased ROS production but those
remained outside (Aß 40) not changed ROS generation. No significant
differences were found between Aß 40 and Aß 42 regarding the extent of the
deleterious effects of both peptides on neuronal viability or synaptophysin
expression. However, Aß 40 elicited a clear delocalization of PSD-95 and
synaptotagmin from prospective synapsis to the neuronal soma suggesting
the occurrence of an immediate effect of Aß 40 on synaptic disassembling.
The formation of Aß 40- or Aß 42-serum albumin complexes avoided the
effects of these peptides on neuronal viability, synaptophysin expression
and PSD-95/synaptotagmin disarrangement suggesting that sequestration
of Aß by albumin prevents deleterious effects of these peptides in neurons.
This suggests that Aß borne by albumin can be safely transported through
body fluids.
P02r-47
DeUbiquitinating enzymes (DUBs) as new
regulators of the Hypoxia Pathway
Teresa Martín-Mateos, Encarnación Pérez-Andrés, Onintza
Carlevaris, Sara Pozo, Edurne Berra
CIC bioGUNE, Derio, ES
Oxygen homeostasis is crucial for aerobic organisms as oxygen is the final
electron acceptor to generate ATP through the oxidative phosphorilation.
Accordingly, low-oxygen availability (or hypoxia) can produce irreversible
damages, even when transient. Adaptation to reduced oxygen availability
is indeed a major physiologic challenge but it is also associated with
pathologies such as ischemic diseases, inflammatory and metabolic
disorders, Alzheimer and cancer. The adaptive response is triggered by
the hypoxia-signalling pathway, which is under exquisite control through
the ubiquitin-proteasome system (UPS). The family of DeUbiquitinating
enzymes (DUBs) specifically deconjugates ubiquitin from targeted
proteins, playing major roles in the UPS control. Because of the reversible
ubiquitination’s crucial role to fine tune the hypoxia pathway and in the
light of DUBs being druggable enzymes, we carried out an unbiased loss-of
function screen. Of the nearly 79 DUBs encoded by the Human genome
and predicted to be active, we individually inhibit the expression of 66
DUBs using pools of small hairpin RNAs (shRNAs) and analyzed for their
effect in hypoxia-driven luciferase reporter activity. Here, we will discuss
the results of our screen, the validation of the selected HITs measuring
the effect of DUBs silencing on endogenous hypoxia-inducible target gene
expression as well as our more recent data regarding the study of the new
hypoxia specific DUBs we have identified.
P02-48
P02r-46
Aberrant co-localization of synapsis proteins
promoted by Alzheimer amyloid-ß peptides.
Protective effect of human serum albumin
Marta Domínguez-Prieto, Ana Velasco, Arantxa Tabernero,
José María Medina
Instituto de Neurociencias de Castilla y León (INCyL), Universidad
de Salamanca, Salamanca, ES
Amyloid-ß (Aß) 25-35, Aß 40 and Aß 42 significantly decreased neuronal
viability although the higher effect was showed by Aß 35-25. This may be
due to a more penetrating ability performed by Aß 25-35 since under our
experimental circumstances Aß 25-35 was internalized by the neuron and
it reached mitochondria. However, Aß 40 did not entry neuron cytosol and
Aß 42 was internalized by neuron but not reached mitochondria. Under
56
Validation of pharmacological chaperones
targeting the oncoprotein nucleophosmin
Andoni Cuevas, Marián Alonso-Mariño, Sonia Bañuelos,
María Ángeles Urbaneja
Biofisika Institute (UPV/EHU, CSIC), Dpt Biochemistry & Molecular
Biology, University of the Basque Country, Leioa, ES
Nucleophosmin (NPM), a highly and ubiquitously expressed protein,
mainly localized in nucleoli but able to shuttle between nucleus and
cytoplasm, plays crucial roles in ribosome maturation and export,
centrosome duplication, cell cycle progression, and response to a variety of
stress stimuli. Much interest in this protein has arisen since the discovery
that heterozygous mutations in the terminal exon of the NPM1 gene are
responsible for the unfolding of NPM C-terminal domain and consequently
its nucleolar release, which leads to massive and aberrant cytoplasm
Salamanca 2016
accumulation of the protein. Given that those genetic and cellular alterations
are the most frequent in acute myeloid leukemia (AML), NPM is considered
as a promising target for this disease. We have validated the effects of two
HTS-based selected hits on HeLa cells transfected with AML-related
NPM mutants. Both candidates interfere with unfolding-related protein
aggregation and prevent aberrant cytoplasm localization of NPM mutants,
prompting them as promising pharmacological chaperones for AML
therapy.
Pósters / Posters
rápida de oligómeros sin periodo de latencia, con una amplia distribución
de tamaños moleculares. Las especies oligoméricas formadas se han
separado mediante cromatografía de exclusión molecular y ultrafiltración
y se ha caracterizado su morfología y su estructura mediante AFM y FT-IR.
La toxicidad de los oligómeros sobre cultivos de células SH-SY5Y se ha
determinado utilizando el ensayo WST-1. Las diferencias estructurales
observadas entre los oligómeros tempranos de Abeta40 y Abeta42 pueden
ser responsables de sus diferentes propiedades citotóxicas.
P02-49
P02r-51
p38γ/p38δ and TPL2 are new components of the
Dectin-1 signalling pathway regulating Candida
albicans infection
Identification and functional characterization
of C14ORF39, a novel synaptonemal complex
protein essential for meiotic recombination and
mouse fertility
Alejandra Escós López1, Miguel Ángel Martín-Serrano1, Dayanira
Alsina-Beauchamp2, Ana Risco1, Ana Cuenda1
1
Centro Nacional de Biotecnología, Madrid, ES, 2Mount Sinai
Hospital, Nueva York, US
Candida albicans is a frequent etiologic agent in sepsis associated
with a high mortality in immune compromised patients. p38γ/p38TM
modulate bacterial lipopolysaccharide-induced sepsis by maintaining
the steady-state levels of TPL2, the MKK kinase that mediates ERK1/2
activation after TLR stimulation. However, their contribution in fungal
sepsis remains unknown. Here we show that p38γ/TM deficiency partially
protected against Candida albicans infection, decreased macrophage and
neutrophil recruitment to infected kidneys, and reduced the production of
cytokines and chemokines, such as CCL2 and KC. Moreover, p38γ/p38TM
deletion impaired the innate immune response to Candida albicans and to
the Dectin-1 ligand Curdlan by blocking ERK1/2 activation and reducing
IL-1βproduction in macrophages. We found that TPL2 is essential for
Dectin-1 signalling in mouse macrophages and human monocytes. We
proposethe existence of a new TAK1-TPL2-MKK1-ERK1/2 pathway
activated by Dectin-1 engagement, and regulated byp38γ/p38TM, whose
targetingmay offer novel strategies for antifungal agent design.
P02-50
Caracterización de la estructura y la citotoxicidad
de oligómeros tempranos de Abeta40 y Abeta42
Bertrand Morel1, María Paz Carrasco-Jiménez2, Xiomara
Gálvez-Escolano2, Carmen Marco2, Antonio Andrés García-Valdivia1,
Esperanza Padín1, David Ruzafa1, Francisco Conejero-Lara1
1
Departamento de Química Física e Instituto de Biotecnología,
Universidad de Granada, Granada, ES, 2Departamento de
Bioquímica y Biología Molecular I, Facultad de Ciencias,
Universidad de Granada, Granada, ES
La formación de agregados fibrilares del péptido beta-amiloide (Abeta) en
el cerebro es una de las señas de identidad de la enfermedad de Alzheimer
(EA). Abeta es el componente principal de las placas seniles, que se observan
en los espacios extracelulares del cerebro de enfermos de EA. Sin embargo,
es cada vez más evidente que son las formas oligoméricas solubles de Abeta
las principales responsables de los efectos citotóxicos sobre las neuronas y
en última instancia de la pérdida de la función cognitiva asociada a la EA.
De las dos formas más abundantes de Abeta, Abeta40 y Abeta42, es este
último el que más contribuye a la toxicidad celular. Desafortunadamente,
debido a su pequeño tamaño, su heterogeneidad e inestabilidad, se sabe
aún muy poco sobre las características estructurales de estos oligómeros,
los mecanismos de su formación o las razones fisicoquímicas de su
citotoxicidad. En este trabajo hemos estudiado la formación de oligómeros
de Abeta40 y Abeta42 durante las fases tempranas del proceso de
agregación, partiendo de las formas monoméricas de ambos péptidos. Las
cinéticas de agregación a 37ºC seguidas mediante dispersión dinámica de
luz y fluorescencia de tioflavina T y bis-ANS indican una acumulación
Natalia Felipe-Medina1, Laura Gómez-H.1, Manuel Sánchez-Martín1,
Owen R. Davies2, Isabel Ramos1, Ignacio García-Tuñón1, Dirk G.
de Rooij3, José Luis Barbero4, Ricardo Benavente5, Elena Llano6,
Alberto M. Pendás1
1
Instituto de Biología Molecular y Celular del Cáncer
(CSIC-Universidad de Salamanca), Salamanca, ES, 2Institute for
Cell and Molecular Biosciences, Newcastle University, Newcastle,
UK, 3Reproductive Biology Group, Division of Developmental
Biology, Department of Biology, Faculty of Science, Utrecht
University, Utrecht, NL, 4Centro de Investigaciones Biológicas
(CSIC), Madrid, ES, 5Department of Cell and Developmental
Biology, Biocenter, University of Würzburg, Würzburg, DE, 6Instituto
de Biología Molecular y Celular del Cáncer (CSIC-Universidad
de Salamanca). Departamento de Fisiología y Farmacología,
Universidad de Salamanca, Salamanca, ES
Meiotic recombination generates crossing over between homologous
chromosomes which are essential for genome haploidization. Humans differ
largely in the number of crossover across the genome. The synaptonemal
complex (SC) provides the structural framework for synapsis and crossing
over processing. Recently, one anonymous gene variant (C14ORF39)
influencing human recombination rate was identified. Here, we show that
C14ORF39 is a novel component of the central element of the synaptonemal
complex that interacts with the well-known synaptonemal complex central
element 1 (SYCE1). Structurally, by homology modeling C14ORF39
shows a repeated linear repeats of triple helical bundle and suggests to
act as a structural linker of the SC. Mice lacking C14ORF39 are defective
in synapsis of homologous chromosomes at meiotic prophase I which
provokes an arrest at pachytene-like stage and consequently infertility. In
agreement to be a modifier of recombination rate in humans, C14ORF39
is essential for the appropriate processing of intermediate recombination
nodules just before reciprocal recombination and crossover take place.
P02-52
Genetics of obesity: can an old dog teach
us new tricks?
Giles SH. Yeo
University of Cambridge, Metabolic Research Labs, MRC Metabolic
Diseases Unit, Addenbrooke’s Hospital, Cambridge, UK
It is clear that the cause of obesity is a result of eating more than you
burn.What is more complex to answer is why some people eat more than
others? Over the past 20 years, insights from human and mouse genetics
have illuminated multiple pathways within the brain that play a key role in
the control of food intake. We now know that the brain leptin-melanocortin
pathway is central to mammalian food intake control, with genetic
disruption resulting in extreme obesity. These, however, remain rare, with
the major burden of disease carried by those of us with ‘common obesity’.
In recent years, genome-wide association studies have revealed more
57
Pósters / Posters
than 100 different candidate genes linked to BMI, with most, including
many components of the melanocortin pathway, acting in the CNS and
influencing food intake. So while severe disruption of the melanocortin
pathway results in severe obesity, subtle variations in these same genes
influence where you might sit in the normal distribution of BMI. As we
now enter this ‘post-genomics’ world, can this new information influence
our treatment and management of obese patients?
P02-53
Modelo murino de las alteraciones metabólicas
encontradas en pacientes con mutaciones en
IGF1R
Carmen Patricia Pérez Matute1, Iciar P. López1, Emma
Recio-Fernández1, Raquel Torrens1, Sergio Piñeiro-Hermida1,
Elisa Wirthgen2, Andreas Hoeflich2, José A. Oteo3, José G. Pichel1
1
Fundación Rioja Salud-CIBIR, Logroño, ES, 2Institute for
Genome Biology, Leibniz-Institute for Farm Animal Biology (FBN),
Dummerstorf, DE, 3Hospital San Pedro-CIBIR, Logroño, ES
IGF1R (Insulin-like Growth Factor type 1 Receptor) es un receptor
transmembrana de expresión ubicua con una gran similitud con el receptor
de la insulina y con funciones biológicas esenciales. Pacientes con
mutaciones alélicas en el gen IGF1R presentan alteraciones metabólicas y
endocrinas. El objetivo del presente trabajo fue analizar metabólicamente
un modelo animal de deficiencia de IGF1R. Se han empleado ratones
mutantes UBC-CreERT2; Igf1rfl/fl con deleción de Igf1r inducida
postnatalmente con tamoxifeno. Los ratones fueron sacrificados 9 semanas
después de la inducción de la deleción y la sangre y los diferentes tejidos
fueron extraídos para su análisis. Los ratones hembra mutantes presentaron
mayor peso corporal y grasa periovárica que sus controles, sin observarse
diferencias en los machos. El peso del hígado se vio incrementado en los
ratones mutantes de ambos géneros. Además, el contenido hepático de
triglicéridos fue significativamente superior en los ratones macho mutantes.
A nivel sistémico, los mutantes presentaron elevados niveles plasmáticos
de insulina y del índice de resistencia a la insulina HOMA. Los niveles
plasmáticos de IGF-1 también se vieron incrementados en los mutantes,
al igual que IGFBP3. Los niveles de IGFBP2 se vieron disminuidos
en los mutantes. Este estudio demuestra que los ratones mutantes son
insulino-resistentes, con elevados niveles de IGF-1 y de triglicéridos en
hígado, aunque algunos de estos efectos son dependientes del género.
Nuestros resultados sugieren que este modelo de deficiencia en IGF1R
refleja alteraciones metabólicas observadas en pacientes con deleción en
este gen, y, por tanto, puede ser una herramienta útil para el conocimiento
en profundidad de los mecanismos que subyacen a la fisiopatología de esta
mutación.
P02-54
Estudio de la hemostasia en modelos murinos
de Telangiectasia Hemorrágica Hereditaria
Cristina Egido Turrión, Claudia Ollauri Ibáñez, Laura Ruiz Remolina,
Alicia Rodríguez Barbero, José Miguel López Novoa, Miguel
Pericacho Bustos
Departamento de Fisiología y Farmacología. Universidad de
Salamanca. IBSAL, Salamanca, ES
La Telangiectasia Hemorrágica Hereditaria (HHT) es una enfermedad
rara causada, en un 90% de los casos, por mutaciones en los genes de
endoglina o de ALK1. Clínicamente se caracteriza por la presencia de
telangiectasias y malformaciones arteriovenosas cuya rotura provoca
graves hemorragias muy difíciles de detener que comprometen la vida
del paciente. La frecuencia e intensidad de estas hemorragias hace que los
pacientes necesiten transfusiones periódicas, lo que merma su calidad de
vida e incrementa el gasto sanitario. Tradicionalmente se ha considerado
58
XXXIX Congreso SEBBM
que la HHT es una enfermedad provocada por una angiogénesis deficiente
y que los sangrados son consecuencia de la rotura de unos vasos sanguíneos
frágiles. Sin embargo, nuestra hipótesis es que esa mala angiogénesis
puede ser responsable de la elevada frecuencia de los sangrados, pero
que su gravedad es consecuencia de alguna alteración de la hemostasia en
estos pacientes. Resultados previos de nuestro grupo han demostrado que
el dominio RGD de endoglina interacciona con integrinas leucocitarias y
regula el proceso de transmigración. Por ello nos planteamos que también
pueda interaccionar con las integrinas plaquetarias y regular así la formación
o estabilización del trombo. Para analizar esta hipótesis hemos estudiado la
hemostasia y la estabilización del trombo en 2 modelos murinos de HHT:
ratones heterocigotos en endoglina (ENG+/-) y en ALK1 (ALK1+/-). Nuestros
resultados demuestran que en ambos casos hay un aumento del tiempo de
sangrado que se acompaña de un retraso en la estabilización del trombo,
siendo las diferencias más claras en el caso de los ratones deficientes en
endoglina. Estos resultados permitirían la búsqueda de nuevos enfoques
terapéuticos para el control de las hemorragias de los pacientes de HHT.
P02r-55
Differential DNA damage response through
alternative splicing of the DDR factor RAP80
Erika Zodda1, Francesca Mateo1, Mònica Pons1, Bruna Oriol1,
Inés Izquierdo2, Marta Cascante2, Timothy M. Thomson1
1
IBMB-CSIC, Barcelona, ES, 2Departamento de Bioquímica y
Biología Molecular, Facultad de Biología, Universidad de Barcelona,
Barcelona, ES
Alternative splicing governs cell-type, lineage-specific or environmentally
regulated expression of variant forms of mRNAs and their encoded
proteins that exert differential functions specifically and exquisitely
adapted to a particular cellular context. By employing a cell model in
which distinct tumor cell subpopulations display clearly differentiated
epithelial or mesenchymal phenotypes and gene programs, we have
identified alternatively spliced mRNAs with potential impact on the
self-renewal capacities of these cell subpopulations. For this, we have
applied RNAseq followed by analysis of mRNA isoforms differentially
expressed between the two subpopulations and shRNA-mediated transcript
knockdowns. This has led us to discover that mRNAs for RAP80, an
adaptor protein that tethers BRCA1 to sites of DNA damage through an
ubiquitin-interacting motif (UIM), are expressed as either predominantly
epithelial or predominantly mesenchymal isoforms, and that these
isoforms switch their expression in epithelial or mesenchymal cells as a
function of expression of the alternative splicing regulators ERSP1/2. We
have found that the predominantly epithelial isoform of RAP80 mRNA
encodes a functional protein that provides more efficient repair in response
to DNA damage. We have also found that the expression of RAP80 is
essential for the maintenance of self-renewal phenotypes of the epithelial
tumor cell subpopulation of our dual-cell model, which displays traits of
cancer stem cells (CSCs). In summary, we have discovered that alternative
splicing regulates efficient DNA damage repair mediated by RAP80,
associated with an epithelial gene program, and that RAP80 is necessary
for the maintenance of CSC properties. These findings represent the first
description of differential DDR linked to epithelial CSC characteristics
regulated through alternative splicing.
P02-56
Efecto de la Restrición Calórica (RC) en el
metabolismo de cerámidas en el hipotálamo.
Relación con la lipotoxicidad y el desarrollo de
resistencia a insulina con la edad
Cristina Pintado Losa1, María Rodríguez Pérez1, Virginia
Gómez-López Carreño2, Carmen Arribas Mocoroa1, Eduardo Moltó
Pérez1, Antonio Andrés Hueva2, Nilda Gallardo Alpizar2
Salamanca 2016
1
Facultad de Ciencias Ambientales y Bioquímica. UCLM, Toledo,
ES, 2Facultad de Ciencias y Tecnologías Químicas. UCLM, Ciudad
Real, ES
El hipotálamo vincula las señales de hormonas y nutrientes al control
de la ingesta, el metabolismo energético y la sensibilidad a insulina. El
desarrollo de resistencia a insulina con la edad es resultado de una serie
compleja de alteraciones, incluyendo un desequilibrio en el flujo lipídico
que conduce a la síntesis de ceramidas en neuronas hipotalámicas que
afectaría al mantenimiento de la homeostasis energética a través de la
activación de vías inflamatorias y/o la inducción de estrés de retículo
endoplásmico (RE) y lipotoxicidad. Un mejor conocimiento de la
regulación del metabolismo lipídico en el hipotálamo podría conducir a
la identificación de nuevas estrategias para la prevención y tratamiento
de la obesidad y la diabetes. Se estudió mediante RT-PCR a tiempo real
la variación con el envejecimiento de la expresión de genes implicados
en la síntesis, degradación y metabolismo de ceramidas, así como de
marcadores de estrés de RE, respuesta a proteínas mal plegadas (UPR)
e inflamación en el hipotálamo de ratas alimentadas ad libitum o en
restricción calórica (RC). La RC produjo un aumento en la expresión
de genes de degradación de ceramidas y descenso en los de síntesis.
Por otro lado la RC provocó la disminución de la expresión de CHOP,
mientras que no se observaron variaciones ni con el envejecimiento
o la RC en la expresión de Grp78 y PDI. El patrón de expresión en
marcadores y citoquinas pro-inflamatorias se altera con la RC,
disminuyó la expresión de iNOS, IL-1β y TNFα en ratas de 8 meses,
pero se obtuvieron efectos opuestos en las de 24 meses. Los resultados
sugieren que la RC en edades tempranas provocaría una disminución en
la generación de ceramidas y en las vías inflamatorias en el hipotálamo,
siendo ésta una posible intervención terapéutica que mejorase la
sensibilidad central a la insulina.
Pósters / Posters
P02-58
p75NTR antagonists attenuate photoreceptor
cell loss in murine models of retinitis pigmentosa
María Platón-Corchado1, Pablo F. Barcelona2, Miguel Marchena1,
Alberto M. Hernández-Pinto1, Sean Jmaeff2, Catalina
Hernández-Sánchez1, H. Uri Saragovi2, Enrique J. de la Rosa1
1
Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, ES,
2
Lady Davis Institute-Jewish General Hospital, McGill University,
Montreal, Quebec, CA
Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that
courses with photoreceptor cell degeneration and death. ProNGF signaling
through p75NTR has been associated to neurodegenerative conditions.
Thus, we have explored the possible role of p75NTR in the course of RP, as
well as its value as pharmacological target for RP treatment. Quantitative
RT-PCR analysis showed no differences in the expression levels of the
proNGF/p75NTR system components between the RP model rd10 and wild
type mouse retinas. However, unprocessed proNGF levels, measured by
ELISA, increased in the rd10 retina at early degenerative stages prior
to the peak of photoreceptor cell death, and remained elevated for the
period of major of photoreceptor cell loss. Reduced proNGF processing
concurs with increased α2-macroglobulin expression, an inhibitor of that
processing, as well as with increased levels of pro-inflammatory cytokines
and GFAP expression. Retinal explants treated with p75NTR antagonists for
24 h showed significantly reduced levels of photoreceptor cell death, as
determined by TUNEL assay. This effect was accompanied by a decrease
in retinal reactive gliosis. A single intravitreal or subconjuntival injection of
the p75NTR antagonist THX-B in rd10 and RhoP mouse RP models elicited a
neuroprotective effect on photoreceptor cells, observable five days later as
an increase in the thickness of the outer nuclear layer, where photoreceptor
nuclei are located. p75NTR-antagonists supported photoreceptor cell
survival both on retinal explants and in vivo, in two different RP models,
thus providing an initial proof of concept on a possible therapy for RP.
P02-57
RasGRF2 controls nuclear trafficking
in photoreceptor cells
David Jimeno García1, Camela Gómez1, Nuria Calzada1,
Pedro de la Villa2, Concepción Lillo3, Eugenio Santos1
1
Centro de Investigación del Cáncer-Instituto de Biología Molecular
y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca,
ES, 2Departamento de Fisiología, Universidad de Alcalá, Alcalá
de Henares, ES, 3INCYL, IBSAL (Universidad de Salamanca),
Salamanca, ES
Analyses of RasGRF1 knockout (GRF1-KO), GRF2-KO and the
GRF1/2 double-KO mouse retinas, revealed specific alterations in cone
photoreceptors in the GRF2-KO and GRF1/2-DKO. These alterations
consist in the specific accumulation of misplaced “ectopic” nuclei in the
photoreceptors segments area. The pathological displacement of cone
nuclei occurred postnatally and peaked between postnatal day 11 (P11)
and P15 coinciding with the so called “late migratory phase” of cone
nuclei. The late migratory phase is a physiological process whereby cone
nuclei move towards the outer limiting membrane (OLM) where they
reach their final position. In the GRF2-KO and GRF1/2-DKO retinas
this movement is deregulated and cone nuclei are located closer to the
OLM and in some regions of the retina even trespass it. Using detailed
inmunocytochemical analyses we have identified disruption of the OLM
and accumulation of activated, phosphorylated forms of PAK, MLC2 and
VASP, molecules known to participate in nuclear migration and cytoskeletal
reorganization, in cone photoreceptors of GRF2-KO and GRF1/2-DKO
retinas. Electroretinographic recordings show specific impairment of cone
photoreceptor cells in GRF1-KO and GRF1/2-DKO retinas. These data
support a critical role of GRF2 in cone nuclear migration and proper retinal
development and functioning.
P03. Biología del desarrollo /
Developmental biology
P03. Biología del desarrollo / Development biology
P03-1
A novel function of aPKC controlling apical cell
trafficking
Sol Sotillos Martín
Centro Andaluz de Biología del Desarrollo/CSIC/UPO,
Dos Hermanas, ES
Cell polarity is a basic characteristic of any cell, required for their proper
physiological function. To establish cell polarity a number of determinants are
required (reviewed in (1)) to restrict the different functional domains in the cell.
When establish and for maintenance of cell polarity a proper trafficking to and
recycling from the membrane is required. And, vice versa, once established
cell polarity is necessary to define the cell domains for trafficking delivery.
Thus, an intermingled between cell polarity and cell trafficking is essential
to maintain each other and for the proper cell physiology. Recently we have
shown a new interacting point between cell polarity and trafficking at the
level of the polarity determinant aPKC and the Rab11-adaptor protein Nuclear
fallout (Nuf, (2)). aPKC is a Ser/Thr kinase of the Par complex essential in most
of the polarity processes. Nuf belongs to the Rab11-family interacting proteins
(Rab11-FIP (3)) that function as an adaptor of Rab11 to the microtubule motor
protein kinesin and the dynein complex (2, 4). We have demonstrated that
active aPKC interacts directly with and phosphorylates Nuf, modifying its
59
Pósters / Posters
XXXIX Congreso SEBBM
cellular distribution. Furthermore, aPKC is a cargo of the Nuf-Rab11 recycling
endosomes and it seems to be regulating its own recycling in the cell by
phosphorylation and displacement of Nuf from the apico-lateral cortex.
References
[1] E. Rodriguez-Boulan, I. G. Macara: Organization and execution of the
epithelial polarity programme. Nat Rev Mol Cell Biol 15, 225-242 (2014).
[2] F. J. Calero-Cuenca et al.: Nuclear fallout provides a new link between
aPKC and polarized cell trafficking. BMC Biol 14, 32 (2016).
[3] C. P. Horgan, M. W. McCaffrey: The dynamic Rab11-FIPs. Biochem
Soc Trans 37, 1032-1036 (2009).
[4] B. Riggs et al.: The concentration of Nuf, a Rab11 effector, at the
microtubule-organizing center is cell cycle regulated, dynein-dependent,
and coincides with furrow formation. Mol Biol Cell 18, 3313-3322 (2007).
GTPases, are widely deemed as potential therapeutic targets owing to their
protumorigenic functions. However, the sparse use of animal models has
precluded the full understanding of their in vivo pathophysiological roles.
Here, we report that the hematopoietic-specific Vav1 GEF unexpectedly
acts as a tumor suppressor by buffering Notch1 signaling in lymphocytes.
This noncanonical function entails the nucleation of cytoplasmic complexes
between Cbl-b and the active Notch1 intracellular domain (ICN1) that favor
the ubiquitinylation-mediated degradation of ICN1. Genetic ablation of
Vav1 upregulates ICN1 signaling in immature T cells and, in collaboration
with ancillary mutations, triggers the rapid development of T cell acute
lymphoblastic leukemia (T-ALL). This pathway is downregulated at
the transcriptional level in human T-ALL of the TLX+ subtype, further
underscoring its potential tumor suppressing roles. These results call for an
overall reevaluation of Rho GEF function in cancer.
P03-2
P03-4
From buds to paws, a temporal transcriptome
analysis
When to decide to divide: a Soxist point of view
1
1
2
Marc Fernández Guerrero , Rocío Pérez Gómez , Fabrice Darbellay ,
Lucille Delisle2, Denis Duboule2, Marian Ros1
1
Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC),
Consejo Superior de Investigaciones Científicas, Universidad de
Cantabria (CSIC), Santander, ES, 2School of Life Sciences, Federal
Institute of Technology, Lausanne, Lausanne, CH
Limbs develop from limb buds composed of a core of limb progenitor
cells covered by the surface ectoderm. A variety of experiments in chick
and mouse embryos indicate that the fate of the distal limb progenitors is
progressively restricted: early progenitors can form the whole proximo-distal
elements of the limb while late progenitors can only form the digits.
Here we have used RNA-seq to reveal the transcriptional features
associated with this developmental transition. The analysis was performed
in mouse forelimbs of 4 different stages (E9.5, E10.5, E11.5 and E12.5)
and restricted to the distal 150μm with ectodermal and mesodermal
components analyzed separately. Two biological replicates were used for
each condition.
Of 46,480 Ensembl-annotated genes, 3,416 were differentially expressed
between ectoderm and mesoderm. In each tissue, the time serie analysis
revealed that major changes occur between E9.5 and E10.5. The accuracy
of the expression patterns was confirmed by in situ hybridization and by
the truthful coincidence with published expression patterns. According
to their temporal expression, six different dynamics of gene expression
were identified. In general, the average expression of transcription factors
decreased with age, whereas the average expression of extracellular
molecules increased with age.
The differentially expressed genes were further subjected to Gene Ontology
(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis
and showed diverse functional categories and signaling pathways. We
are exploring their relevance in orchestrating age-specific developmental
capacities of each tissue.
P03-3
An unexpected tumor suppresor role for
the Rac1 exchange factor Vav1 in T cell acute
lymphoblastic leukemia
Javier Robles Valero1, Luis Francisco Lorenzo-Martín1, Mauricio
Menacho Márquez1, Antonio Abad1, Lluis Espinosa2, Anna Bigas2,
Xosé R. Bustelo1
1
Centro de Investigación del Cáncer (CIC-CSIC), Universidad
de Salamanca, Salamanca, ES, 2Institut Hospital del Mar
d’Investigacions Mèdiques, Barcelona, ES
Rho GDP/GTP exchange factors (GEFs), the enzymes that stimulate Rho
60
Aixa V. Morales1, Mar Muñiz1, Elena Calleja1, Alejandra C. Quiroga1,
Marco A. Cañizares1, Lingling Li1, Silvia Nicolis2, Véronique
Lefebvre3
1
Instituto Cajal, CSIC, Madrid, ES, 2University of Milano-Bicocca,
Milano, IT, 3Cleveland Clinic Lerner Research Institute, Cleveland,
Ohio, US
During the development of the nervous system, the generation of
hundreds of subtypes of neurons and glial cells relies upon the relatively
fast production, amplification, specification and differentiation of a
pool of neural progenitors and neural stem cells (NSCs). Surprisingly,
this strategy is retained to some extent in niches in the adult nervous
system throughout lifetime under physiological conditions. Although the
molecular mechanisms involved in both embryonic and adult neurogenesis
are conserved, it is not clear how the differences in the cell production
rates and in the temporal extent of neurogenesis can be attained. Genes of
the Sox family of transcription factors are essential during neurogenesis.
In the developing spinal cord, we have determined that Sox5 controls cell
cycle exit of neural progenitors and the specification of subtypes of dorsal
interneurons, counteracting the Wnt signalling pathway (Martínez-Morales
et al., 2010; Quiroga et al., 2014). More recently, we have characterized
that both Sox5 and Sox6 are expressed in the majority of NSCs in one of the
longer lasting neurogenic niches, the subgranular zone (SGZ) of the dentate
gyrus of the adult mouse hippocampus.Using inducible targeted deletions:
Sox5fl+/fl+ and Sox6fl+/fl+ mice crossed to a transgenic Sox2-cre-ERT2 line
inducible by tamoxifen, we have determined that Sox6 is required for the
self-renewal ability of the NSCs of the DG, while Sox5 is required for
restricting the proliferation of the NSCs. These results suggest that Sox5
and Sox6 control the activation, proliferation and/or stemness of NSCs
during adult hippocampal neurogenesis.
P03-5
Generación de mutantes específicos para
distintas isoformas de polychaetoid mediante
la tecnología CRISPR/Cas9 en Drosophila
Marta Carrasco-Rando, Mar Ruiz-Gómez
CBMSO, Madrid, ES
Uno de los principales componentes de la barrera de filtración glomerular
implicada en el ultrafiltrado de la sangre en vertebrados es el diafragma
de filtración (DF), un complejo multiproteico que actúa como filtro
molecular y plataforma de señalización intracelular. En Drosophila las
células encargadas de la filtración de la hemolinfa (nefrocitos) presentan
una estructura análoga, tanto molecular como funcionalmente, al DF de
vertebrados. El objetivo principal de nuestro laboratorio es el empleo
de Drosophila como modelo para caracterizar nuevos componentes y
Salamanca 2016
reguladores del DF. Así estamos llevando a cabo el análisis funcional de
polychateiod(pyd), el ortólogo de ZO-1, en la formación y mantenimiento
de los DF en los nefrocitos. Este gen da lugar, por splicing alternativo, a
varios transcritosque codifican para proteínas que presentan los dominios
comunes PDZ, SH3 y Guk, conservados en ZO-1, pero difieren en otros
dominios alternativos. En la deficiencia para pyd los nefrocitos aparecen
aglutinados y presentan una expresión y localización aberrante de los
principales constituyentes del DF. El análisis ultraestructural de estos
mutantes confirma que carecen de diafragmas de filtración. Para analizar
la contribución de los distintos dominios a la función de Pyd en nefrocitos
hemos empleado la tecnología CRISPR/cas9 para generar nuevas líneas
mutantes específicas para diferentes isoformas.
Pósters / Posters
E2, lo cual sugiere que el cAMP ejerce un efecto sobre la inducción de leptina
por E2. Por otro lado se demostró que la sobreexpresión de CBP (proteína
de unión a CREB con función de acetiltransferasa) produce un aumento
en el efecto de E2 sobre la expresión de leptina. También se observó, en
transfecciones transitorias, que la sobreexpresión de HDAC-1, una histona
deacetilasa, disminuyó la inducción de E2 sobre leptina sugiriendo que las
modificaciones en acetilaciones de histonas podrían estar involucradas
en su acción, y el tratamiento con TSA (Triscjostatin A, inhibidor de
deacetilasas) contrarresta este efecto, sugiriendo que las modificaciones
de las histonas en el DNA podrían estar influyendo en su acción. Estos
resultados proveen nuevas evidencias acerca de los mecanismos por los
cuales el E2 regula la expresión de la leptina placentaria. Futuros ensayos
permitirán estudiar en detalle los complejos proteicos formados entre en el
promotor de leptina en respuesta a E2.
P03r-6
Sprouty1 and Sprouty2 cooperate to pattern
internal genitalia independently of Ret signaling
Marta Vaquero Susagna1, Carlos Anerillas Aljama1, Sara Cuesta
Sancho2, Joan Ribera Calvet1, Mario Encinas Martín1
1
Universitat de Lleida, Lleida, ES, 2IRB Lleida, Lleida, ES
Sprouty proteins are feedback inhibitors of receptor tyrosine kinase
signaling pathway. Deletion of Spry1 in mice leads to defects in the
development of the genito-urinary system due to hyperactivation of Ret
signaling. On the other hand, targeted deletion of Spry2 causes craniofacial
and enteric innervation defects owing to excessive FGFR and Ret signaling,
respectively. Besides these distinct roles in mouse organogenesis, recent
data indicate that the different Spry genes play overlapping roles during
development. In the present work we describe that Spry1; Spry2 double
heterozygous mice exhibit internal genitalia abnormalities that are not
caused by dysregulated Ret signaling. Mutant females exhibit imperforate
vagina and hydrometrocolpos, whereas males show seminal vesicle
duplication. These observations are consistent with abnormal Wolffian
duct development, which shows strong Ret expression on its caudal end
from E14.5 to E16.5, embryonic ages between which Müllerian duct fusion
and seminal vesicle formation occur. However, Ret deletion does not affect
vagina or seminal vesicle development. Moreover, unlike kidney defects
found in Spry1 knockout mice, removal of both alleles of Ret in the context
of Spry1; Spry2 heterozygosity do not rescue vaginal defects or seminal
vesicle duplication. Taken together, these observations indicate that Spry1
and Spry2 cooperate during the development of the caudal Wolffian duct in
a Ret-independent manner.
P03-8
Tyrosine 53 is essential for Sprouty1 function
during genitourinary development
Sara Cuesta Sancho1, Marta Vaquero Susagna2, Carlos Anerillas
Aljama2, Mario Encinas Martín2
1
IRB Lleida, Lleida, ES, 2IRB Lleida/Universidad de Lleida, Lleida, ES
Sprouty family genes are feedback inhibitors of receptor tyrosine kinase
(RTK) signalling. Mice lacking Sprouty 1 have defects in urogenital
development reminiscent of human CAKUT (congenital abnormalities of
the kidney and lower urinary tract). The mechanisms of action of Sprouty
proteins are controversial but several in vitro studies point to a conserved
tyrosine in the N-terminus of Spry family members (Tyr53 in Spry1) as a
master regulator of their activity. To address the role of Tyr 53 in vivo, our
group has generated knockin mice bearing a Tyrosine to Alanine mutation in
residue 53 of Spry1. Despite expressing normal levels of the protein, these
mice present genitourinary abnormalities indistinguishable from those
found in Spry1 knockout mice such as fused supernumerary kidneys, and
hydroureter/megaureter. However, unlike Spry1 knockout mice, Spry1Y53A
mice show reduced weight and viability at weaning. Summing up, our
results indicate that Tyr53 is required for Spry1 function in genitourinary
development of mice displaying the same effect characterized in Spry1
knockout mice.
The research leading to these results has received funding from the People
Programme (Marie Curie Actions) of the European Union’s Seventh
Framework Programme (FP7/2007-2013) under REA grant agreement n°
609396.
P03-7
Efectos de la vía del AMPc sobre la acción del
estradiol en la expresión de leptina placentaria
Malena Schanton1, Antonio Peréz Peréz2, José Luis Dueñas3, Victor
Sanchez Margalet2, Cecilia Laura Varone1
1
Departamento de Química Biológica, FCEN, UBA, IQUIBICEN,
CONICET, Buenos Aires, AR, 2Departamento de Bioquímica Médica
y Biología Molecular, Universidad de Sevilla, Sevilla, AR, 3Hospital
Universitario Virgen Macarena, Sevilla, ES
La leptina es una proteína expresada en placenta que durante la gestación
regula la implantación y el desarrollo embrionario. Resultados previos de
nuestro laboratorio han demostrado que el estradiol (E2) regula la expresión
de leptina placentaria a nivel transcripcional involucrando efectos
genómicos y no genómicos. Previamente demostramos que CREB regularía
la expresión basal de leptina, el objetivo del presente trabajo es estudiar el
efecto de vía AMPc sobre la acción del E2 en la expresión de la leptina
placentaria. Se utilizaron células BeWo y explantos de placenta humana
a término como modelos experimentales. Al utilizar los inhibidores de la
vía de PKA, H89 y SQ22536 se observó por Western blot y por ensayos de
genes reporteros, una disminución de la expresión de leptina mediada por
P03-9
Efecto de la inhibición de la quinasa activada por
AMP, AMPK, en la función del espermatozoide
humano
Violeta Calle Guisado1, Ana Hurtado de llera1, David
Martín-Hidalgo1, Lauro González Fernández2, José Mijares Gordún3,
Mari Cruz Gil Anaya1, Ignacio Santiago Álvarez Miguel4, Luis Jesús
García Marín1, María Julia Bragado González1
1
Facultad de Veterinaria, Universidad de Extremadura, Cáceres,
ES, 2Centro de Estudos de Ciência Animal/Instituto de Ciências,
Tecnologias e Agroambiente da Universidade do Porto (CECA/ICETA),
Oporto, PT, 3Centro de Cirugía de Mínima Invasión Jesús Usón, Clínica
Norba, Cáceres, ES, 4Centro de Cirugía de Mínima Invasión Jesús
Usón, Instituto Extremeño de Reproducción Asistida, Cáceres, ES
La proteína quinasa activada por AMP, AMPK regula el metabolismo
celular dependiendo de la carga energética. La forma activa de la AMPK
está fosforilada en la treonina 172. Nuestro grupo ha identificado
previamente esta proteína y su función en el espermatozoide porcino.
61
Pósters / Posters
Nuestro objetivo es estudiar la presencia, localización intracelular y
la función de la AMPK en el espermatozoide humano. Eyaculados
procedentes de donantes sanos se separaron mediante gradiente de
densidad en dos poblaciones de alta y baja calidad espermática. La
identificación y localización de la proteína se llevó a cabo mediante
Western blotting e inmunohistoquímica. La motilidad y viabilidad
espermática se evaluaron en presencia o ausencia del compuesto C
(CC), inhibidor específico de la AMPK, mediante el sistema ISAS de
análisis espermático o citometría de flujo, respectivamente. La AMPK
se localiza en los diferentes compartimentos de espermatozoide humano
con una intensidad variable. La forma activa, p-Thr172-AMPK, presenta
una mayor intensidad en la población espermática de alta motilidad. La
inhibición de la actividad de AMPK con CC reduce significativamente
todos los parámetros evaluados: porcentaje de espermatozoides mótiles,
velocidades (VCL, VSL o VAP), progresividad y otros coeficientes de
motilidad, todo ello sin afectar la viabilidad espermática. Este trabajo
identifica por primera vez la AMPK y su forma activa en el espermatozoide
humano y muestra su implicación en la motilidad espermática. Nuestros
resultados nos permiten seguir trabajando en las posibles implicaciones
de la AMPK en la función del espermatozoide y en su uso en la mejora de
las técnicas de reproducción asistida.
P03-10
Inhibition of APAF-1 with LPT99 prevents
cisplatin-induced apoptosis in HEI-OC1
auditory cells
Blanca Cervantes1, Isabel Sánchez-Pérez2, Carmen Herrero3, Isabel
Varela-Nieto4
1
Centro de Investigación Biomédica en Red de Enfermedades
Raras. Instituto de Investigaciones Biomédicas Alberto Sols
CSIC-UAM, Madrid, ES, 2Dpto. Bioquímica. Fac. Medicina.
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM,
Madrid, ES, 3Spiral Therapeutics, San Francisco, US, 4Centro de
Investigación Biomédica en Red de Enfermedades Raras. Instituto
de Investigaciones Biomédicas Alberto Sols CSIC-UAM. IdiPAZ,
Instituto de Investigación Hospital Universitario La Paz , Madrid, ES
HEI-OC1 (House Ear Institute – organ of Corti 1) is an epithelial
otic cell line that was derived by Kalinec et al. (2003) from the
cochlea of a transgenic mouse called Immortomouse™, which
harbors a temperature-sensitive mutant of the SV40 large T antigen.
The modulation of culture conditions allows the progression of the
progenitor cells to a differentiated hair cell-like with a phenotype
similar to that of the adult organ of Corti. One the most interesting
characteristic of these cells is their sensitivity to ototoxic drugs such as
aminoglycoside antibiotics or antineoplastic agents as cisplatin, which
can cause sensorineural hearing loss. Cisplatin is a highly effective
chemotherapeutic agent, but it has significant ototoxic side effects.
Apoptosis is an important mechanism of cochlear hair cell loss following
exposure to cisplatin. The present study examined the effects of
LPT99, a second generation of APAF-1 inhibitors on cisplatin-induced
apoptosis. Viability and growth rate of HEI-OC1 cells were determined
using a crystal violet based staining method and the activation of
Caspase-3, a biochemical marker for apoptosis cell death was evaluated
with immunocytochemistry. We observed a dose-dependent decrease
in cell viability after challenge with cisplatin (0-5 μg/mL); however
survival rate increased in the presence of 1 μM LPT99. According,
cells treated with cisplatin showed an IC50 of 4.47±1.94 μg/mL, which
increased dramatically to 10.51 ± 3 μg/mL in the presence of LPT99.
In addition, immunofluorescence studies suggest that the activity of
Caspase-3 after cisplatin treatment decreased in the presence of LPT99.
These results suggest that LPT99 protected the cells HEI-OC1 against
cisplatin-induced apoptosis by inhibiting of APAF-1.
Supported by the FP7-PEOPLE-2013-IAPP-TARGEAR.
62
XXXIX Congreso SEBBM
P03r-11
The early epaxial enhancer of Myf5 has a dual
role during embryonic development
Macarena López-Mayorga1, Natalia Moncaut2, Cristina
Vicente-García1, Lydia Teboul3, Peter W. J. Rigby2, Jaime Carvajal
García-Valdecasas1
1
Centro Andaluz de Biología del Desarrollo, Sevilla, ES,
2
The Institute of Cancer Research, London, UK, 3MRC Harwell,
Oxfordshire, UK
The determination and specification of skeletal muscle in vertebrates is
orchestrated by the myogenic regulatory factors (MRFs): Myf5, Mrf4,
MyoD and Myogenin. Myf5 is the first to be expressed in the embryo,
initiating and co-ordinating the myogenic cascade. In absence of Myf5,
progenitors fail to be specified; activation of MyoD rescues the phenotype
and myogenesis progresses. In Myf5/MyoD KO animals rescue does not
take place and no skeletal muscle is formed. Transcription of the Mrf4/Myf5
locus is controlled by over 25 elements. The Early Epaxial Enhancer (EEE)
operates in the dermomyotomal dorsomedial lip (DML) and is the first to
activate Myf5 at E8.5 dpc. We have generated a mouse allele in which
the EEE has been selectively deleted, abrogating early Myf5 expression
in the DML. Homozygous animals do not show an overtly phenotype, as
is the case for the full Myf5 KO lines. Transcriptome comparison between
WT and EEE-KO animals reveals a series of genes downregulated in the
KO, providing – for the first time – an insight into the Gene Regulatory
Networks downstream of Myf5. Two major pathways are affected: one
involved in the myogenic process; the second related to chondriogenesis.
To unravel the full phenotype of the EEE-KO, we have crossed this line
with the MyoD-KO, thus abolishing the rescue by the second MRF. No
muscle groups are absent in these animals, indicating that not a single
muscle derives exclusively from the DML, but bost of the body muscles
show marked atrophy. The EEE element contains two peaks of sequence
conservation; transgenic analyses show only one to be essential for DML
expression. Our results suggest that the EEE has thus a dual function: to
drive the early expression of Myf5 in the DML, and to maintain expression
levels at later stages.
P03-12
DLK2 favorece la diferenciación y maduración
condrocítica en la línea celular ATDC5
A. Ruiz-García1, Laura Alguacil Camacho1, S. Rivero2, J. Laborda1,
J.J. García-Ramírez1
1
Departamento de Química Inorgánica, Orgánica y Bioquímica.
Facultad de Medicina/CRIB, Universidad de Castilla-La Mancha,
Albacete, ES, 2The Chidren’s Research Institute Department
of Clinical Neurology, NW Washinton DC, US
La proteína de membrana DLK2, y su homóloga DLK1, pertenecen al
grupo de ligandos no canónicos de los receptores NOTCH. Ambas proteínas
DLK presentan una región extracelular con seis dominios EGF-like, una
región transmembrana y una corta región intracelular. Las proteínas DLK
desempeñan un importante papel en la proliferación y la diferenciación
celular, en procesos como la adipogénesis, hematopoiesis, miogénesis,
condrogénesis y osteogénesis. Desde hace tiempo se conoce que DLK1
promueve la maduración condrogénica de las células mesenquimáticas,
aunque curiosamente impide su maduración final a condrocitos.
Además, DLK1 ha sido descrito como un inhibidor de la diferenciación
condrocítica de la línea celular de ratón ATDC5 sometida a estímulos
diferenciadores. Estudios previos realizados en nuestro laboratorio revelan
que los genes Dlk1 y Dlk2 se expresan de forma complementaria durante
la diferenciación condrocítica en embriones de ratón. Así, Dlk1 se expresa
en las células menos maduras, como los precondrocitos y los condrocitos
columnares, mientras que Dlk2 se expresa en fases más diferenciadas,
como los condrocitos hipertróficos y los condrocitos terminales. En este
Salamanca 2016
trabajo analizamos el papel de la proteína DLK2 durante la diferenciación
condrogénica de la línea ATDC5 en respuesta a insulina. Para ello, hemos
obtenido transfectantes estables en esta línea celular, con mayor y menor
expresión de DLK2, y hemos analizado varios parámetros específicos de
la diferenciación condrocítica. Nuestros resultados muestran que DLK2
es capaz de estimular tanto la expresión de marcadores condrocíticos
tempranos, como Col2a1 y AGC, como de marcadores tardíos, como
Col10a y Runx2. Además, DLK2 potencia la formación de condensaciones
celulares y la producción de matriz extracelular. Nuestros resultados
muestran que, al contrario que DLK1, DLK2 es un potente activador de la
diferenciación y de la maduración de condrocitos. Estos resultados servirán
de base para investigar en profundidad los mecanismos mediante los cuales
esta proteína regula la diferenciación condrocítica.
P03-13
Functional analysis of transcriptional regulators
during auditory development
Thomas Schimmang
Instituto de Biología y Genética Molecular, CSIC-Universidad
de Valladolid, Valladolid, ES
Transcriptional regulatory networks are essential during the formation
and differentiation of organs. The transcription factor N-myc is required
for proper morphogenesis of the auditory organ and to control correct
patterning of its sensory epithelium, the organ of Corti. We have recently
shown that the Otx2 gene, a mammalian ortholog of the Drosophila
orthodenticle homeobox gene, is a crucial target of N-myc during inner ear
development. Otx2 expression is lost in N-myc mouse mutants, and N-myc
misexpression in the chick inner ear leads to ectopic expression of Otx2.
Furthermore, Otx2 enhancer activity is increased by N-myc misexpression,
indicating that N-myc may directly regulate Otx2. Inactivation of Otx2
in the mouse inner ear leads to ectopic expression of prosensory markers
in non-sensory regions of the cochlear duct. Upon further differentiation,
these domains give rise to an ectopic organ of Corti, together with the
re-specification of non-sensory areas into sensory epithelia. Therefore, the
Otx2-positive domain of the cochlear duct shows a striking competence
to develop into a mirror-image copy of the organ of Corti. Taken together,
these data show that Otx2 acts downstream of N-myc and is essential for
patterning and spatial restriction of the sensory domain of the mammalian
auditory organ.
P04. Biología molecular computacional /
Computational molecular biology
P04-1
Formation and maintenance of nitrogen-fixing
cell patterns in filamentous cyanobacteria
Javier Muñoz-García, Saúl Ares
Grupo Interdisciplinar de Sistemas Complejos (GISC) y
Departamento de Matemáticas, Universidad Carlos III de Madrid,
Leganés, ES
Cyanobacteria forming one-dimensional filaments are paradigmatic
model organisms of the transition between unicellular and multicellular
living forms. Under nitrogen-limiting conditions, in filaments of the
genus Anabaena, some cells differentiate into heterocysts, which lose
the possibility to divide but are able to fix environmental nitrogen for
the colony. These heterocysts form a quasiregular pattern in the filament,
representing a prototype of patterning and morphogenesis in prokaryotes.
Pósters / Posters
Recent years have seen advances in the identification of the molecular
mechanism regulating this pattern. We use these data to build a theory
on heterocyst pattern formation, for which both genetic regulation and
the effects of cell division and filament growth are key components.
The theory is based on the interplay of three generic mechanisms: local
autoactivation, early long-range inhibition, and late long-range inhibition.
These mechanisms can be identified with the dynamics of hetR, patS, and
hetN expression. Our theory reproduces quantitatively the experimental
dynamics of pattern formation and maintenance for wild type and mutants.
We find that hetN alone is not enough to play the role as the late inhibitory
mechanism: a second mechanism, hypothetically the products of nitrogen
fixation supplied by heterocysts, must also play a role in late long-range
inhibition. The preponderance of even intervals between heterocysts arises
naturally as a result of the interplay between the timescales of genetic
regulation and cell division. We also find that a purely stochastic initiation
of the pattern, without a two-stage process, is enough to reproduce
experimental observations.
P04r-2
Optimizing T cell epitope vaccine formulation
for maximum population coverage
Pedro A. Reche, Magdalena Molero-Abraham, Mario Solis-Lopez,
María E. Lafuente
Departamento de Microbiología I, Facultad de Medicina,
Universidad Complutense de Madrid, Madrid, ES
T cells recognize small peptide antigens when presented in the cell surface
of target cells bound to human leukocyte antigens (HLA) molecules. These
peptides are known as T cell epitopes, and there is potential for using such
epitopes in vaccine formulations. A T cell epitope vaccine will only be
effective on those individuals presenting the HLA molecules capable of
binding and presenting the epitopes included in the vaccine. Thereby,
HLA polymorphism pose a major handicap to the development of T cell
epitope-based vaccines covering the entire population. HLA peptide-biding
specificity is determined by the polymorphisms and HLA polymorphic
variants are expressed at vastly variable frequencies in different ethnic
groups. In response to this problem, some authors, included us have defined
HLA supermotifs to identify promiscuous epitopes binding to groups of
HLA molecules known as supertypes. These HLA supermotifs are however
unspecific and can result in discarding relevant epitopes. In contrast, we
describe here a new algorithm that, by taking peptide binding specificities
of the HLA molecules into consideration, minimizes the potential epitopes
required to develop a broadly protective vaccine.
P04-3
Learning the genome. Artificial Neural Networks
applied to genomic data
Davide Bau
CNAG-CRG, Valencia, ES
The three-dimensional organization of chromatin drives gene expression
by bringing together genes and their interacting partners.
Different genes in different cell types are expressed differently, despite
sharing the same DNA sequence. Epigenetic modifications defines cell
specificity; specific patterns of covalent modifications of the histone
tails impact gene expression by altering chromatin structure or recruiting
histone modifiers in a cell specific way. Learning how the epigenomic
landscape contributes to the cellular outcome could help understanding
how the genome is regulated.
Using machine learning techniques, I have integrated histone modifications,
chromatin accessibility and gene expression data to study the impact of
post-translational modifications on genome architecture. My results
show that learning from a subset of all the chromosomes is sufficient to
63
Pósters / Posters
accurately predict a set of features on the remaining chromosomes. My
predictions provide a useful tool to help understanding how the genome is
organized and to complement incomplete genomic data.
Keywords: Genome architecture, Structural biology, Epigenetics, Genomics,
Integrative modeling, Bioinformatics, Machine Learning.
XXXIX Congreso SEBBM
splicing patterns that recapitulate those of undifferentiated cells, are
controlled by MBNL1 and involve multiple cancer drivers, including
the mitotic gene NUMA1. We show that NUMA1 alternative splicing
induces enhanced cell proliferation and centrosome amplification in
non-tumorigenic mammary epithelial cells.
P04-4
Decomposing variation in heterogeneous
clinical omic data
Francisco José Campos-Laborie, José Manuel Sánchez-Santos,
Javier de las Rivas Sanz
Centro de Investigación del Cáncer (CiC-IBMCC, CSIC/USAL/IBSAL),
Salamanca, ES
Individual diversity is one of the most complex issues to deal with in
omic studies of large populations. Most of the current approaches to
detect differences using new generation omic-wide data are based on
the analyses of significant mean or median changes that reflect average
alterations in the whole population studied versus their controls or
references. In this situation the biomarkers that are only related to a subset
of samples are difficult to detect and often wrongly assigned, but in many
occasions such sample subsets are quite relevant for the biological study
performed. Considering that technical and batch-associated variations are
mostly treated and corrected by robust normalization methods developed
in recent years (RUV, SVA) (PMIDs: 25150836, 22257669), we explore
different methodologies to understand the frequent heterogeneity in
disease sample sets that come from specific clinical or biological
features only associated to a subset of the population. Moreover, the
classical normalization approaches often apply ways to reduce or
remove unwanted variation (PMID: 26286812), but our scope is not to
decrease or alter the sample signals but to identify omic biomarkers that
are modified only in a sub-population of the clinical cohorts studied.
The first method proposing the identification of disease outliers using
genomic data was COPA (PMID: 16895932), and several other more
recent approaches have tried to tackle this problem. One efficient
approach to identify features associated to subsets of a population can
be to explore in a recursive way the existing dependent relationships
between features and samples provided by large-scale omic datasets.
This type of comprehensive omic data (genomic, transcriptomic, etc) can
facilitate the finding of new significant biomarkers related to hidden or
not clear phenotypic conditions. The stratification of patients according
to their omic profiles achieved using recursive heuristic methods is a way
that we propose to assign new biomarker features between closely related
states and to subset samples depending on their omic patterns.
P05. Biomembranas y bioenergética /
Biomembranes and bioenergetics
P05-1
Molecular dynamics study of the interaction
of the HIV gp41 LLP2 segment with model
membranes
Emmanuel Fajardo-Sánchez1, David Carrillo-Trevinyo1,
Vicente Galiano2, José Villalaín1
1
IBMC-UMH, Elche, ES, 2Dpto. Física y Arq. Computadores-UMH,
Elche, ES
The construction of new antivirals to prevent the infection of the human
immunodeficiency virus (HIV) is one of the biggest biomedical challenges
nowadays, and the complexity of the interactions between the virus
and the host cell is one of the main problems to achieve that objective.
The basic knowledge of those interactions is one of the most important
backgrounds which can describe this process properly. The present study
aims to understand the molecular interaction between the membrane and
one of the endodomain segments of HIV protein gp41 known as LLP2.
LLP2 plays a key role in the infection process, but little is known about
the molecular interactions between this segment and the membrane.
We have designed a model membrane specifically composed of POPC/
POPE/POPI14/POPS/OSM/CHL1 to study the membrane interaction of
the LLP2 segment by molecular dynamics (MD). The peptide is initially
located at a distance of 5Å of membrane surface without touching any of
the membrane lipids. At the end of the MD simulation, LLP2 seems to
interact stronger with POPI14 than with the other lipids in the membrane.
These data would imply that this gp41 segment interacts specifically
with negatively-charged phospholipids and therefore gp41 function could
depend on specific lipid interaction. These data should help us in our
understanding of the molecular mechanism of HIV life cycle as well as
making possible the future development of potent inhibitor molecules,
which might target gp41 segments involved in membrane binding.
P05r-2
P04-5
The role of RNA processing alterations
in cancer biology
Eduardo Eyras
Universitat Pompeu Fabra, Barcelona, ES
Alternative splicing of mRNAs profoundly influences almost all
biological processes during neoplastic transformation. However, the
specific alternative splicing mechanisms that are disrupted in tumors are
not yet exhaustively characterized. We systematically analyzed mutation,
copy number and gene expression patterns of 1348 RNA-binding protein
(RBP) genes in 11 solid tumor types, together with alternative splicing
changes and the enrichment of binding motifs in the alternatively spliced
sequences. Our comprehensive study reveals widespread alterations in
the expression of RBP genes, as well as novel mutations and copy number
variations in association with multiple alternative splicing changes in
cancer drivers and oncogenic pathways. In particular, we found altered
64
CD98hc at the crossroad of oxidative stress
and amino acid availability
Sara Cano Crespo, Laura Rodríguez de la Ballina, Manuel Palacín
Institute for Research in Biomedicine (IRB Barcelona); Spanish
Biomedical Research Center in Rare Diseases (CIBERER);
Department of Biochemistry and Molecular Biology, University
of Barcelona, Barcelona, ES
CD98hc, the only ubiquitously expressed heavy subunit of Heteromeric
Amino acid Transporters (HATs) is overexpressed in highly proliferative
cells, in both physiological and pathological conditions. CD98hc associated
transporters (i.e. xCT, LAT1 and y+LAT2 in wild type fibroblasts) support
cell survival in vitro and cell proliferation in vitro and in vivo. Cell survival
depends on the capacity of the cell to counterbalance the oxidative stress
via CD98hc-xCT. Indeed, the deficiency of CD98hc-xCT can not be
compensated, leading to cell death by ferroptosis. Supplementation of
culture media with beta mercaptoethanol (β-ME) rescues CD98hc deficient
Salamanca 2016
cell survival. In such conditions, CD98hc-null cells suffer modulation of
other CD98hc-independent amino acid (AA) transporters (for instance
induced expression of peptide transporter 1 (PEPT1)), which triggers
intracellular AA imbalance with reduced levels of branched chain AA
(BCAA) and aromatic (ARO) AAs. Moreover, CD98hc ablation have
an impact in mitochondria at both metabolic and morphology levels and
ablated cells show oxidative stress. Along with all of the aforementioned
adaptations CD98hc-null cells present limited proliferation which
interestingly, is rescued by external supply of dipeptides containing
BCAAs and ARO AAs which can enter via PEPT1. This supplementation
also recovers part of the mitochondrial phenotype, suggesting a strong
dependency on BCAAs and ARO AAs availability for cell proliferation
and energy metabolism and therefore a strict requirement of CD98hc-LAT1
and CD98hc-y+LAT2in our cell model.
P05-3
The role of HIV-1 gp41’s lentivirus lytic peptides
in the interaction of the envelope protein with
viral membrane cholesterol
Jon Ander Nieto Garai1, Eneritz Bilbao Eraña1, Francesc Xabier
Contreras Gómez2, Maier Lorizate Nogales1
1
Departamento de Bioquímica y Biología Molecular, Universidad
del País Vasco and Instituto Biofisika (CSIC, UPV/EHU), Leioa,
ES, 2Instituto Biofisika (CSIC, UPV/EHU) and Ikerbasque Basque
Foundation for Science, Leioa, ES
The HIV-1 envelope protein (env) is involved in the fusion between
the virus and the host cell, a key process in virus infectivity. Env’s
recruitment mechanism to the nascent virion’s lipid bilayer is
yet unknown. The elucidation of env’s recruitment and its lipid
environment would point out new therapeutic targets to hamper virus
entry and therefore the spread of the infection, and would aid in the
generation of immunogens against different envelope epitopes. It has
been suggested that the targeting of env to budding regions requires
lipid raft like membrane domains, the gag polyprotein and the three
Lentivirus Lytic Peptide(LLP) domains in the cytoplasmic tail (CT) of
the protein. In this regard, LLPs they have been described to play a role
in the expression of env to plasma membrane, its fusogenicity and in
the incorporation of env to the viral particles. Therefore, the aim of this
work is to study the env-cholesterol interaction in viral context, more
precisely the role of the LLP domains in such interaction. HEK 293T
cells were transfected with proviral plasmids including two envdeletion
mutants: ΔLLP1 and ΔLLP1-3. Transfected cells were incubated with
radioactively labeled photoactivatable [3H]-photo-cholesterol. This
lipid allows the study of specific in vivo interactions as it covalently
binds to any molecule closer than 3Å. The cell culture supernatant was
then collected and ultra-pure viral particles were purified. Our results
suggest that env is associated to cholesterol in the viral membrane.
Nevertheless, in the ΔLLP1-3 mutant the env-cholesterol interaction
is significatively reduced. Therefore, env seems to be associated to
raft-like lipids in the viral membrane and the LLP domains could play
a key role in this interaction, changing protein structure or affecting
the direct interaction of the CT with cholesterol. We plan to study the
biological significance of the loss of env-cholesterol interaction. We
also plan to study the interaction of lipids with other viral proteins and
even cellular accessory proteins present in the budding site that may
play a role in env recruitment.
P05-4
Mechanism of binding of C1B domain of PKCε to
lipid membranes
Juan Carmelo Gómez-Fernández, Antonio L. Egea-Jiménez, Senena
Corbalán García
Pósters / Posters
Departamento de Bioquímica y Biología Molecular A, Universidad
de Murcia, Murcia, ES
C1 domains are members of the Cys-rich domains superfamily, formed by
50-51 amino acids residues present in many types of proteins, as it is the
case of the classical and novel Protein Kinases C (PKCs). Both types of
PKCs, classical and novel isoenzymes, possess two C1 subdomains, C1A
and C1B, although it is not totally clear why two modules are needed.
C1 domains are known to interact with diacylglycerol and exogenous
agents like phorbol esters. In a previous report, we demonstrated the
importance of not only diacylglycerol but also of anionic phospholipids,
specifically 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) and
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), for the
interaction of the C1B domain with lipid membranes, among which
the C1Bε domain had the highest binding affinity than any other C1B
domains of novel isoenzymes. In this work we have studied the role
of positively charged amino acids residues located on top of the C1Bε
domain and their involvement in the interaction membrane-protein.
Some anionic residues were replaced by alanine and we characterized the
effect of single, double and triple mutations. Results show that binding is
decreased by increasing of the number of residues mutated in the domain,
and it be even abolished in the presence of diacylglycerol. In conclusion,
the electrostatic interactions derived of these positively amino acids
residues is important to give place to membrane docking which is further
stabilized by interaction with the diacylglycerol.
This work was supported by grants from Ministerio de Economía y
Competitividad-FEDER (BFU2014-52269-P) and from Fundación Séneca
(Comunidad Autónoma de Murcia) (19409/PI/14).
P05-5
Intercellular communication in the adult carotid
body germinal niche
Valentina Annese, Elena Navarro-Guerrero, Aida Platero-Luengo,
Verónica Sobrino, Ricardo Pardal
Instituto de Biomedicina de Sevilla (IBiS). Hospital Universitario
Virgen del Rocío/CSIC, Universidad de Sevilla. Dpto. de Fisiología
Médica y Biofísica, Sevilla, ES
Neural stem cells rise high interest among scientists due to their possible
use against neurodegenerative disease. Nervous tissue-specific stem cells
reside in specialized niches that allow them to respond to physiological
demands. A profound knowledge of the functioning of these niches would
allow us to improve the use of stem cells for cell therapy. We have studied
the biology of a subpopulation of neural crest-derived stem cells that persist
in an adult peripheral chemoreceptor organ: the carotid body. This organ
has the capacity to adapt to a hypoxemic situation by increasing in size
and cell number. The carotid body parenchyma contains a subpopulation of
neural stem cells able to give rise to neurogenesis in response to hypoxia,
activating proliferation and completing differentiation into new neuronal
cells. Recent data in our group show that these stem cells are even more
versatile, being able to participate also in the angiogenic process that
takes place in the organ in parallel to neurogenesis. Carotid body stem
cells are able to convert into new vascular cells in response to the hypoxic
stimulus. We are studying in detail the biology of this population of adult
carotid body neural stem cells. We have identified restricted progenitors
within both neuronal and mesenchymal differentiation lineages. We are
particularly interested in the membrane mechanisms by which carotid
body germinal niche cells interact to each other, since these communication
abilities determine the functioning of the niche and therefore the behavior
of stem cells. Altogether, our results on the characterization of the carotid
body germinal niche might have high impact on the use of these stem cells
for cell therapy.
65
Pósters / Posters
P05-6
Mitochondrial permeabilization by Bax
at the single molecule level
Ana J. García Saez
University of Tuebingen, Tuebingen, DE
Bax is a key regulator of apoptosis that mediates mitochondrial
fragmentation and permeabilization of its outer membrane through a
poorly understood mechanism. We have combined electron paramagnetic
resonance with single molecule microscopy and superresolution
microscopy to determine the structural organization, oligomeric state and
nanoscale assembly of active, full-length Bax in the membrane at different
spatial scales. We found that Bax organizes into multiple oligomeric species
based on dimer units. Each Bax molecule in the dimer is characterized by a
stable dimerization domain and a more flexible piercing domain. The most
important structural change during Bax activation is the opening of the
hairpin formed by helices 5 and 6 into a clamp-like structure. Moreover,
in the mitochondria of apoptotic cells, active Bax clusters into a broad
distribution of distinct architectures, including full rings, as well as linear
and arc-shaped oligomeric assemblies. Remarkably, both rings and arc
assemblies of Bax can perforate the membrane, as revealed by atomic
force microscopy. Our data support a new molecular mechanism in which
Bax dimers assemble into oligomers with even number of molecules that
fully or partially delineate pores of different sizes to permeabilize the
mitochondrial outer membrane.
P05-7
Sea anemone actinoporins: Protein molecules
dancing at the water-membrane edge
Álvaro Martínez del Pozo
Departamento de Bioquímica y Biología Molecular, Universidad
Complutense de Madrid, Madrid, ES
Sea anemone actinoporins constitute a fascinating family of multigene
proteins because of their dual behavior at the water-membrane interface.
In water they are mostly monomeric and remain stably folded as soluble
globular proteins. However, upon interaction with lipid membranes
of specific composition they become oligomeric integral membrane
structures resulting in lytic pores that are lethal for their target cells.
Actinoporins pore formation is efficient in sphingomyelin (SM) containing
membranes. However, it is also known that cholesterol (Chol) is required
for effective pore formation as a consequence of the biophysical changes
and intermolecular interactions established within the lipidic membrane.
In spite of the intensive work developed for more than 20 years in this
direction, it is not yet fully understood how the bilayer lipid composition or
the physical state of the membrane affect pore formation. Within this idea,
we have studied how esterols, interfacial hydrogen bonding, membrane
width and the saturation degree of the SM species employed affect
actinoporins pore forming ability. Finally, we have recently shown how
the appearance of actinoporins as multigene families that give rise to many
protein isoforms in the same individual species can be explained in terms
of a synergistic interaction to produce heteropores with highly different
pore-forming activity.
P05-8
Glycosylation-dependent partitioning of IFN-γ
receptor in lipid and actin nanodomains is
critical for JAK/STAT signaling
Ornella Morana1, Cédric Blouin2, Christophe Lamaze2, Xabier
Contreras3
1
Instituto Biofisika (UPV/EHU-CSIC); Departamento de Bioquímica,
Universidad del País Vasco, Leioa, ES, 2Institut Curie - Centre de
Recherche, PSL Research University, Membrane Dynamics and
66
XXXIX Congreso SEBBM
Mechanics of Intracellular Signaling Laboratory; Centre National de
la Recherche Scientifique (CNRS), París, FR, 3Instituto Biofisika
(UPV/EHU-CSIC); Departamento de Bioquímica, Universidad del País
Vasco; IIKERBASQUE, Basque Foundation for Science, Leioa, ES
Understanding how membrane nanoscale organization controls
transmembrane receptors signaling activity remains a challenge. We
studied IFN-γ receptor signaling in fibroblasts from homozygous
patients with a T168N mutation in IFNGR2. This mutation by adding
a neo-N-glycan on IFN-γR2 subunit blocks IFN-γ activity by unknown
mechanisms. We show that the lateral diffusion of IFN-γR2 is confined
by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2
T168N mutant diffusion is confined by distinct actin nanodomains where
conformational changes required for JAK/STAT activation by IFN-γ
could not occur. Removing IFN-γR2 T168N bound galectins restored
lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient
cells whereas adding galectins impaired these processes in control cells.
These experiments prove the critical role of dynamic receptor interactions
with actin and lipid nanodomains and reveal a new function for receptor
glycosylation and galectins. Our study establishes the physiological
relevance of membrane nanodomains in the control of transmembrane
receptor signaling in vivo.
P05r-9
Characterization of the calcium binding mode
of Rabphilin-3A
Jesús Baltanás Copado1, María Dolores Pérez-Sánchez2,
Juan C. Gómez-Fernández2, Senena Corbalán-García2
1
Universidad de Murcia, Úbeda, ES, 2Dpt. of Biochemistry
and Molecular Biology A, University of Murcia, Murcia, ES
C2 domains are ubiquitous conserved Ca+2 activated membranedocking modules present in a wide range of Ca+2-regulated proteins.
They consist of 130 residues andshare a common fold composed of two
four-stranded β-sheets arranged in a compact β-sandwich connected
by surface loops and helices. Many of these C2 domains have been
demonstrated to function in a Ca2+-dependent membrane-binding
manner and hence actas cellular Ca2+ sensors. Calcium ions bind
in a cupshaped invagination formed by threeloops at one tip of the
β-sandwich where the coordination spheres for the Ca2+ ions are
incomplete. This incomplete coordination sphere can be occupied by
neutral and anionic phospholipids, enabling the C2 domain to dock at
the membrane. The binding to Ca+2of the C2A, C2B and C2AB domain
of Rabphilin 3A and different membrane model systems composed of
PtdIns(4,5)P2 and PtdSer have been characterized by using isothermal
titration calorimetry. The C2A domain shows a very low affinity to
bind Ca+2. This interaction relies on a three sequential binding mode
with dissociation constants of 117, 524 and 710 uM. The C2B domain
fitted to a one set ofsites model with a dissociation constant of 2 uM,
indicating that the C2B domain has ahigher affinity for Ca+2 than the
C2A domain. Previous work in our laboratory hasenabled us to capture
an intermediate state of the Ca+2-bound state of the C2A domain.
This suggests a different mechanism of ions interaction with a large
rearrangement inthe calcium binding regions (CBRs) of rabphilin C2A
domain that might explain thelow Ca+2 affinity. Strikingly, when the
calorimetric study was performed with the C2AB domain, the results
fitted into a two set of sites, being the dissociation constants 1.4 uM and
3.8 uM for the first and second components, respectively. Site-directed
mutagenesis of the residues involved in calcium binding has confirmed
that the C2B domain confers Rabphilin 3A with a quick response to
Ca2+ and the C2A with a slow response due to its low affinity. These
results indicate that Rabphilin 3A might play different roles at several
states of the vesicle fusion cycle, depending on the Ca2+ and target
lipids available at each moment at the plasma membrane and transport
vesicles.
Salamanca 2016
Acknowledgements: This work has been sponsored by grants
BFU2014-52269-P (MINECO, Spain-FEDER) and 19409/PI/14 (Fundación
Seneca, Region de Murcia).
Pósters / Posters
no improvements in GABARAP lipidation or GABARAP-PE induced
fusion were observed, perhaps due to structural differences between
yeast and human proteins. The data support the notion that these two
ubiquitin-like systems are involved in driving the biogenesis of the
autophagosomal membrane.
P05r-10
Multifuncional C2 domains
David López-Martínez, Teresa Coronado-Parra,
Juan C. Gómez-Fernández, Senena Corbalán-García
Dpt. of Biochemistry and Molecular Biology A, University of Murcia,
Murcia, ES
The C2 domains are protein modules frequently involved in
targeting proteins to cell membranes. They share a common fold of
two four-stranded-β-sheets arranged in a compact β-sandwich and
surrounded by variable loops and helices, whose variations in the
aminoacidic composition give them the capacity to bind phospholipids
in a calcium-dependent manner. We have shown for numerous C2
domains that they interact differently with Ca2+, phosphatidylserine and
phosphoinositides, conferring different functional abilities to dock at the
plasma membrane with different target messenger affinity mechanism.
Moreover, we have shown that the C2A domain of synaptotagmin 1 is a
non- PI(4,5)P2 responder because of a glutamic residue instead of one
critical lysine. Different affinities to bind different phosphoinositides
in membrane models are controlled by other amino acidic residues
adjacent to the key interacting lysines.
Our discovers shed light on how these domains develop structural
changes to modulate their sensitivity and specificity to various cellular
signals, and provide structural and functional description about in what
manner, C2 domains are controlled by a dual-target mechanism.
P05-11
Autophagosomal elongation: Interplay between
Atg12-Atg5/Atg16 complex and LC3/GABARAP
systems
Marina N. Iriondo, Anton Zuriñe, Ainara M. López-Pardo,
Javier H. Hervás, Ane Landajuela, Alicia Alonso, Félix M. Goñi
Instituto Biofisika (UPV/EHU, CSIC); Departamento de Bioquímica
y Biología Molecular, Universidad del País Vasco, Bilbao, ES
Macroautophagy mediates the degradation of long-lived proteins
and organelles through the de novo formation of double-membrane
autophagosomes (APs) that sequester cytoplasmic material and deliver it
to the vacuole/lysosome. How APs form, especially how the membrane
expands and eventually closes upon itself is an area of intense research.
This process requires the concerted actions of a distinctive set of the
so-called Atg proteins that include two ubiquitin-like proteins, Atg12
and Atg8. Membrane association of the human Atg8 homologues (LC3/
GABARAP) is mediated by the covalent attachment to phosphatidyl
ethanolamine (PE) and has been related to both membrane expansion
and membrane fusion, but the underlying molecular mechanisms
are poorly understood. Furthermore, it has been shown that the
Atg12–Atg5/Atg16 complex in yeast acts as an E3 enzyme for the
conjugation reaction of Atg8, enhancing the E2 activity of Atg3 and
specifying the site of Atg8–PE production. In the present study the two
ubiquitin-like conjugation systems (yeast Atg12 and human Atg8) were
used to analyse the molecular mechanisms by which the presence of
Atg12-Atg5/Atg16 would improve human LC3/GABARAP lipidation
and its ability to induce membrane tethering and fusion, leading to
autophagosome elongation. With this purpose, we have performed
experiments of protein-protein and protein-lipid interaction using
different model membranes and a variety of biophysical approaches i.e.
ultracentrifugation, fluorescence spectroscopy, or confocal microscopy.
Atg12–Atg5/Atg16 improved GABARAP recruitment to lipid
bilayers, in the form of giant unilamellar vesicles (GUVs), however
Acknowledgements: This work has been sponsoredby grants from the
Basque Government (IT838-13) and FEDER/Mineco (BFU2015-66306-P).
P05-12
Alteraciones de la homeostasis de colesterol
por éteres lipídicos antitumorales
Pablo Ríos-Marco1, Marisol Fernández-Ortiz2, Xiomara
Gálvez-Escolano2, José Manuel Jiménez-López2, Antonio Ríos3,
Carmen Marco2, María Paz Carrasco Jiménez2
1
Instituto de Parasitología y Biomedicina López - Neyra, CSIC,
Granada, ES, 2Departamento de Bioquímica y Biología Molecular I,
Facultad de Ciencias, Universidad de Granada, Granada, ES,
3
Departamento de Biología Celular, Facultad de Ciencias,
Universidad de Granada, Granada, ES
Según la Organización Mundial de la Salud, el cáncer es una de las
principales causas de morbilidad y mortalidad en el mundo. Los éteres
lipídicos se presentan como una alternativa terapéutica a utilizar en
quimioterapia contra el cáncer. Se destacan los alquilfosfolípidos
(APL), siendo la edelfosina (1-O-octadecil-2-O-metil-rac-glicero-3fosfocolina) representante de la primera generación de APL, así como
los éteres lipídicos glicosilados (GAEL), en los que se sustituye el grupo
fosfocolina de edelfosina por un grupo monosacárido. Nuestro grupo
de investigación ha demostrado que ambos análogos lipídicos alteran
la homeostasis del colesterol al interrumpir el tráfico intracelular de
este lípido hasta el retículo endoplasmático (RE) en células tumorales
humanas, provocando así una respuesta colesterogénica, que se traduce
en una síntesis de colesterol incrementada. En estos experimentos
hemos utilizado como control positivo el agente U18666A, que bloquea
el transporte de colesterol desde los compartimentos endosomales/
lisosomales al RE. Hemos comprobado que el tratamiento con
β-hidroxipropil-ciclodextrina moviliza el colesterol retenido por
U18666A en los compartimentos endosomales/lisosomales aumentando
el transporte de colesterol a RE. Sin embargo, este efecto no es
observable cuando las células son tratadas con los diferentes éteres
lipídicos, concluyendo que estos agentes no retienen el colesterol
en estos compartimentos. Un efecto diferencial entre APL y GAEL
consiste en que solo edelfosina inhibe significativamente la síntesis
del fosfolípido fosfatidilcolina. Nuestros resultados concluyen que
ambos grupos de éteres lipídicos presentan mecanismos diferentes
responsables de su actividad antitumoral.
Esta investigación ha sido financiada por la Junta de Andalucía
(P11-CVI-7859).
P05-13
Yeast-based system for expression and
purification of functional native human plasma
membrane Ca2+-ATPase isoform PMCA4b
Isaac Corbacho, Francisco F. García-Prieto, Ara E. Hinojosa,
María Berrocal, Ana M. Mata
Depto. de Bioquímica y Biología Molecular y Genética, Facultad
de Ciencias, Universidad de Extremadura, Badajoz, ES
Calcium (Ca2+) plays an important role in cell signaling, and its
homeostasis is implicated in several diseases and neurodegenerative
disorders. Among the different transporters and channels regulating
67
Pósters / Posters
Ca2+ levels, human plasma membrane calcium ATPases (PMCAs)
are responsible for the extrusion of calcium out of the cell. Thus,
understanding PMCAs underlying mechanisms is crucial. However,
there is a limited availability of these proteins for functional and
structural analysis, since traditionally they have been obtained from
either human or pig brain preparations, yielding a heterogeneous
mixture of PMCA isoforms. In this work, using the yeast Saccharomyces
cerevisiae system, we show a new and enhanced method for the
expression of the full-length human PMCA isoform 4b (hPMCA4b).
We have also improved a method for the purification of this native
isoform by calmodulin-agarose affinity chromatography. One of the
most relevant features of this work is that, when compared to PMCAs
purification from pig brain, our method provides a pure single isoform
instead of a mixture of isoforms, essential for fine-tuning the activity
of PMCA4b. Another relevant feature is that the method described in
this work has a superior yield of protein than previously established
methods used to purify PMCA proteins expressed in yeasts.
This work was supported by Ministerio de Economía y Competitividad
(BFU2011-23313 and BFU2014-53641-P); Junta de Extremadura
(GR10108 and GR15139), Fundación Caja de Extremadura and FEDER.
P05r-14
Polyamine-RNA-membrane interactions:
from the past to the future of biology
Carlos Acosta Andrade1, Ibai Artetxe2, Marta G. Lete2, Kepa Ruiz
Mirazo3, Felix M. Goñi2, Francisca Sánchez Jiménez1
1
Departamento de Biología Molecular y Bioquímica, Universidad de
Málaga; and Unit 741 of CIBER de Enfermedades Raras, Málaga,
ES, 2Biofisika Institute (CSIC, UPV/EHU); and Department of
Biochemistry, University of the Basque Country, Leioa, ES, 3Biofisika
Institute (CSIC, UPV/EHU); and Department of Biochemistry,
University of the Basque Country. Department of Logic and
Philosophy of Science, University of the Basque Country, Leioa, ES
Biogenic polyamines (PA), e.g. spermine, spermidine, putrescine, are
widely spread amino acid derivatives, occurring in living cells across the
whole evolutionary scale. Their amino groups confer them a markedly
basic character at the cellular pH. We have tested the interaction of PA
with negatively-charged phospholipids (e.g. PIP2, abundant in the
nuclear membrane), and with nucleic acids (mainly tRNA was used for
practical reasons). PA induced aggregation of lipid vesicles containing
acidic phospholipids, and of nucleic acids. Aggregation was detected
using both spectroscopic and fluorescence microscopy methods (the latter
with giant unilamellar vesicles). PA-liposome complexes were partially
disaggregated when nucleic acids were added to the mixture, indicating
a competition of lipids and nucleic acids for PA, in a multiple equilibrium
phenomenon. Equivalent observations could be made when vesicles
composed of oleic acid and 1-decanol (1:1 mol ratio) were used instead of
phospholipid liposomes. The data could evoke putative primitive processes
of proto-biotic evolution. At the other end of the time scale, the system
may constitute an interesting tool in the development of nanoscale drug
delivery.
Key words: polyamines, lipids, phosphoinositides, membrane dynamics,
LUCA, nanoparticles.
P05-15
Role of PKC alpha in breast cancer progression
Senena Corbalán García, Consuelo Marín-Vicente, Sonia
Reverte-Ródenas, Juan Carmelo Gómez-Fernández
Dpto. Bioquímica y Biología Molecular A, Facultad de Veterinaria,
Regional Campus of International Excellence Campus Mare
Nostrum, Universidad de Murcia, IMIB-Arrixaca, Murcia, ES
68
XXXIX Congreso SEBBM
Cancer cells show a great versatility in signal transduction pathways.
One of the groups of proteins involved in these cellular processes is the
family of Protein Kinase C. These enzymes phosphorylate their targets
in residues of serine or threonine and have been implicated in a myriad
of processes important to tumour progression such as proliferation,
migration or apoptosis. They are ubiquitous and regulated by
phosphorylation, interaction with other proteins and different cofactors
such as Ca2+ and lipids. Depending on the isoenzyme and the cellular
type, their involvement can be pro-oncogenic or anti-oncogenic. So far,
no efficient therapy based on the use of these proteins has been developed
for impairing tumorigenesis. In the present work, we use transcriptomics
to study the effect of supressing the expression of classical PKC alpha
in breast cancer model cells. The Estrogen Receptor+ and Progesterone
Receptor+ MCF7 cells were knocked down in PKC alpha by using
siRNA technology. A total of 13461 unique mRNA transcripts were
analyzed in an Affymetrix microarray. Among them, 168 probes were
significantly up-regulated and 79 probes were down-regulated. Gene
Ontology analyses in several databases identified down-regulated genes
of the MAPK and ErbB families, while the up-regulated genes rendered
inositol phosphate metabolism and phosphatidylinositol signalling as the
most important groups. These results indicate that PKC alpha is directly
involved in the oncogenic process by controlling key regulatory pathways.
Furthermore, its down-regulation promotes the over-expression of many
other proteins indicating that PKC alpha might act as a break to control
their activity.
This work was supported by Ministerio de Economía y Competitividad
(Madrid), Grant [BFU2011-22828] and co-financed by FEDER.
P06. Bioquímica de la nutrición /
Nutritional biochemistry
P06-1
Disminución en parámetros de estrés oxidativo
e inflamación en tejido adiposo visceral y
hepático mediante la suplementación con aceite
de rosa mosqueta, a ratones alimentados
con dieta alta en grasa
Gladys Tapia Opazo, Daniel González-Mañán, Camila Dossi Muñoz,
Amanda D’Espessailles Tapia
Facultad de Medicina, Universidad de Chile, Santiago, CL
Introducción: El aceite de Rosa Mosqueta (RM, Rosa rubiginosa)
contiene ácido alfa-linolénico, precursor de los ácidos grasos omega-3,
el eicosapentaenoico (EPA) y el docosahexaenoico (DHA). EPA y DHA
tienen propiedades antiinflamatorias, antioxidantes y sensibilizadoras de
la insulina, además estimulan la beta oxidación e inhiben la lipogénesis
de novo, por lo cual son agentes protectores del daño inducido por la
obesidad, la resistencia a la insulina, el estrés oxidativo y la inflamación;
alteraciones que se relacionan con la esteatosis hepática y síndrome
metabólico.
Objetivo: Evaluar si la suplementación oral con aceite de RM disminuye
los parámetros de estrés oxidativo e inflamación inducidos por una dieta
alta en grasa, en tejido hepático y adiposo visceral.
Metodología: Se alimentaron ratones (n=9, por grupo) machos C57BL/6J
durante 12 semanas, dividiéndose en 4 grupos: (i) dieta control (20%
proteínas, 70% carbohidratos, 10% lípidos); (ii) dieta control y RM (1,94
mg ALA/ g peso corporal/día, American Bioprocess Ltda); (iii) dieta alta
en grasas (20% proteínas, 20% carbohidratos, 60% lípidos); (iv) dieta alta
en grasas y RM. Se evaluaron parámetros generales (peso corporal, grasa
Salamanca 2016
visceral, histología), estrés oxidativo [niveles séricos de malondialdehido
(ELISA); niveles hepáticos de Nrf2 (IHQ), Hemooxigenasa-1 (Western
blot) y proteínas carboniladas] e inflamación [niveles hepáticos de PPAR-α
(IHQ); niveles séricos (ELISA) y expresión (qPCR) de TNF-α e IL-1β en
hígado y tejido adiposo visceral].
Resultados: Se observó en el grupo con dieta alta en grasa un aumento
significativo (ANOVA unifactorial, Test de Newman Keuls, P<0,05) en
parámetros de estrés oxidativo e inflamación, junto con niveles de Nrf2,
Hemo-oxigenasa1 y PPAR-α disminuidos en comparación con el grupo
alimentado con dieta alta en grasa y suplementado con RM.
Conclusión: La suplementación dietaria con aceite de RM disminuye
el estrés oxidativo y la inflamación hepática y visceral asociado a la
prevención de la esteatosis hepática. FONDECYT 1140547.
P06r-2
La alimentación con dieta de cafetería durante
la lactancia en ratas afecta los niveles de
mir-222 en leche
Catalina Amadora Pomar Oliver1, Heriberto Castro García2,
Catalina Picó Segura1, Juana Sánchez Roig1, Andreu Palou Oliver1
1
Laboratorio de Biología Molecular, Nutrición y Biotecnología
(Nutrigenómica y Obesidad), Universidad de las Islas Baleares (UIB);
y CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN),
Palma de Mallorca, ES, 2Facultad de Nutrición y Salud Pública,
Universidad Autónoma de Nuevo León, Nuevo León, MX
El objetivo de este estudio ha sido analizar en ratas el efecto de la
alimentación con dieta de cafetería durante la lactancia sobre los
niveles en leche de mir-222, seleccionado por su potencial relación con
la obesidad. Para distinguir los efectos debidos a la obesidad per se,
se estudió también un grupo de madres obesas alimentadas con dieta
de cafetería hasta un mes antes de la gestación (madres postcafetería).
Se recogieron muestras de leche a día 5, 10 y 15 de lactancia y se
determinaron los niveles de mir-222. Tras el destete se sacrificaron crías
de los distintos grupos, en condiciones de alimentación y de ayuno de 12 h,
para analizar la expresión de genes relacionados con el metabolismo
glucídico y lipídico en el tejido adiposo blanco (TAB) e hígado. Los
niveles de mir-222 en leche aumentaron durante la lactancia en todos
los grupos, si bien a día 15 los niveles de mir-222 fueron superiores
en las madres del grupo de cafetería, pero no en las de postcafetería,
en comparación con las controles. Tras el destete, las crías control
presentaron niveles de expresión de Prkaa1 (AMPKa1) (una de las
posibles dianas del mir-222) incrementados en respuesta al ayuno,
tanto en hígado como en TAB. Este aumento no se observó en las crías
de madres del grupo cafetería. Las crías de madres del grupo cafetería
mostraron también una alterada respuesta a los ciclos de alimentación/
ayuno en la expresión de genes implicados en vías metabólicas
reguladas por la AMPK: Srebf1, Fasn, Scd1 y Gck en hígado, y Pparg,
Srebf1 y Scd1 en TAB. Estas alteraciones no se observaron en crías
de madres postcafetería. En conclusión, la alimentación con dieta de
cafetería en ratas durante la lactancia resulta en un incremento de los
niveles de mir-222 en leche. Aunque no se conocen los posibles efectos,
el mayor suministro de mir-222 a las crías a través de la leche podría
relacionarse tentativamente con la alteración en la expresión de Prkaa1
y de genes implicados en la lipogénesis y metabolismo de la glucosa en
respuesta a los ciclos de alimentación/ayuno. Si bien hacen falta más
estudios para demostrar la existencia de una relación causa/efecto.
P06-3
Diet and sex hormones regulate hepatic
Synaptotagmin 1 mRNA in mice
Sara Sancho-Knapik1, Clara Gabas-Rivera1, Sonia Gascón1, Luis
Herrera Marcos1, Roberto Martínez-Beamonte2, María A. Navarro1,
Pósters / Posters
Joaquin C. Surra3, Carmen Arnal4, Jesús de la Osada García5
Departamento de Bioquímica y Biología Molecular y Celular,
Zaragoza, ES, 2Departamento de Bioquímica y Biología Molecular,
Zaragoza, ES, 3Departamento de Producción Animal, Zaragoza, ES,
4
Departamento de Patología Animal, Zaragoza, ES, 5Universidad
de Zaragoza. Dpto de Bioquímica y Biología Molecular y Celular,
Zaragoza, ES
1
The expression of Synaptotagmin 1 (Syt1) has been found to be associated
with the lipid droplets in liver. Here, we studied the expression of Syt1in
Apoe-deficient mice receiving cholesterol, Western diet, squalene, and
oleanolic acid. We also studied the influence of sex and impact of surgical
castration. Dietary cholesterol increased hepatic Syt1 expression, an
effect that was enhanced when cholesterol was combined with saturated
fat present in a Western diet. This potentiation was modified by the
administration of 10 mg/kg oleanolic acid or 1 g/kg squalene. Females fed
chow or Western diet showed higher levels of hepatic Syt1 expression as
compared to male mice on the same diet. Surgical castration of males did
not modify the Syt1 expression; however, ovariectomy led to decreased
levels. The data show thathepatic Syt1 expression is influenced by diet and
hormonal milieu.
P06-4
Aplicación de la metabolómica en la búsqueda
de biomarcadores útiles para evaluar la
efectividad de los alimentos funcionales
Manuel Suárez, Susana Suárez-García, Anna Arola-Arnal, Gerard
Aragonès, Francisca Isabel Bravo, Maria Josepa Salvadó, Begoña
Muguerza, Cinta Bladé, Lluís Arola
Grupo de Nutrigenómica, Departamento de Bioquímica y
Biotecnología, Universidad Rovira i Virgili, Tarragona, ES
En los últimos años, el gran avance experimentado por las técnicas
analíticas ha provocado un salto cualitativo en el campo de la nutrición y
salud. En concreto, la aplicación de la metabolómica, la cual nos permite
conocer de forma precisa las moléculas que componen el metaboloma
de un individuo, es de gran interés en la búsqueda de biomarcadores que
confirmen la efectividad de los alimentos funcionales. De este modo,
siguiendo una aproximación no dirigida, se pueden determinar aquellas
moléculas que se ven alteradas en una situación de enfermedad. Una
vez seleccionadas las que pueden servir como biomarcadores, mediante
el uso de técnicas de análisis dirigidas será posible evaluar de forma
directa si la ingesta de un alimento funcional es capaz de revertir
esta situación, retornando el metabolito a sus valores adecuados. Esta
estrategia implica que se inicia la búsqueda del biomarcador sin tener
conocimiento previo de cuáles son las moléculas que pueden verse
modificadas y requiere el uso de equipos altamente precisos y bases de
datos que permitan obtener una lista de todos los metabolitos existentes
para, a continuación, determinar cuáles son los que pueden utilizarse
como biomarcadores mediante el uso de técnicas de análisis estadístico
de tipo multivariante. Así pues, siguiendo este tipo de aproximación,
hemos determinado las moléculas que se modifican tras la ingesta
crónica de una dieta alta en grasas en el proceso de desarrollo de
dislipidemia y que se podrán utilizar como marcadores para evaluar la
efectividad de alimentos funcionales dirigidos a revertir el progreso de
esta enfermedad.
Estudio financiado por el proyecto AGL2013-40707-R del Ministerio de
Educación y Ciencia del Gobierno de España y fondos FEDER.
69
Pósters / Posters
P06-5
Altered levels of circulating
lysoglycerophospholipids are indicative
of early steatosis in hamsters
Susana Suárez-García, Manuel Suárez, Aïda Pascual-Serrano,
Anna Arola-Arnal, Begoña Muguerza Marquínez, Cinta Bladé,
Lluís Arola
Grupo de Nutrigenómica, Departamento de Bioquímica y
Biotecnología, Universidad Rovira i Virgili, Tarragona, ES
The identification of early biomarkers of pathology can enhance clinical
diagnosis and contribute to its prevention. This is especially important
in lifestyle related diseases because early diagnosis allows treatments
based on changes in unhealthy habits, without resort to drugs. In
previous studies, we applied non-targeted metabolomics to identify
differential circulating patterns of lysoglycerophospholipids (LysoPLs)
after chronic intake of high-fat diet (HFD). The aim of the present work
was to determine the LysoPL content of plasma and liver of HFD-fed
animals to investigate if they could be circulating biomarkers of liver
damage. Hamsters were distributed into 2 groups (n=8) and fed either
normal-fat diet (NFD) or HFD. Interestingly, the liver of HFD-fed
animals showed a mild grade of steatosis after 30 days of feeding
without alterations in plasma transaminases. Targeted metabolomics
(UHPLC-MS/MS) was used to quantify LysoPLs. The results showed
significant changes in lysophosphatidylcholines (LysoPCs) and
lysophosphatidylethanolamines (LysoPEs) among groups. Multivariate
analysis showed a clear clusterization among groups and in both tissues
the predictive value of the model was adequate. A strong correlation
across liver and plasma LysoPL content was obtained. Circulating
LysoPCs 16:1, 17:1, 20:5 and 22:5 were decreased in steatotic hamsters
and had the highest number of significant correlations across liver.
Our results suggest that altered levels of plasma LysoPLs, in particular
LysoPCs, indicate an early degree of steatosis in hamsters. LysoPL
evaluation could be a useful tool for the non-invasive diagnosis of
steatosis.
Work funded by AGL2013-40707-R from Spanish Ministerio de Economía
y Competitividad, Fondos FEDER and 2016 FI_B2 00070 and 00107
fellowships.
P06r-6
Caracterización de la actividad glutenásica
de las heces humanas
Sergio Gutiérrez Getino1, Jenifer Pérez-Andrés2, Honorina
Marinez-Blanco1, Miguel Ángel Ferrero1, Javier Casqueiro2,
Leandro B. Rodríguez-Aparicio1
1
Departamento de Biología Molecular, Área de Bioquímica y
Biología Molecular, Universidad de León. Facultad de Veterinaria,
León, ES, 2Departamento de Biología Molecular, Área de
Microbiología, Universidad de León. Facultad de Ciencias
Biológicas y Ambientales, León, ES
La enfermedad celíaca es una enteropatía inflamatoria crónica con
manifestaciones sistémicas que afecta hasta el 3% de la población.
Aunque su etiología es probablemente multifactorial, su desarrollo
depende por completo de la exposición de los individuos, genéticamente
predispuestos, al gluten del trigo y proteínas similares del centeno
y la cebada ingeridos habitualmente con la dieta. Estas proteinas
son resistentes a la degradación completa por las enzimas digestivas
humanas, lo que da lugar a la aparición en el lumen intestinal de péptidos
de alto peso molecular ricos en prolina y glutamina. En los pacientes
celiacos, algunos de estos péptidos, como el 13-mer o el 19-mer, son
responsables de la activación de la respuesta inmune innata y otros,
como el 33-mer, son responsables de la activación de la respuesta
70
XXXIX Congreso SEBBM
inmune adaptativa. Por bioensayo y mediante técnicas electroforéticas
y cromatográficas hemos podido aislar, identificar y caracterizar varias
proteinas presentes en las heces humanas con actividad glutenásica.
Todas ellas son capaces de degradar los péptidos 13-mer, 19- mer y
33-mer que son tóxicos para los individuos celiacos. Estos resultados
permiten establecer las posibles diferencias enzimaticas existentes
entre individuos sanos y celiacos y abren una nueva via en el desarrollo
de estrategias enzimáticas encaminadas tanto al diagnóstico como al
tratamiento de esta enfermedad.
Investigación subvencionada por la Direción general de Investigacion
(SAF2015-64306-R) y por la Junta de Castilla y León (LE283U14).
P06-7
Hipoxia y señalización de insulina en los adipocitos
Maider Varela1, Fermín Milagro Yoldi2, Alfredo Martínez Hernández2,
Carlos De Miguel Vázquez3
1
Universidad de Navarra, Pamplona, ES, 2Centro de Investigación
en Nutrición, Universidad de Navarra; Departamento de Ciencias
de Alimentación y Fisiología, Universidad de Navarra; CIBER
Fisiopatología Obesidad y Nutrición (CIBERObn), Instituto de
Salud Carlos III, Pamplona, ES, 3Departamento de Bioquímica
y Genética, Universidad de Navarra; Centro de Investigación
en Nutrición, Universidad de Navarra; CIBER Fisiopatología
Obesidad y Nutrición (CIBERObn), Instituto de Salud Carlos III,
Pamplona, ES
La hipoxia asociada a la hipertrofia e hiperplasia del tejido adiposo
se ha relacionado con el desarrollo de resistencia a la insulina en los
adipocitos. Estudios previos indican que el receptor de insulina (IR)
funcional se encuentra ubicado principalmente en unas invaginaciones
de la membrana plasmática denominadas caveolas. La caveolina-1
(Cav-1) es la principal proteína estructural de las caveolas y se sabe
que es importante para la correcta activación de la señalización de
la insulina en el adipocito. Sin embargo, se desconoce si la hipoxia
es capaz de alterar la expresión y función de Cav-1 impidiendo la
transmisión de la señal de la insulina. En este trabajo se investigó
la regulación de Cav-1 y el estado de la vía de la insulina inducidos
por la hipoxia en los adipocitos de ratón 3T3-L1. Hemos observado
que la hipoxia altera la expresión de proteínas relacionadas con la
señalización de insulina y reduce significativamente la acumulación
de triglicéridos en el adipocito. Por otra parte, la captación de glucosa
inducida por insulina también disminuyó cuando se incubaron las
células en hipoxia (1%O2) durante 48 horas, mientras que la captación
de glucosa basal aumentó significativamente en comparación con
las células control. Este resultado fue consistente con el aumento de
la expresión del transportador de glucosa independiente de insulina,
Glut1, y con la disminución de la expresión del transportador sensible a
insulina, Glut4. Por otro lado, la hipoxia disminuyó significativamente
la expresión y fosforilación de Cav-1. Con el fin de determinar si Cav-1
está regulada por HIF-1, células 3T3-L1 diferenciadas fueron tratadas
con equinomicina, un potente inhibidor de HIF-1α. Tras 24 horas de
hipoxia, la equinomicina revirtió el nivel de expresión de Cav-1 al
observado en las células control. Un ensayo de inmunoprecipitación de
cromatina demostró que HIF-1 se une directamente a los elementos de
respuesta de hipoxia (HRE) en el promotor de la Cav-1, probablemente
regulando su expresión. En conjunto, estos resultados indican que
Cav-1 puede ser un gen diana de HIF-1 en la hipoxia, contribuyendo al
desarrollo de resistencia a la insulina en adipocitos.
Salamanca 2016
P06-8
Alteraciones metabólicas y marcadores
moleculares asociados a la ingesta de dietas
desequilibradas en macronutrientes
Paula Oliver, Andreu Palou
Universidad de las Islas Baleares (UIB) y CIBER de Fisiopatología
de la Obesidad y Nutrición (CIBEROBN), Palma de Mallorca, ES
El consumo de dietas desequilibradas ha aumentado debido a la mayor
ingesta generalizada de grasas y proteínas, y a la tendencia de reducir
la cantidad de carbohidratos para controlar el peso. Estos desequilibrios
dietéticos podrían estar incrementando el riesgo de sufrir problemas
de salud a largo plazo. Un mayor consumo de grasa se relaciona con
obesidad y síndrome metabólico. Además, se ha descrito un fenotipo
denominado “falso delgado” o “metabolically obese normal-weight”
(MONW), caracterizado por presentar complicaciones metabólicas del
estado obeso pero en ausencia de obesidad. Por otra parte, las dietas
hiperproteicas se han mostrado efectivas a corto plazo para perder peso
y normalizar parámetros metabólicos, pero existen controversias sobre
su inocuidad a largo plazo. Nuestro grupo ha analizado el impacto
sobre la salud de la ingesta crónica de dietas ricas en grasas o proteínas.
Administramos ambas dietas a ratas en cantidad isocalórica a una dieta
control, para evitar el sobrepeso y mimetizar el fenotipo MONW en el
caso de los animales alimentados con dietas hiperlipídicas. Las ratas
MONW, sin ser obesas, presentaron mayor adiposidad y alteraciones
relacionadas con el síndrome metabólico (resistencia a insulina e
inflamación). Por otra parte, la ingesta crónica de dietas hiperproteicas
produjo un descenso en el peso corporal que podría relacionarse con
un menor contenido hídrico pero no con menor adiposidad. Además,
estos animales mostraron signos de riesgo patológico, como incremento
de marcadores de inflamación y mayor tamaño renal. Ambas dietas
desequilibradas incrementaron la deposición de triglicéridos en
el hígado, incrementando así el riesgo de padecer enfermedades
metabólicas. Nuestros resultados muestran también que el desequilibrio
en macronutrientes afectó a la expresión génica de células sanguíneas,
y que este material celular podría usarse para la identificación de
biomarcadores tempranos de alteraciones metabólicas relacionadas
con la ingesta de dietas desequilibradas, como el hígado graso. Debido
al efecto nocivo de la ingesta continuada de dietas desequilibradas es
clave disponer de biomarcadores tempranos para poder prevenir futuros
problemas de salud.
P06-9
Regulación de la expresión de calpaína 1 en el
hígado de rata: importancia del mantenimiento
de los niveles de Vitamina A
Lucía Rodríguez-Fernández1, Rosa Zaragozá Colom2, Concha
García3, Luís Torres3, Joaquin Timoneda1, Juan Viña Ribes3,
Teresa Barber3
1
Dpto. Bioquímica y Biología Molecular, Universidad de Valencia,
Valencia, ES, 2Dpto. Anatomía y Embriología Humana, Universidad
de Valencia; IIS INCLIVA, Valencia, ES, 3Dpto. Bioquímica y Biología
Molecular, Universidad de Valencia; IIS INCLIVA, Valencia, ES
La vitamina A es necesaria para el correcto desarrollo embrionario y la
organogénesis, ejerciendo además importantes efectos sobre la visión,
reproducción, función inmune, proliferación, diferenciación celular y
apoptosis. Muchas de estas funciones son realizadas por el derivado de
la vitamina A, el ácido retinoico, que regula la expresión de una gran
variedad de proteínas, a través de mecanismos transcripcionales y no
transcripcionales. Las calpaínas son un conjunto de cisteín-proteasas
dependientes de calcio implicadas en la regulación de la ruta
mitocondrial y lisosomal de la apoptosis. Las isoformas CAPN1 y 2
son las mejor caracterizadas y difieren en la concentración de calcio
Pósters / Posters
necesaria para su activación. Las calpaínas se encuentra activas
en variados procesos fisiológicos como la diferenciación celular o
patológicos como el daño tisular y el cáncer. Para elucidar el papel que
juega la vitamina A y su derivado el ácido retinoico en la expresión
de las calpaínas, se ha estudiado su expresión en el hígado de ratas
deficientes en vitamina A y en hígado de ratas control tratadas con
ácido retinoico. Tanto en deficiencia de vitamina A y administración de
ácido retinoico a ratas control, se observa un aumento significativo de
la expresión del mRNA de la CAPN1. En paralelo con el aumento de
expresión se observa claramente, en ambos modelos, la autoproteolisis
del extremo Nt, lo que indica que se ha producido la activación de la
CAPN1. Dicha activación podría participar en el daño tisular observado
en la deficiencia en vitamina A y en la administración farmacológica de
ácido retinoico.
Financiación: BFU2013-46434-P; GVPROMETEOII/2014-055.
P06-10
El aceite de rosa mosqueta (Rosa rubiginosa)
preacondiciona al hígado frente al daño por
isquemia reperfusión hepática
Camila Giacinta Dossi Muñoz, Gladys Tapia Opazo
Universidad de Chile, Santiago, Chile, CL
En el tejido hepático la interrupción parcial o total del flujo sanguíneo
(Isquemia=I) seguido de su posterior recuperación (Reperfusión=R) por
largos periodos de tiempo genera daño hepático (evidenciado por aumento
de las transaminasas séricas) y estrés oxidativo. Se han descrito algunas
estrategias que preacondicionan al tejido, disminuyendo o evitando el daño
por IR, con importante aplicabilidad clínica. El aceite de rosa mosqueta
(RM), dado su alto contenido de ácidos grasos esenciales y antioxidantes,
posee propiedades antiinflamatorias y antioxidantes, las cuales podrían
ayudar a disminuir el daño por IR.
Objetivo: Determinar si la administración dietaria de aceite de rosa mosqueta
(Rosa rubiginosa) preacondiciona al hígado, disminuyendo el daño hepático
y el estrés oxidativo inducido por isquemia seguida de reperfusión.
Metodología: Ratas machos, cepa Sprague Dawley (n=9 por grupo) se
dividieron en 4 grupos experimentales: (a) Sham, (b) IR, (c) RM-sham
y (d) RM-IR. Los animales fueron suplementados con aceite de rosa
mosqueta (0,4 mLd/PO) o dosis isovolumétricas de solución salina
(NaCl) durante 21 días. La cirugía IR (1 hora de isquemia y 20 horas de
reperfusión) y laparotomía media sin isquemia (Sham) fueron realizadas
21 días después del tratamiento. Se evalúo: i) peso corporal, peso hepático
y de tejido adiposo visceral, ii) actividad de transaminasas séricas AST
y ALT, iii) histología hepática y iv) estrés oxidativo hepático (proteínas
oxidadas y TBARS).
Resultados: El grupo (b) presentó aumento significativo (ANOVA
unifactorial, Test Newman Keuls p<0,05) en la actividad de transaminasas
séricas, daño histológico (ambos parámetros de daño hepático) y en los
marcadores de estrés oxidativo respecto al grupo (d). Las histologías
hepáticas del grupo (b), presentaron alteración de la histo-arquitectura
hepática, focos de necrosis e inflamación, alteraciones no evidenciadas
en los otros grupos experimentales. No se observaron diferencias
significativas en incremento del peso corporal, peso hepático y de tejido
adiposo visceral.
Conclusión: La suplementación con aceite de RM preacondiciona frente
al daño por IR, asociado a una disminución de parámetros de daño y de
estrés oxidativo.
FONDECYT 1140547
71
Pósters / Posters
P07. Bioquímica perinatal /
Perinatal biochemistry
P07m-1
Calpain-specific proteolytic processing of
adhesion proteins in mammary tissue and breast
cancer cell lines
Rosa Zaragozá1, Lucía Rodríguez-Fernández2, Iván Ferrer-Vicens2,
Concha García2, Sara Oltra3, Elena R. García-Trevijano2, Juan R. Viña2
1
Dpto. Anatomía y Embriología Humana. Facultad Medicina. Univ.
Valencia. IIS INCLIVA, Valencia, ES, 2Dpto. Bioquímica y Biología
Molecular. Facultad Medicina. Univ. Valencia. IIS INCLIVA,
Valencia, ES, 3Servicio Oncología. Fund. Investigación Hosp. Clínico
Universitario. IIS INCLIVA, Valencia, ES
Cleavage of adhesion proteins is the first step for physiological clearance
of undesired cells during postlactational regression of the mammary
gland, but also for cell migration in breast cancer. Calpains (CAPNs)
are known to cleave adhesion proteins. The isoform-specific function of
CAPN1 and -2 was explored in two models of cell-adhesion disruption:
mice mammary gland during involution and different subtypes of breast
cancer cell lines. In both models, E-cadherin, b-catenin, p-120 and
talin-1 were cleaved. Both CAPNs were able to cleave adhesion proteins
from lactating mammary gland in vitro. Nevertheless, CAPN2 was the
only isoform found to co-localize with E-cadherin in cell-junctions at
the peak of lactation. CAPN2/E-cadherin in vivo interaction analyzed
by proximity ligation assay (PLA), was dramatically increased during
involution. Calpain inhibitor administration prevented the cytosolic
accumulation of truncated E-cadherin cleaved by CAPN2. Conversely,
in breast cancer cells, CAPN2 was restricted to the nuclear compartment.
The isoform-specific expression of CAPNs and CAPN activity was
dependent on the breast cancer subtype. However, CAPN1 and CAPN2
knock-down cells showed that cleavage of adhesion proteins and cell
migration was mediated by CAPN1 independently of the breast-cancer
subtype. Data presented here suggest that the subcellular distribution
of CAPN1 and -2 is a major issue in target-substrate recognition and
therefore, it determines the isoform-specific role of CAPNs during
disruption of cell adhesion in either a physiological or a pathological
context.
Grants: BFU-2013-46434-P to J.R.V & R.Z; PI12/02394 to E.R.GT both
of the projects including FEDER grants; and GVPROMETEOII/2014-055
to J.R.V.
P07-2
Neonatal overfeeding alters hepatic insulin
sensitivity during lactation and leads to long-term
insulin resistance and fatty liver in mice: Key role
of Mogat1
Marta Ramon-Krauel1, Thais Pentinat2, Maria Vilà2, Ricky
Pérez-Wienese2, Sílvia Ribó2, Judith Cebrià2, Susana Kalko3, Rubén
Díaz1, Uwe Tietge4, Torsten Plosch4, Josep Jiménez-Chillarón2
1
Hospital Sant Joan de Deu, Esplugues de Llobregat, ES, 2Institut
de Recerca Pediàtrica, Hospital Sant Joan de Deu, Esplugues de
Llobregat, ES, 3IDIBAPS, Universitat de Barcelona, Barcelona, ES,
4
Groningen University, Groningen, NL
Background: Excessive energy intake and rapid weight gain early
in life are associated with obesity, type 2 diabetes, hepatic steatosis
and other features of the metabolic syndrome. The monoacylglycerol
acyltransferase (MGAT) is an enzyme involved in an alternative pathway
72
XXXIX Congreso SEBBM
for triglyceride (TAG) synthesis and storage. It has been recently
proposed to have potential implications in the pathogenesis of hepatic
insulin resistance (IR).
Objective: To understand the mechanisms that contribute to (1) the
development of early IR and (2)the long-term programming of metabolic
disease in a mouse model of childhood obesity.
Method: Here we used a mouse model of neonatal overnutrition (ON)
and accelerated growth that develops obesity, IR, glucose intolerance and
hepatic steatosis with aging. To gain insight about the mechanisms that lead
to IR and steatosis, we performed gene expression profiling (Affymetrix)
in livers from Control and ON Mice.
Results: The Ontology with highest significance was the Lipid
Biosynthetic Pathway. Strikingly, Mogat1 ranked with highest
significance in this list. We confirmed Mogat1 up-regulation in liver
samples from 4-6 month old ON mice. Importantly, Mogat1 was already
elevated in livers of lactating 15-day-old ON mice. Furthermore we found
no changes on other pathways that could contribute to lipid synthesis and
storage in the liver (synthesis de novo, oxidation or VLDL transport).
Mogat1 catalyzes the conversion of Monoacylglycerol to DAG, which
activates PKCε that in turn contributes to IR. In agreement, we found
that Mogat1 over expression resulted in DAG hepatic accumulation and
higher PKCε activation in ON mice.
Conclusion: Mogat1 might be a key player in the development of IR and
hepatic steatosis. Therefore, targeting MGAT activity in the liver might be
a novel potential strategy to improve hepatic insulin sensitivity.
P07-3
Estudio de los mecanismos de regulación
epigenética y su relación con la presencia
de diabetes mellitus gestacional en mujeres
europeas
Eva de la Fuente Luelmo1, María Haro García1, Danuta Dudzik2,
Coral Barbas2, Mª Pilar Ramos Álvarez1
1
Universidad CEU San Pablo, Madrid, ES, 2CEMBIO Facultad
de Farmacia, Universidad CEU San Pablo, Madrid, ES
La diabetes mellitus gestacional (DMG) es una enfermedad multifactorial
en la que participan distintos factores ambientales, genéticos y
epigenéticos y cuya prevalencia sigue aumentando. Asimismo, el
ambiente intrauterino en el que se desarrolla un embrión, puede afectar a
la programación fetal condicionando las enfermedades del recién nacido
en su vida adulta. La enzima metionina adenosiltransferasa (MAT1A)
participa en el ciclo de la homocisteína y es la encargada de crear la
S-adenosilmetionina (SAM), principal donante de grupos metilo en
los procesos de metilación de numerosas moléculas. De dicho ciclo
forma parte también la metionina sintasa (MTR) enzima catalizadora
de la última etapa de la síntesis de metionina. La correcta función de
ambas afectará a la regulación epigenética de la expresión génica. Son
numerosos los polimorfismos descritos sobre ambos genes. El objetivo
de este estudio ha sido obtener las frecuencias alélicas de las variantes
rs7087728 de MAT1A y rs1805087de MTR en una población de mujeres
gestantes con y sin DMG, y observar la existencia de una asociación entre
el fenotipo y el genotipo, mediante el estudio de la metilación total del
DNA. Los resultados muestran que en el grupo de mujeres con DMG son
significativamente más frecuentes el alelo C en MAT1A y el G en MTR.
Por otra parte, el estudio de la cuantificación total del nivel de metilación
del DNA del tejido placentario reveló un mayor nivel de metilación en
las placentas de las mujeres con DMG respecto a las controles. Estos
resultados sugieren una posible relación entre ciertos mecanismos de
regulación epigenética y la presencia de DMG, pudiendo contribuir al
desarrollo de patologías asociadas al síndrome metabólico en los recién
nacidos en etapas posteriores de su vida.
Salamanca 2016
P07-4
Peroxisome proliferator-activated receptor gamma
modulates late pregnancy energy homeostasis
and metabolic adaptations
Patricia Corrales-Cordón1, Yurena Vivas1, Mónica Díez–Hochleitner2,
Adriana Izquierdo-Lahuerta1, Daniel Horrillo1, Ismael Velasco1,
Cristina Martínez-García1, Julio Sevillano2, Mercedes Ricote3,
Manuel Ros1, María Pilar Ramos2, Gema Medina-Gómez1
1
Universidad Rey Juan Carlos, Alcorcón, ES, 2Universidad CEU
San Pablo, Madrid, ES, 3Centro Nacional de Investigaciones
Cardiovasculares, Madrid, ES
Pregnancy requires a progressive adaptation of maternal energy
metabolism, which includes adipose tissue expansion and pancreatic
β-cells adaptation, in the function and structure of these tissues. Insulin
resistance develops predominantly during late gestation, as part of
the metabolic adaptations that support fetus development and growth.
Peroxisome proliferator-activated receptor γ (PPARγ) is involved
in adipogenesis, glucose and lipid metabolism and modulation of
insulin sensitivity. Moreover, PPARγ plays an important role in β-cell
proliferation in other pathologic situations like obesity. Our aim is
to study the role of PPARγ in β-cell adaptation during gestation with
different study conditions. We have created two models: transgenic
PPARγ2 knockout (PPγ2KO-C) mice and specific PPARγ knockout mice
in pancreatic β-cell (βγKO). At D15-D16 of gestation GTT or ITT were
performed, and animals were sacrificed at D18.βγKO females were also
fed with high fat diet 3 weeks before pregnancy. Lack of PPARγ2 induced
higher insulin resistance associated with lower serum adiponectin levels
during late pregnancy. Indeed, ablation of PPARγ2 induced changes in
fat mass distribution, which specially affected the perigonadal deposit.
Moreover, there was an altered expression of genes involved in lipid
metabolism and inflammation in adipose tissue. In the other hand, results
in βγKO mice have shown decreased pancreatic β-cell mass despite high
serum levels of insulin during pregnancy. Their pancreatic weight was
lower compared with the WT animals. There were also differences in
placenta metabolites between βγKO and WT pregnant mice. These data
indicated that an appropriate expression of PPARγ is necessary to ensure
a normal adipose tissue and pancreas metabolism during gestation,
particularly within the late phase of pregnancy when a state of insulin
resistance is established.
Acknowledgements:BFU2012-33594,BFU2013-47384-R,BFU2015-70454-REDT,
BSAF2014-56671-R and 30VCPIGI02.
P08. Bioquímica y biología molecular
de plantas / Biochemistry and
molecular biology of plants
P08-1
Dengue virus capsid peptide binds specifically a
HMMM model membrane
Emmanuel Fajardo-Sánchez1, Vicente Galiano2, José Villalaín1
Instituto de Biología Molecular y Celular, Universidad Miguel
Hernández, Elche-Alicante, ES, 2Dpto. Física y Arq. Computadores,
Universidad Miguel Hernández, Elche-Alicante, ES
1
Pósters / Posters
Dengue virus (DENV) C protein is crucial for viral assembly
in order to ensure specific encapsidation of its genome. DENV
C protein associates with intracellular membranes through a
conserved hydrophobic domain and accumulates in endoplasmic
reticulum-derived lipid droplets which could provide a platform for
capsid formation during assembly. In a previous work (Nemesio et
al., 2013), we described a region in DENV C protein which induced
a nearly complete membrane rupture of several membrane model
systems, which was coincident with the theoretically predicted highly
hydrophobic region of the protein (peptide DENV2C6). In this work
we have performed a molecular dynamics (MD) simulation of the
interaction of a peptide corresponding to this DENV C region with
different membrane model systems. Unrestrained all-atom MD was
carried out using NAMD by using the CHARMM36 force field for the
lipid molecules and the CHARMM general force field for the peptide.
We have used a Highly Mobile Membrane-Mimetic (HMMM) lipid
bilayer model for the MD. We show that DENV2C6 is capable of
binding specifically to membranes and that this binding is modulated
by lipid composition through specific lipid-peptide interactions
(Figure 1). These data corroborate our previous results and open a
new avenue for the development and identification of peptides, which
might be used as new drugs against the DENV life cycle, through the
modulation of membrane structure.
The research conducted in this work was funded by grant BFU2013-43198-P
(Ministerio de Economía y Competitividad, Spain) to J.V.
P08-2
Development of a DNA-based system suitable
for olive oil authentication
Alba Alonso-Rebollo, Natividad Ortega, Sonia Ramos-Gómez,
María D. Busto, David Palacios, María C. Pilar-Izquierdo, Manuel
Pérez-Mateos
Universidad de Burgos, Burgos, ES
Olive oil is recognized worldwide not only for its taste, nutritional
and health benefits, but also for being the most valuable of the
agro-food Mediterranean industry. Mechanical processes to which the
olives are subjected result in the production of olive oil considered
as extra virgin (EVOO) or virgin (VOO). DNA-based methods are
actually considered as one of the most suitable methods for olive oil
traceability and authentication. Currently there are several reports
of molecular markers applied for olive cultivar fingerprinting.
This work presents the design of a DNA-based system suitable
for extra virgin olive oil (EVOO) traceability and authentication,
independently of the cultivar, which allows not only the olive
detection, but also its quantification, by using SYBR-Green qPCR
technology. Chloroplast DNA is more abundant and more resistant
than nuclear DNA. Moreover, the lower mutation rate of this DNA
leads on less intra-cultivar variability and makes it suitable for
species identification. The trnL gene has been applied in species
identification, including olive and, based on this gene, four new
olive-specific amplification systems were designed and analyzed on
basis their sensitivity, specificity and efficiency by qPCR, after their
optimization. One of the systems proved specificity, great sensitivity
and high efficiency, resulting suitable for olive DNA detection in
EVOO. The qPCR analysis of six different commercial EVOOs,
tested by triplicate, showed that system trnL-E allowed not only the
detection of DNA but also its quantification in any of the triplicates
from each EVOO. Thus, we obtained a trnL-based qPCR system
suitable for its application in extra-virgin olive oil authentication
and traceability.
73
Pósters / Posters
P08-3
El silenciamiento del gen Fanpr3.1 de fresa
(Fragaria x ananassa) genera resistencia a la
infección por Colletotrichum acutatum y sugiere
una función en defensa semejante a la de los
genes Atnpr3/Atnpr4 en Arabidopsis
José J. Higuera-Sobrino1, Francisco Amil-Ruiz1, José Garrido-Gala1,
Ayman Lekhbou1, Enriqueta Moyano1, Isabel Arjona-Girona2,
Juan M. Arjona-López2, José A. Mercado3, Fernando Pliego-Alfaro3,
Juan Muñoz-Blanco1, Carlos J. López-Herrera2, José L. Caballero1
1
Departamento de Bioquímica y Biología Molecular, Edif. Severo
Ochoa-C6, Universidad de Córdoba, Córdoba, ES, 2Departamento
de Protección de Cultivos, Instituto de Agricultura Sostenible,
CSIC, Córdoba, ES, 3Departamento de Biología Vegetal, Instituto
de Hortofruticultura Subtropical y Mediterránea La Mayora,
IHSM-UMA-CSIC, Universidad de Málaga, Málaga, ES
Nuestro grupo está interesado en la interacción fresa/Colletotrichum,
como sistema modelo, y en dilucidar y caracterizar genes y mecanismos
moleculares relacionados con la defensa de la planta de fresa a éste
y otros patógenos. Entre los genes que hemos identificado, por su
marcado interés aplicado, está el gen Fanpr3.1, semejante a miembros
de la familia multigénica de factores de transcripción NPR de
Arabidopsis. AtNPR1 es regulador positivo de la ruta de señalización,
mediada por SA, que conduce a la expresión de proteínas PR de defensa
y a la inducción de SAR (Systemic Adquired Resistance). AtNPR3 y
AtNPR4 comparten entre sí el 73% de identidad y tan solo un 34,5% y
36% de identidad, respectivamente, con AtNPR1. AtNPR3 y AtNPR4
son sensores moleculares de SA y se unen a AtNPR1 condicionando
su actividad de una manera compleja, actuando como adaptadores
para su reclutamiento por Cullin 3-based E3 ubiquitin ligasas y su
degradación posterior en el proteasoma. Análisis filogenéticos indican
que FaNPR3.1 está más cercana a AtNPR3 y AtNPR4 que a AtNPR1
y AtNPR2. FaNPR3.1 presenta identidad del 61,6% y del 61,5% con
AtNPR3 y AtNPR4, respectivamente, y del 36,8% y de 37,6% con
AtNPR1 y AtNPR2. Por tanto, no podemos predecir la función de
FaNPR3.1 en fresa mediante comparación de secuencias, ya que
FaNPR3.1 es parcialmente semejante a ambos subgrupos de proteínas
de Arabidopsis. Así, hemos obtenido construcciones plasmídicas,
mediante sistema Gateway, para sobreexpresión/silenciamiento del
gen Fanpr3.1 en la fresa y hemos desarrollado un sistema de ensayo
fitopatológico tras su expresión transitoria en fruto. Los análisis
preliminares indican que el silenciamiento del gen Fanpr3.1 mediante
RNAi provoca resistencia del fruto a la infección por C. acutatum
y la sobreexpresión del mismo en fruto aumenta sensiblemente la
susceptibilidad a dicho patógeno. Estos datos sugieren que FaNPR3.1
podría actuar en fresa de manera semejante a como lo hace su ortólogo
FaNPR4 en Arabidopsis, regulando negativamente la expresión de
proteínas PR de defensa y SAR.
P08-4
Farmacognosia como estrategia terapéutica
para el tratamiento del cáncer de pulmón
Wilber Romero-Fernández1, Israel Manjarres2, Cristian Carvajal2,
Andrés Tayo2, Mileidys Pérez-Alea3
1
Facultad de Ciencias de la Salud. Universidad Técnica de Ambato,
Ambato, EC, 2Facultad de Ciencias e Ingeniería en Alimentos.
Universidad Técnica de Ambato, Ambato, EC, 3Instituto de
Investigación Vall d´Hebron, Barcelona, ES
Las enzimas aldehído deshidrogenasas (ALDH) son una familia de
enzimas que oxidan los aldehídos endógenos y exógenos tóxicos a sus
correspondientes ácidos carboxílicos débiles, jugando un papel clave
en el mantenimiento de la homeostasis celular. Por otra parte, se ha
74
XXXIX Congreso SEBBM
demostrado que las células madre tumorales (CMT) son la fuerza
motriz de la tumorigénesis y la principal responsable de resistencia
a terapia, progresión, recidiva y metástasis. Las CMT se caracterizan
por su acelerada actividad metabólica, dando lugar a una elevada
producción de aldehídos reactivos y radicales libres. Evidencias
experimentales demuestran que las CMT con elevada actividad ALDH
son altamente proliferativas, tumorigénicas, resistentes a la muerte
celular y responsables de la reaparición del tumor y de metástasis en
enfermedades hematológicas malígnas y en tumores de pulmón, mama,
próstata, colon y cerebro.
A todo esto se suma que en la actualidad seguimos utilizando principios
activos derivados de plantas como prototipos para el desarrollo de
fármacos más específicos, eficaces y menos tóxicos. En vista del papel de
las ALDH y concretamente de la isoforma ALDH1A1 en la fisiopatología
del cáncer de pulmón, consideramos que la ALDH1A1 representa una
importante diana terapéutica para el desarrollo de fármacos basados en
la etnofarmacología. Eliminar eficazmente la población de CMT podría
representar una eficiente estrategia para el tratamiento del cáncer de
pulmón. En este trabajo se muestra el uso potencial de la farmacognosia
en la inhibición de la ALDH1A1 que promueve la inducción de apoptosis
e incrementa la eficacia de los tratamientos convencionales del cáncer de
pulmón.
P08-5
Variaciones en el metabolismo oxidativo
de semillas de soja (Glycine max L. Merr.)
en respuesta al ataque de Nezara viridula (L.)
Ivana Sabljic, Karina Balestrasse, Jésica Barneto, Romina
Giacometti, Jorge A. Zavala, Eduardo A. Pagano
Facultad de Agronomía, Universidad de Buenos Aires,
INBA (UBA-CONICET), Buenos Aires, AR
Nezara viridula (chinche verde – southern stink bug) (Hemiptera:
Pentatomidae) es una de las principales plagas del cultivo de soja en
muchas regiones productivas. Su ataque causa una disminución en el
rendimiento, la calidad de la semilla, disminuyendo la viabilidad y el
vigor, y el valor comercial de los granos. Existe información de que el
ataque de insectos provoca la estimulación de la defensa antioxidante
modificando la actividad de varias enzimas. Los principales mecanismos
involucrados comprenden las rutas de señalización celular, la síntesis
de compuestos de defensa y la expresión de enzimas consumidoras de
especies reactivas de oxígeno. El objetivo de este trabajo fue comparar
la respuesta antioxidante de dos genotipos contrastantes: IAC-100
(resistente) y Davis (susceptible) cuando son atacados por adultos
de Nezara viridula (L.). Las actividades de las enzimas Guaiacol
peroxidasa (GPOX), catalasa (CAT) y superóxido dismutasa (SOD)
fueron cuantificadas espectrofotométricamente en extractos de semillas
obtenidas de plantas de soja crecidas en invernadero, en el estadio
R6, después de 72 de iniciado el ataque de los insectos y comparadas
con las de semillas no dañadas. Para analizar las generación de H2O2
las semillas fueron aisladas y sumergidas en una solución 1% de
3,3-diaminobenzidina y analizadas visualmente. Las semillas atacadas
del genotipo Davis presentaron una significativa disminución en el
peso fresco a diferencia de IAC-100 que no mostró cambios entre
control y tratado. El genotipo resistente (IAC-100) fue más activo
que el genotipo susceptible (Davis) en poner en funcionamiento el
metabolismo antioxidante, evidenciando una mayor actividad de todas
las enzimas estudiadas y una disminución en el contenido de H2O2.
En paralelo se realizó un estudio transcriptómico mediante RNA-Seq
para analizar la expresión diferencial de genes en los tratamientos
mencionados. Se encontró una significativa inducción de la expresión
de numerosas proteínas intervinientes en procesos rédox, como, por
ejemplo, peroxidasas, glutatión-S-transferasas y tiorredoxinas.
Salamanca 2016
P08-6
Thymus mastichina from Spain: Aromatic profile
and enantioselective gas chromatography-mass
spectrometry
Ana Belén Cutillas-Gomariz1, Alejandro Carrasco-Ruiz1, Ramiro
Martínez-Gutiérrez2, Virginia Tomás-Martínez3, José Tudela-Serrano1
1
Universidad de Murcia, GENZ-Grupo de Investigación Enzimología,
Departamento de Bioquímica y Biología Molecular-A, Campus de
Excelencia Internacional Regional Campus Mare Nostrum, Murcia,
ES, 2NOVOZYMES SPAIN S.A, Madrid, ES, 3Universidad de Murcia,
Departamento de Quimica Analitica, Murcia, ES
Introduction: Thymus mastichina or wild marjoram is a perennial herb
of the Lamiaceae family. It is endemic from Spain and Portugal andhas
aromatic, food and medical uses. The essential oil of T. mastichina, has
been obtained by Clevenger hydrodistillation.
Experimental (www.um.es/genz/e00605/serv03.htm): Fast gas
chromatography on a non-polar fast column SLB-5ms 15m x 0.1mm x
0.1μm, and an enantioselective chiral column Chiraldex B-DM 30m x
0.25mm x 0.12μm, were carried out using an Agilent 7890 chromatograph,
with hydrogen as gas carrier (PDH), and an Agilent 5975 MSD-EI-SQ.
Sandwich split/splitless injections were performed (Gerstel MPS-2XT).
Results: The main biomolecules of T. mastichina oil from Murcia were
(% concentration, % enantiomers): α-Pinene (2.49,+77,-23), β-Pinene
(3.14,+50,-50), Limonene (2.57,+3,-97), 1,8-Cineole (63.74), Linalool
(20.75,+4,-96), Camphor (0.33,+0,-100), Borneol (0.81,+0,-100),
Terpinen-4-ol (0.94,+100,-0), α-Terpineol (2.86,+66,-34), δ-Terpineol
(0.21), Linalyl acetate (1.55,+5,-95), E-β-Caryophyllene (0.62,+0,-100),
and other minor components.
Discusion: T. mastichina essential oil shows high concentrations of
1,8-Cineole>Linalool>β-Pinene>α-Terpineol>Limonene>α-Pinene>
Linalyl acetate, as main components. Near pure (-)-enantiomers are
Limonene, Linalool, Camphor, Borneol and E-Caryophyllene, whereas
mainly (+)-enantiomers are α-Pinene, Terpinen-4-ol and Terpinyl acetate.
Conclusions: The essential oil of T. mastichina from Murcia has
components, according to ISO4728:2003 standard.
Acknowledgements: Study partially supported by grants from: University
of Murcia, Spain (UMU-15452, UMU-17766) and Fundacion Seneca,
CARM, Murcia, Spain (19545/PI/14). A.B.C. has a fellowship from the
Spanish government (MECD/FPU13-04013).
P08-7
PHB3 and NOA1 interact to maintain the root
stem-cell niche in Arabidopsis
Noel Blanco-Touriñán1, Tamara Lechón2, Virginia Palomares2, Ivett
Barany3, Miguel Ángel Blázquez1, Miguel Moreno-Risueño4, Pilar
Testillano3, Mari Carmen Risueño3, Óscar Lorenzo2, Luis Sanz
Andreu2
1
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV),
Valencia, ES, 2Instituto Hispano Luso de Investigaciones Agrarias
(CIALE). Universidad de Salamanca, Salamanca, ES, 3CIB Centro
de Investigaciones Biológicas, Madrid, ES, 4Centro de Biotecnología
y Genómica de Plantas, UPM-INIA, Madrid, ES
Prohibitins (PHB) are highly conserved proteins across different taxa that
are present in mitochondrial multimeric complexes. The Arabidopsis
thaliana genome contains five PHB genes. The PROHIBITIN3 (PHB3)
gene is broadly expressed in the root and it is necessary for meristematic
cell production (1). Recently, PHB3 has been reported to influence nitric
oxide (NO) levels in different tissues (2). In addition, the mouse Nitric
Oxide-Associated protein 1 (mNOA1) has been reported to interact with
several members of the PHB complex (3). Given the scarce knowledge
on PHB in plants, we have investigated the putative functional connection
Pósters / Posters
between PHB3 and NOA1 in Arabidopsis thaliana. Careful examination
of phb3 roots showed dramatically disorganized meristems, confirming a
function for PHB3 in mitotic cell activity in Arabidopsis primary roots. This
effect seems to be linked to the negative regulation by PHB3 of WUSCHEL
RELATED HOMOEOBOX5 (WOX5) expression, a gene specifically
expressed in the quiescent center (QC) niche which maintains its stem cell
nature. Moreover, meta-analysis of transcriptomic defects in phb3 and noa1
mutants showed that 29% of the genes misregulated in phb3 mutant were
also affected in the noa1 mutant, and most of these genes were regualted
in the same direction by both NOA1 and PHB3, suggesting a positive
correlation between the functions of these two proteins in root development.
In agreement with this, the double phb3noa1 mutant displayed a much more
severe disorganization in the root meristem than the single phb3 mutant,
probably due to the increased expression domain of WOX5. Therefore, we
conclude that PHB3 and NOA1 represent two interacting pathways that
maintain the integrity of the QC by preventing ectopic expression of WOX5.
References
[1] Van Aken et al. (2007) Plant J 52:850-64.
[2] Wang et al. (2010) Plant Cell 22:249-59.
[3] Heidler et al. (2011) J Biol Chem 286:32086-93.
P09. Biotecnología molecular /
Molecular biotechnology
P09-1
Cianómica: una visión holística de la biodegradación
de cianuro y residuos industriales cianurados
Conrado Moreno Vivián, Purificación Cabello, Lara P. Sáez, Isabel
Ibáñez, Mª Paz Escribano, Isabel Manso, Víctor M. Luque Almagro,
Mª Dolores Roldán
Universidad de Córdoba, Córdoba, ES
El cianuro se utiliza en la industria química, en la minería y en la joyería,
actividades que generan residuos muy tóxicos y peligrosos que deben
ser tratados mediante costosos métodos físico-químicos. El cianuro, a
pesar de su toxicidad, puede ser tolerado o incluso asimilado por algunas
bacterias, por lo que la biodegradación es una solución alternativa para el
tratamiento de residuos industriales cianurados. La bacteria Pseudomonas
pseudoalcaligenes CECT5344 utiliza cianuro, cianato o complejos
cianuro-metálicos como única fuente de nitrógeno para su crecimiento a
pH alcalino, lo que evita la formación de HCN volátil. La integración de
aproximaciones ómicas para el estudio de la biodegradación de cianuro y
residuos industriales cianurados por esta bacteria está proporcionando una
visión global del proceso, que resulta de gran interés biotecnológico. La
estirpe CECT5344 ha sido el primer organismo cianotrofo cuyo genoma
se ha secuenciado completamente. Mediante 454-pirosecuenciación se
estableció un borrador del genoma, con un tamaño de 4,65 Mb y 4.321 genes
(1). Mediante secuenciación SMRT (Pacific Biosciences) se ha cerrado
totalmente el genoma, con un tamaño final de 4.696.984 pb y 4.514 genes, y
se ha establecido el patrón de metilación de bases o metiloma (2). También se
ha realizado un análisis transcriptómico de la respuesta al cianuro, al residuo
de la industria joyera y a la carencia de nitrógeno mediante DNA arrays,
que ha permitido identificar genes inducidos por el cianuro y/o el residuo
cianurado y caracterizar la expresión de genes de interés biotecnológico,
como los implicados en la producción de polihidroxialcanoatos utilizables en
la fabricación de bioplásticos (3). Actualmente se están realizando estudios
proteómicos mediante 2D-PAGE y cromatografía líquida-espectrometría
de masas (LC-MS/MS), cuya integración con los resultados del análisis
transcriptómico y la cuantificación de la expresión génica por PCR
cuantitativa proporcionarán una visión holística de las capacidades
degradativas y biotecnológicas de esta bacteria.
75
Pósters / Posters
Agradecimientos: Ministerio de Economía y Competitividad
(BIO2015-64311-R), Junta de Andalucía (P11-CVI-7560) y empresas Magtel,
Gemasur-Ámbito FCC, Saveco y Avenir.
Referencias
[1] Luque-Almagro et al. (2013). Environ. Microbiol. 15:253-270.
[2] Wibberg et al. (2016). J. Biotechnol., en prensa.
[3] Luque-Almagro et al. (2015). J. Biotechnol. 214:171-181.
P09-2
Selección de levaduras salvajes y obtención
de nuevos híbridos con buenas propiedades
para panificación
Rosana Chiva Tomás1, Lorena Celador2, José Antonio Uña2,
Encarna Velázquez2, María Ángeles Santos García2, Mercedes
Tamame González1
1
Instituto de Biología Funcional y Genómica (IBFG). CSIC /
Universidad de Salamanca, Salamanca, ES, 2Departamento
de Microbiología y Genética Universidad de Salamanca Campus
Unamuno, Salamanca, ES
Las levaduras confieren al pan gran parte de sus propiedades organolépticas
y sensoriales. Las cepas comerciales de levaduras de panificación proceden
de híbridos seleccionados por su alta capacidad para fermentar harinas de
trigo y son muy similares genéticamente. Existe otro grupo de levaduras
presentes en masas madre naturales con características fisiológicas y origen
filogenético diferente que están emparentadas con levaduras vínicas.
Las masas madre son mezcla de harina, agua y comunidades simbióticas
de bacterias ácido lácticas (BAL) y levaduras salvajes (LEV), cuya
biodiversidad final viene determinada por varios parámetros físicos
y biotecnológicos. Las masas madre confieren al pan propiedades
organolépticas y sensoriales más apreciadas que las de productos fabricados
a gran escala con levadura industrial. La selección y caracterización de
nuevas cepas BAL y LEV autóctonas de MM desarrolladas con harinas
innovadoras permitirá formular nuevos starter mixtos para conseguir
fermentaciones reproducibles a gran escala y productos industriales más
diferenciables y con propiedades similares a los artesanales.
Para satisfacer la reciente demanda de incrementar la biodiversidad y el
repertorio de las levaduras de panificación disponibles, (i) se han aislado 535
LEV a partir de grano, harinas o masas madre naturales desarrolladas por
expertos; (ii) se han caracterizado genética, bioquímica y fisiológicamente;
(iii) se han seleccionado nuevas cepas de Saccharomyces capaces de
fermentar eficazmente harinas de cereales tradicionales o innovadores
(Tritordeum); y (iv), se han aislado cepas de especies no convencionales
(ncLEV) con potencial interés biotecnológico. Además se han obtenido
mediante “rare mating”, por primera vez, nuevas levaduras híbridas
(LEVHY), entre cepas comerciales de panificación y vínicas; de algunas
de éstas con cepas de laboratorio o/y con LEV aisladas de masas madre.
Las LEV, ncLEV y LEVHY no están modificadas genéticamente y poseen
propiedades deseables para la innovación en la industria agroalimentaria.
XXXIX Congreso SEBBM
sequences or the loss of viral endogenous regions have been described and it
may cause viral attenuation. The CRISPR/Cas9 genome-editing system has
been proposed as a powerful alternative to generate efficiently recombinant
viruses. We have selected the US4 gene from HSV-1 and HSV-2, encoding
glycoprotein G (gG), as the target locus. gG binds chemokines and
neurotrofic factors, enhancing their activities, and represents an excellent
candidate to study viral immune modulation. Knock-in viruses, in which
the US4 gene was replaced by an eGFP cassette as a selection marker, were
generated using the CRISPR/Cas9 tool through the transfection-infection
method. We have specifically replaced the US4 gene by eGFP, selecting
fluorescent plaque-purified recombinant viruses. We have sequenced by next
generation sequencing (NGS) the recombinant viral genomes and confirmed
the correct insertion of eGFP and the absence of alterations elsewhere in the
genome. These results support the CRISPR/Cas9 genome editing system as
a useful approach to generate HSV-1 and HSV-2 recombinants.
P09r-4
Effect of p73 deficiency in adipocyte
differentiation using an in vitro iPSC cell model
Laura Maeso-Alonso1, Marta Martín-López1, Sandra
Fuertes-Álvarez1, Alberto J. Villena2, M. Margarita Marques3, María
C. Marín4
1
Institute of Biomedicine (IBIOMED) University of León, León, ES,
2
Department of Molecular Biology, University of León, León, ES,
3
Department of Animal Production University of León , León, ES,
4
Institute of Biomedicine (IBIOMED) University of León , León, ES
The study of adipocyte differentiation has become very important in the
developed countries due to the increased prevalence of obesity and related
diseases. In particular, the early phases of the adipogenic differentiation
process are not well characterized. Stem cells, either embryonic or
induced pluripotent stem cells (iPSCs) can recapitulate different stages
of differentiation. Therefore, they are emerging as an excellent tool to
improve our understanding of the mechanisms that regulate adipogenesis.
The p53 gene family comprises the transcription factors p53, p73 and p63.
The members of this family share a similar structure and sequence identity
and have been described as regulators of several differentiation processes.
Lack of p73 has been related to myeloid and erythroid differentiation.
In addition, our group has demonstrated that p73 is a positive regulator
of endothelial cell differentiation and vasculogenesis. To further address
p73 role in mesodermal cell fate determination, we analyzed the effect
of p73 deficiency during the adipogenesis process.The effectiveness of
adipogenesis was evaluated in wild type and p73 knockout (KO) iPSCs
by immunofluorescense and transcriptional analysis. Lipid droplet
accumulation was assessed by oil red O staining. Our results indicate
that the dynamics of adipocyte marker expression and lipid droplet
accumulation was impaired in differentiated p73KO-iPSCs, suggesting
that lack of p73 alters the adipocyte differentiation process.
This work was supported by: Grant SAF2015-71381-R (Spanish Ministerio
de Ciencia e Innovación). Laura Maeso-Alonso is a recipient of an AECC
doctoral fellowship.
P09-3
Efficient and specific genome editing of herpes
simplex viruses by CRISPR/Cas9
Alberto Domingo López-Muñoz, Alberto Rastrojo, Rocío Martín,
Antonio Alcamí
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES
Herpes simplex viruses (HSVs) are prevalent human pathogens of clinical
relevance, frequently used as a model to study viral pathogenesis and
modulation of the host immune response. Traditionally, homologous
recombination was used as the main method for the construction of viral
mutants, with low efficient rates. Bacterial artificial chromosomes (BACs)
have also been used to generate recombinant viruses, but residual BAC
76
P09r-5
p73 regulates BMP signaling and its lack
affects the mesenchymal to epithelial transition
during the initiation phase of somatic cell
reprogramming
Marta Martín López1, Laura Maeso Alonso1, Sandra Fuertes
Álvarez1, Inmaculada Diez Prieto2, Pablo Menéndez Buján3, Timo
Otonkoski4, Margarita Marqués Martínez5, M.C. Marín1
1
Institute of Biomedicine (IBIOMED), León, ES, 2Cirugia y Anatomía
Veterinaria, León University, León, ES, 3Josep Carreras Leukaemia
Salamanca 2016
Research Institute, School of Medicine, Barcelona University,
Barcelona, ES, 4Biomedicum Stem Cell Centre, Helsinki, FI,
5
INDEGSAL, León University, León, ES
Somatic cell reprogramming to generate induced pluripotent stem cells
(iPSCs) occurs in three sequential phases: initiation, maturation and
stabilization. The initiation phase starts with a mesenchymal-to-epithelial
transition (MET) triggered by BMP signaling and is characterized by
the activation of an epithelial gene expression program. The maturation
phase is considered the major bottleneck of the process, since cells
must activate endogenous expression of the pluripotency core circuitry
(Oct4, Sox2, and Nanog); thus, only a small number of cells progress
to the final stabilization phase to become fully reprogrammed iPS cells.
p73 is a member of the p53 transcription factor gene family, which also
includes p53 and p63. The members of this family have been involved
in the regulation of stem cell biology. Indeed, p53 acts as an important
barrier to somatic cell reprogramming. However, p73 role in this
process has never been addressed. Therefore, we hypothesized that p73
may play a role in the cellular reprogramming process. To address this,
we reprogrammed WT and p73KO-mouse embryonic fibroblast (MEFs)
and observed that lack of p73 negatively affects the reprogramming
efficiency. Moreover, our data indicates that BMP signaling is impaired
in the absence of p73 affecting the MET transition during the initiation
phase of reprogramming, and resulting in an altered maturation and
stabilization phase. Our results revealed, for the first time, that p73 is
necessary for appropriate BMP signaling during the MET transition of
somatic cell reprogramming.
This work was supported by the grants: SAF2012-36143 and
SAF2015-71381-R.
P09-6
Construcción de levaduras capaces de utilizar
glucosamina
Carmen-Lisset Flores, Carlos Gancedo
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM,
Madrid, ES
La glucosamina es un aminoazúcar (2-amino-2-deoxi-glucosa) que
forma parte de polisacáridos y de proteínas o lípidos glicosilados. En
la levadura Saccharomyces cerevisiae la glucosamina es transportada al
interior y, posteriormente, fosforilada por la hexokinasa. Sin embargo
la levadura no puede usar ese azúcar como fuente de carbono. De
hecho, es un análogo tóxico de la glucosa y se ha usado para obtener
mutantes resistentes a represión catabólica, alguno de los cuales
genera complejas formas priónicas. Nos hemos preguntado por qué S.
cerevisiae no crece en ese aminoazúcar. Una búsqueda in silico de genes
codificantes de enzimas que podrían participar en la vía de utilización de
la glucosamina mostró que S. cerevisiae carece del gen codificante de la
glucosamina-6-P desaminasa que cataliza la desaminación-isomerización
deglucosamina-6-P a fructosa-6-P. Hemos clonado el gen que codifica
esa proteína en la levadura no convencional Yarrowia lipolytica y lo
hemos expresado en S. cerevisiae. La cepa transformada es capaz de
utilizar glucosamina mostrando que la deficiencia de esa enzima es la
responsable de la falta de crecimiento de una cepa silvestre. Estamos
estudiando diversos efectos de la utilización de la glucosamina sobre
la levadura. Curiosamente, aunque Y. lipolytica tiene las enzimas que le
permitirían utilizar la glucosamina, no crece en este azúcar. La causa de
ese defecto podría estar relacionada con las características del equipo
de fosforilacion de azúcares de Y. lipolytica. Estamos trabajando para
dilucidar el defecto metabólico que impide a Y. lipolytica utilizar la
glucosamina.
Agradecimientos: Trabajo parcialmente realizado con la ayuda
BFU2010-19628-C02-02 del MINECO.
Pósters / Posters
P09r-7
Regulación de la expresión de los genes de
síntesis de polifosfatos (ppk2A, ppk2B) y su
degradación (ppx1 y ppx2) en Corynebacterium
glutamicum
Alfonso G. de la Rubia, Laura Marcos-Pascual, Álvaro Mourenza,
José A. Gil, Luis M. Mateos Delgado
Facultad Biología-Ambientales Campus de Vegazana Universidad
de León, León, ES
Corynebacterium glutamicum constituye un buen prototipo para los
análisis moleculares de bacterias corimeformes y otros relacionados:
Mycobacterium, Rhodococcus. Estos suelen presentar resistencia a agentes
generadores de estrés oxidativo por factores tales como (i) pared celular con
ácidos micólicos; (ii) sistemas antioxidantes como los pares tiorredoxina y
su reductasa Trx/TrxR, y el exclusivo par redox de actinobacterias Micotiol
(MSH)/Micorredoxinas (Mrx). Otro mecanismo de resistencia frente
a estrés podría ser el basado en presencia de acúmulos de polifosfatos
(Poli-P), típico de representantes de Corynebacterium. Estos polímeros,
formados por uniones fosfoanhidro de residuos de fosfato inorgánico
(Pi) en supervivencia frente a cambios osmóticos y de pH, formación de
biofilms, procesos de virulencia y acción como chaperoninas, además de los
típicos reserva de fosfato y fuente alternativa de energía. En C. glutamicum
hay dos enzimas polifosfato quinasas (PPK2A y PPK2B) que participan
en la formación de Poli-P, y dos actividades exopolifosfatasas (PPX1 y
PPX2) implicadas en movilizar Poli-P. En mutantes de Corynebacterium
afectado en alguno de estos genes, se observan diferentes niveles de
supervivencia frente a agentes oxidantes (peróxido de hidrógeno) y otros
agentes estresantes. Los análisis de expresión génica usando técnicas de
qPCR han puesto de manifiesto que los genes ppk2A y ppk2B se regulan
de forma coordinada. Además, en este proceso de regulación global,
la carencia de movilización de los gránulos de poli-P (como ocurre al
interrumpir los genes ppx1 y ppx2) parecen reprimir transcripcionalmente
a los genes para PPKs. Esto puede estar relacionado con el tamaño del
gránulo de polifosfato y su posible función de chaperonina primitiva.
Además de los estudios transcripcionales de expresión se está evaluando
su correlación con análisis cuantitativos de polifosfatos mediante técnicas
espectrofotométricas.
P09r-8
Label-free single-molecule analysis
of protein-DNA complexes with nanopores
Garbiñe Celaya1, Jéssica Rodríguez2, Judit Perales-Calvo1,
José Luis Mascareñas2, Arturo Muga1, Fernando Moro1,
David Rodríguez-Larrea1
1
Departamento de Bioquímica y Biología Molecular, Universidad
del País Vasco UPV/EHU and Instituto Biofisika (CSIC, UPV/EHU),
Leioa, ES, 2Centro Singular de Investigación en Química Biolóxica
e Materiais Moleculares (CIQUS), Departamento de Química
Orgánica, Universidade de Santiago de Compostela, Santiago
de Compostela, ES
Protein interactions with specific DNA sequences is crucial in the control
of gene expression and the regulation of replication. Single-molecule
methods offer unique capabilities to unravel the mechanism and kinetics
of these interactions. Here we develop a nanopore approach where the
target DNA sequence is contained in a hairpin followed by a ssDNA
reporter. This system allows to distinguish DNA-protein complexes from
bare DNA molecules, giving both the equilibrium and kinetic constants
of the interaction. We show that this approach can be used to test the
inhibitory effect of small molecules, and their action mechanism. In a
proof of concept, we use different dsDNA-ssDNA reporters to probe the
ability of the nanopore to distinguish the effect of an inhibitor in a complex
mixture of target DNAs and proteins. We anticipate that the use of this
77
Pósters / Posters
technology in chips carrying thousands of pores will led to new inhibitors
of transcription factor binding.
P09-9
Reactivos de transfección de células eucariotas
basados en polietilenimina (PEI) y colorantes
fluorescentes infrarrojos (NIR)
María Dolores Girón González1, Eduardo de los Reyes Berbel2,
Francisco Reche Pérez1, Mariano Ortega Muñoz2, Francisco Javier
López Jaramillo2, Fernando Hernández Mateo2, Francisco Santoyo
González2, Rafael Salto González1
1
Departamento de Bioquímica y Biología Molecular II, Facultad
de Farmacia, Universidad de Granada, Granada, ES, 2Departamento
de Química Orgánica, Facultad de Ciencias, Universidad de
Granada, Granada, ES
La terapia génica se basa en el desarrollo de sistemas de transfección
eficientes que posibiliten el tráfico extra e intracelular de ácidos nucleicos.
La transfección basada en el uso de polímeros catiónicos, una técnica
denominada polifección, es adecuada en la transfección de células en
cultivos así como en animales de experimentación No obstante existe la
necesidad de desarrollar reactivos menos tóxicos y con una alta eficiencia,
fundamentalmente en las aplicaciones in vivo. La posibilidad de incluir
en la estructura de los reactivos de transfección una serie de moléculas
que permitan su seguimiento durante la transfección en animales, lo que
se denominan reactivos teragnósticos, es una poderosa herramienta para el
desarrollo de reactivos de transfección más eficaces. Por ello, en este trabajo
se presenta la evaluación biológica de reactivos de transfección basados
en polietilenimina de diferentes pesos moleculares unidos a fluoróforos
en el infrarrojo cercano. Los reactivos así sintetizados presentan una alta
capacidad de unión y protección del DNA durante la transfección, una
elevada eficiencia de transfección en diferentes tipos celulares en cultivo
y una baja toxicidad. Además, en modelos experimentales de ratones es
posible el seguimiento y localización de los reactivos de transfección
mediante IVIS utilizando el fluoróforo infrarrojo asociado y la eficiencia
de transfección utilizando sistemas reporteros basados en la luciferasa.
En la actualidad estamos extendiendo el uso de estos reactivos para el
transporte de fármacos hacia células diana.
Investigación subvencionada por el Proyecto CTQ2014-55474-C2-2-R
(Ministerio de Economía y Competitividad).
P09-10
Actividad hidrolítica de bacterias halotolerantes
aisladas de pozas salinas
Ángel Wuilder Gárate-Palomino1, Amparo Iris Zavaleta2, Pilar
Foronda Rodríguez3, Basilio Valladares Hernández3, Maria Antonieta
Quispe Ricalde1
1
Escuela profesional de Biología, Facultad de Ciencias. Universidad
Nacional de San Antonio Abad del Cusco, Cusco, PE, 2Laboratorio
de Biología Molecular, Facultad de Farmacia y Bioquímica,
Universidad Nacional Mayor de San Marcos, Lima, PE, 3Instituto
Universitario de Enfermedades Tropicales y Salud Pública de
Canarias. Universidad de La Laguna, La Laguna, ES
Los ambientes salinos son fuentes de microorganismos productores de
enzimas que resisten a procesos biotecnológicos exigentes de salinidad
y temperatura. Así, se han descrito microorganismos halófilos con
gran actividad lipolítica y proteolítica. La búsqueda y aislamiento de
tales microorganismos son el paso inicial para la obtención de enzimas
resistentes a procesos industriales escalables. En el presente trabajo se
describe el aislamiento y la identificación de bacterias halotolerantes
de las aguas salinas de la Provincia de Urubamba, del Departamento de
78
XXXIX Congreso SEBBM
Cusco (Perú). Las muestras fueron obtenidas de 4 pozas saladas y de la
fuente de alimentación. Se realizaron 3 diluciones de cada muestra y el
aislamiento se realizó en medio HM (Extracto de levadura 1%, peptona
0,5%, glucosa 0,1%, MgSO4 0,025%, KCl 0,05%, CaCl 0,009%). La
caracterización de cada aislado se determinó en base a sus características
morfológicas, fisiológicas y bioquímicas. La identificación molecular se
realizó mediante secuenciación del gen ribosómico 16S. Las reacciones
hidrolíticas evaluadas fueron: la proteolisis de gelatina y caseína,
lipólisis de tween 80 y la hidrólisis de almidón y esculina. Las bacterias
aisladas fueron 9 cocos grampositivos, pertenecientes al género
Staphylococcus. Los aislados crecieron en NaCl 5%, 10% y 15% (p/v),
sus temperaturas de crecimiento fueron entre 25ºC a 42ºC. En cuanto
al pH los aislados crecieron entre 5 y 9. Las actividades lipolíticas y
proteolíticas se observaron en 5 y 7 aislados respectivamente, además 4
hidrolizaron la esculina.
Financiación: Científicos INC-Círculos de Investigación en Ciencia y
Tecnología. Convenio Nº007-2014-FONDECYT. CONCYTEC-Perú.
P09-11
The protein phosphatase CnPpz1/CnHal3 system
from Cryptococcus neoformans
Chunyi Zhang
IBB-Universitat Autonoma de Barcelona, Cerdanyola de Vallès, ES
The Saccharomyces cerevisiae protein phosphatase (PPase) Ppz1 is
involved in multiple cellular processes because its important role in the
regulation of monovalent cation (K+ and Na+) homeostasis. In budding
yeast, Ppz1 is inhibited by two regulatory proteins, Hal3 and Vhs3,
which also have moonlighting properties related to CoA biosynthesis.
Ppz1 is found only in fungi, including pathogenic ones and, when
overexpressed in high-copy number Ppz1, it appears to be the most
toxic protein in budding yeast. These characteristics define Ppz1 as
a possible target for antifungal therapies. Cryptococcus neoformans
is a pathogenic fungus responsible for a high rate of mortality in
invasive infections. Exploration of the C. neoformans genome has
identified a single putative gene coding for Ppz1 (CNAG_03673) and
two genes encoding possible Hal3-like proteins (CNAG_00909 and
CNAG_07348). We are in the process of functionally characterize all
three proteins by expression in E. coli and in S. cerevisiae, as a first
step towards the characterization of the function of these protein sin C.
neoformans. Although characterization of CnPpz1 has been hampered
by its extremely G/C rich N-terminal region, we have recently succeeded
in expressing the protein in S. cerevisiae. Preliminary results indicate
that, in high copy number, CnPpz1 can partially replace endogenous
budding yeast Ppz1.
P09-12
Single mutants that tolerate the overexpression
of Ppz1 protein phosphatase
Carlos Alberto Calafí Pascual, María López Malo, Antonio
Casamayor Gracia, Joaquín Ariño Carmona
Institut de Biotecnologia i Biomedicina - Departamento de
Bioquímica, Universitat Autònoma de Barcelona, Cerdanyola
del Vallès, ES
The Saccharomyces cerevisiae PPZ1 gene encodes a protein phosphatase
involved in multiple cellular processes, such as cation homeostasis, cell
cycle progression and cell wall integrity maintenance (Clotet et al., 1996;
Posas et al., 1995). PPZ1 is a non-essential gene, but its overexpression
promotes cell cycle delay (de Nadal et al., 1998) and it appears to be a
highly toxic gene when overexpressed (Makanae et al., 2013). Since
Ppz1 is only present in fungi, including pathogenic ones, it represents a
Salamanca 2016
possible antifungal target. In an attempt to clarify its toxicity mechanism
we are trying to identify genes whose absence abolish or alleviate the
Ppz1-dependent slow growth phenotype. For this purpose we screened
the EUROFAN KO mutant collection transformed with a plasmid in which
the Ppz1 expression iscontrolled by the GAL1-10 inducible promoter.
We expect that single mutants found by this strategy will be involved
in: 1) mechanism of induction of the GAL1-10 promoter by galactose,
or 2) mutants related to the mechanism of toxicity of Ppz1 when it is
overexpressed. The screen of the mutant library has revealed 6 mutants that
tolerate the overexpression of Ppz1 from the used high-copy plasmid. Cells
lacking GAL3 or GAL4 are probably involved in the galactose-dependent
expression of Ppz1. We also found gal11, pgd1, med2 and tcm62 mutant
cells, which are being tested as suppressors of the toxicity triggered by
overexpression of this phosphatase.
P09-13
Caracterización de nanopartículas dirigidas
a marcadores de Cancer Stem Cells en el cáncer
microcítico de pulmón
Carmen Garnacho Montero, Ana Sarrias Giménez, Miguel Ángel
González Reyes, Elena Ábalos Marco, Inés de la Rosa Zurera
Departamento de Citología e Histología Normal y Patológica.
Facultad de Medicina, Universidad de Sevilla, Sevilla, ES
El cáncer de pulmón es la principal causa de muerte por cáncer en todo el
mundo y, a pesar de los avances en el diagnóstico y tratamiento, el pronóstico
de los pacientes sigue siendo pobre. Diversos trabajos han propuesto que
muchos tipos de cáncer, incluyendo el cáncer de pulmón, pueden ser
promovidos por las células madre del cáncer (CSC, del inglés Cancer
Stem Cell), un subconjunto de células indiferenciadas con la capacidad de
autorrenovarse y al mismo tiempo dar lugar a una progenie heterogénea
diferenciada. Estas células serían las responsables no solo de la iniciación
del tumor, sino también de la recurrencia, resistencia a quimioterapia y
radioterapia, y metástasis. Los sistemas de direccionamiento de fármacos
con nanopartículas pueden ofrecer un enfoque innovador para superar la
resistencia de las CSC, así como reducir la frecuencia de recidivas. En este
estudio hemos caracterizado los marcadores ABCG2, CXCR4 y CD44 para
su uso como dianas en un sistema de direccionamiento de fármacos con
nanopartículas que eliminaría selectivamente las CSC en un modelo in vitro
de cáncer microcítico de pulmón. Nuestros resultados revelan la presencia
de los tres marcadores en la superficie de las líneas celulares pulmonares
estudiadas, aunque con diferentes patrones de expresión. Además, tanto
las NP anti-CXCR4, anti-ABCG2, como anti-CD44 se unieron de forma
específica a las células e indujeron la endocitosis de las mismas. Por lo
tanto, los receptores CXCR4, ABCG2 y CD44 podrían ser una buena
diana para el desarrollo de un sistema dirigido de fármacos mediante
nanopartículas para el tratamiento del cáncer microcítico de pulmón.
Financiación: Proyectos de Investigación en Salud. Consejería de
Igualdad, Salud y Políticas Sociales 2013 (PI-0051-2013).
P09r-14
Modulación de la producción de hidrógeno a
través del control del consumo de acetato en
Chlamydomonas
José Luis Jurado Oller, Alexandra Dubini, Aurora Galván, Emilio
Fernández Reyes, David González Ballester
Universidad de Córdoba, Córdoba, ES
Chlamydomonas es un alga verde unicelular ampliamente utilizada como
organismo modelo para el estudio de multitud de procesos relacionados
con plantas (fotosíntesis, metabolismo del nitrógeno y del azufre,
etc.). Sin embargo, en los últimos años ha recibido especial atención
Pósters / Posters
debido a su capacidad para producir triacilglicéridos, hidrógeno y otros
compuestos susceptibles de ser empleados como biocombustibles. Entre
ellos, el hidrógeno (H2) destaca debido a que puede ser combustionado
obteniendo rendimientos energéticos similares a los combustibles
fósiles sin liberar CO2 a la atmósfera. La condición de cultivo más
ampliamente utilizada y estudiada para promover la producción de H2
por Chlamydomonas es la deficiencia de azufre, aunque la ausencia de
otros nutrientes (nitrógeno, fósforo, magnesio, etc.) en la composición
del medio de cultivo también conduce a este fenómeno. Otro factor
importante para la producción de H2 en Chlamydomonas es la presencia
de acetato, una fuente de carbono reducida asimilable por el alga.
Aunque se conoce que la presencia de acetato potencia la producción
de H2 en cultivos adaptados a anoxia y luz, no ha sido realizado un
estudio en profundidad del papel de este compuesto en el proceso. En
este trabajo se discute el papel del acetato en la producción de H2 y se
describe una metodología alternativa a la deficiencia nutricional para
la obtención simultánea H2 y biomasa a través del control del consumo
de acetato. Asimismo, se propone un modelo para la producción de H2
vinculado a la asimilación de acetato con la participación de la cadena
fotosintética.
Founded by MICINN (Proyecto IEO-Plan-E) and BFU2015-70649-P)
with support of the FEDER program, Junta de Andalucía (BIO-128 and
BIO-502).
P09-15
La flora intestinal como fuente de “nuevos”
microorganismos probióticos
Borja Sánchez
Instituto de Productos Lácteos de Asturias (CSIC), Villaviciosa, ES
La definición de probiótico más aceptada actualmente es la de
“microorganismos vivos que cuando se administran en cantidades
adecuadas confieren un beneficio para la salud del hospedador”. El
estudio de los probióticos ha avanzado de manera notable durante los
últimos años, espoleado por los grandes avances en el conocimiento
de la implicación de la microbiota intestinal en el condicionamiento
de la salud y la enfermedad. A pesar de que los microorganismos
comensales de la microbiota intestinal son frecuentemente fuente de
cepas probióticas, éstas no pueden denominarse como tal si no son
convenientemente aisladas, caracterizadas y si no se presentan estudios
clínicos que avalen sus efectos beneficiosos sobre la salud, así como su
seguridad. La evidencia científica disponible a día de hoy sugiere que
ciertas especies de microorganismos comensales representan buenos
candidatos para identificar nuevos probióticos. Muchos estudios
metagenómicos han puesto en evidencia que algunas de estas especies
están subrepresentadas en la microbiota intestinal de grupos con
distintas situaciones fisiológicas y enfermedades, como la enfermedad
inflamatoria intestinal o la obesidad. De hecho, se sospecha que la
ausencia de ciertos de estos comensales, como las bacterias productoras
de ácido butírico, podría predisponer a padecer estas enfermedades,
aunque esta relación causa-efecto es un foco de discusión candente
entre la comunidad científica.
Algunos de estos microorganismos comensales, de los que sin duda
oiremos hablar mucho en el futuro, son las especies Akkermansia
muciniphila, Faecalibacterium prausnitzii, Roseburia spp. o Eubacterium
hallii. El consenso actual entre las distintas asociaciones internacionales
implicadas es considerarlos como probióticos, aunque condicionado a la
presentación de cepas bien caracterizadas, estudios de intervención sólidos
sobre su eficacia y seguridad, y también estudios de ciencia básica donde
se describa el mecanismo de acción preciso a nivel de cepa.
Palabras clave: microbiota intestinal, probióticos, comensales, metagenómica,
salud.
79
Pósters / Posters
P09-16
Syngas as feedstock for polyhydroxyalkanoate
production in Rhodospirillum rubrum
Olga Revelles1, J.L. García2, M.A. Prieto2
Department of Biosystems Science & Engineerin (D-BSSE), Basel,
CH; Centro de Investigaciones Biológicas, CSIC, Madrid, ES,
2
Centro de Investigaciones biológicas, CSIC, Madrid, ES
1
The potential use of municipal solid wastes (MSW) as feedstock for the
production of polyhydroxyalkanoates (PHAs) by a process known as syngas
fermentation is an attractive bio-economic strategy to reduce urban and
agricultural wastes. The anaerobic photosynthetic bacterium Rhodospirillum
rubrum, is an organism particularly attractive for the bioconversion of syngas
into PHAs. In this work, a quantitative physiological analysis of R. rubrum
was carried out by implementing GC-MS and HPLC techniques to unravel the
metabolic pathway operating during syngas fermentation that leads to PHA
production. Further, detailed investigations of the central carbon metabolites
using 13C-labelled substrate showed significant CO2 assimilation (of 40%)
into cell material and PHA from syngas carbon fraction. By a combination
of quantitative gene expression and enzyme activity analyses, the main role
of carboxylases from the central carbon metabolism in CO2 assimilation was
shown, where the Calvin-Benson-Bassham cycle (CBB) played a minor role.
Finally, the potential of this strain as microbial cell factory for the synthesis
of PHA from syngas produced from municipal solid waste is also evaluated.
In summary this work provides valuable information to further optimize
the fermentation process, as well as, it reveals the strong robustness of this
syngas fermentation where the purity of the syngas is not a critical constraint
for PHA production.
XXXIX Congreso SEBBM
warming suggest an increase in the severity and frequency of these adverse
climate events. Developing new biotechnological strategies to increase crop
productivity is essential to face a climate change scenario and secure food
supply to a continuously growing population. Understanding how plants
perceive and transduce adverse environmental conditions into biochemical
signals is critical to fulfill this objective. Here, we report the identification of
RCI5, an Arabidopsis flavine monooxigenase (FMO) whose levels increase
in response to abiotic stress and is implicated in the biosynthesis of TMAO,
a new molecule not described in plants so far. We show that Arabidopsis has
basal levels of TMAO under control conditions and that these levels augment
when exposed to low temperature, high salt or water stress. Analyses of
stress tolerance in Arabidopsis transgenic plants containing constitutive high
levels of TMAO and in wild-type Arabidopsis plants treated with TMAO
demonstrate that this molecule is a positive regulator of plant response to
different abiotic stress conditions. Our data also indicate that TMAO induces
a wide transcriptomic reprograming and a subsequent change in the metabolic
profile that accounts for its role in plant tolerance to abiotic stress. Interestingly,
we have observed a similar role for TMAO in the tolerance of different crops
to abiotic stress. We propose that TMAO is a new plant signalling molecule
with a high biotechnological potential that mediates abiotic stress responses.
P10. Estructura y función de proteínas /
Protein structure and function
P10r-1
P09-17
Explorando la existencia de lipid rafts en
bacterias
Daniel López
Centro Nacional de Biotecnología, CSIC, Madrid, ES
La existencia de microdominios funcionales de membrana en bacterias,
estructural y funcionalmente similares a los conocidos lipid rafts de celular
eucariotas es un campo emergente en microbiología que desarrollaré en mi
presentación utilizando el patógeno Staphylococcus aureus como modelo
bacteriano. Mi presentación delineará tres líneas de trabajo que están
actualmente siendo desarrolladas en mi laboratorio. Una línea estructural,
para dilucidar la composición y el proceso de ensamblaje de estas plataformas
de membrana. Una línea funcional que investiga su significado biológico y,
en este caso particular de S. aureus, su implicación en mecanismos celulares
de infección. Finalmente presentaré una línea más aplicada, utilizando
moléculas capaces de alterar estas plataformas de membrana como terapia
antimicrobiana. La existencia de microdominios funcionales de membrana
en bacterias implica que los organismos procariotas son mucho más
complejos de lo que anteriormente habíamos anticipado.
P09-18
An Arabidopsis flavine monooxygenase provides
new biotechnological strategies to improve crop
tolerance to abiotic stress
Rafael Catalá1, Rosa Mª López-Cobollo2, M.A. Berbis1,
J. Jiménez-Barbero3, J. Salinas1
1
Centro de Investigaciones Biológicas, CSIC, Madrid, ES, 2Imperial
College London, South Kensington Campus, London, UK, 3Centro
de Investigaciones Biológicas, CSIC, Madrid, ES; CIC bioGUNE,
Bizkaia Technological Park, Derio, ES
Drought, salinity and extreme temperatures severely limit crop production
accounting for more than 40% of losses in yield. Predictions about global
80
The ribotoxin α-sarcin cleaves the sarcin/ricin
loop within late 60S pre-ribosomes
Miriam Olombrada1, Cohue Peña1, Olga Rodríguez-Galan2,
Purnima Nerurkar1, Martin Altvater1, José G. Gavilanes3, Álvaro
Martínez-del-Pozo3, Jesús de La Cruz2, Lucía García-Ortega3,
Vikram G. Panse1
1
Institute of Biochemistry, ETH Zurich, Zurich, CH, 2Instituto de
Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/
Universidad de Sevilla, Sevilla, ES, 3Departamento de Bioquímica y
Biología Molecular I, Universidad Complutense, Madrid, ES
The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the
sarcin/ricin loop (SRL), a critical functional rRNA element within the large
ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin
targets the SRL only within mature 60S subunits in vivo remains unresolved.
Here, we show that α-sarcin can cleave SRLs within late 60S pre-ribosomes
poised for nuclear export as well as final cytoplasmic maturation. In vitro
cleavage assays revealed that mature 25S rRNA, but not nucleolar/nuclear
27S pre-rRNA containing 60S pre-ribosomes, are effective substrates of
α-sarcin. Conditional expression of α-sarcin in budding yeast is lethal, but
does not impede nuclear export and/or the cytoplasmic maturation pathway
of 60S pre-ribosomes. Thus, defective SRL containing pre-ribosomes
escape nuclear and cytoplasmic proofreading. Polysome analyses revealed
that these faulty 60S subunits form 80S initiation complexes, but are
impaired in translation elongation. We propose that the functional integrity
of the SRL might be assessed only later during translation.
P10-2
Halophilic protein adaptation results from
synergistic residue-ion interactions in the folded
and unfolded states
Gabriel Ortega Quintanilla1, Tammo Diercks2, Óscar Millet2
UC Santa Barbara, Santa Barbara, US, 2Structural Biology Unit,
CIC bioGUNE, Bilbao, ES
1
Salamanca 2016
The slow but unstoppable progress of evolution has enabled the conquest
of virtually any environment on Earth, even those with conditions far
from “standard” in which the presence of organisms seems unthinkable.
Extremely high salt concentrations exert a huge osmotic stress on living
organisms ultimately leading to their death by dissecation. That is why
salt is a common food preserver, and also why salines and lakes such as
the Dead Sea have been traditionally thought devoted of life. But some
organisms – haloarchaea and some bacteria – are capable of surviving in
these media at near saturating extracellular salt concentrations. They are the
so-called halophiles.How do they do it? They balance the osmotic pressure
at both sides of the cell membrane by accumulating large quantities of
intramolecular cosolutes. In particular, extreme halophilic organisms
accumulate KCl in their cytoplasm up to concentrations of 4M. This is a
huge shock for the intracellular machinery, which has undergone severe
reengineering along evolution in order to remain native and functional
under such harsh conditions. The basis for halo-adaptation relies in a strong
evolutionary selection of certain amino acid types. While maintaining the
same structure as its mesophilic homologues, the amino acid composition of
halophilic proteins is markedly different: acidic residues – especially aspartic
acid – and short, polar side chains – threonine – are favored over bulky,
hydrophobic residues. This reduction of the overall hydrophobic content is
mainly achieved through a drastic decrease in the number of lysines, which
have a long, disordered, aliphatic chain. As a result, halophilic proteins are
very stable, soluble and functional at high salt concentrations. We have
studied the relationship between amino acid composition and halophilic
adaptation. We have mimicked in vitro the evolutionary process in order
to design proteins with different degrees of haloadaptation, and conducted
a comprehensive biophysical characterization thereof under conditions
simulating hypersaline environments. The use of atomic-resolution NMR
has allowed us to study the structural and dynamic properties of folded and
unfolded states of halophilic proteins at the level of individual residues.
We have found how the action of two opposite mechanisms confers great
stability to halophilic proteins in the presence of high amounts of salt:
-The unspecific preferential interactions between highly abundant
carboxylates in the surface and K+ cations in solution stabilize the folded
state,
-Whereas the increase in polar surface – and the concomitant decrease in
hydrophobic area – promotes preferential ion exclusion from the surface of
the unfolded halophilic proteins, enhancing unfolded state destabilization
upon cosolute addition.
These two opposite but complementary mechanisms, both dependent on
the amino acid composition and the amount of the area exposed, constitute
the basis of halophilic adaptation.
P10-3
Tumor specific mutations in the N-terminal
domain of the Tumor Suppressor ING5 decrease
protein stability and cell growth inhibition
Georgina Ormaza Hernandez1, Nekane Merino1, Maider Villate1,
Ignacio Palmero2, Robert Kypta1, María dM. Vivanco1, Francisco J.
Blanco1
1
CIC bioGUNE, Derio, ES, 2IIB (CSIC-UAM), Madrid, ES
The INhibitor of Growth (ING) family of tumor suppressors consists of five
homologous proteins that regulate the transcriptional state of chromatin
by recruiting histone acetyl trasferase and histone deacetylase complexes
to chromatin regions with histone H3 trimethylated at K4 (H3K4me3)1.
This modification is recognized by the Plant HomeoDomain (PHD) that
is present at the C-terminus of the five ING proteins2. ING5 dimerizes
through its N-terminal domain, with a symmetric antiparallel coiled-coil
structure. The PHD fingers are connected with the dimerization domain by
a disordered flexible region that makes them chemically independent and
equivalent as they sample a large volume around the dimerization domain.
Three point mutations have been described for ING5 in oral squamous
carcinoma primary tumors: Q33R, I68V, and, C75R. The three of them are
Pósters / Posters
located in the N-terminal domain, and point to ING5 as a tumor suppressor
in this type of cancer 3. We have found that the N-terminal domains of the
three ING5 mutants form dimeric coiled-coils like the wild type but with
different stability, as measured by thermal denaturation. While the stability
of Q33R mutant is similar to the wild type, mutants I68V and C75R are
strongly destabilized, suggesting a disruption of ING5 function. Cell-based
assays indicate that the structural alterations have a functional impact. We
have found that NIH3T3 cell cultures stably expressing any of the three
ING5 mutants display large changes in their cell cycle profile, compared
to wild-type, most dramatic in the case of C75R mutant, which is also the
most unstable one.
References
[1] Y. Doyon et al. (2006) Mol Cell 21, 51-64.
[2] P.V. Peña et al. (2006) Nature 442, 100-3.
[3] B. Cengiz et al. (2010) Int J Cancer 127(9), 2088-2094.
P10-4
Structural basis for Gαi activation by a
non-receptor protein
Mikel García-Marcos1, Alain Ibáñez de Opakua2, Kshitij Sharma1,
Nekane Merino2, Lien T. Nguyen1, Maider Villate2, Anthony Leyme1,
Arthur Marivin1, Vincent DiGiacomo1, Francisco J. Blanco3
1
Boston University School of Medicine, Boston, US, 2CIC bioGUNE,
Derio, ES, 3CIC bioGUNE, Ikerbasque, Bilbao, ES
Non-receptor Guanine nucleotide Exchange Factors (GEFs) have emerged
as important G protein activators and signaling components in diverse
cellular processes. Their involvement in human disease makes them
attractive therapeutic targets. Here we used nuclear magnetic resonance
(NMR) to study residue-level conformational changes in Gαi3 upon
binding of a non receptor GEF named GIV (Gα-interacting, vesicle
associated protein a.k.a. Girdin). Changes in many NMR signals of Gαi3
could be measured upon GIV binding in solution. Signal perturbations
occurred not only in the predicted binding region (SwII/α3, based on a
molecular model) but also in the nucleotide binding pocket and a GPCR
binding loop. Based on these observations we carried out an extensive
site-directed mutagenesis study from which we conclude that GIV
binding on Gαi does not overlap with the GPCR binding region and that
it allosterically induces conformational changes in the nucleotide binding
pocket. These conformational changes only partially overlap with those
observed upon GPCR activation, indicating that GIV and GPCRs utilize
markedly different molecular mechanisms to activate G proteins. These
results suggest that at least some non-receptor GEFs can be specifically
targeted without affecting GPCR-G protein coupling and explain why
receptor and non-receptor GEFs have different potency in activating G
proteins.
P10r-5
Structural and biochemical characterization
of Mycobacterium tuberculosis replicative
DNA polymerase DnaE1 and its unique
PHP-exonuclease
Soledad Baños-Mateos, Ulla F. Lang, Sarah Maslen, Mark Skehel,
Meindert H. Lamers
MRC Laboratory of Molecular Biology, Cambridge, UK
High fidelity DNA synthesis is essential for all organisms and depends on a
proofreading 3’-5’ exonuclease that is associated with the replicative DNA
polymerase. Contrary to humans and the model organism Escherichia coli,
the proofreading activity in the major pathogenic bacteria Mycobacterium
tuberculosis (Mtb) is not performed by the canonical DEDD exonuclease.
Instead, the exonuclease active site in Mtb is located in an intrinsic
81
Pósters / Posters
domain of the replicative DNA polymerase DnaE1, the PHP domain. The
mechanism of action of the PHP-exonuclease is unknown. Understanding
the mechanisms that control fidelity and mutation rates in M. tuberculosis
is crucial as point mutations drive antibiotic resistance in Mycobacteria,
an issue that is a global health problem. We have solved the crystal
structure of the Mtb DnaE1 polymerase and biochemically characterized
its PHP-exonuclease. The structure shows the presence of a narrow
groove that is likely to bind ssDNA, connecting the polymerase and the
exonuclease active sites. This groove potentially leads the DNA into the
PHP domain and positions it for removal of a misincorporated nucleotide.
Moreover, we have identified a tri-nuclear Zn center in the PHP domain
coordinated by nine conserved residues. Interestingly, the PHP active
site in DnaE1 shows remarkable similarities to the active site of E. coli
endonuclease IV (Endo IV), an enzyme that also cleaves DNA. All these
observations allow us to propose a mechanism for DNA hydrolysis by the
PHP-exonuclease based on the mechanism of action of Endo IV. Finally,
the unique PHP-exonuclease active site of Mtb appears an attractive target
for specific inhibition as is not found in eukaryotes, making the likelihood
of cross reactivity to human exonucleases small. Therefore, this work
provides new insights in understanding replication fidelity in Mtb and will
be a valuable tool in the development of novel treatments that target DNA
replication in Mycobacterium tuberculosis.
P10-6
Recognition of NES cargoes by CRM1 exportin
is altered in certain leukemias
Igor Arregi1, María Ángeles Urbaneja1, Iraia García-Santisteban2,
Marián Alonso-Mariño1, Juan Jesús García-Vallejo3, José Antonio
Rodríguez2, Sonia Bañuelos1
1
Biofisika Institute (UPV/EHU, CSIC) and Department of
Biochemistry and Molecular Biology, University of the Basque
Country (UPV/EHU), Leioa, ES, 2Department of Genetics, Physical
Anthropology and Animal Physiology, University of the Basque
Country (UPV/EHU), Leioa, ES, 3Department of Molecular
Cell Biology and Immunology, VU University Medical Center,
Amsterdam, NL
Nucleocytoplasmic distribution crucially modulates protein function, and
is often deregulated in pathologies. Nuclear export of many proteins relies
on the recognition of their nuclear export signal (NES) by the exportin
CRM1. Nucleophosmin (NPM), a CRM1 cargo that is normally enriched
in nucleoli, is frequently mutated in acute myeloid leukemia (AML) and
aberrantly located in the cytoplasm of leukoblasts from those patients.
The AML-linked mutation of NPM implies acquisition of a novel NES,
responsible for a more efficient nuclear export. A detailed description of the
interaction between CRM1 and different AML-linked mutants should better
explain their pathogenic behavior. In another hematological malignancy,
chronic lymphocytic leukemia (CLL), a recurrent point mutation (E571K)
of CRM1 is found in 5% of patients. The E571K mutation results in a charge
inversion near the NES binding groove of CRM1. However, the functional
consequences of such change remain unexplored. Novel structural insights
into the basis of CRM1/NES interaction allow experimentally addressing the
binding mechanism. We have analyzed the formation of complexes between
wild type and mutant forms of both CRM1 and NES cargoes, trying to
understand the effect of different pathogenic mutations of NPM and CRM1.
P10-7
Analysis of the Binding of the N-terminus
of Neuronal Nitric Oxide Synthase to various
cellular targets
Carlos Costas Insua, Javier Merino Gracia, María Ángeles Canales
Mayordomo, José Ignacio Rodríguez Crespo
Universidad Complutense de Madrid, Madrid, ES
82
XXXIX Congreso SEBBM
Neuronal Nitric Oxide Synthase (nNOS), unlike the endothelial
(eNOS) and inducible isoforms (iNOS), presents an N-terminal
extension of approximately 300 amino acids which contains a PDZ
domain and a beta hairpin, moieties implicated in protein-protein
interactions that regulate its subcellular localization. PDZ domains
consist of ~100 amino acids which share a common beta-sandwich fold
with 6 beta-strands and 2 alpha-helices. Between the beta2-strand and
the alpha2-helix, there is a hydrophobic groove where the C-termini of
interacting proteins bind as antiparallel beta strands. This is typically
directed by the very last four amino acids of the target protein and
has led to a simplistic classification of PDZ domains into class I, II
or III relying on the target sequence. The PDZ domain of nNOS was
initially described as a class III PDZ domain, but recent data have
proposed ligands falling within all classes. Nevertheless, the best
characterized protein known to bind to this motif, NOS1AP, presents a
class II motif. Hence, the molecular basis of these interactions remains
controversial. Using a recombinantly produced PDZ domain, in vitro
binding techniques, NMR spectroscopy and confocal microscopy we
have shed light in these interactions. We have demonstrated that the
PDZ domain of nNOS behaves as a hybrid module, capable of binding
multiple class sequences, but an acidic residue is always necessary.
Furthermore, we suggest a differential binding mode between class
II and class III peptides that would implicate more than the typical
last four residues. Our data has led us to the identification and
characterization of the interaction with the tight-junction protein
Claudin-3. Moreover, we propose a binding regulated through tyrosine
phosphorylation, a mechanism not previously reported for any nNOS
PDZ ligand. Finally, we have developed an affinity chromatography/
mass spectrometry approach which will allow us to identify novel
binding partners not only to the nNOS PDZ domain but also to the
nNOS beta-hairpin.
P10-8
Crystallography captures catalytic steps in human
methionine adenosyltransferase enzymes
Ben Murray1, V. Svetlana Antonyuk2, Alberto Marina3, Shelly C. Lu4,
José M. Matod5, S. Samar Hasnain3, Adriana L. Rojas3
1
Molecular Biophysics Group, Institute of Integrative Biology,
Faculty of Health and Life Sciences, University of Liverpool,
Liverpool, UK; Structural Biology Unit, Center for Cooperative
Research in Biosciences, Derio, ES, 2Molecular Biophysics
Group, Institute of Integrative Biology, Faculty of Health and
Life Sciences, University of Liverpool, Liverpool, UK, 3Structural
Biology Unit, Center for Cooperative Research in Biosciences,
Derio, ES, 4Division of Gastroenterology, Cedars-Sinai Medical
Center, Los Angeles, US, 5CIC bioGUNE, CIBERehd, Parque
Tecnológico Bizkaia, Derio, ES
The principal methyl donor of the cell, S-adenosylmethionine
(SAMe), is produced by the highly conserved family of methionine
adenosyltranferases (MATs) via an ATP-driven process. These
enzymes play an important role in the preservation of life, and their
dysregulation has been tightly linked to liver and colon cancers. We
present crystal structures of human MATα2 containing various bound
ligands, providing a “structural movie” of the catalytic steps. High- to
atomic-resolution structures reveal the structural elements of the enzyme
involved in utilization of the substrates methionine and adenosine and in
formation of the product SAMe. MAT enzymes are also able to produce
S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an
S-ethyl analog of the amino acid methionine, is known to induce steatosis
and pancreatitis. We show that SAE occupies the active site in a manner
similar to SAMe, confirming that ethionine also uses the same catalytic
site to form the product SAE.
Salamanca 2016
Pósters / Posters
P10-9
P10-11
Repositioning Tolcapone as a potent inhibitor of
transthyretin amyloidogenesis and its associated
cellular toxicity
Architecture of hemidesmosomes
Ricardo SantAnna1, Natàlia Reixach2, María Rosario Almeida3, Raul
Insa4, Adrián Velázquez-Campoy5, David Reverter1, Nuria Reig4,
Salvador Ventura1
1
Institut de Biotecnologia i Biomedicina and Departament de
Bioquímica i Biologia Molecular, Universitat Autònoma de
Barcelona, Bellaterra, ES, 2Molecular and Experimental Medicine
Department, The Scripps Research Institute, La Jolla, US,
3
Molecular Neurobiology, IBMC- Instituto de Biologia Molecular e
Celular, ICBAS- Instituto de Ciências Biomédicas de Abel Salazar,
Universidade do Porto, Porto, PT, 4SOM-Biotech, Barcelona, ES,
5
Institute of Biocomputation and Physics of Complex Systems
(BIFI), Joint Unit IQFR-CSIC-BIFI, Universidad de Zaragoza,
Zaragoza, ES
Background: Transthyretin (TTR) is a plasma homotetrameric protein
implicated in fatal systemic amyloidoses. TTR tetramer dissociation
precedes pathological TTR aggregation. Native state stabilizers are
promising drugs to treat the TTR amyloidoses.
Material and Methods: Here, we used biophysical, cell biology and in
vivo studies to repurpose Tolcapone, an FDA-approved molecule for
Parkinson’s disease, as a very potent TTR aggregation inhibitor.
Results and Discussion: Tolcapone binds specifically to TTR in human
plasma, stabilizes the native tetramer in vivo in mice and humans and
inhibits TTR cytotoxicity. The crystal structures of Tolcapone bound
to wild type TTR and to the V122I cardiomyopathy-associated variant
explain why this molecule is a better amyloid inhibitor than Tafamidis, so
far the only drug in the market to treat the TTR amyloidoses.
Conclusions: Overall, Tolcapone, already in clinical trials, is a strong
candidate for therapeutic intervention in these diseases, including those
occurring in the central nervous system, for which no small molecule
approach exist.
José Antonio Manso1, Arturo Carabias1, María Gómez-Hernández1,
Inés García Rubio2, Arnoud Sonnenberg3, José María de Pereda1
1
Instituto de Biología Molecular y Celular del Cáncer (CSIC-USAL),
Salamanca, ES, 2Centro Universitario de la Defensa, Academia
Militar General, Zaragoza, ES, 3Netherlands Cancer Institute,
Amsterdam, NL
Hemidesmosomes (HDs) are junctional complexes that mediate stable
attachment of epithelial cells to the basal lamina, linking the extracellular
matrix to the cytokeratins. In stratified epithelia HDs are composed of
integrin α6β4, the bullous pemphigoid antigen 2 (BPAG2), tetraspanin
CD151, and two proteins of the plakin family: plectin and BPAG1e.
The integrin α6β4 is a receptor for laminins and a central hub of the
HD protein network. The intracellular interactions of α6β4 are mediated
by the cytodomain of the β4 subunit. Plectin and BPAG1e bind to β4
via their N-terminal regions and to cytokeratins via their C-terminal
domains. Defects in HD-proteins cause several types of the blistering
disease epidermolysis bullosa. Our long-term goal is to understand the
organization and regulation of HDs. Previously, we had characterized the
structure of the plectin-integrin β4 complex. Now we have elucidated the
structural basis of the BPAG1e-β4 interaction. Binding occurs between
an N-terminal segment of BPAG1e and the third and fourth fibronectin
type III domains (FnIII-3,4) of β4. Ser residues in BPAG1e are involved in
the binding interface, suggesting that the interaction may be regulated by
phosphorylation. Complementing the analysis of hetero-interactions, we
have elucidated the structure of dimeric plakins. Similarly to most plakins,
plectin has an N-terminal “plakin domain” formed by spectrin repeats that
adopt an elongated structure, which is followed by a central coiled-coil rod
domain that mediates dimerization. The plakin domains are arranged in
parallel in the homo-dimer. Thus, the distance between the β4 binding-sites
is restricted, suggesting that the avidity-mediated stabilization of the
interaction might be linked to the clustering of α6β4 in the membrane.
P10-12
P10-10
Structural changes induced by acidic pH
in human Apolipoprotein B-100
César Martín1, José Ángel Fernández-Higero1, Asier Benito-Vicente1,
Aitor Etxebarria1, José Carlos G. Milicua2, Ostolaza Helena1, José
Luis R. Arrondo1
1
Instituto Biofisika (UPV/EHU, CSIC) y Dpto. Bioquímica y Biología
Molecular, UPV/EHU, Bilbao, ES, 2Dpto. Bioquímica y Biología
Molecular UPV/EHU, Bilbao, ES
Acidification in the endosome causes lipoprotein release by promoting a
conformational change in the LDLR allowing its recycling and degradation
of LDL. Not with standing conformational changes occurring in the LDLR
has expanded considerably, structural changes occurring in LDL particles
have not been fully explored yet. The objectives of the present work were
to study structural changes occurring in ApoB100 by infrared spectroscopy
(IR) and also LDL size and morphology by dynamic light scattering (DLS)
and electron microscopy (EM) at both pH 7.4 and 5.0. We found that pH
acidification from 7.4 to 5.0, resembling that occurring within endosomal
environment, induces a huge reversible structural rearrangement of
ApoB100 that is characterized by a reduction of beta-sheet content in
favor of alpha-helix structures. These structural changes, which are likely
implied in particle release from lipoprotein receptor, also compromise the
apoprotein stability what would facilitate LDL degradation. In conclusion,
the obtained results reveal a more dynamic picture of the LDL/LDLR
dissociation process than previous perceived and provide new structural
insights into LDL/LDLR interactions than can occur at endosomal low-pH
milieu.
Towards a specific cADPR phosphohydrolase by
designed mutagenesis of human ADPRibase-Mn
María Jesús Costas1, José Canales1, Alicia Cabezas1,
Rosa María Pinto1, Joaquim Rui Rodrigues2, João Meireles Ribeiro1,
José Carlos Cameselle1
1
Grupo de Enzimología, Departamento de Bioquímica y Biología
Molecular y Genética, Facultad de Medicina, Universidad de
Extremadura, Badajoz, ES, 2Escola Superior de Tecnologia e Gestão,
Instituto Politécnico de Leiria, Leiria, PT
The NAD+ metabolite cyclic ADP-ribose (cADPR) is a messenger for
Ca2+ mobilization. cADPR turnover is believed to occur by enzymatic
glycohydrolysis to ADP-ribose(1). Mn2+-dependent ADP-ribose/
CDP-alcohol diphosphatase (ADPRibase-Mn) is the only enzyme known
to act as cADPR phosphohydrolase (kcat/KM ≈ 4 × 103 M-1s-1) but it is much
less efficient (150-, 40- or 6-fold lower kcat/KM) than its major activities
on, respectively, ADP-ribose (A), CDP-choline (B) and 2´,3´-cAMP (C).
Mutagenesis of the Phe37, Leu196and Cys253 residues of ADPRibase-Mn
changes enzyme specificity in relative terms, such that the best substrate
of the triple mutant F37A+L196F+C253A is cADPR, with 3-, 1.6- or
9-fold higher efficiencies than on A, B and C. By comparison to the
F37A+L196F mutant, C253A is essential for acquisition of preference
for cADPR(2). Aiming to improve this preference, we have tested new
mutations of Cys253 and Val252. The triple mutant F37A+L196F+C253G,
with a smaller side chain at residue 253 (Ala>Gly), showed an efficiency
increase with cADPR and decreases with A, B and C. The quadruple
mutant F37A+L196F+V252A+C253G, with another side chain made
smaller (Val>Ala), showed additional increase (cADPR) and decreases
(A and C) of efficiencies. The quadruple ADPRibase-Mn mutant
83
Pósters / Posters
displayed kcat/KM ≈ 6 × 104 M-1s-1 on cADPR, which was about 60-, 16or 57-fold higher than on A, B and C, respectively, and represents a
15-fold efficiency increase compared to the wild type. This specificity
was demonstrated in incubations with multiple nucleotides. Further
enhancing of cADPR phosphohydrolase specificity may be obtained by
design of new mutations.
Support: Junta de Extremadura and FEDER (GR15143).
References
[1] Lee, 2012, J Biol Chem 287:31633.
[2] Cabezas et al., 2015, PloS ONE 10(2):e0118680.
P10-13
The principle of microscopic reversibility and
detailed balance for cyclic and acyclic systems
Milagros Molina Alarcón1, Francisco García-Sevilla1, María Llanos
Amo-Saus1, Manuela García-Moreno1, Francisco García-Cánovas2
1
UCLM, Albacete, ES, 2Universidad de Murcia, Murcia, ES
In this contribution, we do not examine neither genesis of the microscopic
reversibility and detailed balance nor their equivalence or difference, in
its conventional and classical expression, which has been widely studied
in the literature(1-3). Considerable confusion and contradictory opinions
surround these concepts in cyclic systems. So, in this communication,
we suggest a general and global formalism, supported by the well-known
Botts and Morales’s cyclic mechanism of enzyme systems. Our analysis
attempts to unify the different positions about these concepts, by means
of generalised valid interpretations of formal enzyme kinetics. The
limitations of the present approach are discussed, and consequences for
universally valid interpretations of phenomenological chemical kinetics
are suggested. As a particular case of this general formalism results the
classical and conventional expression of the microscopic reversibility and
detailed balance. Finally, some considerations about the rapid equilibrium
and steady-state approaches are made. Although our analysis is focussed
to enzyme systems, it could be extended to any cyclic or acyclic system,
enzymatic or not.
XXXIX Congreso SEBBM
ppGpp(1). This molecule takes part in the transition from a regular
transcriptional program to a stress-adapted one. It is known that RelA
is activated to synthesize (p)ppGpp in complex with ribosomes bearing
deacylated-tRNA(2). It is also known that RelA binds to the ribosome
stabilizing an unusual distorted form of the deacylated-tRNA in the
A-site(3). As stringent response is crucial for bacteria survival in stress
condition and virulence, and (p)ppGpp mediated stringent response
system is absence in eukaryotes(4), the enzymes involved in this
mechanism are new targets for drug development, and structural studies
of their activation are needed.
We present a cryo-EM structure of RelA in complex with Thermus
thermopilus 70S ribosome in presence of the antibiotic relacin with
an average resolution around 4 Ǻ. Despite the low occupancy of the
factor, the structure reveals RelA bound to the distorted tRNA in the
A-site of the ribosome. Besides, the map reveals a unique interaction
between RelA and the t-RNA, in which a region of the factor encloses
the deacyalted-tRNA.
References
[1] Potrykus, K. et al., Annu Rev Microbiol (2008) 62: 35-51.
[2] Cashel, M. Annu Rev Microbiol. (1975) 29, 301-318.
[3] Aguirrezabala, X. et al., EMBO Rep. (2013) 14, 811-816.
[4] Hauryliuk,V. et al., Nat. Rev. Microbiol. (2015) 13, 298-309.
P10-15
Molecular characterization of a novel catalytic
macrodomain from Fusobacterium sp
Rubén Zapata Pérez1, Ana Belén Martínez Moñino1, Antonio Ginés
García Saura1, Fernando Gil Ortiz2, Álvaro Sánchez Ferrer1
1
University of Murcia. Campus of International Excellence
“Mare Nostrum”. Departament of Biochemistry and Molecular
Biology. Faculty of Biology, Espinardo, Murcia, ES, 2ALBA
Synchrotron Radiation Facility, Cerdanyola del Vallès, Barcelona, ES
Miguel Zamora Porras, Xabier Agirrezabala, Mikel Valle
CIC bioGUNE, Bilbao, ES
Post-translational modification of proteins by lysine acylation or
deacylation has profound effects on the physiological function
of enzymes. This deacylation, carried out by sirtuins, renders
O-acyl-ADP-ribose (OAADPr) as a product. This latter compound
is substrate for macrodomains, which are conserved structural
domains found in proteins with the ability to bind NAD+ metabolites.
Macrodomains substrate (OAADPr) has been implicated as a signalling
molecule, modulating several cellular processes. A deep in silico
analysis of macrodomain proteins reveals the presence of a putative
macrodomain protein from Fusobacterium sp (FspMacroD) similar
to the human OARD1. In the present poster, the cloning, expression,
purification and activity of the recombinant macrodomain from this
microorganism is described. The gene was cloned first in pET28a
vector, but low expression was achieved. Thus, a multi-tag cloning
approach was carried out, finding that, with a maltose binding protein
tag, its soluble expression was high at 25 ºC. An affinity chromatography
purification method was developed. The molecular mass of purified
FmMacroD was determined by SDS-PAGE (60 kDa). The enzyme
activity was analyzed by HPLC, showing that this macrodomain was
active towards OAADPr, rendering ADPr, the final product of the
reaction. A comparative analysis at sequence level was carried out to
shed light on the classification of this enzyme.
The stringent response is a central adaptation mechanism to the
environment in gram-negative bacteria. This response links nutrient
starvation with the transcriptional control of genes. When levels of
amino-acid are low in bacteria, the amount of deacylated-tRNAs
increases in the cell, and those are detected by stringent factor RelA
which converts ATP and GTP/GDP to the signaling molecule (p)
This study was partially supported by MINECO-FEDER (BIO2013-45336-R)
and by Fundación Séneca (Ayudas Grupos de Excelencia Científica Región
de Murcia, project 19893/GERM/15). R.Z.-P. and A.G.G.-S. are holders of
a predoctoral research contract (FPU) from University of Murcia, Murcia,
Spain. A.B.M.-M. is a holder of research contract associated with the above
MINECO-FEDER project.
In Memoriam. We want to recognize to our fellow, friend and father in
science Ramon Varon who, unexpectedly, passed away when he was
working in finishing this contribution. Rest in peace.
References
[1] Cornish-Bowden, A., Cardenas, M.L. Control of metabolic processes,
Plenum Press, New York, 1990.
[2] Gorban, A.N., Radulescu, O. (2007). Dynamic and static limitation
in multiscale reaction networks, revisited. Physics.Chem-ph arXiv:
physics/0703278v3.
[3] Purich, D.L. Enzyme kinetics. Catalysis and control: A reference of
theory and best-practice methods. Academic Press, London, 2010.
P10-14
Structural study of restriction factor RelA
interaction with bacterial ribosome 70S
84
Salamanca 2016
P10-16
Structural basis for the substrate channel
formation upon dimerization of the
hyperthermophilic Pf2001 esterase
Nathalia Varejao1, Rafael De Andrade2, Rodrigo Almeida2, Cristiane
Ano Bom2, Débora Fogel3, David Reverter1
1
Institut de Biotecnologia i de Biomedicina and Departament
de Bioquimica i Biologia Molecular, Cerdanyola del Valles,
ES, 2Instituto de Química, Programa de Engenharia Química,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, BR,
3
Instituto de Bioquímica Médica Leopoldo de Meis, Programa de
Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, BR
Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes
spread among all forms of life that catalyze the hydrolysis or synthesis
of ester bonds. Among members of esterases, a major biotechnological
interest constitutes the group of hyperthermophilic esterases from bacteria
and archaea, due to its high intrinsic stability at high temperatures. Here,
we describe the structural and functional properties of the Pf2001 esterase
from Pyrococcus furiosus. The crystal structure of the Pf2001 esterase has
been solved at high resolution in two different conformations: monomer
and dimer. Our results reveal important rearrangements in the structure of
the “cap” domain between monomer and dimer, by the formation of an
extensive intertwined helical interface. Moreover, contacts in the dimer
interface are essential for the formation of the hydrophobic substrate
channel in the active site and for the substrate selectivity displayed by
the Pf2001 esterase for medium-size fatty acids (C7-chains). Mutagenesis
and kinetic analysis confirm the major role of the dimer interface in the
enzymatic properties of this esterase. Thus, we propose an activation
mechanism of the Pf2001 esterase by dimerization, which probably occurs
at high temperatures and is necessary for the correct formation of the
substrate selectivity channel in the active-site cleft.
P10-17
Proteínas quiméricas de Chs3 como modelo
para entender el tráfico intracelular de proteínas
de la membrana
Noelia Sánchez Núñez, Carlos Antón Plágaro, Rosario Valle Vicente,
César Roncero Maíllo
Universidad de Salamanca, Salamanca, ES
La proteína Chs3 de S. cerevisiae se ha revelado como un paradigma en
el estudio del tráfico intracelular de proteínas ya que su transporte está
regulado a todos los niveles: salida del retículo endoplásmico (RE),
tráfico desde el Complejo de Golgi, endocitosis, reciclaje endocítico o
degradación en la vacuola. Chs3p es una proteína de membrana politópica
con 6 dominios transmembrana que definen 3 dominios citosólicos y uno
extracelular, cada uno con funciones específicas en la regulación de su
transporte y función.
Para abordar el estudio de estas funciones se han construido una serie
de proteínas quiméricas derivadas de Chs3p que contienen los diferentes
dominios de la proteína fusionada a la GFP. La mayoría de éstas se acumulan
en el RE, confirmando la importancia de la estructura terciaria de Chs3 en
su plegamiento y salida del RE. En muchos casos se observaba también
una tinción citoplásmica, comprobándose que ésta estaba asociada a su
degradación en el citosol. La caracterización de estas proteínas en diversos
mutantes de S.cerevisiae ha permitido demostrar como su degradación es
dependiente en algunos casos de la ruta ERAD (ER-associated protein
degradation) y como la interacción de las quimeras con la proteína silvestre
puede prevenir parcialmente esta degradación. Excepcionalmente, una de
las proteínas mostró una distribución punteada a lo largo de la célula, que
no co-localizaba significativamente con marcadores de cis-Golgi, TGN
(Trans-Golgi Network) o ERES (ER exit sites), por lo que su localización
Pósters / Posters
precisa esta todavía por determinar. Los conocimientos obtenidos de estas
construcciones podrían ofrecer nuevas ideas en los modelos de plegamiento
proteico, extrapolablesa otros eucariotas.
P10-18
Aislamiento y caracterización de albúminas 2S:
desarrollo de herramientas clínicas para
el diagnóstico de la alergia
Cristina Bueno Díaz1, Laura Martín-Pedraza1, Sara Abián1, Pablo
San Segundo-Acosta1, Juan Carlos López-Rodríguez1, Rodrigo
Barderas1, Eva Batanero1, Javier Cuesta-Herranz2, María Teresa
Villalba1
1
Departamento de Bioquímica y Biología Molecular, Faculta de
Ciencias Químicas, Universidad Complutense de Madrid, Villanueva
del Pardillo, ES, 2Departamento de Alergología, Fundación Jiménez
Díaz, Madrid, ES
Objetivos: Identificación de diez albúminas 2S de semillas y frutos secos,
caracterización de su estructura y ensayos inmunológicos con sueros de
pacientes alérgicos.
Métodos: Las albúminas 2S se aislaron por métodos cromatográficos
a partir del extracto proteico. Tras la confirmación de su naturaleza
química por espectrometría de masas MALDI-TOF, se realizó su
caracterización estructural con métodos electroforéticos (PAGE-SDS
mono y bidimensional); la estructura secundaria y su estabilidad térmica
fueron analizadas mediante dicroísmo circular. Por último, se llevaron a
cabo inmunoensayos para comprobar su capacidad alergénica, empleando
sueros de pacientes alérgicos a la mostaza.
Resultados: Las proteínas fueron purificadas a partir de los extractos en
dos pasos cromatográficos, una cromatografía de penetrabilidad seguida de
una de fase reversa por HPLC. Los estudios de espectrometría de masas las
identificaron como albúminas 2S y se determinó la secuencia de algunos
péptidos. Presentan una estructura secundaria generalmente helicoidal,
siendo estables a 85ºC y con intervalos de pI y tamaño muy variados
Discusión: Las proteínas purificadas exhiben características similares a otras
albúminas 2S previamente descritas. La caracterización estructural reveló su
baja masa molecular y pI variados; algunas de ellas presentan la estructura
típica de las proteínas de esta familia, con dos cadenas polipeptídicas de
diferente tamaño, mientras que otras, como la de la pipa de girasol, la de
cacahuete y la de piñón están formadas por una sola cadena. Los estudios
de dicroísmo circular pusieron de manifiesto la alta resistencia de estas
proteínas al tratamiento térmico, a excepción de la albúmina 2S de pistacho.
Conclusiones: Se disponen de 10 albúminas 2S caracterizadas que pueden
ser empleadas como herramientas clínicas en Diagnóstico por Componentes
Puros (CRD) con la tecnología ADVIA-Centauro, en poblaciones alérgicas
españolas, lo que permitirá un diagnóstico más eficaz y un tratamiento más
efectivo de las alergias a alimentos vegetales.
P10-19
Pulmonary surfactant protein SP-B promotes
purinergic-mediated exocytosis of surfactant
by type II pneumocytes
Marta Martínez-Calle1, Bárbara Olmeda1, Paul Dietl2, Manfred
Frick2, Jesús Pérez-Gil1
1
Departamento Bioquímica y Biología Molecular I. Facultad
Biología. Universidad Complutense de Madrid, Madrid, ES,
2
Institute of General Physiology. Ulm University, Ulm, DE
Pulmonary surfactant is a lipoprotein complex essential to lower surface tension
at the respiratory air-liquid interface, stabilizing the lungs against physical
forces tending to collapse alveoli. Surfactant complexes are synthesized by
alveolar type II cells and stored in specialized secretory organelles, called
85
Pósters / Posters
lamellar bodies (LBs). Surfactant is stored in LBs in the form of multilamellar
membrane arrays, where surfactant phospholipids and the hydrophobic
proteins SP-B and SP-C sustain highly packed and dehydrated states.
Stimulation of alveolar type II cells leads to LB exocytosis and surfactant
release. Secreted pulmonary surfactant forms a surface active film along the
air-liquid interface in the alveoli. Surfactant protein SP-B is crucial to promote
formation of the surface active film, and to maintaining their proper structure
along the respiratory dynamics. We report a novel activity of SP-B that may
suppose an important contribution to the homeostasis of pulmonary surfactant
at the alveolar spaces. Membrane-associated SP-B is able to efficiently induce
secretion of pulmonary surfactant from alveolar type II cells. The presence in
the extracellular medium of lipid-protein complexes containing SP-B activates
the P2Y2 purinergic signaling pathway that ultimately triggers exocytosis of
lamellar bodies from alveolar type II cells. This implies that SP-B may not
only be an essential component for the biophysical function of surfactant but
a central element to ensure that a proper density of surfactant complexes is
developed at the respiratory surface.
P10-20
Hydrophobic pores and lipid transfer: a model
for the structure of pulmonary surfactant protein
SP-B and the saposin-like proteins
Barbara Olmeda1, Begoña García-Alvarez1, Manuel J. Gómez2,
Marta Martínez-Calle1, Antonio Cruz1, Jesús Pérez-Gil1
1
Universidad Complutense de Madrid, Madrid, ES, 2Centro de
Astrobiología (INTA-CSIC), Madrid, ES
Surfactant protein SP-B is essential to facilitate the formation and proper
performance of surface active pulmonary surfactant films at the respiratory
air-liquid interface, decreasing the work of breathing and avoiding collapse
of alveoli at expiration. Despite its physiological importance, neither a
structural model nor a molecular mechanism of SP-B is available. SP-B
belongs to the saposin-like family of proteins, which share a common
structural fold even though they show a broad range of different functions,
including colipase, cytolytic and antibiotic activities.
In the present work we have purified native SP-B supramolecular
assemblies using detergent-solubilized surfactant and atomic force and
electron microscopy reveals the presence of ring-shaped particles. This
structure of SP-B assemblies agrees with the structure predicted by a
theoretical modeling based on the crystallographic structure of saposin B,
supporting a model that is consistent with most structure-function features
described for SP-B.
Docking of rings from neighboring surfactant membranes would lead to
formation of SP-B-based hydrophobic tubes, competent to facilitate the rapid
flow of surface active lipids both between membranes and between surfactant
membranes and the interface. The existence of these SP-B complexes not
only sustain the dynamic behavior and mechanical stabilization of the film
required to support breathing, but also explain the permeability of native
surfactant membranes to both polar and non polar molecules. Besides
this, the SP-B oligomeric structure provides a common pattern to sustain
the mechanism of action of other members of the saposin family, based on
potential oligomerization-triggered activities of these proteins.
P10r-21
dUTPasas: la necesidad de lo no esencial
J.Rafael Ciges-Tomas1, Suzanne Humphrey2, José R. Penadés2,
Alberto Marina1
1
Instituto de Biomedicina de Valencia-CSIC, Valencia, ES, 2Institute
of Infection, Immunity and Inflammation. University of Glasgow,
Glasgow, UK
Las dUTPasas (Duts) son enzimas ubicuas que catalizan la hidrólisis de
dUTP generando dUMP y PPi. Anteriormente demostramos que las Duts
de fagos de Staphylococcus aureus actúan como proteínas moonlighting,
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XXXIX Congreso SEBBM
participando en la derepresión de islas de patogenicidad (SaPI). Dicha
actividad señalizadora requiere de un motivo propio de este grupo de
Duts, que denominamos motivo VI, para diferenciarlo de los cinco
motivos catalíticos conservados en todas las Duts. Este motivo VI no
está conservado a nivel de secuencia entre las Duts de fagos de S. aureus,
pero sí a nivel estructural. En base a esta observación hipotetizamos
que la actividad moonlighting de las Duts está regida por la adquisición
de inserciones específicas, motivos VI, cuyas diferencias en secuencia
deben ser responsables de la especificidad por las proteínas diana a
las que regula en cada tipo celular. Para demostrar nuestra hipótesis,
y generalizar la capacidad señalizadora de las Duts, se analizaron las
secuencias disponibles en bases de datos en búsqueda de aquellas que
presentasen inserciones propias de especie. En esta búsqueda se observó
que las Duts de enterococos presentaban una inserción localizada en una
zona característica, aunque varía en tamaño en función de la especie. Esta
característica nos ha hecho pensar que las Duts de estos organismos deben
presentar actividad señalizadora, y que esta inserción debe jugar un papel
importante en esa función. Para confirmar nuestra propuesta llevamos a
cabo una aproximación inicialmente estructural, resolviendo la estructura
tridmensional de las Duts de Enterococcus faecalis y E. faecium, así como
el pariente cercano Lactococcus lactis que carece de dicho motivo VI. Estas
estructuras nos permitieron conocer y delimitar la topología del motivo VI
que, de forma similar a lo observado en las Duts de S. aureus, presenta
una arquitectura estructural similar entre ambos enterococos aunque
su secuencia proteica es radicalmente diferente. En base a estos datos
estructurales hemos podido diseñar una batería de mutantes deleccionales,
así como quimeras entre ambas especies de enterococos, que han sido
evaluadas in vitro e in vivo. Los resultados funcionales y estructurales, que
serán presentados en la comunicación, apoyan el papel señalizador de las
Duts y el papel que juega el motivo VI en esta función.
P10-22
EgcA, bases moleculares de la actividad de
una D-L endopeptidasa implicada en infección
bacteriana
Laura Miguel-Romero1, Gadea Rico-Pérez2, Patricia Casino3,
Francisco García-del Portillo2, Alberto Marina1
1
Instituto de Biomedicina de Valencia-CSIC, Valencia, ES,
2
Centro Nacional de Biotecnología-CSIC, Madrid, ES,
3
Departamento de Bioquímica, Universitat de València. Estructura
de Recerca Interdisciplinar en Biotecnologia i Biomedicina
(ERI BIOTECMED), Burjassot, ES
En las bacterias, procesos tan importantes como el crecimiento, la división,
y la adaptación al medio o infección requieren de cambios constantes de
su pared celular, donde el peptidoglicano es su componente principal.
En el patógeno bacteriano intracelular Salmonella enterica serovar
Typhimurium, la DL-endopeptidasa EcgA (endopeptidase responding
to cessation of growth) es una de las enzimas encargadas de remodelar
esta pared. EcgA hidroliza el enlace γ-D-glutamil-meso-diaminopimélico
(D-Glu-m-Dap) de las cadenas laterales aminoacídicas del peptidoglicano.
EcgA se comporta como un factor de virulencia, ya que su expresión
esta aumentada en bacteria intracelular durante la infección de células
epiteliales y fibroblastos, y es necesaria para producir enfermedad en
el modelo animal de ratón. Como otros factores de virulencia de S.
Typhimurium ausentes en bacterias no patógenas, la expresión de EcgA
está regulada por el sistema de dos componentes PhoQ-PhoP. Para conocer
las bases moleculares de la actividad de este enzima y su participación
en los procesos de virulencia, abordamos su caracterización estructural.
Utilizando técnicas de cristalografía de proteínas hemos resuelto la
estructura tridimensional de la proteína salvajeasí como del mutante
C350A donde la Cys catalítica ha sido substituida por Ala. El análisis
comparativo de estas estructuras entre sí y con otras proteínas de la misma
familia nos han dado indicaciones sobre el reconocimiento y especificidad
por substrato, mecanismo catalítico y regulación del enzima, así como
Salamanca 2016
de su posible mecanismo de regulación. Estas propuestas están siendo
evaluadas por diferentes aproximacionesque van desde análisis in silico
hasta la generación de variantes y su caracterización funcional y biofísica.
Los resultados y su interpretación en el contexto de la actividad del enzima
y su participación en la biología del S. Typhimurium serán presentados en
la comunicación.
Pósters / Posters
given concentrations can rescue the stability of EGFP-UROS mutants. All
together, this indicates that pharmacological chaperones can be effectively
used as a novel therapeutic intervention line against rare diseases, when
derived from protein misfolding.
P10-25
P10-23
Designer Optical Neurotransmitter Sensors
(DOTS)
Inmaculada Sánchez Romero, Harald Janovjak
Institute of Science and Technology Austria (IST Austria), Viena, AT
The foundation of learning lies in synaptic plasticity, the ability of
synaptic connections between nerve cells to change in strength. In
its most common form, activation of N-methyl-D-aspartate receptors
(NMDARs) by chemical neurotransmitters Glutamate, Glycine and or
D-Serine plays a critical role in synaptic plasticity. The limited knowledge
of neurotransmitter signaling at NMDARs prevents us from completely
understanding synaptic plasticity and in turn learning. Our goal is to
engineer a suite of Designer Optical neuroTransmitter Sensors (DOTS) that
can quantitatively and non-invasively visualize these neurotransmitters.
With the exception of Glutamate, no sensors for neurotransmitters exist.
Following the previous approaches to develop sensors for Glutamate, we
have generated novel sensors for D-Serine and Glycine. These specific
and non-invasive optical sensors would provide novel and highly effective
means to directly ‘watch’ neurotransmitter signaling during synaptic
plasticity in situ and in real-time.
P10-24
Pharmacological chaperones as a novel
therapeutic approach against congenital
erythropoietic porphyria (CEP)
Pedro Urquiza Ortiz1, Ana Laín1, Arantza Sanz-Parra1, JM. Blouin2,
E. Richard2, Juan M. Falcon-Perez1, Oscar Millet1
1
CIC BioGUNE, Bilbao, ES, 2Biothérapies des Maladies Génétiques
et Cancers, Institut National de la Santé et de la Recherche
Médicale U1035, Université Victor Segalen, Bordeaux, FR
Congenital erythropoietic porphyria (CEP) is a rare genetic disease
produced by deleterious mutations in UROS gene. Among the most
aggressive mutations, C73R drastically reduces the activity and stability of
uroporphyrinogen III synthase enzyme (UROS), the fourth enzyme of the
haem biosynthetic pathway and it is present in almost one of third of all the
reported CEP cases. As we demonstrated in previous studies, we can restore
the catalytic activity UROS by incorporating residues prone to interact with
R73. Based on this proof-of-concept now we propose a new therapeutic
approach based on the use of pharmacological chaperones to intracellularly
modulate and increase the kinetic stability of the enzyme. Ideally, the
molecules should be able to upregulate the protein’s homeostasis upon
binding the enzyme and stabilizing the folded conformation. Currently, we
have screened a library of 2500 compounds and, interestingly, some hit
compounds stabilize the hostspot C73R in vitro, as monitored by circular
dichroism (CD) and nuclear magnetic resonance (NMR). Additionally, the
intracellular activity for a set of hit compounds was monitored through
the analysis of EGFP-tagged version of UROS(C73R) mutant in M1
Fibroblast human cells. The results obtained at HC automated fluorescent
microscope suggest that the compounds enter into the cells and are able
to interact with the protein UROS(C73R). This result has been further
confirmed using two deletereous mutations resulting in a loss of kinetic
stability (C73R and P248Q) and in a plethora of cells lines (M1, Human
Embryonic Kidney 293 cells (HEK 293) and erythroleukemia k562 cells).
In all cases, we found that the treatment with the selected molecules at
Cloning, purification and characterization
of human Nicotinamide N-Methyltransferase
Ana Belén Martínez Moñino, Rubén Zapata Pérez, Victoria García
Plana, Antonio Ginés García Saura, Álvaro Sánchez Ferrer
University of Murcia. Campus of International Excellence Mare
Nostrum. Departament of Biochemistry and Molecular Biology.
Faculty of Biology, Espinardo, ES
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of
nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine
as donor (EC 2.1.1.1). NNMT plays a significant role in the regulation of
metabolic pathways and is expressed at markedly high levels in several kinds
of cancers, presenting it as a potential molecular target for cancer therapy.
This work describes the cloning, overexpression, purification as well as
the molecular characterization of a soluble human NNMT (hNNMT). We
cloned the NNMT gene in pET28a with an N-terminal His-tag and expressed
it under several expression conditions. After that, an efficient purification
chromatography method was designed with a good enzyme yield. The
molecular mass of purified hNNMT was determined by SDS-PAGE (~ 25
kDa), and by gel filtration (~ 25.10 kDa), confirming the monomeric nature
of hNNMT. The enzyme was active towards nicotinamide in the presence
of S-adenosyl-L-methionine as donor, rendering 1-methylnicotinamide
and S-adenosyl-L-homocysteine as its products, when assayed by HPLC.
The enzyme was also kinetically characterized with both substrates, and its
optimum temperature and pH determined. In addition, a thermal melt assay
was developed using SyPro Orange in order to test new modulators.Finally,
a phylogenetic analysis of this enzyme and other close related NNMTs
found in the databases was also carried out to found conserved blocks.
This study was partially supported by MINECO-FEDER (BIO2013-45336-R)
and by Fundación Séneca (Ayudas Grupos de Excelencia Científica Región
de Murcia, project 19893/GERM/15). R.Z.-P. and A.G.G.-S. are holders of
a predoctoral research contract (FPU) from University of Murcia, Murcia,
Spain. A.B.M.-M. is a holder of research contract associated with the above
MINECO-FEDER project.
P10-26
A mechanism for cargo-specific sorting
from endosomes
Maria Lucas1, David Gershlick2, Ander Vidaurrazaga1,
Adriana Rojas1, Juan Bonifacino2, Aitor Hierro1
1
CIC bioGUNE, Derio, ES, 2Eunice Kennedy Shriver National
Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, US
Endosomes undergo extensive spatiotemporal rearrangements as proteins
and lipids flux through them in a series of fusion and fission events.
These controlled changes enable the concentration of cargo for eventual
degradation while ensuring the proper recycling of other components. A
growing body of studies has now defined multiple recycling pathways
from endosomes to the trans-Golgi network (TGN) which differ in their
molecular machineries. The recycling process requires specific sets of lipids,
coats, adaptors, and accessory proteins that coordinate cargo selection with
membrane deformation and its association with the cytoskeleton. Retromer
is a multi-protein complex that associates with the cytosolic face of
endosomes, where it functions to recycle receptors, transporters, adhesion
87
Pósters / Posters
molecules, and other proteins to the trans-Golgi network (TGN) and the
plasma membrane, through sorting into tubular-vesicular carriers. Defects
in retromer alter the subcellular distribution of its transmembrane cargos
and underlie some forms of Alzheimer’s disease and Parkinson’s disease.
The retromer complex comprises a VPS26-VPS29-VPS35 heterotrimer that
was previously implicated in cargo recognition, and various combinations
of sorting nexin (SNX) proteins that contribute to membrane recruitment
and formation of recycling tubules.
P10-27
A comparative study of assay methods
for screening human Poly(ADP-ribose)
Polymerase inhibitors
Antonio Ginés García Saura, Rubén Zapata Pérez, José Francisco
Hidalgo Céspedes, Ana Belén Martínez Moñino, Álvaro Sánchez
Ferrer
University of Murcia. Campus of International Excellence
Mare Nostrum. Departament of Biochemistry and Molecular Biology.
Faculty of Biology, Murcia, ES
Poly(ADP-ribosyl)ation is a post-translational modification of proteins
involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR)
is a large, structurally complex polymer of repeating ADP-ribose units, and
it is biosynthetized from NAD by poly(ADP-ribose) polymerases (PARPs).
PARP1 is activated by DNA damage and plays a pivotal role in the repair
of DNA strand breaks. The inhibition of PARP1 could have therapeutic
interest in treatment of cancer and ischemia. Thus, the developing of
high-throughput assays (HTS) to screening libraries of potential inhibitors
is of biomedical interest. This work describes the cloning, overexpression
and purification of human PARP1 (hPARP1) to be used in a comparative
study of HTS screening methods due to the high discrepancy between
results found in the bibliography. Two methods, one chemical and another
enzymatic were tested. The first based on reaction with acetophenone at high
temperature and the second, based on the use of alcohol dehydrogenase/
diaphorase with resazurin. The first chemical method was useful in the
NAD+ concentrations range between 0-10000 nM, whereas the enzymatic
was useful between 0-100 nM. The enzyme coupled method was simple
and with less data dispersion. Therefore, the enzymatic method was tested
with two known hPARP1 inhibitors (Rucaparib and Veliparib), finding
a good correlation with described values. Finally, the enzymatic method
was used to test new inhibitors previously selected by molecular docking,
obtaining a new set of hPARP1 inhibitors.
This study was partially supported by MINECO-FEDER (BIO2013-45336-R)
and by Fundación Séneca (Ayudas Grupos de Excelencia Científica Región
de Murcia, project 19893/GERM/15). R.Z.-P. and A.G.G.-S. are holders of
a predoctoral research contract (FPU) from University of Murcia, Murcia,
Spain. A.B.M.-M. is a holder of research contract associated with the above
MINECO-FEDER project.
P10-28
Visión sobre los mecanismos de direccionalidad
cis/trans en la autofosforilación de histidinas
quinasas
Cristina Mideros Mora1, Alberto Marina1, Patricia Casino2
Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, ES,
2
Departamento de Bioquímica. Universitat de València. Estructura
de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI
BIOTECMED), Universitat de València, Valencia, ES
1
Las histidinas quinasas (HK) son proteínas sensoras y transductoras
de señales que junto con las proteínas efectoras denominadas
reguladores de la respuesta (RR), forman los denominados sistemas
88
XXXIX Congreso SEBBM
de dos componentes (TCS). Los TCS son el principal mecanismo de
señalización en bacterias generando respuestas adaptativas a cambios
ambientales; también están presentes en hongos y plantas pero
ausentes en mamíferos. Para transducir la señal las HK se autofoforilan
utilizando ATP en un residuo conservado de His, un proceso regulado
por el estímulo. Después, el grupo fosforilo es transferido a un
residuo de Asp en el RR, activando así la transcripción de genes.
Los TCS se apagan cuando el RR es desfosforilado, un proceso que
se da por autohidrólisis o mediado por la HK. Recientemente se ha
demostrado que las HK pueden autofosforilarse en cis o trans, una
característica derivada de su naturaleza dimérica. De este modo unas
HK pueden autofosforilarse dentro de su propia subunidad, es decir
en cis, mientras otras HK se autofosforilan entre subunidades dentro
del dímero, es decir en trans. Nuestro grupo ha demostrado que la
direccionalidad cis/trans está relacionada con la conexión entre las
dos hélices que forman el dominio DHp, donde se encuentra la His
fosforilable. Más aún, hipotetizamos que esta diferente conectividad
podría tener repercusiones en el reconocimiento entre HK y RR,
afectando a las reacciones catalizadas por los TCS. Para profundizar
en la relación entre la direccionalidad de la autofosforilación y el resto
de reacciones catalizadas por los TCS hemos diseñado y analizado
una bateria devariantes deleccionables del dominio DHp en la HK de
Thermotoga maritima HK853. Hemos observado que la eliminación
de tan solo 2 residuos es capaz de cambiar la direccionalidad de
dicha reacción y que dichos cambios afectan la fosfotransferencia y
defosforilación de su RR. Finalmente hemos analizado como estos
cambios afectan las constantes de unión entre la HK y el RR. Este
estudio nos permite obtener una visión de cómo las reacciones
catalizadas por los TCS afectan al mecanismo de reconocimiento
entre HK y RR.
P10r-29
Structural insights of the calcium mediated
reorganization of the calmodulin/Kv7.2 channel
complex
Eider Nuñez Viadero1, Ganeko Bernardo-Seisdedos1, Carolina
Gomis-Pérez1, Alessandro Alaimo1, Beatriz Peñalver1, Pilar Areso2,
Álvaro Villarroel1
1
Instituto Biofisika (UPV/EHU, CSIC), Leioa, ES, 2Pharmacology,
UPV/EHU, Leioa, ES
The M-current, which is a key controller of neuronal excitability, is
under dynamic control by the phospholipase C cascade, which causes
reduction on PIP2 levels and release of Ca2+ from IP3 sensitive
intracellular stores. Both branches can be used independently by
different G-protein coupled agonists. For instance, whereas M-current
inhibition by muscarinic agonists in sympathetic neurons is mediated
by PIP2, regulation by bradykinin depends on Ca2+ elevation. While
the action of PIP2 in gating is thought to be direct on the channel,
Ca2+regulation is thought to be mediated by calmodulin (CaM), which
binds to an intracellular site of the channel known as helix A + helix
B. The current hypothesis regarding Ca2+ gating posits that CaM wraps
around helix B under resting conditions, and, when CaM becomes loaded
with Ca2+, it embraces both A+B helices simultaneously, causing a large
structural rearrangement. We have examined this issue by monitoring
conformational changes triggered by Ca2+ using FRET. Small FRET
changes between fluorophores linked to the N- and C-terminus of the
same AB domain were observed, which were consistent with minimal
differences on the AB module measured by NMR (see communication
by Bernardo-Seisdedos). In contrast, Ca2+ did not cause changes on the
transfer of energy between fluorophores located at helices A+B (donor)
and CaM (acceptor). Our results suggest that the AB module behaves as
a rigid body around which CaM accommodates, both loaded with and
without Ca2+
Salamanca 2016
P10-30
The Role of RsgA in Ribosomal Maturation
Sean Connell, Jorge Pedro López-Alonso, Paola Fucini, David Gill
CIC bioGUNE, Derio, ES
The bacterial (70S) ribosome consists of 3 RNA molecules and dozens of
ribosomal proteins (r-proteins) which assemble together in a complex and
highly coordinated process. Although in vitro ribosomes can be assembled
by co-incubating their constitutive RNA and protein components, in vivo
the assembly process is facilitated by several trans-acting factors called
ribosome assembly factors. These factors can serve as checkpoints to
monitor the assembly process and/or actively promote rRNA-folding
and r-protein/rRNA interactions to shape the folding landscape. Here we
present a ~5 Å cryo-EM structure of the ribosome assembly factor, RsgA,
bound to the bacterial 30S subunit allowing us to clearly position RsgA on
the ribosome and to build a backbone model of RsgA in its ribosome bound
state. Most obvious in the reconstruction is that the body and platform
domains of the 30S subunit have much higher local resolution than the
head. This stems from the fact that RsgA binding to the 30S subunit
promotes the release of several r-proteins in the head which increases
the flexibility of the rRNA resulting in a loss of order. Notability RsgA
releases r-protein S3, a protein whose premature addition leads to inactive
30S subunits. With regards to the structure of RsgA our reconstruction
suggests that the ordering of the catalytic GTPase centre of RsgA is
dependent on contacts with several distinct rRNA elements (h44, h45,
and h24a) where for example residue A790 (h24a) inserts into a pocket
in the RsgA structure. Therefore, the maturation checkpoint monitored by
RsgA is likely the correct confluence of rRNA helices 44/45/24a and is a
prerequisite for activation of RsgA’s GTPase activity which promotes its
release from the ribosome.
P10-31
Architecture of heteromeric AMPA glutamate
receptors
Beatriz Herguedas, Javier Garcia-Nafria, Ingo H. Greger
MRC Laboratory of Molecular Biology, Cambridge, UK
AMPA-type glutamate receptors (AMPARs) are ligand-gated cation
channels that mediate fast excitatory transmission and play a role in
synaptic plasticity and memory formation. AMPARs predominantly occur
as heteromers of the subunits GluA1 to GluA4. So far, AMPAR structures
have been limited to GluA2 homomers. Here we report the first structures
of AMPAR heterotetramers determined by X-ray crystallography and
electron cryo-microscopy. First, we determined the crystal structures of
the GluA2/3 and GluA2/4 N-terminal domains (NTD), which constitute
the most sequence-diverse half of the receptor and project towards the
synaptic cleft. Structures of NTD heterotetramers revealed a novel compact
conformation with an alternating arrangement of the four subunits around
a central axis. This organization was confirmed by cysteine cross-linking
and permitted us to obtain the structure of an intact GluA2/3 heteromer
by cryo-EM. Two models at resolutions of 8.25 and 10.3 angstroms
were obtained, corresponding to the resting and desensitized states of
the receptor. Our data reveal the organizational features of heteromeric
AMPARs and provide snapshots of their gating transitions in the absence
of ligand.
P10r-32
Structural organization and regulation
of the guanine nucleotide exchange factor C3G
Arturo Carabias del Rey, María Gómez-Hernández, Beatriz
Martín-Gracia, Carmen Guerrero, José M. de Pereda
Instituto de Biología Molecular y Celular del Cáncer (CSIC-USAL),
Salamanca, ES
Pósters / Posters
C3G is a guanine nucleotide exchange factor (GEF) for some members of
the Ras family of GTPases including Rap1 and R-Ras. C3G is involved in
the regulation of a wide range of cellular processes such as proliferation,
differentiation, cytoskeletal remodelling, transformation, and apoptosis.
C3G (120 kDa) is a tripartite protein according to its structural and functional
features: (i) The N-terminal part contains an α-helical rich region that binds
to the cytoplasmic tail of E-cadherin. (ii) The central segment contains five
Pro-rich sequences (P0-P4) that mediate binding to the SH3 domains of at
least Crk, p130Cas, Grb2, Hck and c-Abl proteins. Furthermore Tyr 504
in this region is phosphorylated by Src-family kinases during activation
of C3G. (iii) The GEF activity of C3G lies in the C-terminal third, which
consists of a Ras Exchange Motif (REM) and a catalytic Cdc25H domain.
Here we show that C3G adopts a closed conformation stabilized by
a head-tail interaction. We have identified some residues involved in
this intramolecular interaction. Other GEFs of the Cdc25H family are
autoinhibited by analogous head-tail interactions. We are currently
analyzing the relationship between the conformational state of C3G
and its GEF activity. In this context we are also analyzing the effect of
post-transductional modifications on the structural organization of C3G
and its GEF catalytic activity.
P10-33
Identification of an extranucleolar site
of ribosome production in yeast
Giulia Moriggi, Sonia G. Gaspar, Blanca Nieto, Mercedes Dosil
Centro de Investigación del Cáncer, CSIC/Universidad
de Salamanca, Salamanca, ES
In budding yeast the nucleolus is a crescent-shaped structure that abuts the
nuclear envelope and occupies up to one-third of the nucleus. In mitosis,
the nucleolus remains intact and splits just when the rDNA is segregated,
at the end of anaphase. Thus, the nucleolar ribosome synthesis machinery
persists as a recognizable region throughout mitosis and streams from the
mother into the daughter during telophase. We have unveiled that in early
mitosis there is a site of ribosome production that is separate from the bulk
of the nucleolar material. We found that, during a short time in metaphase,
several ribosome-maturation proteins are present both in the nucleolus
and at a punctate body located in the vicinity of the nuclear envelope in
the emerging daughter cell. Such body is well-separated from the bulk
of the rDNA, which is mostly located in the mother cell, and also from
the daughter spindle pole body. It is a body that contains rDNA-binding
proteins, pre-rRNAs and ribosome maturation factors characteristic of
nucleolar and nucleoplasmic pre-ribosomes, but is devoid of late maturation
factors. The focal accumulation of pre-export 40S subunits at this discrete
site is being used as a tool to study when and how different factors are
incorporated into the 40S subunit synthesis pathway.
P10-34
New insights into ribosome formation
in human cells
Blanca Nieto, Giulia Moriggi, Sonia G. Gaspar, Xosé R. Bustelo,
Mercedes Dosil
Centro de Investigación del Cáncer, CSIC/Universidad
de Salamanca, Salamanca, ES
The nucleolus is a highly structured subnuclear compartment that is
formed around actively-transcribed ribosomal RNA gene repeats. Together
with pre-rRNA synthesis, the nucleolus houses the other major processes
involved in ribosome formation: the processing of pre-rRNAs and the
assembly of ribosomal proteins. Studies in yeast have yielded most of the
current knowledge on the pathway. More than 200 proteins (trans-acting
factors) are known to be essential for specific steps of ribosomal subunit
maturation. The majority of those factors have been found and characterized
89
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in yeast. The functions of the putative human homologs and of other
ribosome biogenesis proteins present only in humans remain largely
unexplored. In recent years, a renewed interest for ribosome synthesis
in humans has emerged by some efforts to find new cancer treatments
thorough inhibition of ribosome production, and by the discovery that
several rare human diseases, the so-called ribosomopathies, are caused by
defects in ribosome maturation. Progress in the field is, however, being
hampered by the lack of success in applying to human cells the techniques
successfully used in yeast. In our laboratory we have optimized a series
of procedures to study pre-ribosomal complexes in human cells. We are
using a combination of microscopy studies, sucrose gradient sedimentation
analysis and size-based separation experiments, to study normally-growing
cells and cells blocked at specific steps of ribosome maturation. Here we
will show our findings on the functions of three trans-acting factors that are
essential for nucleoplasmic maturation and nuclear export of 40S subunits
in humans.
P10-35
Mutations in KCNQ2 causing early infantile
epileptic encephalopathy alter Kv7.2 channel
PIP2 sensitivity
Carolina Gomis-Pérez1, A. Marcé-Grau2, C. Malo1, E. Cuenca-León2,
A. Felipe-Rucián2, M. Raspall-Chaure2, A. Macaya2, P. Areso1, A.
Villaroel1
1
Instituto de Biofisika (CSIC UPV/EHU), Leioa, ES, 2Hospital
Universitari Vall d’Hebron, Barcelona, ES
Mutations in the KCNQ2 gene lead to several neonatal onset seizure disorders
including an epileptic encephalopathy, with adverse neurodevelopmental
outcome. We have found the latter in four patients, carrying de novo (E130K,
W270R and G281R) or maternally inherited(L246F) KCNQ2 mutations .
Here we describe the functional consequences in cells expressing these
channels, alone or in combination with the partner subunit Kv7.3 to mimic
the allelic balance found in humans. All mutations exerted a dominant
negative effect in homomers, whereas Kv7.3 rescued the function of
mutants to different extent. The mutations caused a reduction on current
density with no major changes on voltage-dependency or gating kinetics.
The use of a voltage-dependent phosphatase revealed that the sensitivity to
the essential PIP2 phospholipid is affected in all four mutations to varying
degree. Thus, current density and PIP2 sensitivity may contribute to the
pathogenic mechanism.
XXXIX Congreso SEBBM
hCPOΔC in complex with Nerita versicolor carboxypeptidase inhibitor
(NvCI) was solved by X-ray crystallography at 1.85 Å resolution. The
asymmetric unit of the crystal reveals 2 identical polypeptide chains of
the enzyme (but the biological unit is a monomer, based on gel filtration
experiments) bound asymmetrically to 1 molecule of NvCI. Comparing the
active and inactive forms of the enzyme, we were able to study in detail the
structural features of its catalytic mechanism and the arrangement of the
active site in both situations. As expected, we can observe the movement
of the side chain of Tyr248 of almost 180° from the “up” to the “down”
position upon binding of NvCI. Theoverall structure of hCPO displays
the classic α/β hydrolase fold typical of MCPs and presents all important
residues within the active site involved in substrate binding and catalysis.
Remarkably, the key residue responsible of the substrate specificity
of MCPs (Ile255 in hCPA1, Ile251 in hCPA2 and Asp253 in hCPB) is
replaced in hCPO by an arginine residue (Arg275), which explains the
acidic substrate preference of this enzyme unlike other known human
digestive MCPs. Finally, inhibition constants (Ki) were calculated for
complexes of hCPOΔC andvarious proteinaceous tight-binding inhibitors
of M14A subfamily of MCPs.
P10r-37
Structural insides of Calmodulin/Kv7.2 channel
complex: the calcium effect
Ganeko Bernardo-Seisdedos1, Carolina Gomis-Pérez1, Alessandro
Alaimo1, Araitz Alberdi1, Eider Nuñez-Viadero1, Covadonga Malo1,
Oscar Millet2, Álvaro Villarroel1
1
Instituto Biofisika (CSIC, UPV/EHU), Leioa, ES, 2CIC-bioGUNE,
Derio, ES
Calmodulin (CaM), a bi-partite protein, binding to the A-B module, a
bi-partite target, underlie multiple mechanisms governing the function of
Kv7.2 subunits, which are the main component of the non-inactivating K+
M-current, a key controller of neuronal excitability. Simultaneous binding
to helix A and B is crucial for trafficking to the plasma membrane, and
mediates Ca2+ inhibition. We have resolved [CaM]/[AB-Kv7.2] complex
by NMR and characterized the influence of calcium on the structure.The
result suggest that the complex structure only suffers minor conformational
changes caused by Ca2+ binding. However, the possible mechanism by
which the calcium signaling is mediated in the Kv7.2 channel will be
discussed.
P10-38
P10-36
Enzymatic and structural characterization
of human carboxypeptidase O, a novel digestive
metallocarboxypeptidase with acidic C-terminal
specificity
María del Carmen García Guerrero1, Javier García Pardo1, Roberto
Fernández Álvarez1, Francesc Xavier Avilés Puigvert1, Peter Lyons2,
David Reverter Cendrós1, Julia Lorenzo Rivera1
1
Institut de Biotecnologia i Biomedicina and Departament de
Bioquímica i Biologia Molecular. Universitat Autònoma de
Barcelona, Bellaterra, ES, 2Department of Biology. Andrews
University, Berrien Springs, US
Human carboxypeptidase O (hCPO) belongs to the M14A subfamily of
metallocarboxypeptidases (MCPs) and is an N-glycosylated, membrane-bound
enzyme that participates in the hydrolysis of C-terminal acidic residues from
dietary proteins and peptides, thus complementing the digestive actions of the
well-known pancreatic MCPs hCPA1, hCPA2 and hCPB.
In this study we report the enzymatic and structural characterization of
a recombinant soluble form of hCPO (hCPOΔC). The 3D-structure of
90
Biophysical studies on the interaction among
scFv-h3D6, Aβ and apolipoproteins E and J
Laia Montoliu-Gaya, Sandra Villegas
Protein Folding and Stability Group. Departament de Bioquímica
i Biologia Molecular, Universitat Autònoma de Barcelona,
Cerdanyola del Vallès, ES
The antibody fragment scFv-h3D6 has been shown to be effective
at the behavioral, cellular and molecular levels in the treatment of
the triple-transgenic 3xTg-AD mouse model of Alzheimer’s disease
(AD). Previous studies demonstrated that its ability to aggregate into
a worm-like fibril pathway make this molecule able to withdraw Aβ
oligomers from the amyloid pathway and, in this way, to prevent
its toxicity. Since the important role apolipoproteins E (apoE) and
J (apoJ) appear to play in Aβ clearance and also the fact that the
levels of both apolipoproteins were restored in the 3xTg-mice-AD
after scFv-h3D6 treatment, we aimed to study the interactions among
scFv-h3D6, Aβ and apolipoproteins E and J. To do so, we have used
mimetic peptides (MP) composed by essential structures within these
apolipoproteins for binding to other molecules. We have performed
biophysical characterization of these proteins/peptides alone or in
Salamanca 2016
combination with each other. Our studies show that apoE-MP interacts
with Aβ but no with scFv-h3D6, and when the three are combined
worm-like fibrils cannot be formed. In the apoJ-MP case, it does not
interact with Aβ or neither scFv-h3D6, but when the three molecules
are together there is an interaction and the formation of worm-like
fibrils is potentiated. More studies are necessary, but with these
results we are getting closer to understand how the scFv-h3D6 is able
to reverse AD symptoms in vivo and to set the proper treatment with
such a promising therapy.
Funding: Instituto de Salud Carlos III [FIS-PI113-01330], Generalitat
de Catalunya [SGR-GRC-2014-00885], & Generalitat de Catalunya +
FEDER (50%) [2014-PROD00032]. PIF-UAB student grant (LMG).
P10r-39
From single-molecule protein unfolding to protein
degradation by the proteasome
Alejandra Aguado, Pablo Martín, David Fernández, David
Rodríguez-Larrea
Departamento de Bioquímica y Biología Molecular, Universidad
del País Vasco UPV/EHU and Instituto Biofisika (CSIC, UPV/EHU),
Leioa, ES
Biological nanopores are frequently found within many biological
structures involved in protein folding/unfolding. In the case of the
proteasome, protein degradation requires unfolding and translocation
of protein substrates through a narrow pore into the internal catalytic
chamber. How protein stability relates to protein degradation remains an
unsolved question. Single molecule approaches using protein nanopores
with dimensions comparable to the size of biological molecules have
the potential to provide insight into the underlying physics of the
process. Here we measure the kinetics and energy consumption of the
bacterial proteasome ClpXP during degradation of a battery of mutants
of SsrA-tagged thioredoxin. Single-molecule measurements of protein
unfolding during translocation through the α-hemolysin pore showed
excellent correlation with protein degradation. The thermodynamic
stability as measured with differential scanning calorimetry and the
kinetic stability obtained with urea induced unfolding did not show this
degree of correlation. Our results suggest that the unfolding observed
during nanopore one-end pulling efficiently captures the denaturation
mechanism as it occurs during protein degradation.
P10-40
The spindle-stabilizing function of Cdc14 is
required to promote recombinational DNA repair
María Teresa Villoria López, Facundo Ramos Ochoa, Encarnación
Dueñas, Andrés Clemente Blanco
Instituto de Biología Funcional y Genómica, Consejo Superior de
Investigaciones Científicas (CSIC), Salamanca, ES
Eukaryotic cells are constantly threatened by innumerable sources
of genotoxic stresses that cause DNA damage. In order to maintain
genome integrity, cells have developed a coordinated signaling network
known as the DNA Damage Response (DDR). While numerous kinases
have been thoroughly studied during the activation of the DDR, the role
of protein phosphatases remains elusive. Previous data coming from
our group have revealed the importance of the phosphatase Cdc14 in
promoting recombinational DNA repair. However, how this phosphatase
exerts its molecular function in the DDR is still unknown. Here we show
that a DSB (double strand break) induced by the expression of the HO
endonuclease is actively recruited to one of the SPBs (Spindle Pole
Bodies). Microtubules destabilization by nocodazole treatment during
the induction of the DNA break disrupts SPB-DSB interaction and
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impairs HR (homologous recombination), indicating that SPB integrity
and SPB-DSB binding are essential features of the DNA repair process.
Curiously, inactivation of Cdc14 during the induction of the DNA lesion
causes continuous misalignment of the metaphase spindle, increases
oscillatory SPBs movements, and impairs DSB-SPB tethering. The
DSB-SPB interaction stimulated by Cdc14 requires the N-terminus domain
of the SPB protein Mps3. Overall, this suggests a role of Cdc14 in DNA
repair by promoting spindle stability. Supporting this hypothesis, Cdc14
is released to the nucleoplasm upon HO expression and a distinct focus of
the phosphatase overlaps with the DSB at the SPBs. In a screen looking
for Cdc14 substrates at the SPBs after the induction of a DNA break, we
identified Spc110, the intranuclear receptor for the g-tubulin complex. Loss
of Cdk-dependent Spc110 phosphorylation during DSB induction causes
the same phenotypes as cdc14-1 mutants. Together, our results point to
the function of Cdc14 in DNA repair by promoting SPB stabilization and
SPB-DSB interaction, and suggest that the relocation of damage sites to the
SPBs plays an important role in a naturally occurring repair process that
minimizes genome instability.
P10-41
Cdc14 is released from the nucleolus under
DNA damage and is required for DNA repair
by homologous recombination
Facundo Nehuén Ramos Ochoa1, María Teresa Villoria López2,
Encarnación Dueñas2, Andrés Clemente-Blanco2
1
Instituto de Biología Funcional y Genómica, Consejo Superior de
Investigaciones Científicas (CSIC), Salamanca, ES, 2Instituto de
Biología Funcional y Genómica, Consejo Superior de Investigaciones
Científicas (CSIC), Salamanca, ES
Endogenous metabolic products, such as reactive oxygen species, and
exogenous physical and chemical genotoxic stress, constantly assault
the genetic material in the cell. It has been estimated that there are about
105 lesions per cell per day in humans. In response to such a high levels
of DNA damage, cells have developed a coordinated signalling network
known as the DDR (DNA damage response), which coordinates cell
cycle progression with DNA repair. When this system fails or the rate
of DNA damage exceeds the capacity of the pathway to repair them,
the accumulation of errors can overwhelm the cell and result in early
senescence, apoptosis, or cancer. Recently, multiple lines of evidences
have suggested a complex role for several kinases in the regulation of the
DDR (including the CDK), but little is known about the phosphatases
that revert the effects of these kinases. The serine/threonine phosphatase
Cdc14 was firstly identify in S. cerevisiae as an essential cell cycle
phosphatase required for Cdk inactivation. One special feature of this
phosphatase is its predisposition to dephosphorylate targets of the Cdk,
therefore is reasonable to think that Cdc14 could be a key candidate to
counterbalance the effect imposed by the Cdk in the DDR. In favour
of this hypothesis, Cdc14 is required for cell viability under different
DNA damage conditions. To determine the role of the phosphatase
in the DDR, we used two different recombinational repair pathways:
SDSA (Synthensis dependent strand annealing) and dHJ (double
Holliday junction repair). We observed that cells lacking Cdc14 activity
presented defects in DNA repair in both SDSA and dHJ. Supporting the
role of the phosphatase in DNA repair, we observed that Cdc14 was
released from the nucleolus after induction of a single DSB or treatment
with phleomycin. Finally, by using mass spectrometry in a screen to
identify targets of the phosphatase exclusively in DNA damage, we
have detected several targets directly implicated in DNA damage
repair. Altogether, we have placed Cdc14 at the DNA damage response
context by collaborating in the repair throughout the modulation of
the homologous recombination repair pathway, and providing new
evidences about the role of this phosphatase in the repair of a DNA
lesion.
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Pósters / Posters
P11. Genómica y proteómica /
Genomics and proteomics
P11-1
Estudio molecular y genómico de la contribución
de WHSC1 a la patología del Síndrome de
Wolf-Hirschhorn en pacientes y en modelos
animales
César Cobaleda Hernández1, María Luisa Martínez-Frías2, Sergi
Beltran3, Elena Campos-Sánchez1
1
Centro de Biologia Molecular Severo Ochoa, Madrid, ES,
2
Centro de Investigación sobre Anomalías Congénitas (CIAC),
Estudio Colaborativo Español de Malformaciones Congénitas
(ECEMC), Instituto de Salud Carlos III, Ministerio de Economía
y Competitividad, Madrid, ES, 3Centro Nacional de Análisis
Genómico (CNAG), Barcelona, ES
El Síndrome de Wolf-Hirschhorn (WHS) es una enfermedad poco
frecuente causada por la pérdida de material genético en hemizigosis
en el cromosoma 4(p). Los pacientes presentan numerosos problemas
(malformaciones craneofaciales, discapacidad intelectual, epilepsia,
retraso en el crecimiento, etc.) de bases moleculares aún poco claras. Una
importante causa de mortalidad en el WHS son las infecciones, causadas
por deficiencias del sistema inmune. Uno de los genes delecionados
en 4p es el Wolf-Hirschhorn-Syndrome-Candidate-1(WHSC1), un
regulador epigenético que también participa en reparación del daño al
DNA y cuya sobreexpresión o activación también está implicada en
mieloma múltiple y en leucemias linfoblásticas agudas infantiles. En
la actualidad estamos estudiando el exoma completo de una muestra
de pacientes con WHS, con el fin de determinar si existe acumulación
de daño debida a la pérdida de WHSC1 en 4p, lo que podría hacer
que los pacientes fuesen más susceptibles a desarrollar tumores con
la edad. Además, estamos caracterizando, mediante citometría de flujo
y mRNA-seq, el desarrollo hematopoyético y la función inmune en
modelos de ratón de pérdida y ganancia de función de Whsc1. De esta
manera, combinando la potencia del estudio mediante secuenciación
masiva de muestras de pacientes humanos y de modelos animales
avanzados, esperamos poder identificar los mecanismos moleculares
subyacentes a las alteraciones inmunes y de otros tipos descritas en los
pacientes. Esto permitiría, entre otras cosas, mejorar la calidad de la
asistencia médica a los pacientes de WHS y conocer su susceptibilidad
al daño genético.
P11-2
Heterogeneous susceptibility and evolution
to breast cancer: analysis using a systems biology
approach
Jesús Pérez Losada
Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC),
Universidad de Salamanca/CSIC. Instituto de Investigación
Biomédica de Salamanca (IBSAL), Salamanca, ES
An essential question in cancer is why patients with the same disease
have different clinical outcome. Progress toward a more personalized
medicine in cancer patients requires new strategies to analyze
underlying factors determining heterogeneous clinical evolution. We
integrated genetic, transcriptomic, cell signaling and metabolic profiles
to predict clinical outcomes in a population of mice with variable
susceptibility to breast cancer. Tumors originated in the heterogeneous
population of mice showed similarities with human breast cancer
at those different levels highlighting the importance of studies in
92
XXXIX Congreso SEBBM
heterogeneous populations of mice to extrapolate additional knowledge
to the human population. Consequently, a common gene expression
signature between human and mouse ERBB2 positive breast cancer
predicted evolution in both species. The global architecture of the
studied signaling pathways was similar in breast tumors and livers, and
defined both tumor pathophenotypes and mouse clusters of prognosis.
Mice with intrinsically high levels of AKT and ERK pathways were
more reluctant to develop tumors. Levels of pAKT1(S473) modified
tumor dissemination capability. We identified a metabolic profile
before the onset of the disease that anticipated breast cancer evolution.
We identified the Quantitative Trait Loci associated with the variability
at those different levels. With this approach, we generated prediction
models that better define the heterogeneous behavior of the disease
among individuals considering several molecular layers and tumor
traits simultaneously. Here, we demonstrate by a Systems Biology
approach an effective strategy to identify the heterogeneous behavior
of the ERBB2 positive breast cancer among individuals. This strategy
would help to unravel cancer variability, an essential issue to develop
more individualized medical approaches.
P11-3
Identification of a DNA methylation signature
in liquid biopsy for early non-small cell lung
cancer (NSCLC) diagnosis
Juan Sandoval del Amor1, Ángel Díaz Lagares2, Jesús
Mendez-González3, David Hervás4, María José Pajares5,
María Saigi3, Diana García4, Ana Belén Crujeiras6, Ruben Pío5,
Luis M. Montuenga5, Javier J. Zulueta5, Ernest Nadal3, Antoni
Rosell3, Manel Esteller3
1
IISLaFe, Valencia, ES, 2University Hospital of Santiago
de Compostela (IDIS-CHUS), Santiago de Compostela, ES,
3
IDIBELL, Barcelona, ES, 4Medical Research Institute La Fe,
Valencia, ES, 5University of Navarra, Pamplona, ES, 6Health
Research Institute of Santiago (IDIS-CHUS) and CIBERobn,
Santiago de Compostela, ES
Lung cancer is the leading cause worldwide, mainly due to late
diagnosis. Patient outcome is closely linked to tumor stage at
diagnosis and unfortunately, most lung cancer patients are diagnosed
at late stages when a curative treatment is no longer possible.The
aim of this work was to identify a panel of epigenetic biomarkers for
improving early diagnosis of lung cancer patients using minimally
and non-invasive biological fluids. DNA hypermethylated biomarkers
were identified performing a Genome-wide DNA methylation analysis
(Infinium 450K array) in non-small cell lung cancer (NSCLC) primary
tumors from two different public databases: CURELUNG FP7
Consortium and The Cancer Genome Atlas (TCGA). DNA methylation
levels of selected candidates were analyzed by pyrosequencing in
non- or minimally invasive samples from three independent cohorts
of stage I NSCLC patients and non-tumoral controls: bronchoalveolar
aspirates, bronchoalveolar lavages and sputum. Combined Receiver
Operating Characteristic (ROC) curve was obtained to evaluate the
diagnostic utility of the epigenetic signature. A nomogram was used
to represent the final predictive model for lung cancer disgnostic.
The herein identified DNA methylation signature could improve, in
combination with current diagnostic protocols, the early diagnosis
and outcome of NSCLC patients. The high diagnostic accuracy of
this signature obtained in liquid biopsy offers a minimally invasive
and easy accessible tool for early lung cancer diagnosis. A nomogram
based on the results of this model is proposed as a predictive tool for
clinical diagnostic use.
Salamanca 2016
P11r-4
Histology and transcriptional responses
of biotransformation and antioxidant enzymes
in the liver and testis of Mus spretus mice
exposed to p’,p-DDE
Noelia Morales Prieto1, Isabel Lourdes Pacheco2, Sara López Bellón1,
Ana Luna Alcántara1, José Pérez2, Carmen Pueyo1, Nieves Abril1
1
Department of Biochemistry and Molecular Biology. University of
Córdoba, Córdoba, ES, 2Department of Anatomy and Comparative
Pathology. University of Córdoba, Córdoba, ES
Prohibited in the early 1970’s, the pesticide DDT is still being used to
control insects that carry diseases such as malaria and the Zica virus.
Thus, human and animal populations worldwide are exposed to DDT,
directly or by consuming contaminated food. DDT metabolism generates
p’,p-DDE (1,1-dichloro-2,2-bis(p-chlorophenil)-ethilene), a byproduct
described as an androgen receptor antagonist that can produce male
genital abnormalities and associated with oxidative stress. The molecular
mechanisms and pathological processes underlying the p’, p-DDE toxicity
are still poorly understood.
We have analyzed the impact of p’,p-DDE on Mus spretus hepatic and
reproductive health at histological and transcriptional levels. Mus
spretus, the Algerian mouse, diverged just ~3 million years ago from
the lab mouse Mus musculus and is emerging as particularly appropriate
for studying multigenic diseases, including inflammation and cancer.
Histomorphological analysis of testicular sections revealed normal structure
of seminiferous tubule with well-organized arrangement of germ cells and
somatic cells in control group. The testicular sections of p’,p-DDE exposed
mice were, in contrast, discolored and, on histological examination, showed
tubular damage. In the testes of the exposed mice the seminiferous tubules
were significantly (P<0.001) higher and showed a reduction >30% in the
area occupied by germinal cells and spermatozoids. In the liver, p’,p-DDE
caused cell swelling with small clear vacuoles in the cytoplasm, probably
caused by failure of the Na+/K+ pump. p’,p-DDE differentially altered
the testicular and hepatic mRNA abundance profiles ofgenes involved in
biotransformation pathways (cytochromes and glutathione S-transferases)
and the antioxidant defense (peroxiredoxins, glutathione peroxidases,
catalase, superoxide dismutases), but exerted minimal effects on apoptotic
genes expression. This work will contribute to uncover the involvement
of some detoxifying and antioxidant enzymes on hepatotoxicity and the
impairment of male reproductive health by p’,p-DDE.
P11-5
Efecto de una dieta funcional enriquecida con
probióticos y extracto de dátil sobre la expresión
proteica de la dorada (Sparus aurata)
María José Prieto Álamo1, Ana Belén Plata Gómez1, Esther Donoso
Contreras1, María Ángeles Esteban Abad2, Juan Jurado Carpio1
1
Departamento de Bioquímica y Biología Molecular, Universidad
de Córdoba, Cordoba, ES, 2Departamento de Biología Celular
e Histología, Universidad de Murcia, Murcia, ES
La acuicultura intensiva es una actividad productiva cuyo desarrollo está
condicionado por diversas patologías infecciosas. La prohibición en la
Unión Europea del uso de muchos de los fármacos veterinarios clásicos
exige la búsqueda de alternativas naturales efectivas y seguras. En este
sentido, los probióticos y los extractos vegetales pueden desempeñar un
importante papel. Se ha investigado el efecto a nivel proteómico de una
dieta suplementada con los probióticos Shewanella putrefaciens Pdp11
y Bacillus sp en combinación con un extracto de dátil de conocidas
propiedades antioxidantes sobre especímenes de dorada (Sparus aurata)
cultivados tanto en condiciones normales como en presencia de una
infección experimental por Photobacterium damselae sbsp. piscicida, uno
de los patógenos que diezman las explotaciones de esta importante especie
Pósters / Posters
para la acuicultura española. El análisis de la expresión proteica diferencial
mediante 2-DE ha puesto de manifiesto cambios significativos en 18
proteínas. Algunos son efecto de la dieta mientras que otros son provocados
por la infección. En un análisis preliminar de los cambios de expresión
se han identificado actinas, conocidos sensores redox fisiológicos, lo que
podría relacionarse con el estrés oxidativo inducido por la infección y/o con
el papel antioxidante atribuido a la dieta funcional. Así, se ha comparado
el grado de oxidación reversible de los tioles de las proteínas sin observar,
globalmente, cambios cuantitativos significativos, aunque los patrones de
bandas indican oxidación diferencial en proteínas específicas. También se
ha investigado la actividad de enzimas relacionadas con el estado redox
celular y las defensas antioxidantes: GOR, G6PDH, catalasa y SOD.
Financiación: AGL2011-30381-CO3-03.
P11-6
Breast cancer phenotypic variability is affected
by the biological age
María del Mar Sáez Freire1, Adrián Blanco Gómez2, Sonia Castillo
Lluva3, Chris Lauber4, María Carmen Patino Alonso5, Purificación
Galindo Villardón5, Carmen Martín Seisdedos6,
María Isidoro García6, María Eugenia Muñoz Bermejo7, Julie Milena
Galvis Jiménez8, Trent Northen9, Andrés Castellanos Martín10, Lars
Kaderali11, Jesús Pérez Losada2
1
Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC).
Universidad de Salamanca/CSIC; Instituto de Investigación
Biosanitaria de Salamanca (IBSAL) y Departamento de Fisiología
y Farmacología. Universidad de Salamanca, Salamanca, ES,
2
Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC).
Universidad de Salamanca/CSIC; Instituto de Investigación
Biosanitaria de Salamanca (IBSAL), Salamanca, ES, 3Departamento
de Bioquímica y Biología Molecular I. Facultad de Biología.
Universidad Complutense de Madrid, Salamanca, ES, 4Instituto
de Biometría y Estadística Médica. Facultad de Medicina.
Universidad Tecnológica de Dresden, Dresden, DE, 5Departamento
de Estadística. Universidad de Salamanca, Salamanca, ES,
6
Servicio de Bioquímica Clínica. Hospital Universitario de
Salamanca, Salamanca, ES, 7Departamento de Fisiología y
Farmacología. Universidad de Salamanca, Salamanca, ES,
8
Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC).
Universidad de Salamanca/CSIC; Instituto de Investigación
Biosanitaria de Salamanca (IBSAL), Salamanca, ES; Instituto
Nacional de Cancerología de Colombia, Colombia, CO, 9Department
of Bioenergy/GTL & Structural Biology, Life Sciences Division,
Lawrence, Berkeley, California, US, 10Institute for Research in
Biomedicine (IRB), Barcelona, ES, 11Institute for Bioinformatics.
University Medicine Greifswald, Greifswald, DE,
Breast cancer incidence rates considerably increase with age and young
age at diagnosis correlates with worse prognosis due to a more aggressive
breast cancer behaviour.(1,2) Biological age estimates the functional status of
individuals comparing to other individuals of the same chronological age.
(3)
We defined the biological age integrating phenotypes of oxidative stress,
and calculated Δ biological age as the difference between chronological and
predicted (biological) age. Mice with predicted ages older than chronological
age, were considered biologically older; and mice with predicted ages
younger than chronological age, were considered biologically younger.(4) Our
main goals were (i) define biological age using processes that are common
to cancer and ageing, such as the oxidative stress, and (ii) analyze breast
cancer phenotypic variability according to the biological age. We generated a
cohort of mice with different susceptibility and evolution to ERBB2-induced
breast cancer, using a backcross strategy, and dissected the disease into
different pathophenotypes. We also measured intermediate phenotypes of
oxidative stress. Linkage analysis was carried out to identify quantitative
trait loci (QTL) associated with these phenotypes, and used multivariate
93
Pósters / Posters
models to define biological age. We identified that biologically older mice
developed more aggressive disease than biologically younger mice. We also
identified QTL simultaneously associated with Δ biological age and tumor
pathophenotypes. We identified several genetic and molecular markers that
define biological age and observed that ERBB2 breast cancer phenotypic
variability is affected by the biological age.
References
[1] McPherson K, et al., Bmj. 2000; 321(7261):624-8.
[2] Anders CK, et al., Journal of clinical oncology: official journal of the
American Society of Clinical Oncology. 2008; 26(20):3324-30.
[3] Karasik D, et al., Biological sciences and medical sciences. 2004;
59(3):218-26.
[4] Cho IH, et al., Mechanisms of ageing and development. 2010;
131(2):69-78.
(#)
(*)
Equally contribution as second authors.
Equally contribution as senior authors.
P11r-7
Connection between PKP1 and oncogenic
pathways involving PI3K, NF-κB and H-Ras
detected in lung squamous cell carcinoma cell
lines by gene expression microarrays
Álvaro Andrades1, Joel Martín-Padrón2, Laura Boyero3, Mª Esther
Fárez-Vidal4, Pedro P. Medina1
1
Centre for Genomics and Oncological Research (GENYO);
Department of Biochemistry and Molecular Biology I, School of
Sciences, Granada University, Granada, ES, 2Centre for Genomics
and Oncological Research (GENYO); Department of Biochemistry
and Molecular Biology III and Immunology, School of Medicine,
Granada University, Granada, ES, 3Centre for Genomics and
Oncological Research (GENYO); Department of Biochemistry and
Molecular Biology I, School of Sciences, Granada University;
Department of Biochemistry and Molecular Biology III and
Immunology, School of Medicine, Granada University, Granada,
ES, 4Department of Biochemistry and Molecular Biology III and
Immunology. School of Medicine. Granada University, Granada, ES
Previous studies have identified the plakophilin-1 gene (PKP1) as
a specific biomarker of lung squamous cell carcinoma (LSCC) in
comparison with other types of lung cancer. In order to gain more insight
into the role of plakophilin-1 in LSCC, PKP1 expression was modified
in three LSCC cell lines for subsequent differential expression studies by
gene expression microarrays. In particular, PKP1 expression was inhibited
using siRNA in SK-MES-1 and EPLC-272H, which have comparatively
high basal PKP1 expression, whereas it was overexpressed by means of
lentiviral vectors in NCI-H2170, which has low basal expression of PKP1.
The data generated by the microarrays was analyzed statistically and
functionally. In the analysis, a positive correlation between PKP1 and key
nodes in concogenesis, such as PI3K, NF-κB and H-Ras, was observed.
These results suggest a possible connection between PKP1 and oncogenic
pathways in LSCC cell lines.
XXXIX Congreso SEBBM
of Molecular Biology, Massachusetts General Hospital and Harvard
Medical School, Boston, US, 4Centro Nacional de Análisis Genómico
y Centro de Regulación Genómica, Barcelona, ES
Induction of pluripotency in somatic cells by the Yamanaka factors OSKM is
typically an inefficient and slow process and demethylation of pluripotency
gene promoters has only been detected after about 2-3 weeks. We recently
found that B cells exposed to an 18h pulse of C/EBPα and subsequently
exposed to OSKM efficiently reprogram into iPSCs. We have therefore taken
advantage of this ultra-fast reprogramming system to generate genome wide
nucleotide resolution maps for 5mC and 5hmC (its oxidized derivative) at
different time points during the path towards pluripotency. Already after the
pulse C/EBPα treatment, we observed a gain of 5hmC and a concomitant
loss of 5mC at enhancers of pluripotency genes directly bound by C/EBPα
(including Klf4, Lefty2, Id1 and Tet2). Furthermore, at these sites chromatin
becomes accessible and histones decorated by active marks. The mechanism
underlying C/EBPα local demethylation is likely mediated through the
recruitment of Tet2. This enzyme mediates oxidation of methylcytosine
residues and has been shown to be important for transdifferentiation and
reprogramming. Strikingly, only 24 hours after activation of the Yamanaka
factors methylation at the promoters of the key pluripotency gene Pou5f1
(Oct4) decreases, correlating with the gene’s rapid activation. Our
reprogramming system showing highly dynamic methylation changes may
shed new light on mechanisms of early embryo development and cancer.
P11-9
Mapping and characterization of R-loops causing
genetic instability in S. cerevisiae
J. Carlos Cañas, Belén Gómez-González, Andrés Aguilera
Centro Andaluz de Biología Molecular y Medicina Regenerativa
CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de
Olavide-Junta de Andalucía, Sevilla, ES
R-loops, structures formed by a RNA-DNA hybrid and a displaced
single-stranded DNA molecule, are naturally involved in many different
cellular processes, such as the replication of E. coli plasmids or mitochondrial
DNA, as well as in class-switch recombination of immunoglobulin genes in
B-cells. In certain abnormal circumstances, the co-transcriptional formation
of R-loops can lead to replication fork impairment with deleterious
consequences for genome integrity. To date, such aberrant DNA-RNA
hybrids have been detected through a battery of both indirect and direct tools.
However, at present, neither their length or the frequency nor whether these
hybrids are continuous or discontinuous is known. With the aim of further
characterizing R-loops, we have mapped and counted the DNA molecules
containing DNA-RNA hybrids at the molecular level by the bisulfite
modification assay. We will present the results of the length of R-loops
and the frequency at which they are formed in several mutant strains of S.
cerevisiae, containing mutations in genes involved in different processes
such as mRNA processing or chromatin remodelling. The implications for
the role of R-loops in transcription-associated genetic instability will be
discussed.
P11r-10
P11-8
Rapid demethylation of pluripotency gene
regulatory regions during reprogramming of
C/EBPα-primed B cells into iPSCs
José Luis Sardina1, Antonio Gómez1, Samuel Collombet2, Bruno Di
Stefano3, Ralph Stadhouders1, Clara Berenger1, Carolina Segura1,
Tian V. Tian1, Simon Heath4, Thomas Graf1
1
Centro de Regulación Genómica, Barcelona, ES, 2Institut de Biologie
de l’Ecole Normale Supérieure (IBENS), París, FR, 3Department
94
Proteomic and phosphoproteomic analysis
of the rat pancreas identifies novel targets
involved in the onset of acute pancreatitis
Violeta García-Hernández1, Carmen Sánchez-Bernal1, Domitille
Schvartz2, José Julián Calvo3, Jean-Charles Sánchez2, Jesús
Sánchez-Yagüe1
1
Department of Biochemistry and Molecular Biology, University
of Salamanca, IBSAL, Salamanca, ES, 2Translational Biomarker
Group, Department of Human Protein Sciences, University Medical
Center, Geneva, CH, 3Department of Physiology and Pharmacology,
Salamanca 2016
University of Salamanca, IBSAL, Salamanca, ES
Acute pancreatitis (AP) is an increasingly frequent disorder of the
pancreas that can sometimes be lethal. The development of AP would
be based on early changes in protein expression and signalling pathways
modulating at least the phosphorylation of proteins. We reported here
a Tandem Mass Tag (TMT)-based proteomic and phosphoproteomic
study of rat subcellular fractions of the pancreas during the early phase
of experimental AP. This phase was induced by two s.c. injections of 20
μg cerulein(Cer)/kg body weight at hourly intervals. Changes in protein
expression were validated by Western blotting, and phosphorylation
patterns by Phos-tag SDS-PAGE followed by immunoblot. A
bioinformatics enrichment analysis was performed to characterize
the modulated processes.Out of the around 1000 identified proteins, a
total of 353 unique proteins were found differentially expressed during
AP. 84 and 261 of them were respectively over or underexpressed by
the Cer-treatment, while 8 proteins pointed to a probable subcellular
redistribution. These proteins were mainly related to inflammation
and apoptosis (haptoglobin, clusterin, MCTS-1), trafficking (coatomer
subunits, Rab-related proteins), serine protease inhibition (serpinA1, A6,
B1, A3K) or cytoskeleton impairment (actin, profilin-1). Also, from the
onset of AP we have determined changes in the phosphorylation pattern
of several interesting targets such as fetuin-A or tristetraprolin. We are
currently testing the most promising candidates –including fetuin-A or
various members of the SERPIN superfamily- as early biomarkers of
post-ERCP pancreatitis in human plasma. These data would provide
valuable information for deciphering AP at the molecular level, that may
be potentially useful for its clinical management.
Support: FIS(PI15/01156).
P11-11
Estudio comparativo del estado oxidativo
del tejido adiposo subcutáneo y omental de
pacientes obesos y su contribución al desarrollo
de resistencia a insulina
María del Carmen Navarro Ruiz1, José Alberto Díaz-Ruiz Ruiz2,
José López Miranda3, Rafael Vázquez Martínez1, Rocío Guzmán
Ruiz1, María del Mar Malagón Poyato1
1
IMIBIC / Hospital Reina Sofía / Universidad de Córdoba,
CIBERobn, Córdoba, ES, 2Experimental Gerontology Section,
Translational Gerontology Branch, National Institute on Aging,
National Institutes of Health, Baltimore, US, 3Unidad de
Arteroesclerosis y Lípidos del Hospital Universitario Reina Sofía,
Córdoba, ES
La obesidad se caracteriza por un aumento del tejido adiposo que
predispone al desarrollo de resistencia a insulina. A nivel molecular,
la disfunción de los adipocitos en obesidad se relaciona, entre otros
procesos, con estrés oxidativo. En este trabajo profundizamos en el
estudio de los mecanismos que contribuyen a este proceso y en la
búsqueda de posibles proteínas diana que actúen como marcadores de
disfunción del tejido adiposo en obesidad, teniendo en cuenta la distinta
contribución de los dos depósitos grasos mayoritarios, subcutáneo (SC) y
omental (OM), a la resistencia a insulina. Para ello, realizamos estudios
comparativos de muestras de tejido adiposo SC y OM de pacientes obesos
con distintos grados de sensibilidad a insulina [normoglucémicos (NG),
insulino-resistentes (IR) y diabéticos tipo II (T2DM)] y evaluamos: i)
carbonilación de proteínas mediante derivatización con DNP y análisis
2D-PAGE, ii) peroxidación lipídica mediante trata la medida de 4-HNE
por inmunoblot, iii) especies reactivas de oxígeno (ROS) utilizando la
sonda 2,7’-diclorofluoresceina diacetato y iv) enzimas antioxidantes
[superóxido dismutasa 1 (SOD1), glutatión sintasa (GS)] por inmunoblot.
Observamos que el contenido total de proteínas carboniladas fue mayor
en IR y T2DM vs NG en tejido adiposo OM mientras que no se hallaron
Pósters / Posters
diferencias en el depósito SC. El análisis 2D-PAGE también reveló
perfiles diferentes de proteínas carboniladas entre depósitos. De igual
manera, los niveles de ROS intracelular fueron significativamente
diferentes entre los grupos en OM, pero no en SC, aunque los niveles
de enzimas antioxidantes muestran un perfil similar en ambos depósitos.
En conjunto, nuestros resultados sugieren una respuesta diferencial
dependiente de depósito en relación al desarrollo de enfermedad
metabólica en obesidad y proporcionan nueva información sobre la
contribución fundamental del depósito omental en este proceso.
Financiación: MINECO/FEDER (BFU2013-44229-R; BFU2015-70454-REDT);
JJAA/FEDER (PI-0200/2013); FIS (PIE14_00005) y CIBERobn (ISCIII)
P11r-12
GRAPES: A web-based tool for analyzing,
annotating and filtering structural variants from
Whole-Exome Sequencing
Bernat del Olmo1, Jesús Matés1, Irene Mademont1, Carles
Ferrer-Costa2, Catarina Allegue1, Vincenzo Pascali3, Antonio Oliva3,
Ramon Brugada4
1
Cardiovascular Genetics Center, Biomedical Research Institute
of Girona, IDIBGI, Salt, ES, 2Gendiag.exe SL, Esplugues del
Llobregat, Barcelona, ES, 3Institute of Public Health, Section of
Legal Medicine, Catholic University, Roma, IT, 4Cardiovascular
Genetics Center, Biomedical Research Institute of Girona, IDIBGI;
Department of Medical Sciences, School of Medicine, University
of Girona; and Cardiac Genetics Unit, Cardiology Service, Hospital
Josep Trueta, Girona, ES
Although the advent of Next Generation Sequencing (NGS) has led
already to breakthrough discoveries, two of the most challenging and
limiting aspects that often limit the discovery of new findings using this
technology continues to be data analysis and data integration, especially
for the thousands of laboratories that lack of computational power and
expertise. Here, we report the development of a new web-based platform
to analyze, annotate, and filter Structural variants (SVs) for users with only
basic computational skills. SVs are genetic variants longer than 50 bp that
include Copy Number Variants (CNVs), inversions, tandem duplications
and translocations. CNVs have been associated with various diseases,
including cancer, cardiovascular disease and neurologic disorders.
Whole-Exome Sequencing (WES) is an effective tool to discover common
and rare variants within the coding regions of all human genes. Due to
its widespread adoption, many tools have been designed to detect CNVs
from WES, but only a few include SV discovery support and often they
require advanced bioinformatic skills. Our approach, known as GRAPES
(Genomic Rearrangement Algorithm for Paired-End Sequencing) integrates
coverage signals and misalignment signatures to improve the sensitivity
and sensitivity from WES datasets. We predict that this application will
improve and facilitate the detection of SVs from WES in the clinical and
research fields.
Bernat del Olmo and Jesús Matés: Equally contributing authors.
P11-13
Análisis del secretoma del tejido adiposo
y su papel en la obesidad
D. Pérez-Sotelo1, A. Roca-Rivada2, SB. Bravo3, A. Castro4, J.
Baltar5, I. Baamonde5, FF. Casanueva6, M. Pardo7
1
Grupo Obesidómica, Instituto de Investigación Sanitaria de
Santiago de Compostela (IDIS), Xestión Integrada de Santiago
(XXIS/SERGAS), Santiago de Compostela, ES, 2CIBER Fisiología
de la Obesidad y Nutrición, Instituto de Salud Carlos III, Santiago
de Compostela, ES, 3Unidad de Proteómica (IDIS), (XXIS/SERGAS),
95
Pósters / Posters
Santiago de Compostela, ES, 4CIBER Fisiología de la Obesidad
y Nutrición, Instituto de Salud Carlos III; Grupo Obesidómica,
Instituto de Investigacion de Santiago de Compostela (IDIS),
Xestion Integradad de santiago (XXIS/SERGAS); y Laboratorio
de Endocrinología Molecular y Celular, Instituto de Investigación
Sanitaria de Santiago (IDIS), (XXIS SERGAS), Santiago de
Compostela, ES, 5Servicio de Cirugía General, (XXIS/SERGAS),
Santiago de Compostela, ES, 6CIBER Fisiología de la Obesidad
y Nutrición, Instituto de Salud carlos III; Laboratorio de
Endocrinología Molecular y Celular, Instituto de Investigación
Sanitaria de Santiago (IDIS), (XXIS SERGAS), Santiago de
Compostela, ES, 7CIBER Fisiología de la Obesidad y Nutrición,
Instituto de Salud carlos III; Grupo Obesidómica, Instituto de
Investigación Sanitaria de Santiago de Compostela (IDIS), Xestión
Integrada de Santiago (XXIS/SERGAS), Santiago de Compostela, ES
La obesidad constituye en la actualidad el mayor problema sanitario
a nivel mundial adquiriendo dimensiones epidémicas; se estima que
a este ritmo podría alcanzar un 20% de la población adulta en 2025.
Hasta el momento el número de fármacos que han sido desarrollados
para el tratamiento de la obesidad es muy limitado y no muy exitoso,
siendo la cirugía bariátrica el tratamiento más efectivo hasta la fecha.
Por esta razón, para el desarrollo de nuevos y mejores tratamientos
anti-obesidad, que junto con la dieta y el ejercicio contribuyan a
una reducción del peso corporal, se requiere un mejor conocimiento
de los mecanismos a nivel molecular que controlan el balance
energético. Bajo este contexto, el análisis del perfil secretor del tejido
adiposo, como un actor principal en el desarrollo de la obesidad, se
considera fundamental para entender los mecanismos de regulación
energética. Además del tejido adiposo per se, el lugar anatómico
de su acumulación clasificada como central o periférica, tiene gran
influencia sobre el metabolismo sistémico y sobre el desarrollo de
las patologías asociadas a la obesidad. En el grupo Obesidómica
hemos aplicado técnicas proteómicas al estudio del secretoma de
explantes completos de tejido adiposo omental, subcutáneo y gonadal
procedente de modelos animales de obesidad y anorexia. Además
de adipoquinas clásicas, hemos identificado nuevas proteínas y
descrito un perfil secretor característico en función de su distribución
anatómica. Más recientemente, hemos puesto a punto la técnica
CILAIR (Comparison of Isotope-Labeled Amino acid Incorporation
Rates) al estudio del secretoma de tejido adiposo humano visceral
y subcutáneo de pacientes obesos. Además del alto porcentaje de
proteínas identificadas previamente relacionadas con la obesidad
o sus comorbilidades, el CILAIR se muestra como muy útil para
el estudio del microambiente del tejido adiposo obeso permitiendo
analizar factores recientemente implicados en esta patología como la
remodelización de la matriz extracelular y el perfil inflamatorio.
P11-14
Proteómica funcional para el descubrimiento
de biomarcadores y nuevos fármacos
Manuel Fuentes
Centro de Investigación del Cáncer, Universidad de
Salamanca-CSIC, Salamanca, ES
En la era post-genomica, una vez secuenciado el genoma humano,
uno de los grandes retos en Biomedicina, es comprender la función
de las proteínas codificadas por dichos genes. Aunque, el genoma
posee las instrucciones codificadas de cómo y cuándo expresar las
proteínas, son estas últimas quienes realizan gran parte de las funciones
celulares; principalmente mediante su regulación, interacciones entre
ellas y pequeñas moléculas (ej. cofactores,…), y el desarrollo de los
procesos bioquímicos requeridos para ejercer su función. A pesar de
los grandes avances realizados en genómica y biología molecular,
solamente una pequeña parte del proteoma se ha caracterizado desde
96
XXXIX Congreso SEBBM
el punto de vista bioquímico. Biología de sistemas y proteómica se ha
centrado en la creación de modelos predictivos de rutas de señalización
en base al comportamiento cuantitativo de las proteínas. De hecho, el
conocimiento de estas redes dinámicas de proteínas permitirá describir
interacciones aberrantes relacionadas con procesos patológicos
como el cáncer,… Históricamente, los métodos empleados para la
caracterización bioquímica de estas proteínas solo permiten estudiar de
forma simultánea un número relativamente bajo y, en ocasiones, sin
aportar información dinámica de dichas interacciones. Nuestro objetivo
es mostrar una aproximación metodológica que permite estudiar
cientos/miles de proteínas simultáneamente y en tiempo real, mediante
la combinación de aproximaciones proteómicas y nanotecnológicas,
con el objetivo de identificar biomarcadores y/o nuevos fármacos de
utilidad en medicina personalizada.
P12. Metabolismo del nitrógeno /
Nitrogen metabolism
P12r-1
El óxido nítrico en plantas se produce
por la Mo-enzima ARC con NR
Alejandro Chamizo Ampudia, Ángel Llamas Azua, Aurora Galván
Cejudo, Emilio Fernández Reyes
Universidad de Córdoba, Córdoba, ES
La proteína ARC (Componente de Reducción de Amidoxima) fue
descubierta en 2006 como una nueva enzima que contiene cofactor de
molibdeno, la primera actividad enzimática que se le asigno fue la de
reducción de compuestos N-hidroxilados como la prodroga Amidoxima.
Las proteínas de esta familia se distribuyen a lo largo de los tres reinos de
los seres vivos.
La proteína ARC de plantas se ha caracterizado en Chlamydomonas,
donde se demostró que es una proteína capaz de desarrollar su función
con una NADH-citocromo b5 reductasa y un citocromo b5-1 para
elsuministro de electrones. Curiosamente la estructura formada por
estas 3 proteínas muestra similitud con la nitrato reductasa (NR), ya
que ambas tienen un grupo cofactor de molibdeno, un grupo hemo y
otro grupo FAD, diferenciándose en el sitio activo, donde se encuentra
el cofactor de molibdeno, particular para cada enzima, y el dominio de
dimerización, que solo está presente en la NR. Además se observó que la
NR con Cytb5-1 y Cytb5-R presentan una alta homología de secuencia,
lo que hizo pensar que la NR podría reemplazar a las Cytb5-1 y Cytb5-R
e interactuar con la ARC. En este sentido, se caracterizó esta interacción
y presentó una típica cinética de Michaelis. Además, se estudió como el
nitrito inhibe competitivamente la actividad ARC con benzamidoxima, lo
que indica que el nitrito puede ser un sustrato de ARC en la producción
de óxido nítrico (NO).
Se ha determinado que no hay producción de óxido nítrico in vivo
en un mutante por deleción de la NR, en cambio sí que hemos visto
producción de oxido nítrico en un mutante puntual en la actividad
terminal de NR (actividad dependiente de cofactor de molibdeno), que
es incapaz de reducir nitrato, pero que sí tiene su actividad diaforasa
funcional. Por quimioluminiscencia, se observó que la NR puede
producir óxido nítrico a partir de nitrito, pero no en presencia de nitrato
(mM). Sin embargo, en esta condición de nitrito más nitrato, ARC más
NR sí que puede producir de manera eficiente óxido nítrico a partir de
nitrito, con los electrones proporcionadas por NAD(P)H-diaforasa-NR
hacia la ARC. Además, toda esta producción de óxido nítrico se ha
localizado en el citoplasma.
Salamanca 2016
P13. Neurobiología molecular /
Molecular neurobiology
P13-1
Impact of sexual dimorphism in human
hippocampus during brain ageing
Daniel V. Guebel1, Néstor V. Torres2
1
Biotechnology Counselling Services, Buenos Aires, AR, 2Systems
Biology and Mathematical Modelling Group. Departamento de
Bioquímica, Microbiología, Biología Celular y Genética. Universidad
de La Laguna, San Cristóbal de La Laguna, ES
At the brain level, the effects of sexual dimorphism and normal ageing
seem to overlap. Therefore, to better understand the pathogenesis of many
important neurodegenerative diseases, a robust discrimination between
these effects is required. In this communication we present the results of the
transcriptomic data analysis of samples from human hippocampus, which
map the transition from adulthood to ageing. For this purpose we have used an
optimized technique for microarray post-processing (Q-GDEMAR; Guebel
et al. 2015(1)) extended to a factorial design. We have been able to identify
large sets of differentially expressed genes. The sets of genes operating only
under sex-dependence were disaggregated from those working only under
age-dependence. In addition, both sets of gene types were well-separated
from those operating under sex and age interaction-dependence. The
identified genes were validated against three external, independent sources
of data. The highly significant differences observed among the three types
of genetic dependences lead to the conclusion that sex dimorphism and
sex-for-age interaction should be taken explicitly into account in the studies
of the transition from normal ageing to early neurodegenerative pathologies.
Acknowledgments: This work has been supported by a project grant from
MINECO (ref. BIO2014-54411-C2-2-R) and the IMBRAIN project (ref.
FP7-REGPOT-2012-CT2012-31637-IMBRAIN). DVG was awarded with a
fellowship for visiting professors of Universidad de La Laguna (ref. 597423).
The authors thank Dr. Catalina Feledi for her valuable collaboration.
References
[1] Guebel DV, Perera-Alberto M, Torres NV. Molecular Biosystems.
2015, 12(1):120-32.
P13r-2
Combination of T7 Phage Display and Protein
Microarrays for the characterization of the
humoral response in Alzheimer Disease
Pablo San Segundo Acosta1, Ana Montero Calle1, María Garranzo
Asensio1, Carmen Oeo Santos1, Juan Carlos López Rodríguez1,
Laura Martín Pedraza1, Cristina Bueno Díaz1, Laura Ruiz2, Alberto
Rábano2, Eva Batanero1, Mayte Villalba Díaz1, Rodrigo Barderas
Manchado1
1
Complutense University of Madrid, Madrid, ES, 2Fundación CIEN,
Madrid, ES
Alzheimer’s disease (AD) is a progressive, chronic and neurodegenerative
disorder with a high socioeconomic impact. New approaches to study AD
are necessary to identify new diagnostic biomarkers and therapeutic targets.
Serum autoantibodies from AD patients against altered proteins during the
course of the disease might become a new source of AD biomarkers, useful
for early diagnosis.
A combination of phage display and protein microarrays has been used to
identify AD-specific autoantibodies and their target proteins as biomarkers
of the disease. We have screened two T7 phage display libraries displaying
the cDNA repertoire of the brain of AD patients and healthy individuals’ with
Pósters / Posters
serum from AD patients and controls provided by the CIEN foundation. Both
libraries were iteratively biopanned using serum from healthy individuals
to remove non-specific phages and subsequently with AD patients’ sera to
enrich the libraries in AD-specific phages. After biopanning, 768 phages
from both libraries were printed on nitrocellulose microarrays, probed with
sera from AD patients at different Braak stages and controls to identify
specific phages displaying peptides and proteins recognized by IgGs
from AD patients. Then, specific phages will be sequenced to identify the
displayed peptides and proteins; and their immunoreactivity analyzed by
complementary immunological approaches using a higher cohort of sera.
The identified target proteins will permit the discovery of new
disease-specific biomarkers and the elucidation of new altered pathways
and cellular processes. Further validation, functional analysis and
quantification of the target proteins in sera are planned to determine their
usefulness as blood-based biomarkers and to identify new potential targets
of intervention.
P13-3
Developing a human celular model for the
bacterial amyloidosis RepA-WH1
Aída Revilla-García1, María Moreno-del Álamo2, Juan F.
Giménez-Abián1, Rafael Giraldo1
1
Department of Cellular and Molecular Biology, CIB-CSIC, Madrid,
ES, 2Department of Microbial Biotechnology, CNB-CSIC, Madrid, ES
Protein amyloids arise from the conformational conversion of a soluble
protein into fibrillar aggregates with a crossed b-sheet backbone, leading
to human neurodegenerative and systemic proteinopathies. In bacteria,
amyloids assemble as functional extracellular scaffolds but no natural
proteinopathic amyloidosis has been found in microorganisms yet. The
bacterial protein, RepA can assemble into amyloid fibres upon binding to
plasmid-specific DNA sequences.
RepA-WH1 causes in E. coli an amyloid proteinopathy, which is vertically
transmissible but not infectious, enabling conformational templating by
cross-seeding in vitro and in vivo. Bacterial lineages maintain two mutually
exclusive types (strains) of RepA-WH1 amyloids. The bacterial Hsp70
chaperone DnaK modulates the vertical propagation of these amyloid strains.
We are developing new tools to studying amyloid toxicity elicited
by the synthetic RepA-WH1 prionoid in mammalian cells. For this
purpose, we are now constructing SH-SY5Y based cell lines for the
stable, tetracycline-induced expression of RepA-WH1, targeting it to
either the cytoplasm or the nucleus (the latter depending on a nuclear
localization signal). This objective will identify the route(s) involved in
programmed death of the neuroblastoma cells upon the stable expression
of RepA-WH1(A31V)-TagGFP2. This is the most cytotoxic variant of the
prionoid both in bacteria and, according to our preliminary data on transient
transfections, in human cultured cells too. In parallel, controls carrying just
the fluorescent marker (TagGFP2) and two less cytotoxic variants of the
prionoid (WT and ΔN37) will be also expressed from stable cells lines.
The results presented here empower the bacterial RepA-WH1 prionoid as a
synthetic minimalist model system for amyloid proteinopathies.
P13-4
The WRAP53 protein modulates neuronal
survival after ischemia
Irene Sánchez Morán, Cristina Rodríguez, Ángeles Almeida
Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital
Universitario de Salamanca; Instituto de Biología Funcional y
Genómica (IBFG), CSIC/Universidad de Salamanca, Salamanca, ES
The WD40 domain-containing protein Wrap53 (WD40 encoding RNA
Antisense to p53) is a scaffold protein implicated in Cajal Bodies
maintenance, telomere elongation and DNA repair. WRAP53 loss of function
97
Pósters / Posters
has been related to carcinogenesis and premature aging. Double-strands
breaks may result from ischemic stroke, thereby contributing to neuronal
death and subsequent brain dysfunction. An adequate DNA damage response
is essential to survive after cerebral ischemia and preserve the integrity of the
transcribed genome in neurons. Although DNA repair pathways are active
after ischemia, the molecular mechanisms underlying neuronal survival
remain unknown.
To investigate the role of Wrap53 in neuronal survival after ischemia,
primary mouse cortical neurons were subjected to an experimental
protocol of ischemia in vitro (oxygen and glucose deprivation) for 3
hours and were further incubated in regular medium (reoxygenation). We
first observed that ischemia promoted DNA damage, as revealed by the
accumulation of γH2AX and 53BP-1 in the neurons. Furthermore, we
found a time-dependent increase in WRAP53 gene expression and protein
abundance from 4 hours after the ischemic insult. In parallel, ischemia
induced the traffic of Wrap53 to the nucleus, which has been associated
to cell survival in tumor cells. Moreover, depletion of Wrap53 by siRNA
increased neuronal susceptibility to ischemia-induced apoptosis. Our
results demonstrate that ischemia-induced Wrap53 nuclear accumulation
plays an essential role in neuronal survival.
Funded by ISCIII (PI15/00473; RD12/0014/0007), FEDER, and Junta de
Castilla y León (ISM).
P13-5
Alterations in the ribbon synapse of the first
Pomt1 conditional knockout mouse model of
dystroglycanopathies
Marcos Rubio-Fernández1, Mary Luz Uribe2, Javier Vicente-Tejedor3,
Cristina Susín Lara1, Francisco Germain3, Pedro de la Villa Polo3,
José Martín Nieto2, Jesús Cruces Pinto1
1
Departamento de Bioquímica, Instituto de Investigaciones
Biomédicas Alberto Sols UAM-CSIC; Facultad de Medicina,
Universidad Autónoma de Madrid; Instituto de Investigación
Hospital Universitario La Paz - IdiPAZ, Madrid, ES, 2Departamento
de Fisiología, Genética y Microbiología, Facultad de Ciencias.
Universidad de Alicante, Alicante, ES; Instituto de Investigación
Hospital Universitario La Paz - IdiPaz, Alicante, ES, 3Departamento
de Biología de Sistemas, Facultad de Medicina, Universidad de
Alcalá, Alcalá de Henares, ES
Protein O-mannosyltransferase 1 (POMT1) is one of the causative genes
of dystroglycanopathies (DGPs), a heterogeneous group of congenital
recessive neuromuscular diseases. The most severe DGPs course
with important muscular, brain and ocular anomalies derived from
hypoglycosylation of α−dystroglycan (α−DG), a key protein component
of the dystrophin-glycoprotein complex (DGC) in muscular and
neural cells. α−DG interacts with extracelular matrix (ECM) proteins
by means of its O-glycosylated residues, specifically O-mannosyl
glycans, whose initial mannose is covalently added by POMT1. Our
group has previously evidenced the embryonic lethality of a Pomt1
constitutive knockout (KO) mutation. Consequently, in this work we
generated, by using the Cre-loxP system, a retinal conditional KO
(cKO) mouse model selectively undergoing a Pomt1 intragenic deletion
in developing photoreceptors. Lack of POMT1 in the retinas of these
mice correlated with a loss of α−DG glycosylation and laminin-binding
ability. Electroretinographic (ERG) records in Pomt1 cKO mice showed
a significantly reduced b-wave amplitude and increased implicit time.
By immunohistofluorescence, β-DG and pikachurin (a retinal ECM,
α−DG-interacting-protein) were found to be absent in the outer
plexiform layer (OPL), and a sprouting of bipolar cell dendrites into
the outer nuclear layer was observed. Finally, at the ultrastructural level
ribbon synapses exhibited an anomalous organization, with bipolar
cells processes being barely visible in the synaptic cleft of cones and
rods axon terminals.
98
XXXIX Congreso SEBBM
In conclusion, our findings are strongly suggestive of a pivotal role of
POMT1 and α−DG glycosylation in the formation and function of ribbon
synapses between photoreceptors and bipolar cells in the OPL.
P13-6
The function of alpha-CaMKII is altered
in a mouse model of trisomy 21
Juan José Casañas, Beatriz Galán-Rodríguez, Alexandra
Alves-Sampaio, José Antonio Troca-Marín, María Luz Montesinos
Dpto. Fisiología Médica y Biofísica. IBiS-Universidad de Sevilla,
Sevilla, ES
Local dendritic translation plays an important role in synaptic plasticity.
RNA binding proteins such as the Cytoplasmic Polyadenylation
Element-Binding protein 1 (CPEB1) and the Fragile X Mental Retardation
Protein (FMRP) not only mediate the transport of a number of messengers
into dendrites, but also regulate their translation in response to synaptic
activity. We previously found that local translation is affected in the trisomy
21 (T21) mouse model Ts1Cje (Alves-Sampaio et al., 2010; Troca-Marín
et al. 2011). Now, we have found that the levels of CPEB1 and FMRP
are increased in the Ts1Cje hippocampus. Accordingly, the dendritic levels
of several localized mRNAs, including the alpha-CaMKII messenger,
were augmented. Finally, signalling through alpha-CaMKII in response to
synaptic activity was affected, which could be relevant for the synaptic
plasticity phenotype of this T21 model.
References
[1] Alves-Sampaio, Troca-Marín and Montesinos (2010). J Neurosci
30:13537-13548.
[2] Troca-Marín, Alves-Sampaio and Montesinos (2011). J Neurosci
231:9445-9455.
P13r-7
Is ARMS/Kidins220 involved in the effect
of Syt-IV on BDNF release?
Ana Julia Sánchez Sánchez, Saray López Benito, Juan Carlos
Arévalo Martín
Instituto de Neurociencias de Castilla y León, Universidad
de Salamanca, Salamanca, ES
Neurotrophins are essential for a proper development and functioning of
the nervous system. BDNF, one of the members of the neurotrophin family,
plays a crucial role in physiological and pathological conditions although
the mechanisms underlying its regulated secretion are not completely
understood yet. Previously, it has been proposed that Synaptotagmin-IV
(Syt-IV) acts as a mediator of the BDNF release in neurons. To check
the reliability of Syt-IV in BDNF regulated secretion, we performed
secretion assays under depolarising conditions, in cultured cortical neurons
where Syt-IV levels were reduced. We observed that Syt-IV depletion
potentiated depolarisation-mediated secretion of BDNF. Our studies were
extended to NT-3 and NT-4 treatment and we also observed an enhanced
BDNF release. Since it has recently been reported that ARMS/Kidins220
modulates regulated secretion (López-Benito, 2016), we decided to study
if there was any relationship between this protein and Syt-IV. We have
seen an interaction between them both in over-expression (HEK293
cells) and endogenous conditions (cultured cortical neurons), which has
been further supported by co-localisation assays. Interestingly, an inverse
correlation between their expression and an increased BDNF regulated
release in differentiated cultured cortical neurons was noticed. The
relevance of the interaction of ARMS and Syt-IV is currently under study.
Further experiments will be required to disclose its potential role to fully
understand BDNF regulated secretion.
Salamanca 2016
P13-8
The MDM2-p53 signalling pathway is involved in
neuronal ischemic tolerance
Rebeca Vecino Pérez1, Ángeles Almeida Parra2, María Delgado
Esteban2
1
Instituto de investigación biomédica de Salamanca (IBSAL),
Hospital Universitario de Salamanca, Salamanca, ES; Instituto
de Biología Funcional y Genómica (IBFG), Zamora, ES, 2Instituto
de investigación biomédica de Salamanca (IBSAL), Hospital
Universitario de Salamanca; Instituto de Biología Funcional y
Genómica (IBFG), Salamanca, ES
Brain ischemic preconditioning (IPC) refers to a state of transient tolerance
of the brain tissue to a lethal insult that can be evoked by a prior mild
insult. It is thought that IPC may induce different pathways responsible
for neuroprotection. These mechanisms can involve the attenuation of
cell damage pathways including the apoptotic cell death. In this context,
p53 is a stress sensor that accumulates during brain ischemia leading
to neuronal death. The mouse double minute2 homolog (MDM2), a
p53-specific E3 ubiquitin ligase, is the main cellular antagonist of p53,
mediating its degradation by the proteasome. Increased levels of MDM2
inhibit p53 stability and inactivate apoptotic function, although the role
of MDM2 in neuroprotection is largely unknown.Here, we study the role
of MDM2 and p53 interaction on IPC-induced neuroprotection. Primary
cortical neurons were exposed to a moderate subtoxic concentration of
N-methyl-D-aspartate (NMDA; 20μM NMDA; IPC condition) for 2
hours, followed by incubation for further 90 min in normoxic (presence of
oxygen and glucose) or ischemic (oxygen and glucose deprivation; OGD).
In parallel, control neurons were not stimulated with NMDA. After 4 hours
of incubation in culture medium (reoxygenation condition), neuronal
apoptosis (Annexin-V-staining) was analyzed by flow cytometry. Gene
expression and protein levels were determined by RT-qPCR and western
blotting, respectively.Our results show that IPC induced the expression
of MDM2 in neurons, leading to MDM2 accumulation at 4 hours after
OGD. Moreover, IPC prevented OGD-induced p53 stabilization, caspase-3
activation and apoptosis, which were prevented by neuronal treatment with
the MDM2 inhibitor nutlin-3. These finding demonstrate the key role of the
MDM2-p53 signalling pathway in neuroprotection induced by IPC against
a subsequent ischemic insult and poses MDM2 as an essential target in
ischemic tolerance.
This work was funded by grants from The Instituto de Salud Carlos III
(Miguel Servet I CP0014/00010; RD12/0014/0007) and FEDER (European
regional development funding).
P13-9
Regulation of AMPA receptor trafficking by CPT1C
Maria Casas Prat1, Rut Fadó Andrés1, José Rodríguez Álvarez2,
Núria Casals Farré3
1
Basic Sciences Department, Facultat de Medicina i Ciències
de la Salut, Universitat Internacional de Catalunya, Sant Celoni,
ES, 2Institut de Neurociències and Dpt. Bioquímica and Biología
Molecular, Universitat Autònoma de Barcelona, Cerdanyola del
Vallès, ES, 3Basic Sciences Department, Facultat de Medicina i
Ciències de la Salut, Universitat Internacional de Catalunya, Sant
Cugat del Vallès, ES
Carnitine Palmitoyltransferase 1C (CPT1C) is an isoform exclusively
found in the brain that, unlike the other two mitochondrial isoforms, is
localized in the endoplasmic reticulum and has a residual catalytic activity.
Although CPT1C is not involved in mitochondrial transport of fatty
acids for their oxidation, this isoform is also able to bind malonyl-CoA,
an intermediary metabolite of fatty acid synthesis that decreases during
fasting conditions. It has been suggested that CPT1C might be a sensor of
malonyl-CoA levels in the brain.
Pósters / Posters
Despite the biochemical function of CPT1C remains unknown; recent
studies have demonstrated that this protein is one of the constituents of
AMPA type glutamate receptors (AMPARs) involved in learning processes.
Here, we determined whether basal GluA1 trafficking changed during
metabolic stressed conditions in cortical neurons, and whether CPT1C was
involved in this regulation. Neither total GluA1 levels nor the strength of
CPT1C-GluA1 interaction were altered after 2 hours of TOFA treatment
(an inhibitor of malonyl-CoA synthesis). However, biotinylation assays
indicated that surface GluA1 levels decreased when neurons were treated
with TOFA for 2 hours. By contrast, CPT1C KO cortical neurons did not
respond properly to changes in malonyl-CoA levels. These results suggest
an interesting role of CPT1C as a sensor of malonyl-CoA and a regulator of
GluA1 trafficking during metabolic stressed conditions.
P13r-10
USP8 deubiquitinates TrkB and modulates
TrkB-BDNF functions
Carlos Martín Rodríguez1, Minseok Song2, Begoña Anta3,
Francis S. Lee4, Juan Carlos Arévalo1
1
Institute of Biomedical Research of Salamanca (IBSAL),
Department of Cell Biology and Pathology, Instituto de
Neurociencias de Castilla y León (INCyL), University of Salamanca,
Salamanca, ES, 2Synaptic Circuit Plasticity Laboratory, Department
of Structure & Function of Neural Network, Korea Brain Research
Institute, Daegu, KR, 3Department of Cell Biology and Pathology,
Instituto de Neurociencias de Castilla y León (INCyL), University
of Salamanca, Salamanca, ES, 4Department of Psychiatry, Weill
Medical College of Cornell University, Department of Pharmacology,
Weill Medical College of Cornell University, Nueva York, US
Ubiquitination of the TrkA neurotrophin receptor is a well-recognized
mechanism that plays an important role in nerve growth factor (NGF)
functions. Although TrkB ubiquitination has been also reported, the
molecular machinery involved in the ubiquitination/deubiquitination
of the receptor in response to brain derived neurotrophic factor (BDNF)
is completely unknown. Here we describe the identification of the
ubiquitin-specific protease 8 (USP8), as a deubiquitinase for TrkB. TrkB
and USP8 interact in cultured cortical neurons through the c-terminus of
TrkB and the MIT domain of USP8. Overexpression of USP8 increases the
total amount of TrkB protein and its deubiquitinating activity is required
for this effect. Indeed, in vitro deubiquitination assays show that USP8
deubiquitinates the receptor and kock-down of USP8 protein in neurons
increases TrkB ubiquitination in response to BDNF. In addition, USP8
depletion impairs TrkB endocytosis and BDNF-mediated signaling,
resulting in a reduced dendritic arborization of hippocampal granule
cells in vivo. Altogether these data indicate that the modulation of TrkB
ubiquitination by USP8 regulates the trafficking and the signaling of
the receptor and, consequently, TrkB-BDNF functions, highlighting the
relevance of TrkB ubiquitination.
P13r-11
Regulación de las fosfatasas de especificidad
dual, DUSP, durante la diferenciación de las
neuronas granulares en cultivo. Implicación
del receptor P2X7
Mª José Queipo García, Juan Carlos Gil Redondo, Raquel Pérez Sen,
Esmerilda García Delicado, Mª Teresa Miras Portugal
Departamento de Bioquímica y Biología Molecular. Facultad de
Veterinaria. Universidad Complutense de Madrid, Madrid, ES
Las neuronas granulares de cerebelo de rata se caracterizan por ser un
excelente modelo para estudios de neuroprotección. Éstas constituyen
nuestro principal modelo de estudio y presentan una amplia expresión
99
Pósters / Posters
de receptores de nucleótidos. Nuestro grupo ha podido comprobar
que el receptor ionotrópico de nucleótidos P2X7 es un activador de la
señalización de las quinasas activadas por mitógenos (MAPK) en las
neuronas granulares. Las MAPK están implicadas en procesos celulares
tan trascendentes como proliferación, diferenciación y supervivencia,
motivo por el que están sometidas a una estricta regulación,
principalmente a través de fosfatasas capaces de inactivarlas. Entre
estas fosfatasas cabe destacar las fosfatasas de especificidad dual
selectivas de MAPK (DUSP o MKP), capaces de desfosforilar tanto
residuos de Ser/Thr como de Tyr.
Concretamente, el receptor P2X7 modula de manera significativa la
señalización a través de la vía ERK1/2. Por tanto, centramos nuestros
estudios en las fosfatasas específicas de ERK1/2, como la DUSP6 que
actúa a nivel citoplásmico, y la DUSP1, de carácter nuclear e inducible.
Hemos comprobado que la expresión de DUSP6 y del receptor P2X7
se incrementa de manera paralela a lo largo del desarrollo del cultivo,
mientras que los niveles de DUSP1 disminuyen. Además, la estimulación
del receptor P2X7 con el agonista farmacológico BzATP, modula de
manera bifásica la fosfatasa DUSP6. En una primera fase se observa
una caída de los niveles de DUSP6 consistente con una degradación
inicial vía proteasoma, seguida de una fase de recuperación debida a su
inducción transcripcional. Ambos procesos son dependientes del estado
de activación de ERK1/2, que a su vez se ven moduladas por DUSP6 en
un proceso de retroalimentación negativa. Por otro lado, el receptor P2X7
parece no regular la fosfatasa DUSP1. Sin embargo, su expresión se
encuentra potentemente inducida por la estimulación con la neurotrofina
BDNF, que también activa de manera muy potente las ERK1/2 en las
neuronas granulares.
XXXIX Congreso SEBBM
P13-13
Resveratrol modulates adenosine receptors
endogenously expressed in C6 glioma cells
Alejandro Sánchez-Melgar, José Luis Albasanz, Mairena Martín
Departamento de Química Inorgánica, Orgánica y Bioquímica.
CRIB. Facultad de Ciencias y Tecnologías Químicas / Facultad
de Medicina de Ciudad Real. Universidad de Castilla-La Mancha,
Ciudad Real, ES
Resveratrol (RV) is a polyphenol present in the red wine and others
vegetables products. Epidemiological studies have revealed that resveratrol
could exhibit some neuroprotective role but the mechanisms by which this
polyphenol is acting are still unclear. Adenosine has also a very important
role as a neuromodulator through its receptors. Adenosine receptors are
G protein coupled receptors (GPCR) that inhibit adenylyl cyclase (AC)
activity (A1 and A3) or activate this enzymatic activity (A2A and A2B).
The aim of the present work was to study whether resveratrol was able
to modulate the adenosine receptors in a rat glioma C6 cell line model
which endogenously express these receptors. Our results show a clear
and significant modulation of adenosine receptors gene expression after
resveratrol treatment at 100 μM during 24 hours assessed by real-time
PCR. While A2AR and A2BR genes expression was significantly increased,
A1R and A3R genes were decreased. This was accompanied by a significant
increase in A1R, but not changes in A2AR level, determined by radioligand
binding assays. AC and PKA levels were analyzed by Western blotting
assay. While AC was not significantly affected, PKA was also upregulated
after RV treatment. Results show that resveratrol modulates adenosine
receptors and theirs transduction pathways and suggest a possible role of
these receptors in the effect of resveratrol on cells.
P13-12
Los receptores nucleotídicos P2X7 y los
receptores de EGF regulan los niveles de
la proteína fosfatasa DUSP6 en astrocitos
cerebelosos de rata
Juan Carlos Gil Redondo, María José Queipo García, Esmerilda
García Delicado, Raquel Pérez Sen, María Teresa Miras Portugal
Departamento de Bioquímica y Biología Molecular IV, Facultad
de Veterinaria, Universidad Complutense de Madrid, Madrid, ES
En la regulación de la homeostasis, diferenciación y supervivencia de las
células destaca la vía de las proteínas quinasas activadas por mitógenos
(MAPK). La regulación de la fosforilación y activación de estas serina/
treonina quinasas depende de varias fosfatasas, entre las que encontramos
las proteínas fosfatasas de especificidad dual (DUSP). En concreto, la
fosfatasa DUSP6 (o MKP3) es específica del grupo de MAPK denominadas
ERK. En el presente trabajo, realizado en astrocitos de cerebelo de rata, se
demuestra cómo la estimulación del receptor purinérgico P2X7 es capaz
de regular los niveles de proteína de la fosfatasa DUSP6 de una manera
similar a la realizada por la estimulación del receptor EGF. La estimulación
del receptor P2X7 con el agonista BzATP, o del receptor de EGF, provoca
una caída inicial en los niveles de la fosfatasa, con un pico mínimo en torno
a los 15-30 minutos, seguido por una recuperación a niveles basales tras
una hora de estimulación, superándolos en estimulaciones más largas. La
caída inicial es consecuencia de la degradación proteasomal de la proteína
fosfatasa, como se pudo comprobar al emplear el inhibidor del proteasoma
MG-132. Asimismo, el aumento posterior de los niveles de proteína se
debe a la inducción transcripcional del gen dusp6, como se comprobó
mediante experimentos de PCR cuantitativa. La disminución inicial de los
niveles de fosfatasa coincide en ambos casos con un aumento en los niveles
de fosforilación de las ERK. Tanto la degradación inicial como la posterior
inducción transcripcional son consecuencia de la fosforilación y activación
de las ERK mediante la MAPK quinasa (MEK), como se demostró al
emplear su inhibidor U-0126. Asimismo, se comprobó la participación de
la proteína quinasa C (PKC) en dichos procesos.
100
P13r-14
ARMS/Kidins220 protein controls regulated
secretion of BDNF
Saray López Benito1, Ana Julia Sánchez Sánchez1, Laura Calvo
Enrique2, Cristina Vicente García1, Verónica Inés Brito3, Seila
Fernández Fernández4, Juan Pedro Bolaños4, Silvia Ginés Padrós3,
Lino Tessarollo5, Juan Carlos Arévalo Martín1
1
Department of Cell Biology and Pathology, Instituto de
Neurociencias de Castilla y León (INCyL), University of
Salamanca; Institute of Biomedical Research of Salamanca
(IBSAL), Salamanca, ES, 2Scheeles väg 2, MBB departement,
Karolinska Institutet, Solna, Stockholm, SE, 3Departament de
Biomedicina, Facultat de Medicina, Universitat de Barcelona;
Instituto de Investigación Biomédica August Pi i Sunyer (IDIBAPS);
Centro de Investigación Biomédica en Red sobre Enfermedades
Neurodegenerativas (CIBERNED), Barcelona, ES, 4Department
of Biochemistry and Molecular Biology, University of Salamanca;
Institute of Functional Biology and Genomics (IBFG), University of
Salamanca-CSIC; Institute of Biomedical Research of Salamanca
(IBSAL), Salamanca, ES, 5Neural Development Group, Mouse
Cancer Genetics Program, Center for Cancer Research, National
Cancer Institute, Frederick, Maryland, US
Among the growth factors required for the development and correct
functioning of the nervous system, BDNF is one of the most studied due to
its implication in both physiological and disease conditions. However, the
mechanisms controlling the regulated secretion of BDNF are not completely
understood yet. Here we describe that BDNF secretion induced by
depolarization, NGF, NT-3 or NT-4 is potentiated upon depletion of ARMS/
Kidins220 protein in cultured neurons. To address in vivo the role of ARMS/
Kidins220 in BDNF release, we generated a mouse model to knock-down
its expression in an inducible manner. Cortical slices with reduced levels
of ARMS/Kidins220 showed enhanced BDNF secretion in response to the
aforementioned stimuli. In addition, BDNF levels in the striatum, which come
Salamanca 2016
from cortical and hippocampal projections, are increased in mutant mice with
reduced ARMS/Kidins220 in cortex and hippocampus. Moreover, the levels
of BDNF were further increased in the striatum and hippocampus, but not in
the cortex, of mice depleted of ARMS/Kidins220 that experienced physical
activity with respect to control mice. Since BDNF secretion is impaired
in mouse models of Huntington disease, we sought to check ARMS/
Kidins220 levels in these mice. ARMS/Kidins220 protein is increased
in the hippocampus of HD mice and reduction of ARMS/Kidins220 levels
rescued the secretion defect increasing BDNF release to that one of control
mice. Altogether this data indicated that ARMS/Kidins220 protein controls
regulated secretion of BDNF and may play a crucial role in the pathogenesis
of Huntington´s disease.
P13-15
Effects of febrile seizures on adenosine
receptors in neonates
María Crespo Gutiérrez, David Agustín León Navarro,
Mairena Martín López
Facultad de Ciencias y Tecnologías Químicas. Dpto de Química
Inorgánica, Orgánica y Bioquímica, Facultad de Medicina de Ciudad
Real. CRIB. Universidad de Castilla-La Mancha, Ciudad Real, ES
Pósters / Posters
and deposits of β amyloid caused by aberrant cleavage of its precursor
(APP). Adenosine receptors are G-protein coupled receptors distributed in
the CNS. They can inhibit or stimulate adenylyl cyclase activity, mediating
different physiological responses in cells, and have a neuroprotective role.
It is also known that protein kinase A (PKA) is implicated in learning and
memory processes. Previous results of our group showed that adenosine
A1 and A2A receptors are significantly increased in plasma membranes
from frontal cortex brain in AD. The aim of the present work was to study
different components of transduction pathway in the same cortical area.
To this end, cytosolic fraction of post-mortem frontal cortex brain from
different stages of AD patients and age-matched controls were extracted.
PKC activity was significantly increased from early stages of AD (I-II
of Braak). However, PKA activity revealed a biphasic profile decreasing
in early-medium and increasing in advanced stages of AD. A1 and A2A
receptors analysed by Western-blot were both significantly increased in
early stages of AD and AC1, the main isoform coupled to these receptors,
was also increased in the same stages. Moreover, βA1-40 was significantly
increased from early stages while βA1-42 was increased only in advanced
stages in AD. These results show modulation of transduction pathways
mediated by adenosine and suggest A1 and A2A receptors, ACI, PKA and
PKC as promising pharmacological targets in AD.
P13r-17
Febrile seizures have been associated with epilepsy development but the
underlying mechanism isstill poorly understood. Although febrile seizures
are commonly related to the brain cortex, several studies have suggested
that other sub-cortical areas, such as the cerebellum, could be also involved.
Febrile seizures are one of the most typical convulsive disorders in the
children between 3 months and 6 years corresponding to 2 weeks of life in
rodents, stageat which the cerebellum is still under development. Adenosine
is a nucleoside widely recognized asan endogenous modulator of neuronal
excitability, which exerts neuroprotective and anticonvulsantactions
in the brain. We previously showed short term modulation ofA1R and
A2AR and 5’-nucleotidase (5’-NT) activity in cortex from rat brain
following hyperthermia-induced seizures, suggesting a neuroprotective
role of adenosine. The aim of the present work was to study whether
both receptors and 5’-nucleotidase activity could also be modulated in
the cerebellum in response to hyperthermia-induced seizures. Neonates
were sacrificed 48 hours, 5 and 20 days after hyperthermia seizures and
cerebellar plasma membranes were isolated. The effect of fever seizures on
A1R and A2AR was studied by radioligand binding assays using[3H]DPCPX
and [3H]ZM241385 as radioligands, respectively. A1Rs were significantly
increased after 48h of hyperthermia and no significant differences were
observed after 5 or 20 days. However, A2ARs were affected in a biphasic
manner being decreased after 48 h and increased after 5 and 20 days of
hyperthermia. Changes on receptors were accompanied by affectation
of 5’-NT activity. These results suggest that adenosine could exhibit a
possible neuroprotective role on hyperthermia seizures alsoin cerebellum.
P13-16
Modulation of PKA and PKC in the frontal cortex
of Alzheimer’s disease human brains
Patricia Alonso Andrés1, José Luis Albasanz Herrero1, Isidro Ferrer
Abizanda2, Mairena Martín López1
1
Faculty of Sciences and Chemical Tecnologies. Faculty of Medicine
of Ciudad Real. Inorganic, Organic Chemistries and Biochemistry
Department. CRIB. Castilla-La Mancha University, Ciudad Real,
ES, 2Institute of Neuropathology Pathological Anatomy Service.
University Hospital of Bellvitge, L’Hospitalet de Llobregat, ES
Alzheimer’s disease (AD) is the main neurodegenerative disease in aging
that affects to 24 millions of people around the world. AD is characterized
by cognitive, behaviour and memory alterations. In human brain, it has been
found neurofibrillary tangles with abnormal phosphorylated tau proteins,
p73 is required for ependymal cell ciliogenesis
and Planar Cell Polarity
Sandra Fuertes Álvarez1, Natalia Robledinos-Antón2,
Laura González-Cano3, Isabel Fariñas4, M. Margarita Marqués5,
María C. Marín6
1
IBIOMED, Universidad de León, Valencia de Don Juan, ES,
2
Institute of Biomedicine (IBIOMED), University of León (Current
address: Alberto Sols Institute, CSIC, University of Madrid, León,
ES, 3Institute of Biomedicine (IBIOMED), University of León
(Current address: Luxembourg Centre for Systems Biomedicine,
University of Luxembourg, Esch-Belval, Luxembourg), Leon, ES,
4
Departament of Cellular Biology and CIBERNED, University
of Valencia, Burjassot, ES, 5Department of Animal Production,
University of León, Leon, ES, 6Institute of Biomedicine (IBIOMED),
University of León, León, ES
p73 is a transcription factor that belongs to the p53 family, together with p53
and p63. These genes regulate important processes, like cell differentiation
or stem cell renewal. In particular, p53 and p73 are essential regulators of
Neural Stem Cell biology. Examination of Trp73 knockout mice revealed
TP73 as a key regulator of central nervous system development. These
animals have several neurological abnormalities such as hippocampal
dysplasia and reduced cerebral cortex. Our group has previously
demonstrated the role of TP73 in neural stem/progenitor cells self-renewal.
However, this p73-function does not explain some of the neurological defects
observed in p73KO mice, like hydrocephalus. The adult subventricular zone
contains one of the neurogenic niches of the brain, comprised of neural stem
cells (B-cells), proliferating progenitor cells (C-cells), neuroblast (A-cells)
and ependymal cells (ECs). This germinal centre is organized in structures
called pinwheels in which ECs surround B-cells. We have has demonstrated
that p73 deficiency halts ECs maturation hindering SVZ neurogenic
cytoarchitecture and disturbing the correct neurogenic function.EC ciliary
function is fundamental for appropriate brain function. In this regard, EC
ciliogenesis is dependent upon the establishment of Planar Cell Polarity
(PCP). We have addressed whether lack of p73 affected EC ciliogenesisand
have observed that cilia development is altered in p73KO mice. Strikingly,
ECs lacking p73 fail to establish PCP. Our data have revealed a novel p73
function in ependymal cell maturation and ciliogenesis, and moreover,
identify p73 as a key regulator of PCP.
Work supported by: Grant LE310U14 (Junta de Castilla-León), Grants
SAF2012-36143 and SAF2015-71381-R (Ministerio de Ciencia e Innovación).
101
Pósters / Posters
P13-18
Alterations in hippocampal cells genome
are required for memory formation
Ángel Manuel Carrión Rodríguez
Universidad Pablo de Olavide, Sevilla, ES
The memory formation phenomenon is a complex process that involve
information codification and storage. During this process acquired
information goes from an initial and labile memory state to and stable
memory state by a protein synthesis-dependent mechanism called as
consolidation. Alterations of protein synthesis requires of chromatin
remodelling, through epigenetic alterations, together with transcription
factor activation. In the last decade, we have study the role of some
epigenetic modifications in learning and memory processes. Histone
acetylation and poly-ADP-ribosylation (PARylation) are processes link to
chromatin remodelling and gene expression activation. We demonstrated
that histone H3 acetylation and histone H1 PARylation are involved in
hippocampal-dependent memory storage. In addition, both epigenetic
modifications happen sequentially after training session in order to
promote IEG expression. In addition to de novo protein synthesis, it has
been postulated that neuronal genome sequence alterations may encode
memory trace. This hypothesis seems to be corroborate by two recent
discoveries: a, physiologic electric activity provoke transient DNA breaks
in hippocampus; and b, neuronal genome is a mosaic. During the last
years, we discovered that both DNA metabolism, DNA breaks and repair
mechanisms, and LINE1 retrotransposition are induced after a training
session. Interesting, pharmacologic or genetic inhibition of both processes
produced long-term memory formation impairment. All these data together
indicate that transient alterations in epigenetic factors as well as stable
DNA sequence alteration contribute to the de novo new protein expression
programs required for stabilized the long-term memory trace to from
persistent memories.
P13-19
Expression and regulation of the methionine
cycle genes in the mouse cochlea
Néstor Vallecillo1, María A. Pajares2, Steven Zeisel3,
Lourdes Rodríguez de la Rosa4, Isabel Varela-Nieto4
1
Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM),
Madrid, ES, 2Instituto de Investigaciones Biomédicas Alberto Sols
(CSIC-UAM); Instituto de Investigación Sanitaria La Paz (IdiPAZ),
Madrid, ES, 3Department of Nutrition, School of Public Health,
University of North Carolina Chapel Hill, North Carolina, Chapel
Hill, US, 4Instituto de Investigaciones Biomédicas Alberto Sols
(CSIC-UAM); Instituto de Investigación Sanitaria La Paz (IdiPAZ);
CIBERER, ISCiii , Madrid, ES
The impairment in cochlear homocysteine (Hcy) metabolism has been
associated with hearing loss(1). Epidemiological studies revealed a
correlation between hearing loss and increased plasma Hcy(2). Hcy
represents a key point in the remethylation pathway of the methionine
cycle. Betaine homocysteine S-methyltransferases (BHMT) are
responsible for the remethylation of Hcy. Cochlear BHMT and Hcy
metabolism have been reported to have organ-specific traits(1). Hearing
was evaluated by auditory brainstem response (ABR) recording of 2-3
month-old mice of different strains. Cochlear samples were used for
analysis of selected methionine cycle enzymes by RT-qPCR and Western
blotting.
The methionine cycle was studied in control and VS noise-exposed
C57BL/6J mice (105 dB SPL, 2-20 kHz, 30 min). Two days post-noise,
exposed mice showed increased ABR thresholds and reduced cochlear
expression of Bhmt and Bhmt2. No changes were observed inMat2a, Mat2b
and Ahcy expression or in BHMT, BHMT2, MATα2, MATβ and AHCY
levels. As reported(1), a BHMT band of 68 kDa was found in C57BL/6J
102
XXXIX Congreso SEBBM
cochleae. In contrast, the canonical BHMT band of 45 kDa was detected in
Ola:MF1 mice, suggesting a strain-specific regulation.
To further explore the role of BHMT, we studied the hearing phenotype of
Bhmt+/+, Bhmt+/-and Bhmt-/-C57BL/6x129sv mice(3). Noise exposure caused
a significant and irreversible increase in ABR thresholds in Bhmt-/- with
respect to Bhmt+/+ mice. Bhmt-/-but notBhmt+/+ mouse cochleae showed
significantly increased levels of Bhmt2, no differences were detected in
Mat2a, Mat2b and Ahcy. These data suggest that BHMT plays a central
role in cochlear methionine metabolism and that Bhmt2 could carry out
a compensatory role in cochlear protection against noise injury in the
absence of Bhmt.
This work was supported by Spanish “Ministerio de Economía y
Competitividad” SAF2014-53979-R and FP7-INNOVA-2-AFHELO grants
to IVN.
References
[1] Martínez-Vega et al., FASEB J. 29: 418–432. 2015.
[2] Gok et al., Auris Nasus Larynx, 31:19-22. 2004.
[3] Teng et al., J Biol Chem. 286(42):36258-67. 2011.
P13-20
The APC/C-Cdh1-Rock2 pathway maintains
neuronal network integrity in the adult brain
Verónica Bobo-Jiménez, María Delgado-Esteban, Juan P. Bolaños,
Ángeles Almeida
Institute of Biomedical Research of Salamanca, University Hospital of
Salamanca-University of Salamanca and Institute of Functional Biology
and Genomics, University of Salamanca-CSIC, Salamanca, ES
Dendrite network disruption contributes to the pathology of
neurodegenerative disorders, such as Alzheimer’s disease (AD). However,
the underlying molecular mechanisms are unknown. Here, we show that
postnatal deletion of Cdh1, a cofactor of the anaphase-promoting complex/
cyclosome (APC/C) ubiquitin ligase, in neurons (Cdh1 cKO), disrupted
dendrite arborization and caused dendritic spines and synaptic loss in the
cortex and hippocampus, concomitantly with memory impairment and
neurodegeneration during mouse adulthood. We found that the dendrite
destabilizer Rho protein kinase-2 (Rock2), which accumulates in the
brain of AD patients, is an APC/CCdh1 substrate in vivo, and that Rock2
protein and activity increased in cortex and hippocampus of Cdh1 cKO
mice. In these, inhibition of Rock2 activity, using the clinically approved
drug Fasudil, prevented dendritic network disorganization, memory loss
and neurodegeneration. Thus, APC/CCdh1-mediated degradation of Rock2
maintains dendritic network, memory formation, and neuronal survival
thereby pharmacological inhibition of aberrantly accumulated Rock2 is a
suitable therapeutic strategy against neurodegeneration.
P13r-21
The p53 Arg72Pro single nucleotide
polymorphism determines amyloid-ß neurotoxicity
upon Cdk5-induced p53 stabilization
Rebeca Lapresa, Juan Pedro Bolaños, Ángeles Almeida
Instituto de Biología Funcional y Genómica (IBFG),
CSIC-Universidad de Salamanca and Instituto de Investigación
Biomédica de Salamanca (IBSAL), Hospital Universitario de
Salamanca, Salamanca, ES
The p53 tumor suppressor protein functions as a key regulator of cell
apoptosis and has been described to accumulate in affected brain areas
from Alzheimer’s disease (AD) patients. However, the role of p53 in AD
is controversial. This protein naturally occurs in humans in two functional
variants with single nucleotide polymorphism (SNP) resulting in Arg or Pro
Salamanca 2016
at residue 72 that modulates the apoptotic activity of the p53 protein. Here, we
evaluated the impact of the p53 Arg72Pro SNP on amyloid-ß (Aß)-induced
neuronal apoptosis. Exposure of cortical neurons in primary culture to Aß
triggered cyclin dependent kinase-5 (Cdk5)-induced p53 phosphorylation and
stabilization, leading to mitochondrial dysfunction and neuronal apoptosis,
which were prevented by genetic inhibition of Cdk5 and p53. In cortical
neurons from humanized Tp53 Arg72Pro knock-in mice, we found that neurons
expressing the Arg72-p53 polymorphic variant showed a higher susceptibility to
Aß-mediated mitochondrial depolarization and neuronal death when compared
with the Pro72-p53 variant. This effect was abrogated by ectopically expressing
apoε4 –a well-known major risk factor for AD- but not apoε3 and apoε2.These
results indicate that neuronal susceptibility to Aß toxicity is conditioned by the
Tp53 Arg72Pro SNP, hence suggesting that this SNP may be considered as a
genetic biomarker for AD risk in apoε4 non-carriers.
P13-22
L-lactate mediated neuroprotection against
glutamate-induced excitotoxicity requires
ARALAR-MAS pathway
Beatriz Pardo Merino, Irene Llorente-Folch, Carlos Rueda,
Irene Pérez-Liébana, Jorgina Satrústegui
Centro de Biología Molecular Severo Ochoa (CBMSO), CSIC-UAM;
Centro de Investigación Biomédica en Red en Enfermedades Raras
(CIBERER); Instituto de Investigación Sanitaria Fundación Jiménez
Díaz, Madrid, ES
ARALAR/AGC1 is the aspartate-glutamate carrier in neuronal mitochondria
and constitutes the regulatory step in the malate-aspartate NADH shuttle (MAS)
in brain. The lack of ARALAR expression provokes, in mice, neurological
disturbances such as epilepsy, hypomyelination, and deficits in motor
coordination; and in human, the rare disease “global cerebral hypomyelination”
with similar characteristics to those described for mice. Glutamate
excitotoxicity plays a key role in the induction of neuronal cell death occurring
in many neuropathologies, including epilepsy. ARALAR deficiency did not
aggravate glutamate-induced neuronal cell death in vitro; however, L-lactate
acting as an additional metabolic source rescued from glutamate-induced
neuronal death only control, but not ARALAR-deficient neurons. L-lactate
ameliorated glutamate-stimulated neuronal respiration partially preventing the
decrease in the cytosolic ATP/ADP ratio induced by glutamate, and drastically
reduced reactive oxygen species formation in mitochondria (measured as
accumulation of 8-oxoguanosine) only in ARALAR-expressing neurons.
Systemic administration of the glutamatergic agonist kainic acid (KA) is a
well characterized model to study epilepsy-induced brain damage. The loss of
half-a-dose of ARALAR in aralar+/- mice produced a normal lifespan with no
apparent phenotypic disturbances in basal conditions. However, these aralar+/mice suffered enhanced KA-induced seizures and hippocampal neuronal cell
death with respect to control animals, in an in vivo model of excitotoxicity
in which increased L-lactate levels and L-lactate consumption have been
proven. Failure to use lactate is possibly responsible for the exacerbated in vivo
excitotoxicity in aralar+/- mice.
Pósters / Posters
of trisomy Ts16 and euploid mice. We report that in the plasma membrane
of euploid cells an increase in phosphatidylcholine concentrations occurs in
the presence of oleic acid. However, in trisomic cells oleic acid failed to
increase phosphatidylcholine incorporation into the plasma membrane. Gene
expression analysis of trisomic cells revealed that the phosphatidylcholine
biosynthetic pathway was deregulated. Taken together, these results
suggest that the overdose of specific genes in trisomic lines delays
differentiation in the presence of oleic acid. The dual-specificity tyrosine (Y)
phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human
chromosome 21. DYRK1A contributes to intellectual disability and the early
onset of Alzheimer’s disease in DS patients. Here we explored the potential
role of Dyrk1A in the reduction of phosphatidylcholine concentrations in
trisomic cells in the presence of oleic acid. The downregulation of Dyrk1A
by siRNA in trisomic cells returned phosphatidylcholine concentrations up
to similar levels to those of euploid cells in the presence of oleic acid. Thus,
our results highlight the role of Dyrk1A in brain development through the
modulation of phosphatidylcholine location, levels and synthesis.
P13-24
Toxins and pathogenic LRRK2 cause alterations
in secretory vesicle age
Antonio Jesús Lara Ordóñez1, Mar Martínez-Salvador1, Jesús
Madero-Pérez1, Elena Fernández1, Evy Lobbestael2, Veerle
Baekelandt2, Sabine Hilfiker1
1
Instituto de Parasitología y Biomedicina López-Neyra, Granada,
ES, 2Laboratory for Neurobiology and Gene Therapy, Leuven, BE
Neurons and neuroendocrine cells are packed with secretory vesicles, only a
few of which seem to be releasable upon appropriate stimuli. Previous studies
have shown that vesicles display functional and spatial segregation according
to age, whereby newly synthesized vesicles seem to be preferentially released.
Here, using various fluorescent cargo proteins which change colour over time
and fused to neuropeptides, we evaluated effects of toxins associated with
Parkinson´s disease on secretory vesicle age and distribution in dopaminergic
cells in culture. We found alterations in the percentage of old versus newly
synthesized vesicles largely consistent with previously observed toxin-induced
effects on secretion and/or axonal transport. Various reagents which modulate
either proteasome function or macroautophagy indicate that preferentially older
secretory vesicles are subject to degradation by autophagy. In addition, older
vesicles were preferentially excluded from neurites, indicating the existence
of a cellular mechanism able to distinguish vesicles according to their age
for intracellular transport as well as degradation. Pathogenic LRRK2 caused
similar alterations in vesicle age, with implications for previously described
LRRK2-mediated dopaminergic transmission deficits.
P13-25
Analysis of the Akt/mTORC1 pathway after
glucose deprivation versus oxygen-glucose
deprivation in neurons and nervous system
Ana Velasco Criado, Marian Hijazi, José María Medina
Universidad de Salamanca, Salamanca, ES
Mario Villa González1, Carlota Gil Martín1, Laura Mateos2,
Francisco Wandosell3, María José Pérez Álvarez4
1
Centro de Biología Molecular Severo Ochoa, CSIC-UAM,
Cantoblanco, ES, 2Centro Nacional de Biotecnología. CSIC-CNB,
Cantoblanco, ES, 3Centro de Biología Molecular Severo Ochoa,
CSIC-UAM y CIBERNED, Cantoblanco, ES, 4Centro de Biología
Molecular Severo Ochoa, CSIC-UAM Dpto. Biologia (Fisiología
Animal), Facultad de Ciencias, UAM, Cantoblanco, ES
Aberrant formation of the cerebral cortex could be attributed to the lack of
suitable substrates that direct the migration of neurons. Previous work carried
out at our laboratory has shown that oleic acid is a neurotrophic factor. In
order to characterize the effect of oleic acid in a cellular model of Down’s
syndrome (DS), here we used immortalized cell lines derived from the cortex
Ser/Thr kinase mTOR (mammalian target of rapamycin) with a key role
in cell growth and metabolism regulates the balance between anabolic and
catabolic processes, linking up extracellular stimuli: growth factors (GFs)
and nutrients (glucose, amino acids, etc.) with intracellular processes:
transcription, translation, synthesis of lipids/proteins and autophagy. In
P13-23
Restrained phosphatidylcholine synthesis in a
cellular model of Down’s syndrome is associated
with the overexpression of Dyrk1A
103
Pósters / Posters
XXXIX Congreso SEBBM
neurons, the activation of the Akt/mTOR induces increase of lipids/proteins
synthesis and attenuate autophagy; and its downregulation may increase
autophagy exacerbated in neurodegeneration processes such as Alzheimer
Disease´s (AD). mTOR comprises two different complex: mTORC1
and mTORC2, with differences in their functions and susceptibility to
rapamycin. Both mTOR kinases are stimulated by a plethora of GFs,
through membrane receptors RTKs or GRPC´s/PI3K/PDK1/Akt at different
levels, while mTORC1 is regulated by Akt whereas mTORC2 regulates
Akt. Extracellular glucose (Glu), via ratio AMP/ATP, activates AMPK that
also regulates mTORC1. We are interested in the analysis of the relative
contribution to mTORC1 activation of glucose and/or oxygen in models
of cerebral ischemia. Our initial data indicated a differential coupling of
AMPK, Akt and mTORC1 in ischemia models. To evaluate the neuronal
contribution we asses the status of Akt/mTORC1 activity after deprivation
of different combinations of GFs, Glu or oxygen at distinct time points
using SHSY5Y neuroblastoma cells. MTORC1 activity levels was inferred
by phosphorylation of p706SK and p-EBP1 and Akt activity was assessed
by phosphorylation of Thr308. Our data indicated a differential response of
Akt, mTORC1 and AMPK after deprivation of GF, oxygen and/or glucose
(OGD). These results will be discuss in the context of brain ischemia model.
P13r-26
Role of gabra2, GABAA receptor alpha-2 subunit,
in CNS development
Verónica González Núñez
Universidad de Salamanca, Salamanca, ES
P13-28
The role of PTEN in Alzheimer’s pathology
gabra2 gene codes for the alpha-2 subunit of the GABAA receptor, one of
the ionotropic receptors which has been related to anxiety, depression and
other behavioural disorders, including drug dependence and schizophrenia.
GABAergic signalling also plays a role during development, by promoting
neural stem cell maintenance and renewal. To investigate the role of gabra2
in CNS development, gabra2 deficient zebrafish were generated. The pattern
of proliferation during the embryonic development was disrupted in morphant
embryos, which also displayed an increase in the number of apoptotic nuclei
mainly at the mid- and hindbrain regions. The expression of several genes
(notch1, pax2, fgf8 and wnt1) known to contribute to the development of the
central nervous system was also affected in gabra2 morpholino-injected
embryos, although no changes were found for pax6a and shh a expression. The
transcriptional activity of neuroD (a proneural gene involved in early neuronal
determination) was down-regulated in gabra2 deficient embryos, and the
expression pattern of gad1b (GABA-synthesizing enzyme GAD67) was clearly
reduced in injected fish. I propose that gabra2 might be interacting with those
signalling pathways that regulate proliferation, differentiation and neurogenesis
during the embryonic development; thus, gabra2 might be playing a role in the
differentiation of the mesencephalon and cerebellum. Given that changes in
GABAergic circuits during development have been related to several psychiatric
disorders, such as autism and schizophrenia, this work might be helpful to
understand the role of neurotransmitter systems during CNS development and to
assess the developmental effects of several GABAergic drugs.
P13-27
The Mdm2 309T>G polymorphism modulates
the MDM2-p53 signaling pathway and conditions
the functional outcome of stroke patients
1
1
María Esther Ramos Araque , Cristina Rodríguez González , Irene
Sánchez Morán1, José Carlos Gómez Sánchez1, Tomás Sobrino2,
José Castillo2, Ángeles Almeida Parra1
1
Instituto de Investigación Biomédica de Salamanca, Hospital
Universitario de Salamanca; Instituto de Biología Funcional y
Genómica, Universidad de Salamanca-CSIC, Salamanca, ES,
2
Instituto de Investigación Sanitaria de Santiago de Compostela,
Hospital Universitario de Santiago de Compostela, Santiago
de Compostela, ES
104
The highly variable prediction of functional outcome after stroke could be
the effect of different genetic backgrounds to apoptosis. MDM2 protein
is the main negative regulator of p53, which plays an important role on
neuronal apoptosis after cerebral ischemia. Recently, we found that the
Arg72Pro single nucleotide polymorphism (SNP) of p53 regulates the
pro-apoptotic activity of the protein and conditions neuronal vulnerability
to ischemia-induced apoptosis and functional outcome of stroke patients.
In MDM2 a SNP in the promoter gene (309T>G) modulates levels of
Mdm2 expression. However, the role of Mdm2 309T>G SNP in stroke
prognosis remains unknown.
Here we study the association of the Mdm2 309T>G SNP and the functional
prognosis after stroke in blood samples from 408 patients with ischemic
stroke and 206 with intracerebral hemorrhage. Functional outcome at 3 and
12 months was evaluated by the modified Rankin scale. Mononuclear cells
from healthy individuals were collected to measure levels of MDM2 mRNA
and protein. We found that mononuclear cells harboring the Mdm2 TT
genotype have lower levels of MDM2 mRNA and protein than those with the
Mdm2 GG and Mdm2 TG genotypes, which may affect p53 stabilization and
then cell vulnerability to ischemia-induced apoptosis. Furthermore, patients
harboring the Mdm2 TT genotype showed poor functional outcome at 3 and
12 months following ischemic (p=0.003) and hemorrhagic (p<0.0001) stroke.
Our results suggest that the Mdm2 309T>G SNP modulates the MMD2p53
signaling pathway and then cell susceptibility to apoptosis, which conditions
the functional outcome of patients after stroke.
Shira Knafo
Ikerbasque, Basque Foundation for Science, Bilbao, ES
Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic
malfunctions leading to cognitive deficits ranging from mild impairment to
full-blown dementia in Alzheimer’s disease. Aβ appears to skew synaptic
plasticity events toward depression. We found that inhibition of PTEN, a
lipid phosphatase that is essential to long-term depression, rescued normal
synaptic function and cognition in cellular and animal models of Alzheimer’s
disease. Conversely, transgenic mice that overexpressed PTEN displayed
synaptic depression that mimicked and occluded Aβ-induced depression.
Mechanistically, Aβ triggers a PDZ-dependent recruitment of PTEN into the
postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ
motif, and a cell-permeable interfering peptide, we found that this mechanism
is crucial for Aβ-induced synaptic toxicity and cognitive dysfunction. Our
results provide fundamental information on the molecular mechanisms of
Aβ-induced synaptic malfunction and may offer new mechanism-based
therapeutic targets to counteract downstream Aβ signaling.
P14. Parasitología molecular /
Molecular parasitology
P14-1
Aptámeros como herramientas para la detección
de Leishmania
Víctor Manuel González Muñoz1, Celia Pinto Díez1, Valerio Frezza1,
Manuel Soto2, Ana García Sacristán3, M. Elena Martín Palma1
1
IRYCIS-Hospital Ramón y Cajal, Madrid, ES, 2Centro de Biología
Molecular Severo Ochoa (CSIC-UAM), Madrid, ES, 3Aptus Biotech
SL, Madrid, ES
Los parásitos del género Leishmania producen la leishmaniasis, la cual
afecta a millones de personas en todo el mundo. Existen distintos sistemas
Salamanca 2016
diagnósticos para la leishmaniasis, muchos de ellos para uso veterinario.
Los comúnmente utilizados en diagnóstico clínico están basados en la
detección de anticuerpos generados por el sistema inmunológico del
paciente o la amplificación de regiones específicas del genoma del
parásito mediante PCR. Ambos sistemas presentan problemas ya que la
respuesta inmune puede mantenerse elevada aún bastante tiempo después
de que se supere la enfermedad y, por otra parte, aunque la detección
del DNA del parásito indica la presencia del mismo, la baja presencia
de Leishmania en sangre aumenta el número de falsos negativos. La
alternativa actual es realizar PCR de muestras de médula ósea, en las que
el número de parásitos es sensiblemente mayor. Sin embargo, se trata de
una técnica invasiva que produce molestias y puede resultar peligrosa
para el paciente. Los aptámeros son moléculas de RNA o DNA de cadena
sencilla que, debido a la estructura terciaria particular que son capaces
de adquirir en función de su secuencia, interaccionan con su diana con
elevadas afinidad y especificidad. Hasta el momento, hemos obtenido
aptámeros frente a diferentes proteínas de L. infantum (H2A, H3 y PABP)
y demostrado que son capaces de detectar sus dianas en distintos sistemas
como ELONA, western blot o slot blot. Nuestro propósito es desarrollar
nuevos sistemas diagnósticos basados en el reconocimiento de proteínas
del parásito (más abundantes que el propio genoma) por aptámeros
específicos que, eventualmente, pueden ser amplificados por PCR, lo que
podría permitir alcanzar mayor sensibilidad que los sistemas actualmente
disponibles.
P14-2
Predicción de proteínas de membrana en el
intestino del argásido Ornithodoros moubata:
Selección de antígenos para el desarrollo
de vacunas
Prosper Obolo-Mvoulouga, Ricardo Pérez-Sánchez,
Raúl Manzano-Román, Ana Oleaga Pérez
IRNASA, CSIC, Salamanca, ES
El argásido Ornithodoros moubata es el principal vector en África de la
peste porcina africana y de la fiebre recurrente humana y representa un
buen modelo para investigar el proceso digestivo en argásidos a nivel
molecular. En el intestino de las garrapatas se expresan proteínas vitales
para su fisiología y para la infección y transmisión de patógenos, las cuales
son de interés como potenciales dianas para el desarrollo de vacunas
anti-garrapata y vacunas bloqueantes de la transmisión de patógenos.
En la búsqueda de antígenos vacunales, se prioriza a los expresados en
la membrana plasmática del enterocito, porque están expuestos a la luz
intestinal y resultan accesibles a los anticuerpos ingeridos con la sangre del
hospedador inmunizado. En el presente trabajo, el transcriptoma intestinal
de O. moubata se analiza in silico con objeto de definir las proteínas
de membrana expresadas por el enterocito, incluyendo su anotación
funcional y potencial antigenicidad. Se identifican 1725 transcritos con 1
dominio transmembrana y 1348 con 2 o más, anotándose respectivamente
506 y 601 proteínas. Su caracterización funcional revela diferencias entre
ambos grupos. La mayor parte de las proteínas con actividad catalítica
y unidora presentan 1 dominio transmembrana y las que tienen función
transportadora presentan varios dominios. Se identifican 91 proteínas
potencialmente localizadas en la membrana plasmática, la mayoría con
múltiples dominios transmembrana y actividad transportadora y, de ellas,
59 son potencialmente antigénicas (score Vaxijen >0,5). Varias de éstas
(tetraspanina, aquaporina, la homologa a la Bm86) ya han sido definidos
como candidatos vacunales frente a otras garrapatas y otros parásitos y
su expresión en el intestino de O. moubata se ha confirmado mediante
estudios proteómicos. Estos resultados han permitido seleccionar varios
antígenos de interés, que serán validados en pruebas de vacunación en
animales.
Trabajo financiado por el Ministerio de Economía y Competitividad,
Proyecto AGL2013-42745-P.
Pósters / Posters
P14-3
Análisis del transcriptoma del intestino del
argásido Ornithodoros moubata e identificación
de genes implicados en la alimentación,
digestión de la sangre y la defensa frente
a patógenos
Prosper Obolo Mvoulouga, Ana Oleaga Pérez, Raúl Manzano Román,
Ricardo Pérez Sánchez
IRNASA, CSIC, Salamanca, ES
Las garrapatas son parásitos hematófagos de importancia médica y
veterinaria porque transmiten numerosos patógenos que afectan al
ganado, los animales de compañía y las personas. El intestino medio
de las garrapatas es el órgano encargado de digerir la sangre y absorber
los nutrientes imprescindibles para su supervivencia y reproducción.
Además constituye la primera barrera defensiva a la que se enfrentan los
patógenos ingeridos con la sangre. El intestino es pues parte de la interfase
hospedador-garrapata-patógeno y en él se expresan proteínas vitales para
la garrapata y para la transmisión de los patógenos. El estudio de los genes
diferencialmente expresados durante la alimentación permite explorar
nuevas estrategias de control de las garrapatas y conocer la fisiología de la
digestión en argásidos a nivel molecular, asunto sobre el que apenas existen
datos. El objetivo del presente trabajo es la identificación de los genes de
O. moubata diferencialmente expresados durante la digestión de la sangre,
para lo cual se ha secuenciado y analizado el transcriptoma intestinal en
condiciones basales (ejemplares no alimentados) y en las primeras fases
de la digestión (a las 48 horas de la alimentación). Se han identificado
8.026 genes diferencialmente expresados entre garrapatas alimentadas y no
alimentadas. Su clasificación funcional revela que determinados grupos de
genes con actividad peptidasa implicados en la digestión de hemoglobina
y los implicados en respuestas defensivas, detoxificación y respuesta al
estrés asociado a la toma de sangre se encuentran fuertemente regulados
y su nivel de expresión aumenta significativamente tras la alimentación.
Por el contario, el nivel de expresión de genes involucrados en procesos
de endocitosis y transporte intracelular y en el metabolismo y transporte
de carbohidratos y lípidos no varía tras la ingesta de sangre. Este trabajo
proporciona datos inéditos acerca del efecto de la alimentación en el
transcriptoma intestinal de esta especie.
Trabajo financiado por el Ministerio de Economía y Competitividad,
Proyecto AGL2013-42745-P.
P14-4
Análisis de la expresión génica diferencial
mediante RNAseq en los promastigotes
metacíclicos de Leishmania major
Alberto Rastrojo Lastras, José Carlos Solana, Manuel Soto, Begoña
Aguado, José M. Requena
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES
Los protistas del género Leishmania son causantes de las leishmaniasis, un
grupo de graves enfermedades que afectan a humanos y otros mamíferos en
muchas zonas del planeta. Estos parásitos son particularmente interesantes
por su regulación génica postranscripcional. La transcripción policistrónica
y los procesos de trans-splicing y poliadenilación son los responsables de
generar los niveles adecuados de cada uno de los mRNAs en cada una de
las fases de su ciclo vital. El parásito requiere de dos huéspedes, un insecto
vector y un mamífero donde multiplicarse, idealmente de forma crónica.
Para la transmisión exitosa desde el insecto hasta el huésped mamífero
el parásito experimenta un proceso denominado metaciclogénesis en
el que adquiere la capacidad de resistir a la lisis por complemento y a
otros elementos de defensa del sistema inmunitario. Hemos estudiado los
cambios en el transcriptoma del parásito asociados a la diferenciación desde
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las formas procíclicas a las formas metacíclicas, utilizando Leishmania
major, por ser la especie en la que el transcriptoma ha sido detalladamente
anotado. Se prepararon tres réplicas biológicas de parásitos recién
aislados de ratones infectados, separándose promastigotes procíclicos
y metacíclicos. Se aislaron las formas metacíclicas (alrededor del 1%
de los parásitos en la fase estacionaria de cultivo), utilizando el método
de selección negativa con aglutinina de cacahuete. Una vez extraído el
RNA, se procedió a la secuenciación masiva de la fracción de poli-A y las
lecturas se mapearon frente al transcriptoma de referencia, determinándose
los niveles de expresión de cada transcrito. Se han identificado transcritos
cuyos niveles varían significativamente durante la metaciclogénesis. Entre
los transcritos cuyos niveles disminuyen están los codificantes de las
histonas, en consonancia con que en la fase metacíclica el parásito no se
divide. Otros transcritos que varían codifican para proteínas de unión a
RNA y transportadores de membrana.
P14-5
Role of ubiquitin-activating (E1)
and ubiquitin-conjugating (E2) genes
in the infective stage of Leishmania infantum
Jaime Larraga, Ana Alonso, Pedro J. Alcolea, Vicente Larraga
Centro de Investigaciones Biológicas (CSIC), Madrid, ES
Leishmaniasis, a disease caused by protozoa of the genus Leishmania, affects
about two million people all over the world. The main clinical forms are:
cutaneous, mucocutaneous and visceral. In Europe, the visceral disease is
caused by L. infantum and constitutes a zoonosis transmitted through the bite
of sand flies of the genus Phlebotomus. At the infective stage of Leishmania,
within the insect vector, a certain number of genes are overexpressed and
may be related with the infection ability of the parasite. Between these genes
are the ubiquitin-activating enzyme (E1) and the ubiquitin-conjugating
enzyme (E2). The ubiquitin-activating enzyme E1 catalyzes the first step
of the ubiquitination reaction that marks proteins for degradation via the
proteasome. The E2 enzyme accepts ubiquitin from the E1 complex and
catalyzes its covalent attachment to other proteins.Both genes have been
cloned in pQE-30 vector and expression was carried out in Escherichia
coli strain M15. These proteins have been purified using Ni2+ affinity
chromatography. For the detection of the abundance of the protein in the
growth curve of the parasite, a policlonal antibody has been obtained for
both proteins. The modeling of both proteins was performed using PyMol
software and the protein structure has been compared with the corresponding
human ones. The E1 enzyme seems to be a homodimer structure and is
different to the human orthologous. The E2 enzymes is similar to the A
chain of the human orthologous. The alignments against the orthologous
sequences have been made using ClustalW and BlastP. In the case of the
ubiquitin-activating gene (E1), the homology with different species of the
Leishmania genusis approximately 90%. With the related in Trypanosoma
cruzi the homology is 41%. With Homo sapiens the homology drops to
33%. The ubiquitin-conjugating gene (E2) shows a homology of 92% with
L. major and 82% with L. braziliensis. With T. cruzi the homology drops to
68%. In the case of H. sapiens the homology is reduced to 69%.
P14-6
Influencia del microambiente en el transcriptoma
de promastigotes de Leishmania infantum:
cultivo axénico vs flebotomo
Ana Alonso Ayala1, Pedro J. Alcolea1, María A. Degayón1, Maribel
Jiménez2, Ricardo Molina2, Vicente Larraga1
1
Centro de Investigaciones Biológicas, CSIC, Madrid, ES, 2Instituto
de Salud Carlos III, Madrid, ES
Los distintos perfiles de expresión génica se han obtenido comparando
promastigotes extraidos del medio natural, el vector Phlebotomus
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XXXIX Congreso SEBBM
perniciosus (Pro-Pper), con los de cultivo axénico. Hemos seleccionado
tres poblaciones en cultivo: promastigotes en fase estacionaria (Pro-Stat),
promastigotes metacíclicos obtenidos por selección negativa con aglutinina
de cacahuete (Pro-PNA-) y amastigotes intracelulares, obtenidos a partir de
la infección in vitro de células fagocíticas de la línea celular U937 (Amas).
El análisis transcriptómico se ha llevado a cabo mediante microarrays
genómicos de Leishmania infantum; en el caso de la comparación de los
promastigotes obtenidos del medio natural con los de fase estacionaria
de cultivo axénico (Pro-Pper/Pro-Stat), también mediante spliced leader
RNA seq (SL-RNAseq). Los resultados obtenidos indican una tasa de
sobre-expresión menor de genes en amastigotes, lo que apoya la hipótesis de
la pre-adaptación de los promastigotes diferenciados para su paso al medio
intracelular en el hospedador mamífero. También hemos comprobado que
los Pro-Pper son más infectivos que los Pro-PNA-, se sobre-expresan genes
relacionados con la infectividad, como los genes que participan en la síntesis
de GIPL, LPG y PPG, además de obtener un mayor número de células U937
infectadas in vitro. En la comparación Pper/Pro-Stat la mayoría de los genes
diferencialmente expresados están implicados en regulación de la expresión
génica y señalización intracelular. Los datos de SL-RNAseq confirman la
infectividad de Pro-Pper, se sobre-expresan genes directamente relacionados
con la metaciclogénesis como ATG8 (autofagia), HASPA1, HASPB, MBAP,
AMA1 y META2. La influencia del cultivo axénico debe ser evaluada caso
por caso, ya que puede afectar a los resultados de expresión. Los distintos
estudios del transcriptoma permiten laselección de marcadores relacionados
con la infectividad del parásito y su posible utilización como posibles dianas
terapéuticas o nuevos candidatos de antígenos protectores para el desarrollo
de vacunas frente a la leishmaniasis.
P14-7
Obtainment and proteomic characterization of
exosomes from sheep´s fertile cyst hydatid fluid:
definition of potential exosomal markers for
Cystic Echinococcosis
Raúl Manzano Román1, Carlos Sánchez Ovejero1, Adriano Casulli2,
Mar Siles Lucas1
1
Instituto de Recursos Naturales y Agrobiología de Salamanca
(IRNASA-CSIC), Salamanca, ES, 2Istituto Superiore di Sanità,
Rome, IT
Cystic echinococcosis (CE) is a chronic and complex disease with a
limited understanding of parasite-host interactions. These interactions can
be modulated by a dynamic crosstalk between extracellular vesicles (EVs)
harboring proteins and nucleic acids. Diagnosis of CE relies on tools with
no satisfactory performances and prognosis-associated follow-up test are
still lacking. There is, therefore, a need for the identification of specific
markers. The cargo of EVs has been shown to be changed in disease,
as is their abundance in the circulation. Studies showing a correlation
with disease progression have made these vesicles a favorable target for
diagnostic purposes (circulating antigens or miRNAs). In relation with CE,
these novel sources for plasma-derived markers may also serve to improve
viability assessment and the basis for alternative therapeutics. Floatation
into Iodixanol gradients differently separates subtypes of EVs. Thus, in
the present study, an exosome enriched EV fraction was isolated from
hydatid fluid (HF) of fertile sheep cysts by filtration, ultracentrifugation
and further purification by the OptiPrepTM method. GO analyses showed
endosome derived EVs -bona fide exosomes- that were further visualized
by Nanoparticle Tracking Analyses and parasite specific proteins were
further identified by electron microscopy after immuno-gold labelling.
Label-free semi-quantitative proteomic analysis of this fraction determined
specific parasite exosomal markers of potential clinical interest. This
proteomic characterization identified a panel of potentially interesting
diagnostic proteins: unique canonical exosomal markers -like TSG101and vesicle-surface proteins such as Protein DJ 1 or beta enolase of parasite
origin potentially useful as CE biomarkers.
Salamanca 2016
P14r-8
Using attenuated Leishmania parasites lacking
for HSP70-II gene as a vaccine against
leishmaniasis
José Carlos Solana Morcillo, Laura Corvo Villén, José María Requena
Rolanía, Manuel Soto Álvarez
Centro de Biología Molecular Severo Ochoa (CBMSO-UAM), Madrid, ES
Leishmaniases are a group of neglected tropical diseases caused by the
infection with the protozoan parasite Leishmania. Since patients cured
of leishmaniasis develop protective immunity to reinfection, vaccination
should be feasible. The only effective vaccine for controlling the human
disease is leishmanization, a procedure abandoned due to safety concerns.
During the last decade advances in Leishmania molecular genetics
have allowed the design of genetically modified parasites resulting in
attenuated strains. A Leishmania infantum knockout, lacking for HSP70
type II gene (LiΔHSP70-II) was developed in our group and used as a live
vaccine in a resistant model of cutaneous leishmaniasis. C57BL/6 mice
subcutaneously immunized with LiΔHSP70-II did not show any side
effects due to vaccination and were able to control an infective challenge
with a virulent strain of Leishmania major. Vaccinated mice mounted
an early and controlled inflammatory response resulting in the absence
of a lesion in the site of infection (ear dermis). These promising results
support the strategy of generating a new line of ΔHSP70-II attenuated
parasites on cutaneotropic species such as Leishmania major. Two
different approaches have been considered: replacing of both alleles,
after two rounds of transfection with antibiotic-resistance cassettes,
and CRISPR-Cas9-mediated gene knockout. This new technique can be
useful to solve some difficulties frequently observed in the traditional
method due to Leishmania genomic plasticity, mosaic aneuploidy and
creation of extra copies of the targeted gene.
Funding: EU-7th. 603181 (MuLeVaClin). FIS-PI14/00366 (FEDER
FUNDING).
Pósters / Posters
as a higher replication rate within macrophages exposed to Leishmania
overexpressing YinP. All together, these results strongly suggest a relation
between YinP gene expression and leishmaniasis pathogenesis. In order
to localize YinP expression in the leishmanial cell, pXG-mCherry34-YinP
plasmid was constructed. In this vector, YinP gene was inserted directly
near mCherry, to generate fluorescent fusion proteins expressed in the
parasites. Fluorescent microscopy disclosed that red fluorescence of
mCherry-YinP was localized in the nucleus. Further experiments need to
be done to elucidate other cellular location of YinP protein. Finally, we aim
to analyze the involvement of YinP in Leishmania virulence in vivo. To that
end, we will use the integrative constitutive expression vector pLEXSY to
generate new strains (pLEXSY-YinP) also overexpressing the gene. We’ll
then assess the infectivity of those mutants in a murine in vivo model.
Therefore, a study of both, parasitic burden within target organs such as
liver and spleen, and the lesion progression during the infection will be
performed.
P14-10
Epigenetic variation in malaria parasites:
sex, drugs and… adaptation
Alfred Cortés Closas
ICREA y ISGlobal, Barcelona, ES
Two cells with identical genomes can have dramatically different
phenotypes. If the differences are transmissible from one generation
to the next, the differences are considered epigenetic. Recent research
has established that the genes that control key processes in the biology
of malaria parasites, such as nutrient uptake, resistance to some toxic
compounds or sexual conversion, are regulated at the epigenetic level.
Differences between individual parasites in the expression of these genes
confer phenotypic plasticity and can mediate the adaptation of parasite
populations to changes in their environment. In this talk I will present our
latest results in the study of the adaptation of P. falciparum parasites to
changes in their environment, with a focus on pfap2-g, the master regulator
of sexual conversion, and adaptation to heat-shock.
P14-9
YinP gene: New approach for understanding
the mechanism of acquiring infectivity
by Leishmania parasites?
P14-11
The lack of type I IFN may enhance Leishmania
major infection in C57BL/6 mice
Miriam Algarabel Olona1, Andrés Vacas Oleas1, José Peña Guerrero1,
Connar Leeming1, Celia Fernández Rubio1, Ester Larrea2, Socorro
Espuelas3, Paul Nguewa1
1
Instituto de Salud Tropical Universidad de Navarra (ISTUN)/
Department of Microbiology and Parasitology, Pamplona, ES,
2
Instituto de Salud Tropical Universidad de Navarra (ISTUN)/
Pathogen Immunology (CIMA) University of Navarra, Pamplona,
ES, 3Instituto de Salud Tropical Universidad de Navarra (ISTUN)/
Department of Pharmacy and Pharmaceutical Technology, University
of Navarra, Pamplona, ES
Celia Fernández-Rubio1, Estanislao Nistal-Villan2, Miriam Algarabel
Olona1, Andrés Vacas-Oleas1, José Peña-Guerrero1, Connar
Leeming1, Gloria González-Aseguinolaza2, Esther Larrea3, Paul
Nguewa1
1
Institute of Tropical Health University of Navarra (ISTUN),
Department of Microbiology and Parasitology, Pamplona, ES,
2
Gene Therapy and Regulation of Gene Expression Program, Center
for Applied Medical Research (CIMA) , Pamplona, ES, 3Institute
of Tropical Health University of Navarra (ISTUN), Pamplona, ES
Leishmaniasis is a neglected tropical disease caused by protozoan parasites
of the genus Leishmania. Current drug therapies are unsatisfactory due to
their toxicity, long treatment courses and development of resistance. In
order to improve existing treatments, the identification and characterization
of novel therapeutic targets based on parasite genes involved in Leishmania
pathogenesis is essential. One of these genes is YinP. It may play a role in
the acquisition of infectivity by Leishmania promastigotes. This gene is
highly conserved and has demonstrated to be involved in several cellular
processes such as embrionary progress, ribosomal biogenesis, cellular
proliferation, genetic transcription and carcinogenesis. Our first assays
have shown that YinP reaches its highest expression level in metacyclic
promastigotes, the infective stage. Furthermore, we have performed
several experiments to analyze the infectivity of parasites overexpressing
YinP. Our data reveal a dramatic increase of the ratio of infection as well
Leishmania major is the causative agent of the old world cutaneous
leishmaniasis currently re-emerging due to the refugee crisis in the
Middle East and North Africa. It is widely recognized the anti-parasite
role of the type II IFN, but function of type I IFN in the parasite
infection still remains unclear. Toll/IL-1R domain-containing adaptor
inducing IFN-β (TRIF) and IFN-β promoter stimulator-1 (IPS-1) are
protein adaptors implicated in the recognition of double-strand RNA
that enhances adaptive immune responses.The aim of this study
was to investigate the impact of the deficiency in TRIF and IPS-1
protein adaptors during Leishmania major infection. For that, bone
marrow-derived macrophages (BM-DM) from C57BL/6 wild type (wt)
and double TRIF and IPS-1 knock out (ko) mice were infected with
Leishmania major promastigotes. After 6 and 72 hours of infection, a
significant increase in the percentage of Leishmania-infected BM-DM
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Pósters / Posters
and in the amount of Leishmania DNA in BM-DM was detected.
Such alterations were related to the lack of the mentioned adaptors.
Consequently, these results prompted us to study the expression pattern
of certain host genes involved in leishmanicidal activity, such as iNOS
and IFNβ. We observed a significant decrease of iNOS and IFNβ mRNA
expression in BM-DM from double ko mice compared to wt animals.
Furthermore, a reduced nitric oxide (NO) production in BM-DM cells
from doble ko mice was also observed. An in vivo assay was performed
comparing wt and IPS-1 ko mice infected with Leishmania major. Four
weeks post-infection, half of animals showed ulceration (50 % IPS-1 ko
vs 0 % wt). In addition, there was a higher parasite burden in the liver
from the IPS-1 ko group respect to wt. These results point to a role of
type I IFN in Leishmania infection.
P14-12
Analysis of the protein interactome of Leishmania
major ORC1/CDC6
Laura Corvo1, César Poza-Carrión2, María Gómez1, José M. Requena1
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid,
ES, 2Centro Nacional de Biotecnología (CSIC), Madrid, ES
1
Eukaryotic DNA replication involves firing of multiple origins licensed
by the assembly of pre-replication complexes (pre-RCs), composed
of an Origin Recognition Complex (ORC), a six-subunit complex
(Orc1-6) that recruits Cdc6 to the replication origin, and the MCM
(mini-chromosome maintenance) helicase complex (Mcm2-7) which
is loaded, along with Cdt1, onto DNA by the ORC-Cdc6 complex.
The assembly of additional factors as Cdc45 and GINS leads to the
pre-initiation complex formation, which allows the establishment of
bidirectional replication forks. This process is tightly regulated and
highly conserved among eukaryotic and archaeal organisms. While
nuclear DNA replication has been extensively investigated in yeast and
higher eukaryotes, little is known about the replication machinery in
protozoans. Trypanosomatids are a group of kinetoplastid parasites
which includes the human pathogenic genera Trypanosoma and
Leishmania. The latter causes a group of diseases globally known
as Leishmaniasis, responsible of 20,000 to 30,000 deaths annually
worldwide. Several eukaryotic replication protein orthologues have
been annotated in trypanosomatids, although some of them are lacking,
and only one ORC orthologue, related to both Orc1 and Cdc6 proteins,
have been clearly found in genomic databases, indicating that these
parasites may possess a novel or diverged replication machinery.
In order to gain insight into Leishmania origin recognition complex
architecture and function, we have generated a Leishmania major
cell line overexpressing Orc1 fused to mCherry. We have focused
on the analysis of Orc1 subcellular location and the identification
of Orc1-interacting factors in both wild type and Orc1-mCherry
overexpression backgrounds.
P14-13
Análisis funcional mediante RNA-seq
de la proteína fosfatasa tipo 1 LinJ.15.0240
de Leishmania infantum
María A. Degayon1, Ana Alonso1, Pedro J. Alcolea1, Gowthaman
Ramasamy2, Guillermo Padilla1, Peter J. Myler2, Vicente Larraga2
1
Centro de Investigaciones Biológicas. CSIC, Madrid, ES, 2Center
for Infectious Disease Research. Seattle, US
La leishmaniasis visceral es una zoonosis causada por el parásito
Leishmania infantum en la Cuenca Mediterránea. Las proteínas
fosfatasas tipo 1 (PP1) son las principales serina-treonina fosfatasas
presentes en los organismos eucariotas y participan en la regulación
de diversos procesos celulares. El papel de dichas proteínas apenas ha
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XXXIX Congreso SEBBM
sido estudiado en el género Leishmania. De acuerdo a los análisis de
expresión génica diferencial mediante microarrays (Alcolea y cols.
2010), el gen de la PP1 LinJ.15.0240 está diferencialmente expresado
a lo largo del ciclo biológico de L. infantum. El perfil de expresión
génica analizado por qRT-PCR mostró una expresión constante
de la PP1 LinJ.15.0240 a lo largo de la curva de crecimiento de los
promastigotes axénicos, coincidiendo con la abundancia de proteína
detectada por Western blot. Además, se observó la sobre-expresión del
gen LinJ.15.0240 en promastigotes obtenidos del vector P. perniciosus
respecto a promastigotes axénicos. Por otro lado, se generaron
transfectantes estables de sobre-expresión de la PP1 mediante el vector
integrativo pIRmcs. Los perfiles de expresión génica evaluados por
RNA-seq revelaron una sobre-expresión del gen LinJ.15.0240 en todos
los días analizados de la curva de crecimiento. Sin embargo, los análisis
por Western blot mostraron la sobre-expresión de la PP1 únicamente a
día 1. Curiosamente, el 92% de los genes diferencialmente expresados
se encuentran sub-expresados según los datos de secuenciación masiva.
Estos genes están asociados a funciones moleculares como la expresión
génica, transporte y metabolismo de acuerdo a la base de datos Gene
Ontology. Entre los genes diferencialmente expresados cabe destacar la
sobre-expresión en el día 1 del gen A2, característico de amastigotes,
así como la sub-expresión en los días 3 y 5 de genes descritos como
marcadores de metaciclogénesis, SHERP y Meta 1. Estas observaciones
sugieren que la PP1 LinJ.15.0240 puede tener un papel esencial en los
procesos de diferenciación de L. infantum.
P14-14
The Enoyl-CoA hydratase/isomerase
of Leishmania infantum as a potential
therapeutic target
Luis Telles de Carvalho Coimbra Martins, Ana Alonso, Pedro
Alcolea, Vicente Larraga
Centro de Investigaciones Biologicas (CIB), Consejo Superior de
Investigaciones Científicas (CSIC), Madrid, ES
Leishmaniasis are a group of infectious diseases, caused by protozoan
parasites of the genus Leishmania and transmitted by the bite of
phlebotomine sand flies. L. infantum is the responsible strain for the
zoonotic visceral form of the disease in the Mediterranean Basin, being
the dog the main reservoir. The treatments currently available present
several disadvantages such as toxicity, drug resistance and recurrence.
We have selected a specific gene that encodes for a L. infantum
enoyl-CoA protein (LinJ. 29.2420), over-expressed during the most
infective stage of the parasite, obtained, from metacyclic promastigotes
isolated through negative selection with PNA (Alcolea et. al., 2009).
This over-expression may be directly related with the infectivity of
this parasite. This protein has both hydratase and isomerase activities
which are involved in the -oxidation pathway. Enoyl-CoA hydratase
catalyzes the syn hydration of water. Enoyl-CoA isomerase catalyzes
the reaction in which cis-3-enoyl-CoA or trans-3-enoyl-CoA is
converted into trans-2-enoyl-CoA. This gene has been cloned in the
pQE-30 vector and its expression was performed in the Escherichia coli
strain M15. Optimal expression conditions of the encoded protein were
found to be 2 h at 37 °C. This protein has been purified by Ni2+ using
affinity chromatography at native conditions. A polyclonal antibody
has also been obtained in order to study the protein abundance trough
out the growth curve of the parasite. Using the I-Tasser, PyMOL and
BlastP software’s together with the PDB database we were able to
predict a protein 3D model. This protein forms hexamers, made up of
two trimers. The nucleotide sequence was aligned, using ClustalW with
orthologous sequences, giving low percentages of homology with the
mammalian proteins, and very low with the human one (6%). These
values as well as the predicted protein structure suggest that this could
be a good candidate as a therapeutic target.
Salamanca 2016
P14-15
Leishmania and its relationship with human
skin microbiome.- Leishmanicidal activity of
modulins, peptide toxins from Staphylococcus sp
M. Ángeles Abengozar Infantes1, Nuria Davó1, Luis Rivas López1,
David Andreu Martinez2
1
Centro de Investigaciones Biológicas (CSIC), Madrid, ES,
2
Universitat Pompeu Fabra, Parc de Recerca Biomèdica,
Barcelona, ES
Leishmania has been traditionally studied as an isolated organism or at
most as a mandatory intracellular parasite of the macrophage. However, in
cutaneous leishmaniasis (CL), Leishmania is in contact with the components
of skin microbiome. Of special relevance, Leishmania skin ulcers often
become infected with bacteria, often by Staphylococcus epidermidis, a
typical skin commensal, and by S. aureus. Both staphylococci secrete a group
of cationic and amphipathic peptides collectively known as phenol-soluble
modulins (PSM), with a broad microbicidal spectrum. Our goal was to
ascertain the role of modulins in the control of CL as well as their potentiality
as new leishmanicidal agents. As a proof of mechanism, PSMα1 and PSαM2,
and their respective N-terminal dipeptide truncated forms from S. aureus,
together with δ- and γ-toxins from S. epidermidis were assayed both on
L. major promastigotes as well as on axenic and intracellular L. pifanoi
amastigotes. All these modulins lysed promastigotes at low micromolar
concentrations (IC50 <30 μM: ranked in increasing IC50 PSMα2Δ1-2 <pps
mα1=psmα1δ1-2≤psma2<δ-toxin<<γ-toxin), with=”” additive=”” effect=””
in=”” combination.=”” axenic=”” amastigotes=”” psmα2=”” was=””
the=”” most=”” active=”” peptide=”” (ic50=3.3 μM), and with the highest
selectivity index (SI=3). It also reduced intracellular infection by 30% at
sub-IC50 concentrations. </ppsmα1=psmα1δ1-2≤psma2<δ-toxin<<γ-tox
in),>PSMs led to plasma membrane permeabilization by an all-or-none
mechanism, as assessed by in vivo bioenergetic collapse of promastigotes,
plasma membrane depolarization, permeation to vital dyes and transmission
electron microscopy. In addition, PSMs were spotted inside Leishmania
at subIC50, suggesting additional intracellular targets. In combination with
LL-37 or dermcidin, two human skin antimicrobial peptides, PSMs showed
antagonism and no effect, respectively. Altogether, PSMs appear to be
feasible modulators of infection, as well as new leishmanicidal hits. The role
of other microbial actors in the final CL outcome will be discussed.
Projects: FIS ISCIII,PI12-02706& RETICS-FEDER, RD12/0018/0007
(LR) & AGL2014-52395 (DA).
P14-16
Papel inmunomodulador de la doxiciclina
en un modelo murino de malaria cerebral
María Linares1, Javier Peiró1, Gabriela Martínez-Chacón1, Daniel
Sánchez-Melendo1, Susana Pérez-Benavente1, Antonio Puyet1, José
M. Bautista1, Patricia Marín-García2, Amalia Diez1
1
Departamento de Bioquímica y Biología Molecular IV (UCM).
Instituto de Investigación Hospital 12 de Octubre, Madrid, ES,
2
Facultad de Ciencias de la Salud (URJC), Alcorcón, ES
La malaria cerebral (MC) es la complicación más grave de la infección por
Plasmodium falciparum. La variabilidad individual de sus síntomas refleja
que es un síndrome heterogéneo lo que dificulta el manejo de la enfermedad,
impidiendo su diagnosis temprana y un tratamiento rápido y eficaz. Aunque
su patogénesis no está bien definida, su complejidad se relaciona con el
secuestro de eritrocitos en la vasculatura cerebral y la respuesta inflamatoria
del hospedador. La magnitud y el momento de liberación de mediadores
inflamatorios modulan la respuesta inmune a la malaria, resultando en
un control adecuado de la parasitemia o por el contrario provocando el
desequilibrio de tales mediadores causando un daño letal en el hospedador.
En este sentido la inmunomodulación sería una nueva perspectiva para
Pósters / Posters
el tratamiento de la MC. La doxiciclina, como antibiótico, es una terapia
capaz de controlar la malaria, sin embargo, se desconoce si durante el
aclaramiento del parásito posee un efecto potenciador del sistema inmune
en el cerebro. Por ello, hemos evaluado el posible efecto inmunomodulador
de la doxiciclina durante el desarrollo de la malaria cerebral mediante el
análisis de la respuesta inmune en el modelo murino C57BL/6 infectado
con P. berghei ANKA. Los resultados obtenidos muestran que los animales
que desarrollan MC y son tratados con doxiciclina, aumentan los niveles
de IgG específicas tras una segunda infección que se relaciona con un
patrón diferencial de reconocimiento antigénico. Dicha respuesta inmune
adaptativa, aunque es capaz de prolongar la supervivencia de los animales
en la primera infección, no les confiere protección suficiente en las sucesivas
infecciones. Adicionalmente describimos el papel de la IL10 como elemento
condicionante de la MC durante la infección.
P14-17
Inhibition of Plasmodium falciparum
intraerythrocytic cycle by purine analogs
Laura Sánchez Vega1, Susana Pérez Benvente1, Rafael de la
Cámara1, David Monedero2, Daniela Ubiali3, Giovanna Speranza4,
José Manuel Bautista1, Jesús Fernández Lucas2, Antonio Puyet
Catalina1
1
Department of Biochemistry and Molecular Biology IV & Research
Institute Hospital 12 de Octubre, Universidad Complutense de
Madrid, Madrid, ES, 2Applied Biotechnology Group, Universidad
Europea de Madrid, Madrid, ES, 3Drug Sciences Department,
University of Pavia, Pavia, IT, 4Department of Chemistry; University
of Milan, Milan, IT
The purine synthesis pathways are essential for the life cycle of many
pathogenic organisms in mammals. Most parasites lack the necessary
enzymes for de novo synthesis of purines, using instead the salvage
pathways as their only source of these substrates. The transporters and
enzymes involved in the salvage pathways are considered good targets for
chemotherapy agents and purine analogs, which are used extensively in
the treatment of several diseases. The enzymes catalyzing the cleavage of
glycosidic bonds of nucleosides have an essential role in the purine salvage
pathway of Plasmodium, the parasite causing malaria, and can therefore
be considered as potential therapeutic targets for antimalarial drugs. New
inhibitors of the plasmodial adenosine deaminase (PfADA), purine nucleoside
phosphorylase (PfPNP) and hypoxantine-guanosine-xantine phosphoribosyl
transferase (PfHGXPRT) are currently investigated for this purpose. In
addition, PfPNP activity can be used for the activation of pro-drugs, which
can be channeled through PfHGXPRT. The resulting metabolites would
be toxic for the parasite, as they could be incorporated to RNA/DNA
hampering transcription/translation or replication. In this work several
purine base and purine ribonucleoside analogs displaying substitutions at
positions 6, 7 and 8, and chemotherapy agents with substitutions a position
2 of adenine have been tested as potential inhibitors of the intraerythrocytic
cycle of Plasmodium falciparumin vitro. The results suggest that fluorinated
derivatives of adenine, adenosine and adenosine phosphate can be effective
as parasite growth inhibitors at subcytotoxic concentrations.
P14-18
Study of YinP, a novel gene involved
in Leishmania pathogenesis. A potential new
target for drug discovery
Miriam Algarabel Olona1, Celia Fernández-Rubio1, Andrés Vacas
Oleas1, Esther Larrea2, José Peña Guerrero1, Connar Leeming1, Iñigo
Gamboa1, Carlos Berrio1, Carmen Sanmartín3, Socorro Espuelas4,
Paul Nguewa1
1
Institute of Tropical Health University of Navarra (ISTUN),
Department of Microbiology and Parasitology, Pamplona, ES,
109
Pósters / Posters
2
Institute of Tropical Health University of Navarra (ISTUN),
Pamplona, ES, 3Institute of Tropical Health University of Navarra
(ISTUN), Department of Organic and Pharmaceutical Chemistry,
Pamplona, ES, 4Institute of Tropical Health University of Navarra
(ISTUN), Department of Pharmacy and Pharmaceutical Technology,
Pamplona, ES
Leishmaniasis is a neglected tropical disease caused by Leishmania spp.
The improvement of existing treatments and the discovery of new drugs
remain ones of the major goals to control and eradicate this disease. Based
on parasite genome, we have identified and characterized YinP, a novel
therapeutic target involved in Leishmania pathogenesis. YinP is a highly
conserved gene which contains two main domains: YinP (N-terminus)
and ONC. It has been demonstrated that YinP is involved in several
cellular processes such as cellular proliferation and genetic transcription.
Firstly, we analyzed the intracellular location of the protein. Fluorescent
microscopy disclosed that red fluorescence of mCherry fused with YinP
was only localized in the nucleus. In addition, we demonstrated that, in
Leishmania promastigotes, it plays a role in the acquisition of infectivity. In
fact, YinP reaches its highest expression level in metacyclic promastigotes,
the infective forms. After generating mutant parasites overexpressing YinP,
we observed that these clones exhibited a dramatic increase of the ratio of
cell infection and higher replication rate within macrophages. Furthermore,
we are carrying a screening of new compounds that have shown promising
leishmanicidal activity. Interestingly, YinP overexpressing parasites are
more resistant to these drugs. Consequently, it may be a new therapeutic
target. On the other hand, we have obtained more mutant parasites with
different levels of ONC domain overexpression. Preliminary data suggest
that these cells may show lower growth compared to WT parasites.
Moreover in such mutants, YinP gene expression levels seem to be
abrogated respect to control. Finally, we have also generated clones with
high levels of YinP∆ONC in order to study the role of ONC in YinP.
Therefore, further experiments need to be performed to better characterize
YinP function and activity.
P14-19
Epitope mapping of immunodominant antigens
from Plasmodium falciparum as serological
markers of malaria infection
Patricia Marín-García1, Amalia Diez2, Isabel González-Azcárate2,
Alejandra Martínez-Serna2, Ana Abad2, Pedro Reche3, José Miguel
Rubio4, Antonio Puyet2, José M. Bautista2
1
Fac. Ciencias de la Salud, URJC, Alcorcón, ES, 2Depto. Bioquímica
y Biología Molecular IV, UCM e Instituto de Investigación Hospital
12 de Octubre, Madrid, ES, 3Depto. Inmunología, UCM, Madrid,
ES, 4Instituto de Salud Carlos III, Majadahonda, ES
Blood-stage malaria immunity can be partially acquired after years of
exposure to Plasmodium falciparum infection through the elicitation of
protective antibodies. The target antigens of protective antibodies and
the basis of their acquisition remain largely unknown. Addressing these
knowledge gaps could accelerate malaria vaccine discovery and the
development of new rapid diagnostics kits. To this end, we performed
immunomics screening of human sera against a clinical P. falciparum
isolate. Malaria cases from African patients were classified into different
categories according to clinical symptoms and parasitaemia and degree
of immunological protection. After 2D-immunoblotting of P. falciparum
proteins and peptide mass fingerprinting we identified 17 antigens
representing a collection of parasite proteins conferring residual immune
protection against clinical malaria. Interestingly, this data included new
antigens not previously described. We selected five new antigenic proteins
for epitope screening with high-resolution 15-mer overlapping peptide
arrays, and identified high immunoreactive epitopes in all five antigens.
Next, we selected ten 20-mer peptides from these immunoreactive peptides
for further characterization by ELISA with a human sera collection of
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XXXIX Congreso SEBBM
infected and non-infected individuals from endemic areas in Africa. Our
preliminary results suggest that some of these epitopes may be useful for
detection of subclinical malaria.
P14-20
Antimalarial activity of a new family of PfG6PD
inhibitors
María Linares1, Irene Sola2, Nelson Alencar3, Paloma Abad1,
Susana Pérez-Benavente1, Caterina Pont2, Cristina Sampedro2,
Jordi Juárez-Jiménez3, F. Javier Luque3, Diego Muñoz-Torrero2,
José M. Bautista1
1
Department of Biochemistry and Molecular Biology IV & Research
Institute Hospital 12 de Octubre, Universidad Complutense
de Madrid, Ciudad Universitaria, Madrid, ES, 2Laboratory of
Pharmaceutical Chemistry (CSIC-associated Unit), Faculty of
Pharmacy and Institute of Biomedicine (IBUB), Universidad
de Barcelona (UB), Barcelona, ES, 3Department of Nutrition,
Food Science and Gastronomy, Faculty of Pharmacy (Torribera
Campus) and IBUB, UB, Santa Coloma de Gramenet, ES
Worldwide malaria caused by Plasmodium falciparum is responsible for
nearly 1 million deaths annually. Despite the efforts made in the discovery
of new antimalarial agents, the parasite has developed resistance against
the vast majority of available therapies. Therefore, there is an urgent need
to search for new drugs with new mechanisms of action.
In the malaria parasite, the bifunctional glucose-6-phosphate
dehydrogenase-6-phosphoglucono-lactonase (PfG6PD) has been validated
as a target to inhibit the asexual parasite stages. Since PfG6PD is the
evolutionary result of the fusion of the two genes that are separated in most
eukaryotes (including the human host hG6PD), their differences could
enable the rational design of selective inhibitors against the parasite PfG6PD
active site.
We have evaluated the antimalarial activity of a new family of inhibitors
designed and synthesized upon the structural differences between
PfG6PD and hG6PD. A total of nine different molecules have been
screened by enzyme kinetics in the parasite and human enzyme, IC50 in
P. falciparum culture and cytotoxicity in human cells. Most molecules
showed greater selectivity against PfG6PD with IC50 values within the
micromolar range. In addition, the most potent inhibitors also showed
a greater inhibitory activity of the P. falciparum growth in culture.
Furthermore, phenotypic assays at 48h upon treatment determined that
schizont was the most sensitive intraerythrocytic stage to this family of
compounds, which suggests a different mechanism of action than that of
chloroquine, making it interesting for combined antimalarial therapy and
enhanced immunomodulation. Finally, the absence of in vitro cytotoxicity
complements to these new molecules with an added value as promising
antimalarial inhibitors.
P14-21
Iron overload delays malaria infection in mice
and improves immune and antioxidant responses
Sandra Sánchez Jaut1, Isabel González Azcárate1, Patricia Marín
García2, Susana Peréz Benavente1, Javier Uceda Fernández1, Marta
García Sánchez1, Alí Naghi Kamali1, Antonio Puyet Catalina1,
Amalia Díez Martín1, José Manuel Bautista Santa Cruz1
1
Department of Biochemistry and Molecular Biology IV, Universidad
Complutense de Madrid, Facultad de Veterinaria, Madrid, ES, 2Faculty
of Health Sciences, Universidad Rey Juan Carlos, Alcorcón, ES
Anemia is a common public health problem in Africa, where >80%
of malaria prevalence occurs, and where 50% anemia cases are due
to iron deficiency. Consequently, in malaria endemic regions the iron
supplementation is routinely used to prevent iron deficiency anemia and to
Salamanca 2016
treat patients with the disease. However, the association of iron levels with
malaria tolerance is still controversial.
To study the effect of iron supplementation on malaria infection, we
inoculated 100mg/kg/day of iron dextran during 21 days to BALB/c mice
and challenged with lethal Plasmodium yoelii 17XL. This induced iron
overload promoted transitory red skin, weight loss and liver damage, that
resumed once finished iron inoculation. Expectedly, hepcidin-1 expression
was induced in liver to maintain iron homeostasis.
Interestingly, iron overload delayed lethal malaria infection through early
mechanisms related to antioxidant and innate immune responses. Thus,
iron changed the spleen immune-cell pattern with respect to macrophages,
B cells, antigen-presenting cells and dendritic cells (DCs), as well as
induced the expression of the anti-inflammatory heme oxygenase-1 in
liver. Iron treated mice increased their proportion of macrophages during
the parasitemia growth in spleen in comparison to control, providing
advantage against the blood-stage malaria infection. Further experiments
are required to assess whether additional mechanisms induce beneficial
immune responses.
P14-22
Caracterización de factores asociados a AMPK
que regulan la quiescencia en la forma infectiva
de Trypanosoma brucei
Gloria Ceballos Pérez, Manuel Saldivia, Jean-Mathieu Bart, Miguel
Navarro
Consejo Superior de Investigaciones Científicas (CSIC), Granada,
ES
En mamíferos, la señalización a través de AMPK se encuentra asociada
a respuestas celulares frente al estrés oxidativo, metabolismo glicosídico
y diferenciación. En Trypanosoma brucei, nuestro grupo ha observado
mediante herramientas moleculares y farmacológicas que la proteína
AMPK regula de manera específica la diferenciación del parásito hacia
formas quiescentes mediante ensayos in vivo e in vitro. Además, el análisis
proteómico del complejo TbAMPK identificó una posible asociación de esta
quinasa con proteínas responsables de la respuesta oxidativa del parásito.
Con el objetivo de determinar la posible relación entre la regulación de
AMPK, el metabolismo oxidativo y la diferenciación de T. brucei, se
analizó la capacidad de respuesta aestrés oxidativo del parásito mediante
la regulación de AMPK. Nuestros resultados sugieren que la activación
de TbAMPKα1 es capaz de regular la respuesta oxidativa de T. brucei.
Además, se observó mediante RT-qPCR que el tratamiento con H2O2 es
capaz de regular la expresión de genes asociados a diferenciación hacia
formas quiescentes. En conjunto, estos datos sugieren que TbAMPKα1
tiene una función antioxidante asociada a diferenciación en T. brucei.
P14-23
La localización de la RNA polimerasa II mediante
Chip-Seq identifica secuencias promotoras
funcionales en el genoma de Trypanosoma brucei
Carlos Cordón Obras1, Sara Torres Rusillo1, Diana López Farfán1,
Jean Mathieu Bart1, Fabian Lorenzo2, Basilio Valladares2, Mark
Carrington3, Nicholas J. Dickens4, Miguel Navarro1
1
Instituto de Parasitología y Biomedicina López-Neyra, CSIC,
Granada, ES, 2Universidad de Canarias, Las Palmas de Gran
Canaria, ES, 3Department of Biochemistry. University of Cambridge,
Cambridge, UK, 4Wellcome Trust Centre for Molecular Parasitology.
University of Glasgow, Glasgow, UK
La diversificación de los kinetoplástidos, incluyendo Trypanosoma
brucei, a partir de otros eucariotas dio lugar a la adquisición de nuevas
características moleculares y celulares a nivel de transcripción y genoma.
En concreto, la transcripción policistrónica de los genes codificantes de
Pósters / Posters
proteínas revela un sistema ancestral semejante al de los procariotas.
Estudios previos han sugerido que el inicio de la transcripción puede estar
localizado en las regiones conocidas como regiones de cambio de hebra
(Strand Switch Regions –SSR-), situadas entre unidades transcripcionales.
Sin embargo, estas secuencias SSR son de gran tamaño y aún no está
claro su papel en el inicio y regulación de la transcripción. Para estudiar la
ocupación de la ARN polimerasa II (pol II) en el genoma de Trypanosoma
brucei, realizamos ensayos combinando inmunoprecipitación de cromatina
(ChIP) y secuenciación masiva (ChIP-Seq). La alta resolución de esta
técnica permite definir de forma precisa las regiones en las que la pol
II se acumula. Nuestros resultados revelan que existe una acumulación
significativa de la pol II en 306 picos a lo largo del genoma, la mayoría
de ellos asociados con SSRs (52,7%) y regiones intergénicas (27,4%), con
un tamaño medio de 1955 bases. A continuación, analizamos la actividad
promotora de las secuencias ocupadas por la pol II en el cromosoma 7
mediante un ensayo de transfección in transient con un gen reportero
(luciferasa) en formas procíclicas de T. brucei. Estudiamos una de estas
secuencias generando deleciones para definir la mínima región necesaria
para el inicio de la transcripción y mutaciones a través de este mínimo
fragmento. Así, determinamos la posición +1 mediante Primer Extension
e identificamos los principales elementos reguladores, la mayoría de ellos
situados aguas abajo del +1. Nuestro trabajo demuestra el potencial de estas
regiones como secuencias promotores y proporciona la primera descripción
de elementos reguladores asociados a las unidades transcripcionales
policistrónicas de T. brucei.
P15. Radicales libres y estrés oxidativo /
Free radicals and oxidative stress
P15r-1
Absence of PGC-1α induces cellular senescence
and early immortalization
Ignacio Prieto, Alberto Zambrano, Ana Aranda, María Monsalve
Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM),
Madrid, ES
Peroxisome Proliferator Activated Receptor (PPAR) γ Coactivator
1α (PGC-1α) is a master regulator of oxidative metabolism; being
responsible of the Reactive Oxygen Species (ROS) detoxification, and
the lack of PGC-1α leads to oxidative stress due to ROS accumulation.
It is well-known that elevated concentrations of ROS can cause Double
Strand Breaks (DSB) in the DNA. Oxidative stress and DNA damage
are, among others, triggers of Cellular Senescence, a cellular process
characterized by cell cycle arrest in response of aberrant proliferation
and tumor progression. The aim of the study was to determine the
possible role of PGC-1α in cellular senescence. To test this idea, we
used PGC-1a+/+ and PGC-1a-/- MEFs that were serially passed till they
reach cellular senescence and immortalized spontaneously. Our results
show that, in the absence of PGC-1a, MEFs produce higher levels of
mitochondrial reactive oxygen species, accumulate more oxidative DNA
damage measured by DNA damage repair complexes formation and
showed delayed DNA repair, leading to an early induction of senescence
markers and induction of cell cycle inhibitors. However, cell proliferation
rate was not significantly different between PGC-1a+/+ and PGC-1a-/MEFs regardless of passage number. Importantly, PGC-1a-/- MEFs
showed and early escape from senescence, compared to PGC-1a+/+ MEFs.
These results suggest that the lack of PGC-1α leads to enhanced DNA
damage and induction of senescence, but is also associated with an early
escape from cellular senescence, supporting a tumor suppressor role for
PGC-1α.
111
Pósters / Posters
P15-2
PGC-1α downregulation in the steatotic liver
enhances ischemia-reperfusion injury and
impairs ischemic preconditioning
Cristina Sánchez Ramos1, Alberto Tierrez2, Javier Laso2,
Ramón Bartrons3, Joan Roselló-Catafau4, María Monsalve1
1
Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM),
Madrid, ES, 2CNIC, Madrid, ES, 3Universitat de Barcelona,
Barcelona, ES, 4Institut d´Investigacions Biomèdiques de Barcelona
(CSIC), Barcelona, ES
Liver steatosis is associated with mitochondrial dysfunction, elevated ROS
levels and a enhanced sensitivity of ischemia-reperfusion (IR) injury and a
very limited response to preconditioning protocols. In this study we aimed
to determine if the downregulation in the steatotic liver of peroxisome
proliferator activated receptor  co-activator 1 (PGC-1), a master
regulator of mitochondrial metabolism and reactive oxygen species (ROS),
that is known to play a relevant role in liver metabolic control, could be
responsible for the sensitivity of the steatotic liver to ischemic damage.We
found that PGC-1 is induced in the normal liver, following an IR protocol
and this induction correlates with increased levels of antioxidant proteins.
This induction is severely curtailed in the steatotic liver, and results in a
limited induction of antioxidant proteins. PGC-1-/- mice under chow diet
do not have steatotic livers, but show an enhanced sensitivity to IR injury
and a lack of response to ischemic preconditioning, that recapitulates the
behaviour of the steatotic liver in terms of liver damage, although the
inflammatory response differers between both models. We used an in vitro
model to analyze how PGC-1 expression is regulated by changing O2
tensions, and found that hypoxia (1% O2) downregulates PGC-1 while
reoxygenation (3% O2) induces it, and is responsible for the recovery of
antioxidant gene expression following the ischemic period.
Conclusion: PGC-1 plays an important role in the protection against
IR injury in the liver that is likely associated with its capacity to induce
antioxidant gene expression.
P15-3
Daily supplementation with an almond beverage
enriched with docosahexaenoic, vitamin E and
olive oil alters inflammatory state in response
to acute exercise
Xavier Capó1, Miquel Martorell2, Antonio Sureda1, Joan Riera3,
Franchek Drobnic3, Josep Antoni Tur1, Antoni Pons1
1
CIBER: CB12/03/30038 Fisiopatología de la Obesidad la
Nutrición, CIBEROBN, Instituto de Salud Carlos III (ISCIII).
Research Groupon Community Nutrition and Oxidative Stress,
Science Laboratory of Physical Activity, Department of Fundamental
Biology and Health Sciences. University of Balearic Islands, Palma
de Mallorca, ES, 2Departamento de Nutrición y Dietética, Facultad
de Farmacia, Universidad de Concepción, Concepción, CL, 3Sports
Physiology Dept. CAR, Sant Cugat del Valles, ES
Omega 3polyunsaturated fatty acids (n3-PUFA) diet supplementation exerts
healthy effects against oxidative stress-based diseases. High PUFA levels
enhance lipid peroxidation altering oxidative stress balance. Ageing is also
associated with oxidative stress with higher values of oxidative damage
markers in elderly than in young. The aim was to determine the effects of an
almond and olive oil-based DHA and vitamin E-enriched beverage dietary
supplementation, acute exercise and age on circulating inflammatory markers.
The diet of five young and five senior athletes were supplemented during five
weeks with a functional beverage. Athletes performed a maximal exercise
test at the beginning and at the end of the nutritional intervention. Plasma and
erythrocytes were isolated from blood taken immediately before and 1 hour
after the exercise. Functional beverage diet supplementation increased the
DHA erythrocytes content but attenuated the increased plasma non-esterified
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XXXIX Congreso SEBBM
fatty acids levels induced by acute exercise. Dietary functional beverage
supplementation significantly decreased sL-selectin, ICAM3 but increased
TNFα plasma levels; age increased ICAM3 and acute exercise decreased
lipoxin, PGE2 plasma levels. Some interaction between exercise, age and
supplementation were observed in sL-selectin, ICAM, IL6 and TNFα
plasma levels. Young athletes presented the lowest sL-selectin and ICAM3
and the highest IL6 plasma levels after acute exercise after dietary beverage
supplementation respect the other situations. The TNFα plasma levels were
the highest in senior athletes after exercise and dietary supplementation.
Finally, dietary functional beverage supplementation of young athletes
enhances a pro-inflammatory circulating environment in response to acute
exercise that was minus evident in senior athletes.
Acknowledgments: CIBEROBN CB12/03/30038. We hereby acknowledge
the PhD grant provided by the University of the Balearic Islands.
P15-4
Serum and follicular fluid distribution of human
PON1 and PON3 enzymes
Irantzu Pérez-Ruiz1, Susana Meijide1, Zaloa Larreategui2, Marcos
Ferrando2, José Ignacio Ruiz-Sanz1, M. Begoña Ruiz-Larrea1
1
Department of Physiology, Medicine and Nursing School, University
of the Basque Country, UPV/EHU, Leioa, ES, 2Valencian Institute of
Infertility (IVI) – Bilbao, Leioa, ES
Physical and mental health is compromised in women suffering from
infertility and is currently considered a sociological problem. Assisted
reproduction techniques (ART) are helping these women to overcome
this problem, so that improvements in the area are of great interest. It is
known that ROS and antioxidants play an important role in reproduction;
therefore the control of redox balance and the enzymes which are
responsible for its preservation is essential. The human paraoxonase (PON)
gene family consists of three members (PON1, PON2, and PON3) that
exhibit antioxidant activities. The aim of this study was to characterize
the extracellular paraoxonase system (PON1 and PON3) in serum and
follicular fluid (FF). Samples were retrieved from the same women
undergoing an ovarian stimulation cycle. To this end, PON1 (arylesterase
and paraoxonase) and PON3 (simvastatinase) activities were measured by
spectrophotometric and HPLC methods, respectively. Protein expression
was detected by quantitative western blotting using specific antibodies.
The results indicate that all analyzed PON1 activities were significantly
higher (p<0.05) in serum than in FF. However, PON1 expression did
not show significant differences between both fluids. Regarding PON3
activity, no differences were found between serum and FF, but, in contrast,
its expression was significantly higher (p<0.05) in serum than in FF. In
conclusion there are different regulatory mechanisms governing the
specific activity of these extracellular enzymes in serum and follicular
fluid.
This work was supported by the Basque Country Government (Dep. Education,
Universities and Research, ref. IT687-13), and UPV/EHU (CLUMBER
UFI11/20 and PES13/58). The work was approved by the Ethics Committee of
the UPV/EHU (M10_2015_180_RUIZLARREA), and performed according to
the UPV/EHU and IVI-Bilbao agreement (October 5, 2015).
P15-5
Training enhances immune cells mitochondrial
dynamics and its antioxidant capabilities
synergistically with dietary docosahexaenoic
supplementation
Carla Busquets-Cortés1, Xavier Capó1, Miquel Martorell2, Josep
Antoni Tur1, Antonio Sureda1, Antoni Pons1
1
CIBER: CB12/03/30038 Fisiopatología de la Obesidad la
Salamanca 2016
Nutrición, CIBEROBN, Instituto de Salud Carlos III (ISCIII).
Research Groupon Community Nutrition and Oxidative Stress,
Science Laboratory of Physical Activity, Department of Fundamental
Biology and Health Sciences. University of Balearic Islands, Palma
de Mallorca, ES, 2Departamento de Nutrición y Dietética, Facultad
de Farmacia, Universidad de Concepción, Concepción, CL
Exercise training induces adaptations in mitochondrial metabolism,
dynamics and oxidative protection. Omega-3 fatty acids change membrane
lipid composition and modulate mitochondrial function. The aim was
to investigate the effect of 8-weeks training and docosahexaenoic acid
(DHA) supplementation (1.14 g/day) on the mitochondria dynamics
and antioxidant status in peripheral blood mononuclear cells (PBMCs)
from sportsmen. Subjects were assigned to an intervention (N=9) or
placebo groups (N=7) in a randomized double-blind trial (ClinicalTrial.
gov NCT02177383). Nutritional intervention significantly increased the
DHA content in erythrocyte membranes from the experimental group.
No significant differences were reported in circulating PBMCs, in their
capability to produce reactive oxygen species and in Mn-superoxide
dismutase protein levels. The proteins related with mitochondrial dynamics
generally increased after 8-weeks training and tha was enhanced by DHA
supplementation. Mitofusin (Mtf)-1 and -2, optic atrophy protein-1 (Opa-1)
and mitochondrial transcription factor A (Tfam) were significantly higher
in the DHA-supplemented group after intervention. Cytochrome c oxidase
(COX IV) activity and uncoupling proteins (UCP)-2 and 3 protein levels
increased after training, with higher UCP-3 levels in the supplemented
group. In conclusion, training induced mitochondrial adaptations which
may contribute to improved mitochondrial function. This mitochondrial
response is modulated by DHA supplementation.
Acknowledgments: Acción Estratégica en Salud del Ministerio de Ciencia e
Innovación DPS2008-07033-418 C03-03. The authors hereby acknowledge
the FPI/1648/2014 grant provided by Conselleria d’Educació, Cultura i
Universitats, Direcció General d’Universitats i Recerca, Govern de les
Illes Balears.
P15r-6
Dietary polyphenols and their role in coenzyme Q
metabolism
Lucía Fernandez del Rio1, Rosa Mª Nevado García1, María Isabel
Burón Romero1, Anish Nag2, Catherine F. Clarke2, José Manuel
Villalba Montoro1
1
University of Córdoba, Córdoba, ES, 2University of California,
Los Angeles, US
Coenzyme Q (Q) is a lipophilic organic molecule well known as a
membrane-soluble electron carrier and antioxidant. Due to its antioxidant
properties and its use as dietary supplement, the interest in this molecule
has increased in recent years. However, our knowledge of its metabolism
is quite limited. p-Hydroxybenzoic acid (pHB) was the only confirmed
precursor of Q ring for more than 40 years until Pierrel et al. (2010) and
Marbois et al. (2010) characterized p-aminobenzoic acid (pABA) as a novel
Q precursor in yeasts. Later, Block et al. (2014) showed that Arabidopsis
is able to use p-coumarate, but not pABA, as another ring precursor in Q
biosynthesis. Recently, Xie et al. (2015) have described that human and
E. coli cells do not utilize pABA as precursor in the biosynthesis of Q
while p-coumarate and resveratrol, another polyphenol structurally similar
to p-coumarate, serve as ring precursors of Q in E. coli, yeast and human
cells.
We hypothesized that other natural polyphenols could also behave as
biosynthetic precursors of Q in mammalian cells. Mouse cells isolated from
kidney proximal tubule (TKPTS) were used to test seven polyphenols:
resveratrol, kaempferol, quercetin, piceatannol, luteolin, naringenin and
apigenin. Our results showed an increase in Q levels in cells treated with
some of these polyphenols, with kaempferol having the strongest effect.
Pósters / Posters
The increase of Q was related with upregulation of its biosynthetic rate
because it was totally inhibited by pABA, a well-characterized inhibitor of
Coq2 activity in mammalian cells. Additionally, we studied directly the role
of kaempferol in Q biosynthesis by a competitive assay with radiolabelled
14
C-pHB. Kaempferol competed with 14C-pHB as ring precursor of Q
and inhibited incorporation of 14C-pHB while simultaneously increasing
Q levels. Moreover, we have performed studies with U-13C-kaempferol
and show that this polyphenol is efficiently used as ring precursor in the
biosynthesis of Q. Further experiments will be needed to understand the
metabolic steps of how kaempferol is utilized as a ring-precursor in the Q
biosynthetic pathway.
P15r-7
Alterations of mitochondrial biogenesis,
physiology and autophagy flux in an in vitro
model by a dietary polyphenol
Elena Gutiérrez Casado, Lucía Fernández del Río, José Manuel
Villalba Montoro
Departamento de Biología Celular, Fisiología e Inmunología.
Universidad de Córdoba, Córdoba, ES
Many antioxidant substances are able to suppress the
deleterious effect of reactive oxygen species (ROS). Kaempferol
(3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a
flavonoid found in many edible plants and used in traditional medicine,
which can behave as antioxidant or pro-oxidant in a concentration-dependent
manner. At high concentrations it can also induce autophagy and apoptosis,
whereas at low concentrations it is capable of neutralizing superoxide
anion. Recent results obtained by our research group indicate that this
substance produces a significant increase in the levels of coenzyme Q, an
endogenous antioxidant, in mouse kidney cells. The goal of the present
study was to determine how mitochondrial physiology, as well as, the
autophagy flux were affected under the same experimental conditions
previously tested. Moreover, since this polyphenol can also activate the
mitochondrial sirtuin Sirt3, we wanted to test out if in the presence of
nicotinamide, a Sirt1 (NAD+-dependent deacetylase which plays a role
in the autophagy flux) activator at low doses, we observed an increased
response added to the flavonoid treatment. Cultures of a mouse kidney
proximal tubule cell line (TKPTS) were treated with 10 μM kaempferol
during 48 hours in the presence of absence of 10 mM nicotinamide. After
that, different mitochondrial and oxidative stress-related parameters were
measured, including mitochondrial abundance, intracellular levels of
superoxide and peroxides, mitochondrial superoxide and mitochondrial
membrane potential. Proteins related with autophagy were also measured
by western blot. Our results indicate that kaempferol can modulate ROS
levels and is able to modify the expression of different autophagy markers.
In addition this polyphenol can alter some parameters of the mitochondrial
function in TKPTS cells.
P15-8
Efecto de un sazonador de hollejo de uva sobre
vías de señalización asociadas a la hipertensión
en células HUVEC
Mónica Gisela Gerardi, Raquel Del Pino García, Mónica Cavia-Saiz,
Dolores Rivero-Pérez, Mª Luisa González San José, Pilar Muñiz
Rodríguez
Departamento de Biotecnología y Ciencia de los Alimentos,
Facultad de Ciencias, Universidad de Burgos, Burgos, ES
La disfunción endotelial es el principal mecanismo implicado en el desarrollo
de la hipertensión arterial. Es resultado de un desbalance entre los niveles de
óxido nítrico (NO) y de radicales libres (ROS) en el endotelio vascular como
consecuencia del estrés oxidativo. Diversas vías de señalización celular
113
Pósters / Posters
regulan los niveles de estas dos moléculas. Entre ellas, se encuentran las
que incluyen los factores de transcripción NF-κB y Nrf2. Los polifenoles
de la uva poseen efectos moduladores sobre estos factores, lo que le otorgan
efectos protectores manteniendo la buena funcionalidad del endotelio
vascular. Nuestro trabajo consistió en caracterizar un producto de hollejo
de uva con características funcionales para ser usado como sazonador.
Para evaluar sus efectos funcionales el hollejo de uva se sometió a dos
digestiones in vitro (gastrointestinal y fermentación colónica). Se evaluó
la capacidad antioxidante por el método ABTS y su efecto protector en la
línea de células endoteliales (HUVEC) sometida a estrés oxidativo con una
alta concentración de glucosa. Se evaluó la viabilidad celular (mediante el
ensayo de MTT) observándose un efecto citoprotector de las fracciones de
digestión gastrointestinal y de fermentación colónica. Asimismo, el análisis
de la expresión de los factores de transcripción (NF-κB y Nrf2) mediante
real-time PCR y los niveles de activación de las kinasas (p38-MAPK y Akt)
y de la vía del NF-κB (NF-κB e ikk) mediante inmunoblot. Se observó que el
producto digerido en forma completa posee efectos protectores al aumentar
la expresión de Nrf2 y disminuir la de NF-κB en las células sometidas a
estrés oxidativo así como también al aumentar los niveles de p38-MAPK y
Akt fosforilados y disminuir los de NF-κB e ikk.
P15-9
Las glutaredoxinas monotiólicas Grx3/4 regulan
Sir2 mediante glutationilación. Efecto en la
resistencia a estrés y envejecimiento
Núria Vall-llaura, Elisa Cabiscol Català
IRB Lleida. Dept. Ciències Mèdiques Bàsiques, Universitat de
Lleida, Lleida, ES
En S. cerevisiae, las glutaredoxinas (Grx) monotiólicas son esenciales en la
homeostasis del hierro pero, a diferencia de las Grx ditiólicas, se considera
que no presentan una actividad oxido-reductasa relevante, dado que solo
cuentan con 1 cisteína en su centro catalítico. Por otra parte, los mecanismos
de regulación de la sirtuína Sir2, una histona deacetilasa NAD-dependiente
implicada en la respuesta al estrés oxidativo y el envejecimiento, son
desconocidos. En este estudio se investigó la regulación redox de Sir2 en
situación de estrés y el papel e impacto fisiológico de Grx3 y Grx4 como
tiol-reductasas de Sir2. Demostramos tanto in vitro como in vivo, que Sir2
se conjuga con glutatión (glutationilación) bajo estrés, lo que disminuye su
actividad deacetilasa. Niveles fisiológicos de Grx3/4 pueden revertir esta
modificación postraduccional. Grx3/4 interaccionan con Sir2 reduciéndolo
y restableciendo su actividad de silenciamiento en el telómero. Mediante
mutagénesis dirigida, se identificaron residuos de cisteína claves, localizados
en el dominio catalítico de Sir2, como dianas de glutationilación. La
mutación de esos residuos generó cepas con mayor resistencia tanto al estrés
redox como al envejecimiento cronológico. Estos hallazgos ofrecen una
nueva perspectiva, a nivel molecular, de las Grx monotiólicas en los procesos
regulados por cambios redox, siendo Sir2 un sustrato fisiológico regulado
por S-glutationilación. Teniendo en cuenta que la familia de las sirtuínas está
conservada desde bacterias hasta humanos, estos resultados pueden ampliar
nuestra comprensión del envejecimiento y sus enfermedades asociadas.
P15-10
Dietary supplementation with almond and
olive oil based beverage enriched with
docosahexaenoic and vitamin E affects
inflammatory gene expression in immune cells
Antoni Pons Biescas1, Xavier Capó1, Miquel Martorell2, Antoni
Sureda1, Joan Riera3, Francisco Drobnic3, Josep A. Tur1
1
CIBER: CB12/03/30038 Fisiopatología de la Obesidad la
Nutrición, CIBEROBN, Instituto de Salud Carlos III (ISCIII),
Research Group on Community Nutrition and Oxidative Stress,
Science Laboratory of Physical Activity, Department of Fundamental
114
XXXIX Congreso SEBBM
Biology and Health Sciences, University of Balearic Islands, Palma
de Mallorca, ES, 2Departamento de Nutrición y Dietética, Facultad
de Farmacia, Universidad de Concepción, Concepción, CL, 3Sports
Physiology Dept. CAR, Sant Cugat del Valles, ES
Exercise alters the number and function of circulating cells of the immune
system. Omega-3 diet supplementation and exercise influence cytokine
production and protect against chronic inflammation. The aim was to analyse
the effects of an almond and olive oil beverage enriched with α-tocopherol
and docosahexaenoic (DHA) on inflammatory and immune gene expression
in peripheral blood mononuclear cells (PBMCs) and its effect on physical
performance in sportsmen. Five young athletes and five senior athletes were
supplemented for five weeks with a functional beverage. Blood samples
were taken before and after supplementation and immediately before and 1
hour after the exercise test and PBMCs were isolated. Diet supplementation
increased DHA erythrocytes content. Exercise test significantly increased
body temperature, lactate blood levels and fatigue perception (Borg index)
but no effects of dietary supplementation were observed on physical
performance parameters. Age significantly reduced the values of Borg index
during exercise test. NFκβ activation significantly increased as consequence
of acute exercise mainly after dietary functional beverage supplementation;
however no effects of exercise, supplementation or age were observed on
NFκβ gene expression. Dietary beverage supplementation significantly
increased 15LOX2 PBMCs gene expressions mainly in senior group.
IL1β and IL8 expressions were enhanced after supplementation in young
group mainly in post-exercise but not in senior group. To sum up, diet
supplementation do not affect athletic performance. PBMCs are primed to
pro-inflammatory response after exercise as indicated by its increased NFkβ
active levels. Exercise enhances pro-inflammatory genes expressions mainly
in young group after supplementation. This reinforces a pro-inflammatory
effect of the functional beverage supplementation in young athletes.
We hereby acknowledge the PhD grant provided by the University of the
Balearic Islands and CIBEROBN CB12/03/30038.
P15-11
La pérdida de DNA mitocondrial en células
de cáncer hepático desequilibra la autofagia
inducida por hipoxia química mediante
la desregulación de la vía de señalización
AMPK-ULK1 independientemente de HIF-1
Elisa Lozano1, Maitane Asensio2, Silvia Di Giacomo3, Annelies
Noorlander2, Silvia Jiménez2, Felipe Jiménez1, Elisa Herráez1, José
Juán García Marín1, María José Pérez García1
1
Hepatología Experimental y Vectorización de Fármacos
(HEVEFARM), IBSAL, Universidad de Salamanca. Centro de
Investigación Biomédica En Red para el Estudio de Enfermedades
Hepáticas y Digestivas (CIBERehd), Salamanca, ES, 2Hepatología
Experimental y Vectorización de Fármacos (HEVEFARM), IBSAL,
Universidad de Salamanca, Salamanca, ES, 3Dept. Physiology and
Pharmacology Vittorio Erspamer, Sapienza University of Rome,
Roma, IT
Antecedentes: La activación de la autofagia por factores exógenos del
microambiente tumoral, como hipoxia, o endógenos como alteraciones en
el DNA mitocondrial (DNAmt) es frecuente en células tumorales.
Objetivo: Estudiar el efecto de la pérdida del genoma mitocondrial sobre
la autofagia inducida por hipoxia en células SK-Hep-1 de cáncer hepático.
Métodos: La hipoxia se simuló químicamente añadiendo cloruro de
cobalto (CoCl2) o deferoxamina (DFO) al cultivo.
Resultados: En las células silvestres (WT) y deficientes en DNAmt (Rho),
estos agentes aumentaron la actividad lactato deshidrogenasa y la expresión
de HIF-1 y p21, e indujeron la parada del ciclo celular. La hipoxia química
activó la autofagia en las células WT pero no en las Rho. Se reprimió la
Salamanca 2016
expresión de inhibidores de autofagia dependientes de HIF-1, como
Bcl-2 y mTOR, en ambos tipos de células mientras que, solo en las WT, la
hipoxia química activó la ruta pro-autofágica mediada por AMPK-ULK1.
La generación de especies reactivas de oxígeno constitutiva e inducida
por hipoxia química fue menor en las células Rho. En las células WT, el
antioxidante N-acetilcisteína bloqueó la activación de AMPK inducida por
CoCl2 y DFO, pero no el estrés del retículo endoplásmico inducido por
CoCl2. Estos compuestos aumentaron la tasa de muerte en las células WT y
en menor medida en las Rho. Al bloquear la activación de la autofagia con
3-metiladenina, la muerte celular inducida por DFO se redujo, mientras
que la inducida por CoCl2 aumentó en las células WT, pero no en las Rho.
Conclusión: La disfunción mitocondrial debida a la pérdida de DNAmt
interfiere con las rutas de señalización que desencadenan la autofagia en
células tumorales hepáticas, lo que puede favorecer la carcinogénesis.
Pósters / Posters
species (ROS) as metabolic byproducts of oxidative phosphorylation.
Acute hypoxia produces a superoxide burst, which can signal downstream
cell adaptations. Here we show that hypoxia triggers acute deactivation
of mitochondrial complex I, changing its catalytic activity from an
oxidoreductase to a Na+/H+ antiporter. The subsequent stimulation
of mitochondrial Na+/Ca2+ exchanger (NCLX) activity promotes
mitochondrial depolarization and superoxide production and underlies the
participation of both complex I and NCLX in acute oxygen sensing. NCLX
inhibition is associated with the suppression of downstream ROS-driven
responses to hypoxia such as hypoxic pulmonary vasoconstriction, HIF-1α
stabilization and ischemic brain damage in vivo. These results provide
biological explanations for superoxide production in acute hypoxia and
highlight complex I and NCLX as potential therapeutic targets in diseases
involving mitochondrial ROS signalling, hypoxia and oxidative stress.
Cofinanciado ISCIII-FEDER (PI15/00179).
P15-14
P15-12
Las redoxinas y la promiscuidad
de los intercambios redox entre cisteínas
José Antonio Bárcena
Universidad de Córdoba, Córdoba, ES
Existen dos rutas antioxidantes de tioles en la mayoría de los organismos, una
basada en Tiorredoxina (Trx) y otra en Glutarredoxina (Grx) y glutatión (GSH).
Cada una tiene una flavoenzima oxidorreductasa única para mantenerlas
reducidas con el poder reductor del NADPH (también Ferredoxina en plantas).
Originalmente se consideró a la Trx ajena al mundo del GSH, que era exclusivo
de Grx y las Peroxirredoxinas (Prx) realizaban su acción antioxidante con
ayuda exclusiva de Trx. El panorama es distinto hoy día pues se han descrito y
se siguen descubriendo interacciones entre ambos sistemas, el glutatión y las
cisteínas de otras proteínas, dando lugar a una diversidad de mecanismos, no
solo antioxidantes, sino de regulación y señalización.
En esta charla se hará un recorrido sobre los descubrimientos de estas
interacciones “inesperadas”, con énfasis en las contribuciones históricas
y recientes del grupo de investigación sobre redoxinas de la Universidad
de Córdoba y de otros grupos españoles. Entre las primeras cabe destacar
la detección en los ’90 de isoformas de Grx en tejidos de mamífero y su
localización en el núcleo de algunas células; la distribución de la Grx2
de levadura, producto de un único gen con dos sitios de iniciaciónde
la traducción, entre el citosol y la mitocondria; la conexión entre ácido
lipoico, Grx y GSH; la participación de Grx2 en el ciclo catalítico de la
Prx1 de levadura; la resolución de Prx1 por GSH, mediante un mecanismo
novedoso a concentraciones muy bajas, sin consumirse durante el ciclo
catalítico y protegiendo a Prx1 de la sobreoxidación; la actuación de
Trx como desglutationilasa completando el ciclo; finalmente, las dos
actividades contrapuestas desnitrosilante y transnitrosilante de Trx1
humana funcionando simultáneamente.
P15r-13
Acute hypoxia produces a superoxide signal
through oxygen sensing by mitochondrial complex
I and sodium/calcium exchange via NCLX
Pablo Hernansanz Agustín1, Laura Moreno1, Elena Ramos2, Tamara
Villa-Piña2, Elisa Navarro1, Esther Parada1, Alicia Izquierdo-Álvarez2,
Nuria Sánchez-López1, Laura Pelaez-Aguado1, Daniel Tello1, Izaskun
Buendía1, Anabel Marina1, Ángel Cogolludo3, Javier Egea1, Manuela
G. López1, Anna Y. Bogdanova4, Antonio Martínez-Ruiz2
1
Universidad Autónoma de Madrid, Guadalajara, ES, 2Hospital
Universitario de la Princesa, Madrid, ES, 3Universidad
Complutense, Madrid, ES, 4University of Zurich, Zurich, CH
Oxygen is a key molecule of aerobic life, but can give rise to reactive oxygen
Evaluación del efecto citotóxico y bioactividad
de un producto derivado de residuos de
vinificación sobre las células HUVEC
Mónica Gisela Gerardi, Mónica Cavia Saiz, Dolores Rivero Pérez,
Ana Castañeda, Raquel Del Pino, Mª Luisa González San José,
Pilar Muñiz Rodríguez
Departamento de Biotecnología y Ciencia de los Alimentos,
Facultad de Ciencias, Universidad de Burgos, Burgos, ES
La hipertensión arterial (HTA) es uno de los factores de riesgo
cardiovascular más prevalentes en los países occidentales. Numerosos
estudios epidemiológicos sugieren que dietas ricas en polifenoles
mejoran la función endotelial y disminuyen la presión arterial. Estos
efectos positivos se han relacionado con la reducción del estrés oxidativo
por su efecto directo sobre los ROS resultando en un aumento en la
biodisponibilidad de NO y la inhibición de la enzima convertidora de
la angiotensina (ACE), clave para el control de la presión arterial. Una
fuente importante de polifenoles son los productos derivados de residuos
de vinificación, descritos como fuentes de fibra dietética y antioxidantes.
El objetivo de este estudio fue evaluar el efecto sobre biomarcadores de
estrés oxidativo e hipertensión arterial del tratamiento con un producto
derivado de residuos de masas de vinificación sobre células endoteliales de
la vena umbilical (HUVECEA.hy926) normoglicémicas e hiperglicémicas.
Para ello se trataron las células HUVEC con los compuestos liberados
tras la digestión gastrointestinal in vitro de los extractos y se evaluó la
citotoxicidad, el estado redox, cambios en la permeabilidad endotelial,
el efecto inhibitorio sobre el sistema renina- angiotensina. Los resultados
muestran que el producto no presenta citoxocidad y mejora el estado
redox celular en células sometidas a un medio hiperglucémico. Sus efectos
antihipertensivos se asocian a cambios observados en la regulación de las
enzimas ACE y eNOS. Asimismo, el tratamiento con el producto de hollejo
regula la permeabilidad de la monocapa de células endoteliales modificada
por el TNF-α y el INF-γ en condiciones normo como hiperglucémicas.
En resumen, nuestros resultados sugieren que los productos derivados de
residuos de masas de vinificación presentan un alto potencial antioxidante
y un efecto protector de la función endotelial, lo que implica su posible uso
como alimento funcional o nutracéutico para la prevención y/o tratamiento
de patologías cardiovasculares.
P15-15
Control redox de tioles proteicos por redoxinas
en una línea celular de hepatocarcinoma en
un contexto de señalización por óxido nítrico
endógeno
María José López-Grueso1, Raúl González2, Carlos A. Fuentes-Almagro3,
Raquel Requejo-Aguilar1, Emilia Martínez-Galisteo1, Jordi Muntané2,
115
Pósters / Posters
C. Alicia Padilla1, J. Antonio Bárcena1
Dpto. Bioquímica y Biología Molecular e IMIBIC. Universidad
de Córdoba, Córdoba, ES, 2Dpto. Cirugía General, Hospital
Universitario Virgen del Rocío. IBIS. Universidad de Sevilla,
Sevilla, ES, 3Unidad de Proteómica, SCAI. Universidad de Córdoba,
Córdoba, ES
1
Los efectos del óxido nítrico (NO) se deben en su mayor parte a la
modificación por S-nitrosilación de restos de cisteína estructural y
funcionalmente importantes en proteínas clave. La Tiorredoxina (Trx) y la
Glutarredoxina (Grx) son los principales sistemas celulares para el control
del estado redox cisteínico habiéndose demostrado actividad desnitrosilasa
para la Trx. Para definir el papel de Trx y Grx en la homeostasis redox y
sus proteínas diana en condiciones nitrosilantes, se han silenciado Trx1
y Grx1 con siRNA específicos en células HepG2 que sobreexpresan NO
Sintasa-3 (NOS3) y se han determinado cuantitativamente los proteomas
global (Progenesis QIp) y redox tiólico (MRM-Skyline) diferenciales. Se
ha aplicado la técnica de “Biotin Switch” para comprobar que los cambios
redox se deben a S-nitrosilación. La sobreexpresión de NOS3 provoca
muchos cambios a nivel del proteoma global. A nivel de proteoma redox,
se han asignado valores de la relación “Cys reducida/Cys oxidada” para
cada péptido cisteínico identificado. Así se han determinado dianas de la
actividad desnitrosilasa de Trx1. La propia Trx1 se encuentra nitrosilada
en su Cys73. Sorprendentemente, algunas Cys están menos oxidadas
cuando la Trx1 está silenciada, indicando que posiblemente son sustratos
de la actividad transnitrosilasa de Trx1. Por tanto, se demuestra que la Trx1
actúa en sentido antioxidante eliminando nitrosotioles proteicos mediante
el sitio activo que tiene el ditiol Cys32-Cys35 y en sentido prooxidante
transfiriendo un grupo nitroso a otros sustratos cisteínicos desde un segundo
sitio activo en el que está Cys73. Estas actividades contrapuestas de Trx
son conocidas, pero hasta ahora nunca se había descrito su funcionamiento
simultáneo en las mismas condiciones celulares.
P15r-16
Complex I assembly into supercomplexes
regulates mitochondrial ROS production
in neurons and astrocytes
Irene López-Fabuel1, Juliette Le Douce2, Gilles Bonvento2, Andrew
M. James3, Michael P. Murphy3, Angeles Almeida4, Juan Pedro
Bolaños1
1
Institute of Functional Biology and Genomics (IBFG), University
of Salamanca-CSIC, Salamanca, ES, 2CEA, DSV, I2BM, MIRCen,
CNRS UMR 9199, Université Paris-Sud, Université Paris-Saclay,
Fontenay-aux-Roses, FR, 3Medical Research Council Mitochondrial
Biology Unit, Wellcome Trust/MRC Building, Cambridge, UK,
4
Institute of Biomedical Research of Salamanca (IBSAL), University
Hospital of Salamanca, Salamanca, ES
Reactive oxygen species (ROS) abundance is the result of the balance
between their biosynthesis and degradation. In the brain, ROS detoxification
is taken over by astrocytes through a robust antioxidant system that
protects neighbor neurons; however, the mechanisms that control ROS
production are unknown. Here, we found that astrocytes produce ROS
several-fold faster than neurons by mitochondrial complex I. In astrocytes,
complex I is mainly present as an individual complex, while in neurons it
is predominantly assembled into supercomplexes. Using a complexomics
approach, we identified complex I subunit, NDUFS1, to be less abundant in
astrocytes than in neurons. Interestingly, NDUFS1 knockdown in neurons
limited the association of complex I into supercomplexes, leading to
impaired mitochondrial oxygen consumption and increased mitochondrial
ROS. Conversely, over-expression of NDUFS1 in astrocytes promoted
complex I incorporation into supercomplexes leading to decreased ROS.
Thus, dynamic complex I assembly into supercomplexes is unveiled as a
novel biological mechanism governing ROS abundance and contributing to
the bioenergetics shapes of neurons and astrocytes.
116
XXXIX Congreso SEBBM
P15-17
Estudio de las características bioenergéticas
y del estrés oxidativo en líneas de cáncer de
colon de diferentes estadios y su relación
con la sensibilidad a la quimioterapia con
5-fluorouracilo e irinotecán
Mª del Mar Blanquer Rosselló, Jordi Oliver Oliver, Ádamo Valle
Gómez, María del Pilar Roca Salom
Grupo Multidisciplinar de Oncología Traslacional, Institut
Universitari d’Investigació en Ciències de la Salut (IUNICS),
Universitat de les Illes Balears. Centro de Investigación Biomédica
en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn,
CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma
(IdISPa), Palma de Mallorca, ES
Las células tumorales de estadios primarios son más vulnerables al estrés
oxidativo por presentar bajos niveles de enzimas antioxidantes. En tumores
de estadios más avanzados, en cambio, los sistemas antioxidantes se
encuentran incrementados por una presión selectiva hacia la adaptación
al estrés confiriéndoles resistencia a los tratamientos quimioterapéuticos
que inducen estrés oxidativo. El objetivo fue analizar parámetros
bioenergéticos y comparar el efecto citotóxico de los tratamientos
antitumorales 5-fluorouracilo (5-FU) e irinotecán (IRI) y en combinación
con resveratrol (RSV) en líneas de cáncer de colon en diferentes estadios:
tumor primario (Caco-2 y HT29) y metastásica (SW620). Para ello, se
determinó la velocidad de crecimiento, los niveles de estrés oxidativo y
la expresión de proteínas implicadas en la funcionalidad mitocondrial y
metabolismo celular (SIRT1, SIRT3, PGC1α, NRF1, NRF2, TFAM, UCP2,
PDH y LDH). Para determinar los efectos citotóxicos de los tratamientos,
se analizó la viabilidad celular y los niveles de estrés a diferentes dosis y
tiempos. La línea metastásica fue la que presentó un mayor crecimiento,
mayores niveles de estrés, así como mayor expresión de SIRT3 y TFAM.
Se observó que el IC50 del 5-FU es menor que el del IRI en HT29 y Caco-2
mientras que en la línea SW620 se da el perfil opuesto. Además, dosis de
IRI de 5 a 40 μM a 48 h provocaron un 50 % más de estrés que el 5-FU
en las SW620. Estos resultados sugieren que el IRI tiene un mayor efecto
citotóxico en la línea metastásica que en las primarias. Por otra parte, el
tratamiento conjunto de 5-FU y RSV en la línea SW620 resultó en un
efecto citotóxico sinérgico.
Beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y FEDER-Unión
Europea “Una manera de hacer Europa”.
P15-18
La importancia de la UCP2 en el control
del estrés oxidativo en líneas de melanoma
Margalida Torrens-Mas1, Daniel González-Hedström2, Mario
Moreno-Fernández2, Jordi Oliver1, Pilar Roca1, Jorge Sastre-Serra1
1
Grupo Multidisciplinar de Oncología Traslacional, Institut
Universitari d’Investigació en Ciències de la Salut (IUNICS),
Universitat de les Illes Balears. Centro de Investigación Biomédica
en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn,
CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma
(IdISPa), Palma de Mallorca, ES, 2Dept. Biología Fundamental y
Ciencias de la Salud, Universitat de les Illes Balears, Palma de
Mallorca, ES
El receptor de melanocortina 1 (MC1R) es clave para el desarrollo
del melanoma, ya que está implicado en la pigmentación de la piel y la
protección contra la radiación ultravioleta. Cuando se activa, el receptor
induce la expresión de PGC-1α, que a su vez controla genes relacionados
con la biogénesis mitocondrial y las enzimas antioxidantes, así como la
proteína desacoplante UCP2, regulando así los niveles de especies reactivas
de oxígeno (ROS). Estudios recientes sugieren que la UCP2 podría conferir
Salamanca 2016
a las células resistencia frente al estrés oxidativo, siendo importante para
la promoción tumoral. Nuestro objetivo fue estudiar la importancia de la
UCP2 sobre el estrés oxidativo en distintas líneas de melanoma con distintas
características del MC1R (mutado y no mutado). Para ello, se determinó la
expresión de los genes PGC-1α, enzimas antioxidantes y UCP2, así como
las actividades de las enzimas antioxidantes y la producción de ROS en
las líneas HBL, A375 y MeWo. Las líneas HBL y MeWo, no mutadas para
MC1R, presentaron unos mayores niveles de PGC-1α respecto a la línea
con el receptor mutado A375. La producción de ROS fue superior en las
líneas A375 y MeWo comparado con HBL. Sin embargo, las células MeWo
mostraron unos mayores niveles tanto de la expresión como de la actividad
de las enzimas antioxidantes que las otras dos líneas, mientras que la
expresión de UCP2 fue mayor en la línea HBL. Estos resultados sugieren
que la UCP2 puede jugar un papel clave en la producción de radicales
libres en líneas de melanoma y, por tanto, en la evolución del tumor, siendo
un factor importante a tener en cuenta junto a las características de PGC-1α
y MC1R.
Financiado: beca FPU del MECD yAECC (Asociación Española contra
el Cáncer).
P15-19
4-n-Butylresorcinol, a depigmenting agent
used in cosmetics, reacts with tyrosinase
Antonio García Jiménez1, José Antonio Teruel Puche2, Carmen
Vanessa Ortiz Ruiz1, José Berna Cánovas3, José Tudela Serrano1,
Francisco García Cánovas1
1
GENZ-Group of research on Enzymology (www.um.es/genz),
Department of Biochemistry and Molecular Biology-A, Regional
Campus of International Excellence Campus Mare Nostrum,
University of Murcia, Espinardo, ES, 2Group of Molecular
Interactions in Membranes, Department of Biochemistry and
Molecular Biology-A, University of Murcia, Espinardo, ES, 3Group
of Synthetic Organic Chemistry, Department of Organic Chemistry,
Faculty of Chemistry, University of Murcia, Espinardo, ES
Introduction: 4-n-Butylresorcinol (BR) is considered the most
potent inhibitor of tyrosinase, which is why it is used in cosmetics as a
depigmenting agent. However, some of these compounds previously
considered inhibitors have recently been described to act as alternative
substrates.
Materials and methods: Spectrophotometric assays were carried out to
determine the nature of BR, and to characterize it kinetically. Chemical
shift values (δ) in 13C were determined by NMR. The chemical structures
of these ligands were constructed with PyMOL 1.5.0.1.
Results: The values obtained provided the Michaelis-constant (60.26 ±
8.76 μM) and catalytic constant (8.49 ± 0.20 s-1). Studies of BR docking to
the Em (met-tyrosinase) form of the enzyme show that the hydroxyl group
in C-1position is oriented toward the copper atom A (CuA), as in it is
L-tyrosine. As regards Eox (oxy-tyrosinase), BR is oriented with the carbon
in C-6position ready to be hydroxylated.
Discussion: The Em form is not active on BR, but Eox can act on this
molecule, hydroxylating it to o-diphenol. In turn, this is oxidized to an
o-quinone, which isomerizes to a red p-quinone. Thus, for tyrosinase to act
on this compound, a mechanism to generate Eox in the medium is required,
which can be achieved by means of hydrogen peroxide or ascorbic acid.
Conclusion: The results obtained show that BR is an alternative substrate
of tyrosinase, originating o-quinones, which isomerize to p-quinones
that can react with thiol compounds, a finding that could have important
implications in pharmacology and cosmetics.
Acknowledgements: This work was partially supported by: Projects 19545/
PI/14, 19304/PI/14 and 19240/PI/14 (Fundación Seneca, CARM, Spain);
Projects SAF2013-48375-C2-1-R and CTQ2014-56887-P (MINECO);
Projects UMU15452 and UMU17766 (University of Murcia); C.V.
Pósters / Posters
Ortiz-Ruiz has a MEC-FPU fellowship (AP2010-4300), A. García-Jiménez
has a FPU fellowship from University of Murcia.
P15-20
Efecto del resveratrol sobre la proliferación
celular, ciclo celular y estrés oxidativo en la línea
celular de cáncer de colon SW620
María del Mar Blanquer-Rosselló1, José Alberto Guerrero Castillo2,
Reyniel Hernández López1, Ádamo Valle1, Pilar Roca1, Jordi Oliver1
1
Grupo Multidisciplinar de Oncología Traslacional, Institut
Universitari d’Investigació en Ciències de la Salut (IUNICS),
Universitat de les Illes Balears. Centro de Investigación Biomédica
en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn,
CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma
(IdISPa), Palma de Mallorca, ES, 2Dept. Biología Fundamental y
Ciencias de la Salud, Universitat de les Illes Balears, Palma de
Mallorca, ES
El resveratrol (RSV) es un estilbenoide, presente en la piel de las uvas
y otros frutos rojos, del que se han descrito efectos antioxidantes y
antinflamatorios; además de efectos anticancerígenos, al inhibir la
proliferación y la progresión del cáncer. Algunos estudios han relacionado
estos efectos por ser un activador de la función mitocondrial y los
mecanismos de antioxidación. El balance de estrés oxidativo juega un
importante papel en la regulación del ciclo celular.
Por ello, nos planteamos estudiar el efecto de dosis fisiológicas de RSV (10
μM) sobre el estrés oxidativo, el ciclo celular y la proliferación de la línea
de cáncer de colon SW620. Se ha evaluado la viabilidad celular (cristal
violeta), ciclo celular (citometría), el potencial de membrana (TMRM),
la producción de ROS (Amplex red) y los niveles de UCP2, la apoptosis
(Annexin V y PARP/PARP rota) y autofagia (MDC). También se estudió el
efecto del RSV como adyuvante al tratamiento con 5-fluorouracilo (5FU)
sobre la viabilidad y la producción de ROS.
El tratamiento con RSV en las células de colon SW620 tuvo un efecto
inhibidor de la proliferación celular induciendo la apoptosis (ciclo celular
y PARP/PARP rota) sin aumento de la autofagia (MDC). Este efecto podría
ser debido al incremento en la producción de ROS, ligado en parte a la
reducción de la expresión de la UCP2. El efecto del RSV potenció los del
5FU inhibiendo la viabilidad y el nivel de estrés oxidativo.
Los resultados obtenidos permiten concluir que el RSV aumenta la
susceptibilidad de las células cancerígenas frente a la quimioterapia, al
modificar la capacidad de las células de colon SW620 a hacer frente a un
estrés oxidativo.
Financiado: beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y
FEDER-Unión Europea “Una manera de hacer Europa”.
P15-21
HO-1 induction via α7 nicotinic acetylcholine
receptors: implications in the neuroinflammatory
response
Manuela G. López1, Elisa Navarro1, Cristina Fernández-Mendívil1,
Enrique Luengo1, Jonathan Godbout2
1
Instituto Teófilo Hernando. Departamento de Farmacología.
Facultad de Medicina. Universidad Autónoma de Madrid. Instituto
Universitario de Investigacion la Princesa. Hospital de la Princesa,
Madrid, ES, 2Department of Neuroscience. Institute for Behavioral
Medicine Research. Center for Brain and Spinal Cord Repair. The
Ohio State University Wexner Medical Center, Ohio, US
We have previously shown that microglial HO-1 induction, via
α7nAChR, controls inflammation to provide neuroprotection in stroke.
Now, we have evaluated the implication of the α7nAChR/Nrf2/HO-1
117
Pósters / Posters
axis in inflammatory models and its functionality with aging. Exposure
of organotipic hippocampal cultures to the combination of antimycin-A
(0.1 μM) and lipopolysaccharide (1 ng/ml) caused synergic neurotoxicity,
in addition to mitochondrial depolarization, overproduction of reactive
oxygen species, Nox4 overexpression and aberrant protein aggregation.
The α7 nicotinic receptor agonist, PNU282987, prevented all these
alterations by a mechanism that implicated HO-1 induction. The
implications of HO-1 in controlling neuroinflammation, via α7nAChR
stimulation, were further corroborated in WT and LysMCreHmox1∆/∆
mice, that do not express HO-1 in myeloid cells, treated with LPS.
Furthermore, BALB/c mice were injected with LPS and the α7nAChR
agonist, PNU282987 (i.p. or i.c.v). As expected, LPS (i.p.) challenge
reduced body weight and induced lethargy and social with drawal
in adult mice. Peripheral administration of the α7nAChR agonist
improved these parameters. The α7nAChR agonist also reduced
microglial activation with suppression of IL-1β and TNFα mRNA levels
4 and 24 h after LPS injection. Nonetheless, when PNU282987 was
administered (i.p.) two hours after LPS challenge there was no effect.
These data were interpreted to indicate that peripheral enhancement of
α7nAChR signaling after microglia became activated was an ineffective
intervention. When the α7nAChR agonist was administered centrally
(i.c.v.) 2 h after LPS injection in adult and aged mice, in adult mice,
central augmentation of nAChR signaling attenuated LPS-induced
sickness behavior and microglial activation; however, in aged animals
there was no improved recovery or decreased microglial activation
following LPS challenge.
In conclusion, we provide evidence that stimulation of α7nAChR signaling
is anti-inflammatory via HO-1, but this cholinergic-dependent regulation is
absent in microglia of aged mice.
XXXIX Congreso SEBBM
P15r-23
Friedreich ataxia cell model to screen new
therapeutic drugs
Elena Britti, Fabien Delaspre, Joaquim Ros
IRB Lleida, Lleida, ES
Friedreich´s ataxia (FA) is the most common hereditary autosomal recessive
ataxia, affecting one of 50,000 people. It has been linked to expansion of a
GAA-triplet repeat in the first intron of the FXN gene, leading to a reduced
level of frataxin. The main affected cell types are sensitive neurons from
dorsal root ganglia, cardiomyocytes and β cells. There is currently no
effective cure or treatment for Friedreich ataxia. Life expectancy is around
40-50 years. During over 20-30 years the management of individuals
with FA will require a multidisciplinary approach, from requirement of
mobility aids to surgery, significantly impacting upon quality of life. Our
lab has developed a FA cell model using primary culture of dorsal root
ganglion sensory neurons (DRGs) from neonatal rat in which frataxin
level was reduced by lentivirus transduction of shRNA sequences targeting
frataxin mRNA. Frataxin deficiency in DRGs results in altered calcium
homeostasis, neurite degeneration and apoptotic cell death. The analyse
of this model point out several altered Ca2+-dependent pathways in
response to reduced level of frataxin. Based on this model we set up a
drug screen in order to identify potential new therapeutic approaches that
could be used to treat FA patients. We characterized the ability of altered
pathway targeting drugs in protecting frataxin deficient cells from neurite
degeneration, calcium homeostasis alteration and apoptosis. In parallel, we
used lymphoblastoid cell lines from FA patients to decipher the mechanism
of action of the drugs.
P15-24
P15r-22
Redox state of lipoic acid is altered in
Frataxin-deficient cardiac myocytes
Rosa Purroy Lledós, Anna Carrera Salinas, Joaquim Ros Salvador,
Jordi Tamarit Sumalla
Ciències Mèdiques Bàsiques, Universitat de Lleida, IRB Lleida,
Lleida, ES
Friedreich ataxia is a neurodegenerative disease accompanied by
hypertrophic cardiomyopathy. This hereditary disease is caused by
deficient frataxin expression, a mitochondrial protein that has been
related to iron homeostasis, energy metabolism, and oxidative stress.
We have recently set up a cardiac cellular model of Friedreich Ataxia
based on neonatal rat cardiac myocytes and lentivirus-mediated frataxin
RNA interference. As these frataxin-deficient cardiomyocytespresent
signs of oxidative stress, we decided to explore the presence of
protein thiol modifications in this model. With this purpose, the
presence of reversible oxidized cysteine residues was investigated
using the thiol-reactive fluorescent probe Bodipy-iodoacetamide
and 2D-gel electrophoresis. We identified three spots with altered
redox status in frataxin deficient cardiomyocytes that were identified
by mass spectrometry as Electron transfer flavoprotein-ubiquinone
oxidoreductase (ETFD), Dihydrolipoyl dehydrogenase (DLDH) and
ATP synthase subunit alpha (ATPA). As DLDH is involved in lipoic
acid regeneration, we hypothesized that frataxin-deficient cells could
present lipoic acid in a more oxidized from. Lipoic acid is found bound
to the E2 subunit of pyruvate dehydrogenase (PDH) and 2-ketoglutarate
dehydrogenase complexes. Thus, we analyzed the western-blot signal
of lipoic acid in cells treated or not with iodoacetamide. We observed
a clear decrease in the iodoacetamide reactive form of lipoic acid
bound to PDH E2, indicating that lipoic acid was more oxidized in
frataxin-deficient cells.
118
Transformation of plum plants with cytosolic
antioxidants leads to enhanced abiotic stress
tolerance
Pedro Díaz Vivancos
CEBAS-CSIC, Murcia, ES
In order to understand the role of cytosolic antioxidant enzymes in abiotic
stress protection, transgenic plum (Prunus domestica cv. Claudia verde)
plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) and/
or ascorbate peroxidase (cytapx) were produced. First, we examined the
potential functions of cytSOD and cytAPX in in vitro plum plants against
salt stress. Several transgenic plum plantlets expressing cytsod and/or
cytapx showed an enhanced tolerance to salt stress, mainly lines C5-5 and
J8-1 (expressing several copies of sod and apx, respectively). Interestingly,
these plants showed higher regeneration efficiency and enhanced vigour.
Transformation as well as NaCl treatment influenced the antioxidative
metabolism of plum plantlets, including enzymatic and non-enzymatic
antioxidants.
Then we tested the water stress (WS) tolerance under greenhouse
conditions of two transgenic lines: line C3-1, co-expressing both
transgenes simultaneously; and line J8-1, harbouring 4 copies of cytapx.
Line J8-1 exhibited an enhanced stress tolerance that correlated with better
photosynthetic performance and a tighter control of water use efficiency.
Furthermore, this WS tolerance also correlated with a higher enzymatic
antioxidant capacity than non-transformed and line C3-1 plum plants. On
the other hand, line C3-1 displayed an intermediate phenotype between
non-transformed plants and line J8-1 in terms of WS tolerance. Moreover,
proteomic analysis revealed differences between non-transformed and
J8-1 plants, mainly in terms of the abundance of proteins related with
carbohydrate metabolism, photosynthesis, antioxidant defences and protein
fate. Overall, our results suggest that transgenic plum plants are able to
cope more efficiently with the induced oxidative stress under unfavourable
environmental conditions.
Salamanca 2016
P15-25
Sobrecarga de hierro y estrés oxidativo en
la médula ósea de pacientes con síndromes
mielodisplásicos: evolución tras el tratamiento
con deferasirox
Tamara Jiménez Solas1, María Díez Campelo2, Irene Aires Mejía2,
Juan Carlos Caballero Berrocal2, Sandra Muntión Olave2, Félix
López Cárdenas2, Luis García Martín2, Alba Redondo Guijo2, Mª
Consuelo del Cañizo2, Ángel Hernández Hernández3
1
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca. Servicio de Hematología, Hospital Universitario de
Salamanca. Instituto de Investigación Biomédica de Salamanca
(IBSAL), Salamanca, ES, 2Servicio de Hematología, Hospital
Universitario de Salamanca. Instituto de Investigación Biomédica
de Salamanca (IBSAL), Salamanca, ES, 3Departamento de
Bioquímica y Biología Molecular, Universidad de Salamanca.
Instituto de Investigación Biomédica de Salamanca (IBSAL),
Salamanca, ES
Los síndromes mielodisplásicos (SMD) son una serie heterogénea de
desórdenes hematológicos, caracterizados por hematopoyesis inefectiva en
médula ósea asociada a citopenias, incluyendo anemias severas. Un alto
número de pacientes de SMD de bajo riesgo recibe soporte transfusional
como única terapia. Éstos presentan sobrecarga férrica, lo que provoca
aumento en la producción de especies reactivas del oxígeno (ROS), al
ser el hierro un catalizador de las reacciones de Fenton y Harber-Weiss.
Esto podría implicar un alto nivel de estrés oxidativo para las células de la
médula ósea (MO) de los pacientes.
En base a estos datos, nuestra hipótesis de trabajo es la siguiente:
La sobrecarga férrica involucrada en el incremento de ROS en la MO
potencia la disfunción de las células hematopoyéticas (HSPC) y del
estroma. Nuestro objetivo es analizar si el tratamiento quelante con
deferasirox (DFX) puede reducir el estrés oxidativo en HSPC, así como
el daño asociado en el DNA, y estudiar su efecto sobre la capacidad
hematopoyética.
Al comparar muestras de pacientes de SMD antes del tratamiento frente
a controles sanos, nuestros resultados preliminares muestran mayores
niveles de ROS en las poblaciones de HSPC de MO de los pacientes,
mayor daño en el DNA y cierto incremento de la apoptosis temprana. Tras
el tratamiento con DFX, se aprecia una tendencia hacia la reducción del
daño en el DNA en la mayoría de pacientes.
Los ensayos clonogénicos realizados antes del tratamiento indican
una capacidad hematopoyética disminuida de las HSPC de pacientes.
Mientras que tras el tratamiento, la relación cluster/CFU se aproxima a los
valores control. Globalmente, estos resultados sugieren una mejora de la
funcionalidad de la MO de los pacientes tras el tratamiento quelante.
Pósters / Posters
Whilst it is known the neuroprotective benefits of subtle stimulation of
cannabinoid receptor-1 (CB1), its over-stimulation during cannabis abuse
causes psychosis, memory loss and neurodegeneration. Recently, it has been
demonstrated the occurrence of mitochondrial CB1 (mtCB1) in neurons
and astrocytes where, by down-regulating protein kinase A and complex
I activities, neuronal functions are physiologically regulated. However,
whether over-activation of mtCB1 during cannabis abuse contributes to
the detrimental effects of cannabis abuse on neurons is elusive. Given that
neuronal survival depends on astrocyte metabolic support, we studied the
impact that over-stimulation of astrocyte mtCB1 exerts on mitochondrial
energy metabolism of neurons. We show that long-term (24 h) exposure of
the CB1 agonists ∆9-tetrahydrocannabinol and HU-210 to astrocytes causes
dephosphorylation of the complex I-assembly factor NDUFS4 and loss of
complex I protein. In consequence of this, the production of mitochondrial
reactive oxygen species and glycolytic-released lactate significantly
decreased in astrocytes. Co-incubation of the CB1 agonists-pretreated
astrocytes with untreated neurons caused mitochondrial membrane
depolarization, energy impairment and apoptotic death of neurons.
Noticeably, the changes triggered by the CB1 agonists were abolished in
astrocytes obtained from CB1 knockout mice or treated with the plasma
membrane-permeable CB1 antagonists AM251 and SR141716A, but
not the plasma membrane-impermeable CB1 antagonist hemopressin.
Thus, the astrocyte-dependent metabolic support of neurons is disrupted
by over-stimulating mtCB1 in astrocytes, likely contributing to the
neurological disabling caused by cannabis abuse.
(Funded by the NIH/NIDA grant number 1R21DA037678-01).
P15-27
Hydroxytyrosol reduces oxidative damage on
oxidative stress in an experimental model of
multiple sclerosis
Eduardo Agüera1, Cristina Conde1, Evelio Luque2, Macarena Aguilar
Luque3, Manuel LaTorre3, Ana Isabel Giraldo3, Montse Feijoó3,
Rafael Lillo4, Begoña Mª Escribano5, Alberto Galván3, Isaac Túnez3
1
Unidad de Gestión Clínica de Neurología, Instituto Maimónides de
Investigación Biomédica de Córdoba (IMIBIC)/Hospital Universitario
Reina Sofía (HURS), Córdoba, ES, 2Sección de Histología,
Departamento de Ciencias Morfológicas, Facultad de Medicina y
Enfermería, IMIBIC/Universidad de Córdoba (UCO), Córdoba, ES,
3
Departamento de Bioquímica y Biología Molecular, Facultad de
Medicina y Enfermería, IMIBIC/UCO, Córdoba, ES, 4Sección de
Psiquiatría, Departamento de Ciencias Sociosanitarias y Radiología
Clínica y Medicina Física, Facultad de Medicina y Enfermería,
IMIBIC/UCO, Córdoba, ES, 5Sección de Fisiología, Departamento de
Biología Celular, Fisiología e Inmunología, Facultad de Veterinaria,
IMIBIC/UCO, Córdoba, ES
(Col.: Novartis)
P15r-26
Over-stimulation of mitochondrial cannabinoid
receptor-1 (mtCB1) in astrocytes disrupts
astrocyte-dependent metabolic support of
neurons
Daniel Jiménez Blasco1, Mónica Resch-Beucher1, Mónica
Carabias-Carrasco1, Eva Resel2, Manuel Guzmán2, Juan P. Bolaños1
1
Institute of Functional Biology and Genomics (IBFG), University
of Salamanca-CSIC; and Institute of Biomedical Research of
Salamanca (IBSAL), University Hospital of Salamanca, Salamanca,
ES, 2Department of Biochemistry and Molecular Biology I, Instituto
Universitario de Investigación Neuroquímica, Complutense
University, Madrid, ES
Objective: The aim of the present work was studied the effects of
administration of hidroxytyrosol (HT) in a myelin oligodendrocyte
glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis
(EAE) model, similar to reproduce human multiple sclerosis (MS).
Material and methods: Twenty male Dark Agouti rats, weighing 180-200
g and 2 months old were used in the study. The rats were divided into 4
groups of 5 as follow: i) control; ii)vehicle; iii) EAE; and iii) EAE+HT.
Rats were injected with an emulsion containing myelin oligodendrocyte
glycoprotein (MOG) supplemented with heat-inactivated Mycobactirium
tuberculosis H37Ra. HT was administered orally using catheter during 65
days, starting 14 days after inoculation with MOG.
Results: Our data showed a significant decrease score as well as a
reversion towards to normality in the oxidative stress biomarkers after HT
supplementation
Conclusions: The HT administration shows positive and protective effects
in EAE animals. However, more experimental and clinical studies in this
line are required.
119
Pósters / Posters
P15-28
Papel de la proproteína convertasa subtilisina/
kexina de tipo 9 (PCSK9) en el macrófago
César Eduardo Rosales-Mendoza1, Marina Mojena2, Silvia
González-Ramos2, Marta Paz-García2, Emilio Acosta-Medina3,
Paloma Martín-Sanz2, Lisardo Boscá2
1
Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM),
Madrid, ES; Departamento de Bioquímica y Medicina Molecular,
Universidad Autónoma de Nuevo León, Monterrey, MX, 2Instituto de
Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, ES,
3
Centro de Estudios Avanzados de Cuba, La Habana, CU
PCSK9 es una serin proteasa perteneciente a la familia de las proproteínas
convertasas que se libera principalmente por el hepatocito. Su función es
regular la degradación del receptor del colesterol LDL (LDL-R), uniéndose
a este receptor e impidiendo su reciclaje e induciendo su degradación
lisosomal así como la disminución de su expresión en la superficie de
los hepatocitos. Esto impide el aclaramiento hepático del colesterol LDL
(LDL-C) sérico, aumentando sus niveles, lo que supone un importante factor
de riesgo cardiovascular. PCSK9 se ha convertido en una proteína de interés
relevante desde la identificación de su papel en el metabolismo lipídico. A
su vez, PCSK9 ha sido implicada en la inflamación. Se ha documentado
que los niveles de PCSK9 correlacionan con los de LDL-C en condiciones
patológicas como la sepsis. Los macrófagos, que son participantes activos
de la respuesta inflamatoria, así como en los procesos antiinflamatorios y
de pro-resolución de la inflamación, son de gran interés en el desarrollo de
aterosclerosis. El proceso inflamatorio de la aterosclerosis parece estar más
vinculado a PCSK9 que en solo la regulación del LDL-R.
En el presente trabajo presentamos que tanto macrófagos humanos como
murinos no expresan PCSK9. Sin embargo, su presencia modula la
activación y fenotipo de estas células inmunes, tanto a nivel transcripcional
como funcional, entre ellos la disminución del LDR-L, así como la expresión
de genes inflamatorios y la generación de radicales libres. Por tanto,
además de su papel en el metabolismo lipídico, PCSK9 ejerce cambios en
el macrófago que pueden contribuir al desarrollo de aterosclerosis.
P16. Regulación de la expresión
génica y dinámica del genoma /
Regulation of gene expression
and genome dynamics
P16-1
Thermodynamic signature for drug binding
in the minor-groove of DNA
XXXIX Congreso SEBBM
in the minor groove of either C+G-rich or A+T–rich DNA sequences is
entropically driven while intercalation is enthalpically driven. To gain
insights into the different binding signatures, we have used UV-thermal
denaturation and ITC (isothermal titration calorimetry), as well as
computations of the hydrophobic free energy of binding, to determine the
thermodynamic profile of drug binding to G+C-rich DNA sequences. The
thermodynamic analysis shows that the binding in the minor groove of DNA
is, regardless of the DNA sequence involved, always entropically-driven,
dominated by the hydrophobic transfer of drug molecules from solution
to the DNA-binding site. Direct molecular recognition between drugs
and DNA through hydrogen bonding and van der Waals contacts also
contributes significantly to drug-DNA complex formation.
References
[1] Chaires, J. B. (2006) Arch. Biochem. Biophys. 453, 24-29.
[2] Barceló, F., Scotta, C., Ortiz-Lombardía, M., Méndez, C., Salas, J. A. &
Portugal, J. (2007) Nucleic Acids Res. 35, 2215-2226.
P16r-2
Efecto sobre el envejecimiento celular
de la regulación traduccional mediada
por la fosforilación del factor eIF2alfa
Tamara Jiménez Saucedo, Juan José Berlanga Chiquero, Miguel
Ángel Rodríguez Gabriel
Centro de Biología Molecular Severo Ochoa, Madrid, ES
El resultado de la respuesta a estrés (daño, muerte o adaptación y
supervivencia) depende fundamentalmente de los procesos tempranos que
tienen lugar en las células tras la exposición al mismo. Estos eventos tempranos
incluyen la detección de estrés, la transducción de señales bioquímicas y los
cambios transcripcionales y postranscripcionales. La hipótesis central de este
proyecto es que los primeros cambios postranscripcionales de la expresión
génica tras el estrés, especialmente la reprogramación de la traducción
por la fosforilación del factor 2 de iniciación eucariota (eIF2), generan las
señales y el conjunto de proteínas necesarias para promover la adaptación
y la supervivencia de las células y organismos. Tras la aplicación de estrés,
se induce la fosforilación de la serina 52 de la subunidad α del factor eIF2
lo que conlleva una disminución de la tasa de traducción general, así como
la traducción de mRNAs específicos de respuesta a estrés. Además, estos
eventos tempranos pueden condicionar procesos a largo plazo tales como
el envejecimiento celular, y la aparición de patologías asociadas a una
exposición crónica al estrés. En nuestro laboratorio hemos utilizado la
levadura de fisión Schizosaccharomyces pombe como modelo y hemos
llevado a cabo experimentos de privación de nutrientes, lo cual produce
un efecto positivo sobre la longevidad. Además, se ha estudiado el efecto
de varios inhibidores de la traducción sobre el envejecimiento así como
el estado de la traducción en ambas condiciones. Detallamos los análisis
bioquímicos de los resultamos en nuestra comunicación.
Francisca Barceló Mairata1, José Portugal2
Universidad de les Illes Balears, Palma de Mallorca, ES, 2Instituto
de Biología Molecular de Barcelona, CSIC, Parc Cientíific de
Barcelona, Barcelona, ES
P16-3
1
A mechanism for the recruitment
of recombination proteins during DNA
damage tolerance
We have obtained complete thermodynamic profiles consisting of binding
constants, free energy, enthalpy, and entropy for the binding of drugs in
the G+C-rich regions of DNA. Such profiles are important in anticancer
drug design because they provide information on the balance of driving
forces that is not accessible by structural or computational methods alone
[1, 2]. Despite most of the small drugs that bind to C+G-rich sequences are
intercalating agents, a variety of molecules bind directly and selectively
to C+G-rich DNA tracts through the minor groove without intercalation.
Interestingly, many of those molecules are active as chemotherapeutic
agents by inhibiting replication or transcription. The binding of drugs
María J. Cabello-Lobato, María I. Cano-Linares,
Román González-Prieto, Félix Prado
Cabimer-CSIC, Sevilla, ES
120
The recombination proteins Rad52 and Rad51 help the fork to pass
through blocking lesions and to fill in the gaps of ssDNA generated during
the process of lesion bypass. Their recruitment to the ssDNA lesions
has obligatorily to occur during S phase. Here we show that Rad52 and
Rad51 display the same kinetics of chromatin binding as the helicase
Mcm2-7: they accumulate in G1, are released during replication, and
Salamanca 2016
Pósters / Posters
Exploring the contribution of human prefoldin
to gene expression
gene expression. To achieve this PipX establishes physical complexes with
PII or NtcA in response to low or high 2-oxoglutarate levels, respectively.
Transcriptome analyses carried out with mutant strains are consistent with
NtcA-dependent gene expression modulation of PipX. The results also
suggest regulatory roles of PipX in an NtcA-independent way, implicating
novel proteins in its network.
In most cyanobacteria, pipX forms an operon with pipY. PipY is a member
of the widely distributed COG0325 protein family, PLP-binding proteins
of unknown function but related to amino acid metabolism and Vitamin
B6 homeostasis, as inferred by null mutant analyses. To try to understand
the function of COG0325 proteins and the regulatory connections
between PipX and PipY proteins in cyanobacteria, we are characterizing
the pipX-pipY operon of Synechococcus elongatus PCC7942, with an
emphasis on the in vivo functions of PipY. Results from bioinformatics,
transcriptomic and genetic analyses will be discussed.
Laura Payán-Bravo1, Silvia Jimeno-González2, Xenia Peñate1,
José Carlos Reyes2, Sebastián Chávez1
1
Instituto de Biomedicina de Sevilla (IBiS), Hospital Virgen del
Rocío-CSIC-Universidad de Sevilla, Sevilla, ES, 2Centro Andaluz
de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC,
Sevilla, ES
Contribution of TFIIS to nucleosome positioning
revealed by improved MNase-seq
remain bound to chromatin in the presence of replicative blocking lesions.
This coordinated response requires Cdc7-dependent physical interactions
between Rad51/Rad52 and the Mcm2-7 helicases that are not at the fork.
Accordingly, reducing Cdc7 activity impairs sister-chromatid junction
formation and ssDNA filling by recombination. Therefore, the loading of
Rad51 and Rad52 in G1, together with a Mcm2-7-mediated mechanism
that couples the fork advance with Rad52/Rad51 binding to replicative
damage, provides a strategy to ensure the filling of the ssDNA lesions
through an error-free repair mechanism.
P16r-4
Prefoldin is a heterohexameric complex composed of six different subunits
(PFDN1 to PFDN6) present from archaea to higher eukaryotes. It is a
co-chaperone that cooperates with the cytoplasmic chaperonin CCT in the
folding of actin and tubulin monomers. Eukaryotes also display a prefoldin-like
complex, which contains PFDN2 and PFDN6 in addition to other non-canonical
prefoldin subunits.We have previously showed that prefoldin is also localized
in the nucleus of the yeast Saccharomyces cerevisiae where it plays a role
in chromatin dynamics during transcription elongation (Millán-Zambrano,
et al., 2013). We have also demonstrated that defective histone dynamics
provokes changes in transcription elongation and co-transcriptional splicing
in human cells (Jimeno-González et al., 2015). Here, we report that depletion
of the canonical complex-specific PFDN5 subunit caused co-transcriptional
splicing defects in CTNNBL1, a long human gene commonly used to analyze
transcription elongation. However, we did not detect major alteration in the
transcription rate of RNA polymerase II or its processivity (indirectly measured
by pre-mRNA appearance) when PFDN5 was depleted. On the whole, these
results suggest a possible role of prefoldin in human gene expression by
contributing to co-transcriptional events.
References
[1] Millán-Zambrano G, Rodríguez-Gil A, Peñate X, de Miguel-Jiménez
L, Morillo-Huesca M, et al., (2013) The prefoldin complex regulates
chromatin dynamics during transcription elongation. PLoS Genet 9(9):
e1003776.
[2] Jimeno-González S, Payán-Bravo L, Muñoz-Cabello AM, Guijo
M, Gutierrez G, et al., (2015). Defective histone supply causes changes
in RNA polymerase II elongation rate and cotranscriptional pre-mRNA
splicing. Proc Natl Acad Sci U S A. 112(48):14840-5.
P16-6
Gabriel Gutiérrez1, Gonzalo Millán-Zambrano2, Xenia Peñate Salas2,
Sebastián Chávez de Diego2
1
Departamento de Genética, Universidad de Sevilla, Sevilla, ES,
2
Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen
del Rocío/CSIC/Universidad de Sevilla, Sevilla, ES
Understanding chromatin dynamics is a key to other related processes,
including DNA replication and transcription. As a first step, recently, an
increasing amount of effort has been devoted to precisely define nucleosome
positioning in different organisms. The most popular method to do so
involves chromatin digestion by micrococcal nuclease (MNase), nowadays
followed by ultrasequencing of the generated fragments. Although the
sequence bias of MNase has been known for a long time, a data-based way
of correcting this bias for sequencing experiments is still missing. Here we
provide such a method, based on the digestion of naked DNA and the use
of the bioinformatic tool DANPOS. Using Saccharomyces cerevisiae as
a model, we demonstrate that the overall quality of the data is better after
the correction proposed here. The characteristics of the method make it
perfectly applicable to any other organism. We found that the correction
affects more to those genes containing a canonical TATA element in their
promoter than to TATA-like genes. Importantly, the improved method
helped to uncover a new role for the TFIIS transcription elongation factor
in chromatin dynamics. The absence of TFIIS caused a general increase
in nucleosomal fuzziness, and a stronger occupancy of the -1 nucleosome
at some promoters. Considering that TFIIS acts on RNA polymerase II
to solve backtracking events, our results suggest that RNA polymerase II
backtracking stabilizes nucleosomes out of their usual location and, by
preventing it, TFIIS contributes to shape nucleosome positioning.
P16-5
P16r-7
Expanding the roles of PipX, a cyanobacterial
global regulator
Búsqueda de nuevos reguladores de Kin4
y análisis de su función en el punto de control
de posicionamiento del huso
José Ignacio Labella, Javier Espinosa, Raquel Cantos, Asunción
Contreras
Universidad de Alicante, San Vicente, ES
Cyanobacteria are ubiquitous autotrophic microorganisms that perform
oxygenic photosynthesis. They response efficiently to changes in the
Carbon/Nitrogen ratio through the highly conserved signal transduction
protein PII and the global nitrogen regulator NtcA, a cyanobacterial
CRP-like transcription factor. In the model cyanobacterium Synechococcus
elongatus PCC 7942, PipX, a small protein exclusive of cyanobacteria,
works as a mechanistic link to connect PII signalling with NtcA-regulated
Laura Matellán1, Ana María Rincón2, Fernando Monje-Casas3
1
Universidad de Sevilla / CABIMER, Sevilla, ES, 2Universidad de
Sevilla, Sevilla, ES, 3CSIC, Sevilla, ES
Durante la división celular por mitosis es esencial mantener la fidelidad
en la transmisión del material genómico para evitar que las células
resultantes adquieran un cariotipo aberrante. Con este fin, las células
cuentan con distintos mecanismos de vigilancia que controlan la
segregación de los cromosomas. Entre estos sistemas, en Saccharomyces
cerevisiae es particularmente interesante la existencia del punto de control
121
Pósters / Posters
de posicionamiento del huso (SPOC), un mecanismo de vigilancia que
evita que las células con el huso mitótico mal orientado salgan de mitosis,
lo cual es esencial dada la naturaleza asimétrica de la división celular en
la levadura de gemación. La proteína quinasa Kin4 juega un papel central
en el SPOC. Sin embargo, los mecanismos que regulan la actividad de
Kin4 tras la activación del SPOC todavía no se conocen en profundidad.
Nuestro objetivo es la búsqueda de proteínas que interaccionen con
Kin4 de forma dependiente del estado de activación del SPOC, y que
por tanto puedan jugar un papel importante en la regulación de este
mecanismo de vigilancia. Con este fin, hemos generado una estirpe de
levaduras en la que podemos activar constitutivamente el SPOC de forma
condicional. Usando esta estirpe, hemos conseguido purificar Kin4 tanto
en condiciones de crecimiento normal como en condiciones de activación
del SPOC, y hemos estudiado el conjunto de proteínas que interaccionan
con esta quinasa de forma diferencial en ambas condiciones. El análisis
de estas proteínas y de su papel en la regulación de la actividad y/o la
localización de Kin4 nos permitirá descifrar los mecanismos moleculares
de regulación del SPOC. Los resultados de esta investigación serán
importantes para, por medio del estudio de los homólogos de las proteínas
identificadas, entender cómo se coordina el correcto posicionamiento
del huso con la progresión del ciclo celular en divisiones asimétricas en
eucariotas superiores.
P16-8
Histone depletion prevents telomere-telomere
fusions in pre-senescent cells
Marta Barrientos-Moreno1, Marina Murillo-Pineda2,
Ana M. Muñoz-Cabello3, Félix Prado1
1
CABIMER-CSIC, Sevilla, ES, 2Instituto Gulbenkian de Ciência,
Oeiras, PT, 3IBIS, Sevilla, ES
Nucleosome assembly during DNA replication establishes the primary
chromatin structure, which is essential for genome organization and
stability. Defective chromatin assembly causes replication fork instability
and hyper-recombination, and impairs sister-chromatid decatenation
and chromosome bi-orientation during mitosis. The importance of
nucleosome assembly in genome integrity is also highlighted by the
fact that replicative aging in yeast and replicative senescence in yeast
and humans are associated with a severe reduction in the amount of
histones. Telomere shortening can prevent tumour development by
inducing senescence of transformed cells; however, it can also lead to
cancer initiation by inducing chromosomal instability because cells
divide during pre-senescence with critically short telomeres that can
be recognize as unrepaired breaks and be fused to other telomeres
(telomere-telomere fusions; T-TFs) or DSBs, thus causing gross
chromosomal rearrangements. Here we show that histone depletion
prevents T-TFs in mec1∆ tel1∆ cells defective in telomere maintenance.
Since pre-senescence in cells lacking the telomerase has been shown to
be associated with a reduction in the pool of available histones through
a Mec1-dependent mechanism, we hypothesize that histone depletion
is an active mechanism to protect telomeres that is lost in mec1∆ tel1∆
cells. This mechanism is independent of the telomerase but strictly
dependent on homologous recombination. We propose that the increase
in recombination by replication fork collapse as a consequence of the
deficit of histones protects short telomeres during pre-senescence.
P16-9
Sod1-activity is linked to selenite toxicity in yeast
Hayat Heluani Gahete1, Elisabet Fernández García1, Carlos Ruiz
Rodríguez1, Prince Saforo Amponsah2, Bruce Morgan2, Ralf Wellinger1
1
CABIMER, Sevilla, ES, 2TU Kaiserslautern, Kaiserslautern, DE
Selenium (Se) is a trace element that is essential for human health.
Reduction of inorganic selenium (sodium selenite; Na2SeO3) involves
122
XXXIX Congreso SEBBM
glutathione (GSH) oxidation and the formation of reactive oxygen species
(ROS). ROS then have to be detoxified in order to avoid ROS-mediated
DNA damage and genomic instability1. We find that the budding yeast
sod1Δ mutant rescues growth of selenite hypersensitive yeast mutants. This
is a surprising observation, given the fact SOD1 encodes for a superoxide
dismutase needed for the tolerance to ROS by the conversion of ion
superoxide (O2-) into oxygen (O2) and hydrogen peroxide (H2O2). Here, we
will present new data on factors involved in selenite resistance pointing to a
complex interplay between mechanisms involved in selenite detoxification
and oxidative stress response.
Keywords: ROS, Sod1, Sodium Selenite
References
[1] Herrero E, Wellinger RE. Review: “Yeast as a model system to study
metabolic impact of selenium compounds”. Microbiol Cell (2015).
P16r-10
Role of morphine, miR-212/132 and μ opioid
receptor in the regulation of Bdnf in zebrafish
embryos
Ada Jiménez González1, Adrián García Concejo1, Saray López
Benito1, Juan Carlos Arévalo2, Raquel E. Rodríguez3
1
Instituto de Investigación Biomédica de Salamanca; Instituto de
Neurociencias de Castilla y León (Universidad de Salamanca),
Salamanca, ES, 2Instituto de Investigación Biomédica de
Salamanca; Instituto de Neurociencias de Castilla y León
(Universidad de Salamanca); Departamento de Biología Celular y
Patología, Salamanca, ES, 3Departamento de Bioquímica y Biología
Molecular; Instituto de Investigación Biomédica de Salamanca;
Instituto de Neurociencias de Castilla y León (Universidad de
Salamanca), Salamanca, ES
Morphine is one of the first-line therapies for the treatment of pain despite
its secondary effects. It modifies the expression of post-transcriptional
regulators like miRNAs. In the present study, we analyzed miR-212 and
miR-132 and their implication in morphine effects in the zebrafish Central
Nervous System (CNS) through the regulation of Bdnf expression. We
used control and knock-down zebrafish embryos to assess the effects of
morphine in miRNAs212/132 and mitotic or apoptotic cells by qPCR,
immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB
were studied by western blot and through a primary neuron culture. A
luciferase assay was performed to confirm the binding of miRNAs 212/132
to mecp2.
Our results showed that morphine exposure decreases miR-212 but
upregulates miR-132, as wells as Bdnf and TrkB, and it changes the
localization of proliferative cells. However, Bdnf expression was
downregulated when miRNAs 212/132 and oprm1 were knocked-down.
Furthermore, we proved that these miRNAs inhibit mecp2 expression by
binding to its mRNA sequence. The described effects were corroborated
in a primary neuron culture from zebrafish embryos. Taking these results
into consideration we propose a mechanism in which morphine alters
the levels of miRNAs 212/132 increasing Bdnf expression through
mecp2 inhibition. oprm1 is also directly involved in this regulation. The
present work confirms a relationship between the opioid system and
neurotrophins pointing to miRNAs 212/132 as novel regulators of the
effects produced by morphine on the CNS. Both miRNAs have a key role
on morphine effects through the regulation of Bdnf pathway which is also
controlled by oprm1.
Salamanca 2016
Pósters / Posters
P16r-11
P16-13
μ opioid receptor expression after morphine
administration is regulated by miR- 212/132
cluster
Regulación de la replicación por el checkpoint
de fase S en Saccharomyces cerevisiae
Adrián García Concejo, Ada Jiménez González, Raquel E. Rodríguez
Instituto de Neurociencias de Castilla y León (Universidad de
Salamanca); Instituto de Investigación Biomédica de Salamanca,
Salamanca, ES
Since their discovery, miRNAs have emerged as a promising therapeutical
approach in the treatment of several diseases. miR-212 has already been
related to cocaine intake and to addiction, and here we prove that the
miR-212/132 cluster can be regulated by morphine through the activation
of the mu opioid receptor (Oprm1). The molecular pathways triggered after
morphine administration also induce changes in the levels of expression
of oprm1. In addition, miR-212/132 cluster is actively repressing the
expression of mu opioid receptor by targeting a sequence in the 3’’ UTR
of its mRNA. These findings suggest that this cluster is closely related
to opioid signaling and function as a post-transcriptional regulator,
modulating morphine response in a dose dependent manner. The regulation
of miR-212/132 cluster expression is mediated by the MAP kinase pathway,
CaMKII-CaMKIV and PKA, through the phosphorylation of CREB and
methyl cytosine binding protein 2 (MeCP2). In addition, the regulation of
both oprm1 and of the miRNAs cluster promoter is mediated by MeCP2,
acting as a transcriptional repressor on methylated DNA after prolonged
morphine administration. This mechanism explains the molecular signaling
triggered by morphine as well as the regulation of the expression of the mu
opioid receptor mediated by morphine and the implication of miR-212/132
in these processes.
P16r-12
Caracterización bioquímica de la resolvasa
de uniones de Holliday Yen1
Francisco Javier Aguado Domínguez, Vanesa Hurtado Nieves,
Miguel González Blanco
Centro de Investigación en Medicina Molecular y Enfermedades
Crónicas (CiMUS), Universidade de Santiago de Compostela,
Santiago de Compostela, ES
La recombinación homóloga (HR) es un mecanismo esencial para la
reparación de roturas en la doble cadena del DNA. Durante este proceso,
se pueden generar intermediarios recombinatorios ramificados, que en
algunos casos pueden madurar hasta formar uniones estables constituidas
por cuatro cadenas de DNA denominadas uniones de Holliday (HJ).
Dichas conexiones deben deshacerse oportunamente para permitir una
correcta segregación cromosómica durante la mitosis. Para ello, la célula
dispone de varios mecanismos de resolución de HJ, siendo una de las
enzimas implicadas en este proceso la nucleasa selectiva de estructura
Yen1. La actividad de Yen1 está altamente regulada a lo largo del ciclo
celular mediante modificaciones post-traduccionales. Concretamente, su
fosforilación por parte de Cdk en fase S conlleva su expulsión del núcleo
y disminuye su afinidad por el DNA, reduciendo la actividad biológica
del enzima. En anafase, el enzima es defosforilado por la fosfatasa Cdc14,
que permite su reactivación e importación al núcleo, donde podrá eliminar
aquellos intermediarios de recombinación más persistentes. Recientemente
hemos demostrado la toxicidad de expresar una versión mutante de Yen1
constitutivamente activa. Sin embargo, desconocemos cuál de las posibles
actividades nucleásicas de Yen1 puede ser responsable de dicha toxicidad
ya que aún no se han caracterizado a fondo las propiedades bioquímicas de
este enzima. Aquí presentamos un análisis bioquímico de Yen1 que revela
actividades inesperadas de esta nucleasa. Tales actividades distintas de la
resolución canónica de las HJ podrían explicar los efectos deletéreos de la
desregulación de este enzima y, por tanto, de la importancia de su adecuado
control.
Esther C. Morafraile1, Alberto Bugallo1, Mónica Segurado2
Instituto de Biología Funcional y Genómica, Salamanca, ES,
2
Departamento de Microbiología y Genética (USAL), Salamanca, ES
1
Para mantener la integridad genómica, el DNA debe ser protegido del
daño provocado por agentes genotóxicos o por alteraciones surgidas
durante la replicación. El checkpoint de fase S, mediado por Mec1 y
Rad53 en Saccharomyces cerevisiae (ATR y Chk2 en células humanas,
respectivamente) responde a perturbaciones en la replicación y coordina
una respuesta celular global necesaria para mantener la integridad del
genoma. Estamos interesados en entender cómo el checkpoint de fase S
regula la replicación en estas condiciones, y más específicamente, en los
mecanismos moleculares que preservan la estabilidad de las horquillas de
replicación y promueven el reinicio de la síntesis de DNA tras bloqueos
en la replicación. Como la quinasa Rad53 estabiliza las horquillas de
replicación mediante un mecanismo dependiente de Exo1, hemos estudiado
la regulación de esta nucleasa, y nuestros resultados indican que Exo1 es
fosforilada por Rad53 en respuesta a estrés replicativo o daño en el DNA.
Mutantes fosfomiméticos de EXO1 suprimen el colapso de horquillas de
replicación de mutantes rad53, al igual que la deleción de la nucleasa,
lo que sugiere fuertemente que la fosforilación de Exo1 tiene un efecto
inhibitorio en la actividad de la proteína. Sin embargo, la deleción de EXO1
no suprime el defecto de reiniciación de horquillas observado en el mutante
rad53, lo que sugiere que la reactivación de las horquillas bloqueadas es
regulada mediante otro mecanismo. Nuestros resultados indican que Rad53
promueve el reinicio de horquillas de replicación a través de la inducción
transcripcional de genes de respuesta a daño en el DNA. Por tanto, además
de prevenir la degradación de las horquillas, Rad53 promueve el reinicio
de la replicación induciendo la expresión de los genes de la ribonucleótido
reductasa (RNR) y regulando la localización subcelular del complejo tras
estrés replicativo.
P16r-14
Búsqueda de nuevos reguladores de Bfa1 y
análisis de su papel en la inhibición de la salida
de mitosis tras la generación de daños al ADN
Inés García de Oya1, Fernando Monje-Casas2
1
Universidad Sevilla/ CABIMER, Sevilla, ES, 2CSIC, Sevilla, ES
La fidelidad de la transmisión de la información genética durante el ciclo
celular es controlada por la acción de distintos mecanismos de vigilancia,
que previenen la generación de productos aneuploides. En Saccharomyces
cerevisiae, estos puntos de control (checkpoints) controlan, entre otros
aspectos, la integridad del genoma, la unión anfitélica de los cromosomas
al huso mitótico, o el correcto posicionamiento del huso. A pesar de que
los tres mecanismos de vigilancia anteriores responden a distintas señales
y en diferentes momentos del ciclo, todos ellos conducen a una inhibición
de la salida de mitosis a través de la acción del complejo Bfa1-Bub2. Estas
proteínas son reguladores negativos de la ruta MEN (mitotic exit network),
una cascada de señalización que determina la salida de mitosis.
La actividad del complejo Bfa1-Bub2 es regulada mediante su
fosforilación. En concreto, Bfa1 es fosforilada gradualmente a lo largo
del ciclo celular por las quinasas Cdc28 y Cdc5, alcanzando su máximo
nivel de fosforilación en anafase. La fosforilación de Bfa1 por Cdc5 en
anafase inhibe la actividad de Bfa1-Bub2, promoviendo así la salida de
mitosis. Por otro lado, en condiciones de activación del checkpoint de
posicionamiento del huso (SPOC), Kin4 fosforila a Bfa1 para mantenerla
en un estado activo e inhibir la salida de mitosis hasta que el huso se
alinee correctamente. Estudios recientes desarrollados por nuestro grupo
de investigación (Valerio-Santiago et al., 2013) demuestran que, además
de por las quinasas anteriores, el estado de fosforilación de Bfa1 es
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también regulado por una quinasa aún no identificada que mantiene a Bfa1
fosforilado tras la activación del checkpoint de daño al ADN. El objetivo de
mi investigación es la identificación de la quinasa o quinasas responsables
de la fosforilación de Bfa1 tras la generación de daños en el ADN y de su
papel en el control de la ruta MEN en estas condiciones.
P16r-15
Mechanisms underlying gene repression
in hypoxia
María Tiana Cerrolaza, Benilde Jiménez Cuenca, Luis del Peso
Ovalle
Universidad Autónoma de Madrid, Madrid, ES
In response to hypoxia, cells activate a stereotyped gene expression
pattern that allows them to cope with the restricted oxygen supply. This
response involves the induction of hundreds of genes by a mechanism that
requires the recruitment of the hypoxia inducible factors (HIFs) to their
cis-regulatory regions. On the other hand, the adaptation to reduced oxygen
also implies the repression of a large number of genes, but the mechanisms
by which hypoxia leads to gene repression have not been fully elucidated
yet. In this work we aimed to identify the general mechanisms that underlie
gene downregulation in response to hypoxia.
To this end, we firstly determined the relative contribution of transcription
and decay rates to the hypoxic gene expression and found that transcription
is the major factor contributing to the gene expression changes triggered
by hypoxia, whereas modification of half-lives had marginal impact. Next,
by focusing on transcriptional changes, we found that, although EPAS1
binding was restricted to the fraction of the genes induced by hypoxia,
it was required for both positive and negative changes of transcription.
Taken together, these results suggested that transcriptional repression
is indirectly mediated by EPAS1 and consequently we concluded that it
should be mediated by transcription factor(s) downstream of EPAS1. Next,
we exploited the genome-wide transcription factor binding sites generated
by the ENCODE project to identify transcription factors enriched in the
regulatory regions of the transcriptionally repressed genes. This analysis
revealed a reduced set of transcription factors as key molecules that could
act downstream of EPAS1 to repress gene expression in hypoxia.
P16-16
Activation-induced cytidine deaminase interacts
with SUV4-20H and targets H4K20 methylation
to class-switch recombination sites
Virginia Carolina Rodríguez Cortez1, Paloma Martínez-Redondo*2,
Francesc Català-Moll*1, Javier Rodríguez-Ubreva1, Antonio
García-Gómez1, Laura Ciudad Garrido1, Henar Hernando Arrabal1,
Carlos Company1, José Miguel Urquiza1, Javier Di Noia3, Alejandro
Vaquero2, Esteban Ballestar Tarín1
1
Chromatin and Disease Group, Cancer Epigenetics and Biology
Programme (PEBC), Bellvitge Biomedical Research Institute
(IDIBELL), Barcelona, ES, 2Chromatin Biology Group, Cancer
Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical
Research Institute (IDIBELL), Barcelona, ES, 3Institut de
Recherches Cliniques de Montréal, Division of Immunity and Viral
Infections, Montreal, CA
Activation-induced cytidine deaminase (AID) triggers antibody
diversification in B cells by catalysing deamination and subsequently
mutating immunoglobulin genes. The biological roles of this enzyme
with respect to its potential relationship with active DNA demethylation
are controversial. Association of AID with RNA Pol II suggests a possible
relationship with gene regulatory functions. AID is mutated in hyper-IgM
type 2 (HIGM2) syndrome. In this study, we investigated the potential
role of AID in the acquisition of epigenetic changes. We discovered that
124
XXXIX Congreso SEBBM
the binding of AID to the IgH locus, a prominent AID target, causes an
increase in H4K20me3. We also demonstrate for the first time that AID
interacts with the three SUV4-20 enzymes, and that SUV420H1.2, which is
bound to the Sμ site of the IgH locus, is replaced by SUV420H2 upon AID
binding. Analysis of HIGM2 mutants indicates that the interaction with
SUV4-20 enzymes takes places at the N-t of AID, as even a truncated form
of AID lacking two C-t thirds of the protein shows a specific interaction.
Our results provide a new perspective on the interplay between AID and
the histone modification machinery in setting the epigenetic status of
class-switch recombination sites.
*Equal contributors
Correspondence to: Esteban Ballestar, Chromatin and Disease Group,
Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical
Research Institute (IDIBELL) L’Hospitalet de Llobregat, Barcelona, Spain.
Key words: AID, Hyper-IgM, H4K20me3, SUV420.
P16-17
Meiotic recombination variability in male mice:
genetic and environmental effects
Elena de la Casa Esperón1, Esteranía San Martín Pérez2
1
Parque Científico y Tecnológico de Castilla-La Mancha. C.R.I.B.
Universidad de Castilla-La Mancha, Albacete, ES, 2C.R.I.B.
Universidad de Castilla-La Mancha, Albacete, ES
Meiotic recombination is not only important for reshuffling the alleles
passed to the next generation, but also for ensuring proper chromosome
segregation to the gametes. Changes in the frequency and distribution of
crossovers can increase the risk of producing aneuploid gametes that result
in fertility and developmental defects. Hence, recombination is very tightly
controlled. Crossover frequency and location depend on both genetic and
epigenetic factors, but little is know about both of them in mammals. Recent
studies have shown that the endocrine disruptor bisphenol A can modify
the levels of recombination, possibly through epigenetic changes. We
have initiated a study for characterizing the effects of other environmental
factors on male crossover frequency and distribution, particularly of
those that have epigenetic and phenotypic effects through generations,
such as certain diets. Because recombination levels show some degree of
variation in mouse, we expect that the environment might have different
impact on recombination in different genetic backgrounds. Therefore, we
have first characterized the frequency and distribution of crossovers in
males of diverse Mus musculus inbred strains. Our studies confirm that
recombination levels depend on the genetic background and, hence, are
genetically controlled. These levels are particularly high in strains of M. m.
musculus subspecies origin. We are initiating our studies of environmental
impact on recombination by testing whether diverse diets have an effect on
male recombination levels.
P16r-18
Papel de la eIF2alfa quinasa GCN2 en la
supervivencia celular en respuesta a estrés
nutricional y a radiación ultravioleta
Teresa García Prieto, César de Haro Castella, Juan José Berlanga
Chiquero
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES
La respuesta al estrés permite a los seres vivos responder y adaptarse
a un ambiente cambiante que los expone a radiación, cambios de
temperatura, agentes tóxicos y a la eventual carencia de nutrientes.
Una adecuada respuesta al estrés no solo determina las posibilidades
de sobrevivir y adaptarse, sino que también puede afectar al proceso
natural de envejecimiento y al desarrollo de algunas patologías
Salamanca 2016
Pósters / Posters
asociadas al mismo (cáncer, diabetes, enfermedades neurodegenerativas
y cardiovasculares). La naturaleza de la respuesta al estrés en
mamíferos es fundamentalmente adaptativa, y debe de entenderse
como un intento del organismo de restablecer la homeostasis celular
y sistémica que ha resultado alterada. Además, episodios de estrés de
moderada o baja intensidad a lo largo de la vida pueden preparar al
animal para responder mejor a futuras situaciones de estrés agudo. Este
concepto, se ha denominado hormesis. La fosforilación transitoria del
factor de iniciación de la traducción eIF2 en su subunidad alfa, por
eIF2alfa quinasas específicas, provoca una parada instantánea de la
síntesis general de proteínas y, al mismo tiempo, activa la traducción
de algunos ARNm implicados en promover y coordinar la respuesta al
estrés. La eIF2alfa quinasa GCN2 se activa en respuesta a privación
de aminoácidos y a irradiación con luz ultravioleta. En este estudio
hemos comprobado que la falta de GCN2 en fibroblastos embrionarios
de ratón hace a estas células más sensibles a dichas formas de estrés,
disminuyendo su capacidad de supervivencia a corto plazo y su
capacidad proliferativa a largo plazo. Además, hemos tratado de probar
el concepto de hormesis, sometiendo a las células a privación de
aminoácidos previamente a su tratamiento con radiación ultravioleta.
development. Data from our lab indicate that DYRK1A is recruited to
proximal promoter regions of genes characterized by the presence of
a conserved palindromic motif. DYRK1A-dependent transcriptional
regulation of its targets appears to be due to phosphorylation of the
Carboxy-Terminal Domain (CTD) of the RNA polymerase II. However,
we know little on how DYRK1A is recruited to its genomic loci. By using
electrophoretic mobility assays, we have found that bacterially expressed
and purified DYRK1A binds to single-stranded DNA probes containing
the palindromic sequence, suggesting that DYRK1A might bind to
unwound DNA stabilizing single strands at the chromatin level. Analysis
of the ChIP-Seq data from ENCODE reveals that several transcription/
chromatin-related factors can be recruited to the DYRK1A-consensus
motif. The list includes the co-repressor ZBTB33/KAISO and the tumor
suppressor BRCA1. We have validated the presence of ZBTB33/KAISO
and BRCA1 at several DYRK1A-bound regions; in addition, the interaction
of DYRK1A with KAISO or BRCA1 is detected in nuclear extracts by
co-immunoprecipitation experiments. These findings raise the possibility
of a functional cross-talk between DYRK1A and any of these proteins in
the regulation of their target genes.
P16-19
Oncogene or tumor suppressor? New roles
in tumorogenesis for the mitotic kinase Plk1
P16-21
An X family DNA polymerase protects
mitochondrial DNA in the infective filament
of Ustilago maydis
María Tenorio-Gómez, José Pérez-Martín
Instituto de Biología Funcional y Genómica, Salamanca, ES
Ustilago maydis is a phytopathogenic fungus thatinfects the maize plant
to provoke the corn smut disease. A morphological switch from yeast to
infective filament is an indispensable prerequisite for enabling the infection
to take place. This filament is a cell cycle arrested dikaryotic hypha that
arises from the conjugation of two compatible yeasts, grows on the plant
surface and eventually develops an specific structure called appresorium,
required to penetrate the plant tissue. The formation of the filament in U.
maydis is driven by a complex transcriptional programme which is mainly
orchestrated by a transcriptional regulator called b-factor. One of the direct
targets of the b-factor is PolX, a gene encoding a putative DNA polymerase
which is strongly expressed upon filament formation (Brachmann et al.,
Mol Microb 42, 1047). According to its sequence, this polymerase would
belong to the X family of DNA polymerases, implicated in DNA repair
processes. We have analysed the ability of polX null mutant dikaryotic
hyphae to cope with DNA damaging agents and we have found only slight
defects in response to oxidative stress. Nevertheless, we have come across
that PolX seems to be required for the ability of the dikaryotic hyphae to
resume proliferation once the b-programme is repressed. Interestingly, we
have also found that PolX is a mitochondrial protein, most likely involved
in the repair of damaged mitochondrial DNA. In this communication we
will discuss why the induction of the infective hypha needs the presence of
a DNA polymerase to protect the mitochondria.
Pablo Sanclemente1, Rocio Sotillo2, Marcos Malumbres1,
Guillermo de Cárcer Díez1
1
CNIO, Madrid, ES, 2DKFZ, Heidelberg, DE
Mitosis is the ultimate step of the cell cycle, allowing the dividing cell to
segregate their DNA content equally to the two daughter cells, and it is
tightly regulated by many kinases. Interestingly, several mitotic kinases are
overexpressed in different type of tumors. Therefore, mitotic kinases are
bona fide anti-cancer targets, and several of them are currently in clinical
trials. Polo-like kinase 1 (Plk1) is a cell cycle regulator kinase, which
controls a wide variety of process throughout cell cycle progress, being
essential for the mitosis accomplishment. Plk1 modulates centrosome
maturation, spindle assembly, chromosome segregation, and cytokinesis.
When Plk1 kinase activity is inhibited, it leads to mitotic arrest due to
spindle malformation, and eventually to cell death. Since early days,
many efforts have been done to depict the Plk1 relationship with cancer
disease. In 1997 Plk1 was catalogued as oncogene, and subsequent
studies demonstrate its interactions with other cancer-related proteins.
Indeed, Plk1 is overexpressed in many tumor types, and this feature often
confers poor prognosis. However, the possible oncogenic mechanism of
Plk1 is still not well described. We aimed to reevaluate the tumorigenic
potential of Plk1 overexpression, by generating a precise Plk1 induction
knock-in mouse model. Surprisingly, our in vitro results revealed that
when Plk1 is overexpressed, it halts cell proliferation, induces polyploidy
and moreover prevents cell transformation driven by other major
oncogenes. Concomitantly, our in vivo data show that Plk1 does not lead to
transformation events in the mouse. Therefore, our data indicates that Plk1
might play as a tumor suppressor, leading to a completely new scenario for
this mitotic kinase.
P16-20
Chromatin bound-DYRK1A: recruitment
and regulatory mechanisms
Laura Barba Moreno, Chiara Di Vona, Susana de la Luna Gargantilla
Gene Regulation, Stem Cells and Cancer Programme, Centre for
Genomic Regulation (CRG), The Barcelona Institute of Science
and Technology, and Universitat Pompeu Fabra, Barcelona, ES
DYRK1A (Dual-specificity tyrosine-(Y)-Regulated Kinase 1A) belongs
to a highly conserved family of protein kinases -DYRK- present in all
eukaryotes. DYRK1A is known to participate in cell proliferation and
differentiation decisions, and in particular, to fulfill key roles in brain
P16-22
Common genetic polymorphisms are excluded
from functional hypoxia response elements
Daniel Martínez Alcázar, Luis del Peso Ovalle
Instituto de Investigaciones Biomédicas Alberto Sols, Madrid, ES
A wide range of diseases course with an unbalance between the consumption
of oxygen by tissues and its supply. This situation triggers a transcriptional
response, mediated by the hypoxia inducible factors (HIFs), that aims to
restore oxygen homeostasis. Little is known about the inter-individual
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Pósters / Posters
variation in this response and its role in the progression of disease. Herein,
we sought to identify common genetic variants affecting hypoxia response
elements (HREs) and characterize their effect on transcription. To this end,
we constructed a list of genome-wide HIF-binding regions from publicly
available experimental datasets and studied the genetic variability in these
regions by integration of human variability deposited in the dbSNP database.
Our analysis revealed that, although we found 141 SNPs that disrupt potential
HREs in HIF-bound regions, the proportion of functional HREs affected by
variants is significantly lower than expected by chance. These data strongly
suggest that functional HREs are under negative selection.
P16-23
Non-coding variants in human cardiac disease
Mel·lina Pinsach Abuin1, Bernat del Olmo1, Jesús Mates1, Jing
Zhang2, Daria Merkurjev2, Catarina Allegue1, Sergiy Konovalov2,
Farah Sheikh2, Ramon Brugada1, Ivan Garcia-Bassets2, Sara
Pagans1
1
Department of Medical Sciences, University of Girona, Girona, ES,
2
Department of Medicine, University of California San Diego, La Jolla, US
Each human individual is unique in 0.1% of the genome sequence
(known as genetic variation). This small but important fraction of distinct
sequence largely contributes to phenotypic disparity among individuals
and their distinct susceptibility to disease and response to pharmacological
treatments. Thereby understanding how the “0.1% fraction” leads to
phenotypic disparity and predict at least part of its contribution to pathology
would be essential to understand and treat human disease. Here, we present
an integrative and comprehensive approach that combines genomic,
epigenomic, experimental, and deep-learning-based studies to decipher
rules that govern the translation of genetic variation into phenotypic
heterogeneity in noncoding regions, and apply this approach for the study
of human cardiac diseases, in general, but also Brugada Syndrome (BrS),
in particular. Until now, BrS is a heart disease mainly associated with
genetic variation within coding regions of cardiac ion channels. We have
identified and characterized >30,000 genomic regions in human cardiac
cells in which genetic variation is more likely to have a functional impact,
and have sequenced a cohort of BrS patients to associate genetic variation
within some of these regions to disease. We used maps of transcription
factor binding combined with a deep-learning-based approach to predict
functional effects of genetic variation. We captured and genotyped 1,300
(over the total of >30,000) open chromatin regions related to cardiac ion
channels, identifying 6,584 variants that were unique for each individual
and short haplotypes shared by several patients. Focused on transcription
factor CTCF for validation purposes, we found 68 variants overlapping
with CTCF binding sites, and predict that 49/16/3 will decrease/increase/
no-change CTCF binding, respectively. Experimental validation of these
predictions allows us the extrapolation of our approach to the full set of
thousands of noncoding variants, and for the prediction of functionally
relevant variants in any type of cardiac and non cardiac disease.
P16-24
Use Yen1ON as a tool to understand the
dynamics of replication/early recombination
intermediates
XXXIX Congreso SEBBM
must be eliminated to safeguard sister chromatid segregation. In budding
yeast, the Yen1 nuclease acts as a last resource to eliminate JMs that have
persisted until late stages of mitosis. This nuclease is under a strict cell
cycle-dependent control, which regulates both its subcellular location and
biochemical activation. During S phase, Cdk-mediated phosphorylation
of Yen1 promotes its nuclear exclusion and its catalytic inactivation. In
anaphase, Cdc14 drives Yen1 dephosphorylation leading to its nuclear
relocalization and enzymatic activation.
The relevance of such an stringent regulation is underscored by the genome
instability that ensues after the expression of a constitutively active and
nuclear version of Yen1 (Yen1ON). Indeed, overexpression of such mutant
leads to cell death, despite the cause of such lethality remains unclear.
Incidentally, we have observed the appearance of spontaneous suppressors
of this phenotype, which we intend to use as a tool to unveil the cellular
targets of Yen1 unregulated activity and their availability for cleavage.
Here we will lay out the strategy and preliminary results of an unbiased
screening for suppressors of the lethality of Yen1ON overexpression.
P16-25
Caracterización funcional del promotor del gen
humano TIMM8A
Héctor Merenciano González, Guillermo Suay Montagud,
Sandra García Benlloch, Beltrán Ortolá Navarro, Rafael Alís Pozo,
Jesús Ángel Prieto Ruiz, José Rafael Blesa Blesa
Grupo Medicina Molecular y Mitocondrial. Facultad de Medicina
y Odontología. Universidad Católica de Valencia San Vicente Mártir,
Valencia, ES
Fundamentos y objetivos: El síndrome de Mohr-Tranebjaerg (MTS) fue
la primera enfermedad descrita debida a alteraciones en alguna translocasa
mitocondrial. La base molecular corresponde a alteraciones en la proteína
TIMM8A, un miembro de las tiny TIMMs. El objetivo del trabajo es realizar
la caracterización funcional del promotor de TIMM8A para comprender los
mecanismos de expresión génica, con el fin de abrir nuevas vías hacia la
búsqueda de estrategias para la actuación y tratamiento sobre esta enfermedad.
Materiales y métodos: Se realizó análisis in silico mediante el programa
TESS y la base de datos TRANSFAC® 6.0. Mediante técnicas de biología
molecular (PCR, clonación, etc.) se generaron diferentes constructos, que
se transfectaron en células HeLa y SH-SY5Y. Se hicieron ensayos de
Luciferasa y estudios in vitro de retardo en gel (EMSA).
Resultados: En el análisis in silico se encontraron sitios de unión para
los factores de transcripción Sp1, NRF-1 e IRF-2, cuya funcionalidad fue
comprobada mediante EMSAs. De los ensayos con Luciferasa se concluyó
que no existen sitios de unión para factores de transcripción relevantes en
las regiones en dirección 5’ desde el sitio de unión para IRF-2.
Discusión y conclusiones: Los resultados obtenidos nos permiten
afirmar que Sp1 y NRF-1 son los principales activadores de la expresión
de TIMM8A. El hallazgo de la secuencia de unión para IRF-2 y la
comprobación de su funcionalidad abre un nuevo campo de estudio acerca
de la regulación de las translocasas mitocondriales.
Palabras clave: Translocasas de Membrana Mitocondrial Interna (TIMM),
proteína Translocasa de Membrana Mitocondrial Interna 8 (TIMM8), gen
de Translocasa Mitocondrial Interna 8 (TIMM8A), Factor regulador de
interferón 2 (IRF-2), factor de transcripción Sp1, Factor Respiratorio
Nuclear 1 (NRF-1), Terapia génica mitocondrial.
Vanesa Hurtado Nieves
DNA repair and Genome Intregity, Santiago de Compostela, ES
P16-26
Homologous recombination (HR) is one of the main pathways for double
strand break (DSB) repair. HR involves a series of strand exchanges that
result in branched DNA intermediates named joint molecules (JMs).
JMs are usually disrupted and repaired at early cell-cycle stages by
anti-recombinogenic helicases. However, JMs can occasionally mature into
four-way DNA intermediates known as Holliday Junctions (HJs), which
126
Expression of the human TIM23 gene is regulated
by the GABP (NRF-2) transcription factor
Jesus A. Prieto-Ruiz1, Sara Sáez-Atiénzar1, Rafael Alis Pozo2,
Ignacio Ventura1, Sandra Garcia-Benlloch1, Beltran Ortola-Navarro1,
José Hernández-Yago1, José R. Blesa1
Salamanca 2016
1
Facultad de Medicina, Universidad Católica de Valencia San
Vicente Mártir, Valencia, ES, 2Universidad Católica de Valencia,
Valencia, ES
The Tim23 protein is the key component of the mitochondrial import
machinery in all mitochondrion-bearing eukaryotes. It is essential in S.
cerevisiae and N. crassa, and Tim23-/- knockout mice are not viable. In this
paper, we characterize the promoter region of the humanTIM23 gene and
establish the essential role of the GA-binding protein (GABP or NRF-2)
transcription factor in activating TIM23 expression. In particular, we found
four GABP sites cooperating in the global expression of TIM23 although
with different contributions. In addition, expression of TIM23 also appears
to be regulated by the repressor activity of the transcriptional factor
RBP-JKappa. These results interestingly are in the way to complete the
picture where the main subunits of the human mitochondrial translocases
(TOM and TIM) are regulated by GABP transcription factor.
P16-27
Dynamics of cell fate decisions in embryonic
stem cells
Sergi Aranda Aragón
Centre for Genomic Regulation (CRG), The Barcelona Institute
of Science and Technology; and Universitat Pompeu Fabra (UPF),
Barcelona, ES
The self-renewing nature of embryonic stem cells (ESCs) is a consequence
of their ability to proliferate indefinitely while maintaining pluripotency,
by means the capacity to differentiate into all the adult cell types. Despite
remarkable progress in understanding the mechanisms controlling ESCs
pluripotency, little is known about how ESCs cell division is coordinated
with self-renewal and differentiation. ESCs are characterized by a unique cell
cycle structure, with a short G1 phase and enlarged S phase. The singularity
of the ESCs cell cycle is proposed to be a hallmark of pluripotency. Indeed,
recent studies suggest that the shortening of the cell cycle is required for
pluripotency and that the extension of the G1 phase is a cause, rather than
a consequence, of cell differentiation. We have performed comprehensive
proteomic and transcriptomic analyses and have found that pluripotency
gene networks and the activity of chromatin modifiers oscillate during cell
cycle progression. These oscillations correlate with changes in epigenetic
marks throughout the different phases of the cell cycle. Finally, our functional
studies indicate that ESCs are primed for a specific cell fate by a cell
cycle-linked mechanism. In sum, our data suggest that the cell cycle acts as
segregation clock,by dictating stem cell fate towards distinct cell types. Our
findings provide fundamental insight into how cell fate decisions take place
during embryo development, and suggest a potential framework whereby
oscillations during cell cycle can be utilized to differentiate ESCs towards
a specific cellular outcome, which is a major goal in regenerative medicine.
P16-28
eIF5A translation elongation factor function and
post-transcriptional regulation
Esther Gamero1, Verónica Muñoz Soriano2, Carlos Villarroel1, Alice
Stanciu1, Tianlu Li1, María T. Martínez-Pastor3, Sergi Puig4, Nuria
Paricio2, Paula Alepuz1
1
Departamento de Bioquímica y Biología Molecular; ERI
Biotecmed, Universitat de València, Burjassot, ES, 2ERI Biotecmed;
Departamento de Genética, Universitat de València, Burjassot, ES,
3
Departamento de Bioquímica y Biología Molecular, Universitat de
València, Burjassot, ES, 4Departamento de Biotecnología, Instituto
de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, ES
eIF5A is an essential and evolutionary conserved translation elongation
factor, which has been proposed to be involved in the translation of proteins
Pósters / Posters
with consecutive prolines. In yeast, eIF5A is required for the shmoo
formation that initiates cell fusion during mating. In this process, cells
require eIF5A for the localization of polarisome components, induction of
cell fusion proteins and actin assembly. We discovered eIF5A to be needed
for the translation of the formin Bni1, which is involved in the formation
of actin cables from the shmoo tip and, therefore, required for the polarized
growth during shmoo formation. The function of eIF5A in controlling
polarized growth seems to be conserved among eukaryotes. We show
that Drosophila eIF5A is able to restore growth of yeast cells depleted of
endogenous eIF5A, and allows efficient translation of yeast Bni1 formin
and therefore, shmoo formation. In flies, we discovered eIF5A to be
involved in dorsal closure (DC) during Drosophila embryogenesis through
the translation of formin Diaphanus. In humans, overexpression of eIF5A
has been involved in tumor development and associated with poor survival
and metastasis. In many eukaryotes, such as yeast and humans, eIF5A is
encoded by two paralogous genes which are differentially regulated and
contain putative AU-rich elements (AREs) in the 3’-UTR of their mRNAs.
We are investigating the role of these ARE-elements in the regulation of
the expression of eIF5A isoforms and the ARE-binding proteins involved.
This post-transcriptional regulation of eIF5A could be important for the
polarized growth of cells during cancer progression.
P17. Regulación metabólica /
Metabolic regulation
P17-1
Osteopontin: a key regulator of liver
lipid metabolism
Maitane Núñez-García1, Beatriz Gómez-Santos1, Xabier Buqué2,
Juan L. García-Rodríguez1, Marta R. Romero3, José J.G. Marín3,
Beatriz Arteta4, Carmelo García-Monzón5, Wing-kin Syn6, Olatz
Fresnedo1, Patricia Aspichueta2
1
Department of Physiology, Faculty of Medicine and Dentistry,
University of the Basque Country, UPV/EHU, Leioa, ES,
2
Department of Physiology, Faculty of Medicine and Dentistry,
University of the Basque Country, UPV/EHU, Leioa, ES; BioCruces
Health Research Institute, Barakaldo, ES, 3Experimental Hepatology
and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca,
Salamanca, ES; Center for the Study of Liver and Gastrointestinal
Diseases (CIBERehd), Carlos III National Institute of Health,
Madrid, ES, 4Department of Cellular Biology and Histology, Faculty
of Medicine and Dentistry, University of the Basque Country, UPV/
EHU, Leioa, ES, 5Liver Research Unit, Santa Cristina University
Hospital, Instituto de Investigación Sanitaria Princesa; Center
for the Study of Liver and Gastrointestinal Diseases (CIBERehd),
Carlos III National Institute of Health, Madrid, ES, 6Regeneration
and Repair, Institute of Hepatology, Foundation for Liver Research,
London, UK; Division of Gastroenterology and Hepatology,
Medical University of South Carolina, Charleston, US; Section of
Gastroenterology, Ralph H Johnson Veteran Affairs Medical Center,
Charleston, US
Osteopontin deficiency protects from obesity-related hepatosteatosis
and liver fibrosis. Here we have investigated the role of osteopontin
(OPN) as a direct regulator of liver metabolism and whether OPN could
be a serum-biomarker of liver metabolic dysfunction in non-alcoholic
fatty-liver (NAFL) disease patients. For this purpose, metabolic
pathways were analyzed by lipidomic and radiometric techniques in
hepatocytes and total livers from OPN-KO mice and WT littermates and
from WT mice that received recombinant (r)OPN. OPN-KO mice were
also treated with intragastric atorvastatin for two weeks. Liver biopsies
127
Pósters / Posters
taken during programmed laparoscopic cholecystectomy and matched
serum samples from non-obese subjects with NAFL (n=13) or with
normal liver (n=13) were analyzed. We demonstrate that OPN regulates
the crosstalk between liver cholesterol and phosphatidylcholine
metabolism, being the key process the conversion of cholesterol into bile
acids through the action of cholesterol 7-alpha-hydroxylase, frequently
decreased in NAFL. In OPN-deficient mice enhanced cholesterogenesis
in hepatocytes leads to decreased de novo lipogenesis and in vivo
inhibition of cholesterogenesis by atorvastatin normalizes liver
phosphatidylcholine content. Finally, serum OPN levels correlate with
liver phosphatidylcholine and cholesterol concentration in non-obese
NAFL patients, in whom adipose tissue metabolic deregulation is not
evident and conversion of cholesterol into bile acids is decreased. In
conclusion, OPN is a key regulator of liver lipid metabolism and may
be an early serum-biomarker of metabolic deregulation in non-obese
NAFL patients.
Supported by the Basque Government (IT-336-10) and the UPV/EHU
(UFI11/20).
P17-2
Optimization and validation of sample
preparation procedures for high throughput
untargeted liver lipid profiling by
UHPLC-ESI-Q-TOF MSE
Beatriz Abad1, Laura Guevara2, Daniel Bailera2, José Andrés
Fernández3, Begoña Ochoa2
1
Central Analysis Service, Faculty of Science and Technology,
UPV/EHU, Leioa, ES, 2Department of Physiology, Faculty of
Medicine and Dentistry, UPV/EHU, Leioa, ES, 3Department of
Physical-Chemistry, Faculty of Science and Technology, UPV/EHU,
Leioa, ES
Lipids are deregulated in the onset and progression of many diseases,
and are functionally involved in mechanisms of disease etiologies.
This requires the development of high throughput analyses capable of
dealing with large numbers of samples which are necessary to reach
statistical significance in clinical studies. In this work, a rapid, selective
and sensitive untargeted ultra high performance liquid chromatography
coupled to electrospray ionization and quadrupole time-of-flight mass
spectrometry (UHPLC-ESI-Q-TOF MS) strategy using automatic
and simultaneous acquisition of exact mass at high and low collision
energy, MSE have been used for determination the complex profile
of lipid species forming a tissue in a single run injection of 13 min.
However, a lipidomics analysis by UHPLC-MS relies on the quality
and reproducibility of the extraction procedure of lipids. We now know
that there is not a unique procedure able to successfully extract all lipid
classes and molecular species, and also that guidelines are offered in
several published papers and the LIPID MAPS website. Thus, adoption
of one protocol requires neat election of criteria according to the
objective of the analytical study, of the equipment available and of
the staff training. Here we compare reproducibility and efficiency of
extraction of two sample preparation protocols for liver lipid extraction
and profiling by UHPLC-ESI-Q-TOF MSE using 17 glycerolipid and
10 sphingolipid non-natural standards. The protocols are a liquid-liquid
extraction procedure (method A) and a protein precipitation method
(method B). The two methods were then benchmarked using a set of
criteria selected to be adopted in high throughput analytical workflow,
including simplicity and cost, reproducibility, lipid recovery and
coverage. In addition, comparison with benchmarking for targeted
lipid profiling was performed. Our data show that the method of choice
for liver extract preparation depends on the coverage and human and
equipment available and that the two methods A and B meet good
quality levels.
128
XXXIX Congreso SEBBM
P17-3
Las gotas lipídicas como centro coordinador
del metabolismo lipídico y su desregulación
en obesidad
María del Mar Malagón, Yoana Rabanal-Ruiz, Andrés Trávez, Rocío
Guzmán-Ruiz, Rafael Vázquez-Martínez
IMIBIC/Hospital Universitario Reina Sofía, Universidad de Córdoba,
Córdoba, ES
El tejido adiposo juega un papel crucial en la gestión de las reservas
energéticas del organismo, que se almacenan en forma de lípidos neutros
en las gotas lipídicas características de los adipocitos. En obesidad, el
tejido adiposo sufre importantes cambios moleculares que conducen a
la deposición ectópica de lípidos y al desarrollo de resistencia a insulina
por un proceso de lipotoxicidad. La capacidad de los adipocitos para
almacenar y liberar lípidos depende en gran medida de la maquinaría
molecular asociada a las gotas lipídicas, que incluye, entre otras, enzimas
relacionadas con la síntesis de triglicéridos y fosfolípidos, lipasas,
perilipinas, y proteínas relacionadas con el tráfico intracelular y la fusión
de membranas, como las GTPasas de bajo peso molecular Rab y Arf, y v- y
t-SNARE. De hecho, las gotas lipídicas son orgánulos altamente dinámicos
que interaccionan entre sí y con otros orgánulos relacionados con el tráfico
y metabolismo de lípidos, aunque no se conocen los mecanismos que
median dichas interacciones ni la relevancia de las mismas en la función de
los adipocitos. En este contexto, nuestros estudios ponen de manifiesto que
una de las proteínas Rab asociadas a gotas lipídicas, Rab18, que interviene
en la lipólisis mediada por receptores β-adrenérgicos y en la lipogénesis
inducida por insulina, coordina la interacción de las gotas lipídicas con
retículo endoplásmico, mitocondrias y peroxisomas. De esta manera,
Rab18 permitiría ajustar la incorporación, movilización y uso de lípidos
con el remodelado de las gotas lipídicas, tanto de los lípidos neutros del
interior de las gotas como de los fosfolípidos de su cubierta. Los cambios
observados en la expresión de Rab18 y de uno de sus reguladores, GDI2,
en el tejido adiposo en condiciones de obesidad podrían contribuir a la
desregulación del almacenamiento y movilización de lípidos observado en
dichas condiciones.
Financiación: MINECO/FEDER (BFU2010-17116; BFU2013-44229-R;
BFU2015-70454-REDT); J. Andalucia/FEDER (PI-0200/2013; CTS-6606);
FIS (PIE14-00005) y CIBERobn (ISCIII).
P17m-4
Role of MKK3 in High-Fat Diet induced diabetes
Edgar Bernardo Vasco1, Nuria Matesanz1, Leticia Herrera1,
Sonia Pérez-Sieira2, Elena Rodríguez1, Miguel Marcos3, Lourdes
Hernández-Cosido4, Rubén Nogueiras2, Ángel Nebreda5, Guadalupe
Sabio1
1
Fundación Centro Nacional de Investigaciones Cardiovasculares
CNIC, Madrid, ES, 2Department of Physiology, CIMUS, University of
Santiago de Compostela-Instituto de Investigación Sanitaria; CIBER
Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago
de Compostela, ES, 3Department of Internal Medicine, University
Hospital of Salamanca-IBSAL; Department of Medicine, University
of Salamanca, Salamanca, ES, 4Bariatric Surgery Unit, Department
of General Surgery, University Hospital of Salamanca; Department
of Surgery, University of Salamanca, Salamanca, ES, 5Institute for
Research in Biomedicine (IRB Barcelona), Barcelona Institute of
Science and Technology; Institució Catalana de Recerca i Estudis
Avançats (ICREA), Barcelona, ES
Obesity is an imbalance between food intake and energy expenditure
characterized by an excess in fat accumulation and by a chronic
inflammation. Obesity is also related to other diseases such as type 2
diabetes, dyslipidemia and hypertension. MAPK are serine/threonine
Salamanca 2016
specific protein kinases, include ERK1/2, p38 MAPK and c-jun N-terminal
kinase (JNK), they are involved in several cellular processes such as
inflammation, growth and differentiation. JNK is recently described
to have a role in diabetes however the role of p38 in obesity is poorly
understood. p38 is activated by two upstream kinases, MAPK kinase 3 and
MAPK kinase 6 (MKK3 and MKK6). In this work we study the upstream
kinase MKK3 in order to understand the role of p38 MAPK in obesity and
diabetes.
Pósters / Posters
stages of adipogenesis blocks the differentiation. Furthermore it has been
demonstrated that p38 take a part in glucose uptake stimulated by insulin
both in adipocytes and myocytes. Treatment of those cells with p38 alpha
and beta inhibitors moderate the glucose uptake via GLUT4 but has no
effects on the translocation of this transporter to the cell membrane. Since,
the role of p38 delta kinase in the adipose tissue is not well understood, our
goal in this study is to examine the contribution of p38 delta specifically
in the adipose tissue by selectively knocking-down this p38 isoform in
adipose tissue, as well as the impact of p38 delta in the communication of
adipose tissue with other metabolic organs.
P17-5
MKK6 controls T3-mediated browning of white
adipose tissue
Nuria Matesanz1, Edgar Bernardo1, Rebeca Acín-Pérez1, Beatriz
Caballero1, Sonia Pérez-Sieira2, Lourdes Hernández-Cosido3,
María del Valle Montalvo1, Alfonso Mora1, María Elena Rodríguez1,
Luis Leiva-Vega1, Ana Victoria Lechuga1, Clara Álvarez2, Jesús
Ruiz-Cabello1, Jorge L. Torres3, Francisco Centeno4, Miguel Marcos3,
José Antonio Enríquez1, Rubén Nogueiras2, Guadalupe Sabio1
1
Fundación Centro Nacional de Investigaciones Cardiovasculares
Carlos III, Madrid, ES, 2University of Santiago de Compostela-Instituto
de Investigación Sanitaria, A Coruña, ES, 3Hospital Universitario de
Salamanca, Salamanca, ES, 4Universidad de Extremadura, Badajoz, ES
Obesity, a major health problem that has become pandemic, is characterized
by fat accumulation, alteration of different metabolic pathways, and insulin
resistance in peripheral tissues. Increase of body temperature by activating
adipose tissue has emerged as a possible tool to fight obesity. Previous
studies in metabolism showed that p38 MAPK family, a group of kinases
that respond to stimuli such as metabolic and oxidative stress, may be
implicated in brown adipose activation, but little is known about its role
in obesity. We found that expression of MKK6, an upstream kinase that
controls phosphorylation and activation of p38, increased in adipose tissue
of wild-type (WT) mice on a high-fat diet (HFD). To study the participation
of MKK6 in the development of obesity and diabetes, the effect of a HFD
on knockout mice lacking MKK6 (Mkk6-/-) was investigated.
We thank the staff at the CNIC Animal facility. G.S. is an investigator of
the Ramón y Cajal Program.This work was funded by the following grants
to G.S.: ERC 260464, EFSD 2030, MICINN SAF2013-43506-R, and
Comunidad de Madrid S2010/BMD-2326; to L.H.C: Junta de Castilla y
León GRS 681/A/11; to R.N: MICINN BFU2012-35255, Xunta de Galicia
EM 2012/039 and 2012-CP069. The CNIC is supported by the Ministry of
Economy and Competition and the Pro-CNIC Foundation and is a Severo
Ochoa Center of Excellence (MINECO award SEV-2015-0505).
We thank the staff at the CNIC Genomics and Bioinformatics units for
technical support and help with data analysis. G.S. is an investigator of
the Ramón y Cajal Program. I.N. is supported by a CNIC IPP FP7 Marie
Curie Programme (PCOFUND-2012-600396). This work was funded
by the following grants to G.S.: ERC 260464, EFSD 2030, MICINN
SAF2013-43506-R, and Comunidad de Madrid S2010/BMD-2326. The
CNIC is supported by the Ministerio de Economía y Competitividad and
the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence
(MINECO award SEV-2015-0505).
P17m-7
Role of p38 δ in liver metabolism
Valle Montalvo-Romeral, Luis Leiva, Victor Bondía, Juan Antonio
López, Jesús Vázquez, Guadalupe Sabio
Fundación Centro Nacional de Investigaciones Cardiovasculares
(CNIC), Madrid, ES
Obesity is currently a serious health problem throughout the world, which
prevalence has reached epidemic proportion (Mokdad et al. 2001). This is
due to the obesity is associated with others disorders like impaired glucose
tolerance, dyslipidemia and hypertension. All these are collectively known
as “metabolic syndrome” (Isomaa et al., 2001).
Stress activated protein kinases (SAPKs), an important family of kinases
implicated in the transduction of stress signals into the cell, could be key
regulator of the development of diabetes and cancer. While the role of c-Jun
N-terminal kinases (JNK) in metabolic diseases has been study for a long
time (Sabio et al., 2008; Sabio and Davis 2010; Sabio et al., 2010), less
is known about the role of the other stress kinases, p38 mitogen-activated
stress kinases (p38 MAPK). In mammals, four p38 MAPK isoforms have
been identified: p38α, -β, -γ, and -δ. The role of the p38γ/δ remains elusive,
although recently we have founded that these kinases (γ/δ) control liver
steatosis (González-Terán, Matesanz and Nikolic et al., 2016). As liver
is an essential metabolic organ which control body energy metabolism,
playing a key role in controlling blood glucose, protein and fat levels, we
will discuss the role of p38 δ in liver metabolism.
P17r-6
Role of p38 delta protein kinase in adipose
tissue
Ivana Nikolic, Alfonso Mora, Luis Leiva Vega, Victor Bondia, Elena
Rodríguez, Guadalupe Sabio
Stress kinases in Diabetes, Cancer and Cardiovascular Disease,
Myocardial Pathophysiology Area, Spanish National Center for
Cardiovascular Research, Madrid, ES
Mitogen-activated protein kinases (MAPKs) are serine-threonine protein
kinases which detect a variety of extracellular stimuli and response
by activating signaling cascade pathways resulting as specific cellular
reaction. This protein family includes the ERK, predominantly activated
by mitogens and differentiation signals and JNK and p38 MAPKs,
primarily activated by stress stimuli. Up to now it has been identified 4
different isoforms of p38: p38 alpha, beta, gamma and delta. It has been
shown that p38 kinases are involved in differentiation of preadipocytes
into adipocytes and chemical inhibition of p38 alpha and beta in the early
P17m-8
The metabolic pathways involved in maintaining
liver glycerolipid homeostasis are dysregulated
in early stages of obesity promoted hepatocellular
carcinoma
Daniela Mestre Congregado1, Igor Aurrekoetxea1, Larraitz
Fernández-Ares1, Xabier Buqué1, Juan L. García-Rodríguez2,
Beatriz Gómez-Santos2, Diego Sáenz de Urturi-Indart2, Maitane
Núñez-García2, Jon Ander Fernández-Rodríguez2, Ainhoa Iglesias3,
Ana Zubiaga3, Patricia Aspichueta1
1
Dpt. of Physiology, Fac. of Medicine and Nursery, University of
Basque Country and BioCruces Health Research Institute, Cruces
University Hospital, Bilbao, ES, 2Dpt. of Physiology, Fac. of Medicine
and Nursery, University of Basque Country, Bilbao, ES, 3Dpt. of
Genetic, Physical Anthropology and Animal Physiology, Fac. of
Science and Technology, University of Basque Country, Bilbao, ES
129
Pósters / Posters
Obesity prevalence is increasing and with it the patients with non-alcoholic
liver disease and the risk of suffering hepatocellular carcinoma (HCC). HCC,
the primary malignant liver cancer, is the third leading cause of cancer-related
deaths. The aim here was to analyze, during the first stages of liver disease,
the metabolic changes involved in obesity promoted HCC. C57B16:129Sv
mice were injected intraperitoneally with diethylnitrosamine (DEN) (25mg/
kg) or PBS when they were 14 days old and fed with high fat diet (HFD)
or chow diet until sacrificed at 3 (3m) or 9 months old (9m). As expected,
mice exposed to the mix of HFD+DEN exhibited higher number and size
of tumors at 9m than those exposed only to DEN. At 3m, mice treated with
HFD+DEN gained more body weight and white adipose tissue comparing
to any other group. They also lost sensitivity to glucose, responded higher
to piruvate after fasting than any other group and exhibited increased serum
fatty acids, all of it suggesting the development of insulin resistance with
increased gluconeogenesis. HFD+DEN treatment induced a decrease in
liver phosphatidylethanolamine and phosphatidylcholine (PC) content
and a concomitant increase in that of diglyceride and triglyceride (TG).
Besides, liver TG secretion was reduced in mice exposed to HFD+DEN
when compared to any other group. Although 14C-glycerol incorporation
into hepatic glycerolipids maintained unaltered, 3H-choline incorporation
into PC increased in mice treated with HFD+DEN, which suggest PC
synthesis through CDP-choline pathway. As conclusion, the increased fatty
acid uptake, decreased VLDL secretion and altered glycerolipid metabolism
in liver together with the high insulin resistance in the first stages of liver
disease will promote HCC development in obesity.
P17-9
An aging model in calorie-restricted mice
following a range of dietary fats reveals
significant changes of key ultrastructural
and physiological parameters in kidney
Miguel Calvo-Rubio Barrera1, María Isabel Burón Romero1,
Guillermo López Lluch2, Plácido Navas Lloret2, Rafael de Cabo3,
Jon J. Ramsey4, José Manuel Villalba Montoro1, José Antonio
González Reyes1
1
Departamento de Biología Celular, Fisiología e Inmunología,
Campus de Excelencia Internacional Agroalimentario, ceiA3,
Universidad de Córdoba, Córdoba, ES, 2Universidad Pablo de
Olavide-CSIC, Sevilla, ES, 3Translational Gerontology Branch,
National Institute of Aging, National Institutes of Health, Baltimore,
US, 4VM Molecular Biosciences, University of California, Davis, US
The Membrane Theory of Aging proposes that lifespan is inversely related
to the level of unsaturation in membrane phospholipids. Calorie restriction
(CR) has been repeatedly reported to prevent age-related diseases in
a wide range of animals, including nonhuman primates and humans. In
rodents CR also increase life span, which may be related to alterations in
membrane phospholipids fatty acids, constituting a powerful tool to study
the aging process. Here we have studied the effect of these dietary fats on
structural and physiological parameters of kidney from mice submitted to
40% CR for 6 and 18 months. We have established four dietary groups: one
control group fed 95 % of a predetermined ad libitum intake, and three CR
groups fed 40 % less than ad libitum intake. Lipid source for the control
and one of the CR groups was soybean oil whereas the two remaining CR
groups were fed diets containing fish oil, or lard. Analyses were performed
using quantitative electron microscopy techniques and protein expression
in western blots. CR seems to mitigate most of the analyzed parameters
related to aging in kidney. These measured parameters included glomerular
basement membrane and foot processes thickness as well as filtration slits
width in podocytes, along with mitochondrial mass in convoluted proximal
tubules. Markers of mitochondrial dynamics and autophagy were also
evaluated in renal homogenates. The significance of our results in this
study are reinforced by additional approaches developed by our group in
different key organs such as skeletal muscle and liver. These studies have
independently indicated that lard as a fat source shows the optimal effect in
130
XXXIX Congreso SEBBM
all of them, which could be due to significant increases of monounsaturated
fatty acids levels in the tissues.
P17m-10
GLUT1 correlates with aggressiveness while
diabetes is inversely related with prostate cancer
Pedro González Menéndez, David Hevia, Iván González-Pola, Juan
C. Mayo, Rosa M. Sáinz
Instituto de Oncología del Principado de Asturias (IUOPA),
Universidad de Oviedo, Avilés, ES
Cancer cells show an increase in glucose uptake, mainly associated with
an overexpression and/or membrane translocation of GLUT transporters.
GLUT1 is the main responsible in most of the tumors but others can be
involved such as the insulin-dependent GLUT4. Interestingly, diabetes
decreases the risk of prostate cancer (PCa) while the insulin signalling has
an important role in PCa progression. Thus, the aim of this study was to
find evidences in vivo about the participation of GLUT transporters in PCa
progression as well as its relation with diabetes and insulin blood levels.
For that, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice
were employed. First, we found that there is a direct association between
GLUT1 and GLUT4 protein levels and PCa progression. GLUT4 levels
progressively increased with tumour progression while GLUT1 levels
were enhanced predominantly in poorly-differentiated tumours and seemed
associated to agressiveness. This increase correlated with Bcl-2, HIF1α and
cytoplasmatic AR levels. After castration, GLUT1/4 levels decreased and
the treatment with testosterone for 5 days restored both GLUT1 and GLUT4
mRNA levels and GLUT4 protein expression. This recovery was related
with AMPK phosphorylation. TXNIP, an inhibitor of GLUT1 translocation,
was overexpressed in castrated mice. Blood Insulin levels were higher in
TRAMP mice compared to WT mice. Its levels decreased with malignancy,
as well as in castrated mice. In addition, streptozotocin-induced diabetic
TRAMP mice showed an inhibition of tumor development. In conclusion,
our work confirms that diabetes is inversely related with PCa in mice while
GLUT1 protein levels are associated with tumour aggressiveness.
P17-11
Alteración en la homeostasis de los ácidos
biliares por interferencia de los glucocorticoides
con la vía de señalización enterohepática
FXR/FGF19
Faten A. Al-Aqil1, María J. Monte1, María A. Serrano1, Raquel
González2, Carlos J. Aranda2, Borja Ocón2, Olga Martínez-Augustín2,
Fermín Sánchez de Medina2, Iker Uriarte3, Matías A. Ávila3,
José J.G. Marín1, Marta R. Romero1
1
Hepatología Experimental y Vectorización de Fármacos
(HEVEFARM), Instituto de Investigación Biomédica de Salamanca
(IBSAL), Universidad de Salamanca. Centro de Investigación
Biomédica en Red de Enfermedades Hepáticas y Digestivas
(CIBERehd), Salamanca, ES, 2Facultad de Farmacia, Universidad
de Granada. Centro de Investigación Biomédica en Red de
Enfermedades Hepáticas y Digestivas (CIBERehd), Granada, ES,
3
Hepatología, Centro de Investigación Médica Aplicada (CIMA),
IDISNA, Universidad de Navarra. Centro de Investigación Biomédica
en Red de Enfermedades Hepáticas y Digestivas (CIBERehd),
Pamplona, ES
Antecedentes: El tratamiento con glucocorticoides (GC) puede alterar la
homeostasis de los ácidos biliares (AB), regulada por FXR, a través del
péptido intestinal FGF19 (Fgf15 en roedores).
Objetivo: Evaluar los efectos de los GC de uso clínico: dexametasona (DEX),
prednisolona y budesonida, sobre la vía de señalización enterohepática FXR/
FGF19 y su repercusión en el metabolismo de los AB.
Salamanca 2016
Métodos: Se utilizaron ratones C57BL/6, Fxr-/-, Fgf15-/- y GR-/(selectivamente para el receptor de GC intestinal) y células Alexander de
hepatoma humano transfectadas con FXR/RXR.
Resultados: El tratamiento con GC redujo de forma dosis-dependiente
la expresión intestinal de Fgf15 en ratones, mientras que elevó la del
transportador de AB Asbt. A dosis no hepatotóxicas la DEX disminuyó
la expresión intestinal de Fgf15 sin alterar la hepática de Cyp7a1 (clave
en la síntesis de AB), que es inhibida por Fgf15. Tampoco se alteró la de
genes implicados en el transporte (Bsep, Ntcp, Mrp2) y el metabolismo
(Baat) de AB. En ratones Fxr-/- y GR-/-, con baja expresión basal de Fgf15,
la DEX causó una represión adicional de Fgf15. A pesar de lo cual, la
expresión de Cyp7a1 estaba inhibida en ambas cepas. Por el contrario, en
ratones Fgf15-/-, los GC estimularon la expresión de Cyp7a1. En células
Alexander los GC redujeron la expresión de FGF19, aún en presencia de
GW4064 (agonista de FXR), y estimularon, de forma FXR-independiente,
la expresión de CYP27A1 (clave en la síntesis de AB).
Conclusión: Los GC interfieren con la regulación de la síntesis hepática de
AB, alterando el control que ejerce FXR a través de la vía de señalización
mediada por FGF19, lo que puede contribuir a la aparición de alteraciones
en la función hepatobiliar en pacientes que reciben tratamientos
prolongados con GC.
P17-12
Ghrelin reduces GABAergic output through
carnitine palmitoyltransferase (CPT) 1A
Joan Francesc Mir1, Matthieu Lichtenstein2, Judit García-Villoria3,
Minéia Weber1, María Calderón-Domínguez1, Blanca Balanyà1,
David Sebastián4, Antonio Zorzano4, Núria Casals5, Antònia Ribes3,
Cristina Suñol2, Laura Herrero1, Dolors Serra Cucurull1
1
Departament de Bioquímica i Fisiologia, Facultat de Farmàcia,
Institut de Biomedicina de la Universitat de Barcelona. Centro de
Investigación Biomédica en Red de Fisiopatología de la Obesidad
y Nutrición (CIBEROBN), Barcelona, ES, 2IIBB - CSIC. CIBEResp,
Barcelona, ES, 3Sección de Errores Congénitos del Metabolismo –
IBC, Servicio de Bioquímica y Genética Molecular, Hospital Clínic,
IDIBAPS, Centro de Investigación Biomédica en Red (CIBER)
de Enfermedades Raras, Barcelona, ES, 4Institute for Research
in Medicine (IRB Barcelona), Barcelona, ES, 5Departament
de Ciències Bàsiques, Facultat de Medicina i Ciències de la
Salut, Universitat Internacional de Catalunya (UIC). Centro de
Investigación Biomédica en Red de Fisiopatología de la Obesidad
y Nutrición (CIBEROBN), Barcelona, ES
Introduction: Ghrelin modulates neuropeptides in arcuate hypothalamus
and it is essential for the control of feeding and glycaemia. Nonetheless,
little is known about its effect on amino acid neurotransmitters. We assessed
ghrelin effect on gamma-aminobutyric acid (GABA) metabolism and
release and the involvement of carnitine palmitoyltransferase (CPT) 1A asa
downstream effector in primary cortical neurons (CTX) and hypothalamic
GT1-7 cell line. We used a pharmacological inhibition with etomoxir, a
CPT1A-specific inhibitor, and genetic inhibition with Ad-shCPT1A and
total ablation in CTX from CPT1A(loxP/loxP) mice.
Results: We observed that ghrelin reduces GABA release in cortical
neurons under physiological glucose concentration similar to that of
the cerebrospinal fluid. This effect is mediated by CPT1A, since its
pharmacological inhibition with etomoxir or genetic ablation restores
this reduction. Ghrelin also produces an increase in mRNA of cpt1a,
gadx, vgat and GABA shunt genes. Moreover, we observed that ghrelin
induces changes in the levels of TCA intermediates, as well as it modifies
mitochondrial function by reducing oxygen consumption rate and the
production of reactive oxygen species. These three effects are reversed by
genetic inhibition of CPT1A.
Conclusion: All these observations indicate that ghrelin treatment of
cortical/hypothalamic neurons might modulate GABA metabolism and
release and highlight CPT1A as a key mediator of ghrelin’s effect.
Pósters / Posters
P17-13
Generation of a CPT1C mutated protein
insensitive to malonyl-CoA
Cristina Miralpeix1, Rosalía Rodríguez-Rodríguez1, María Casas1,
Laura Herrero2, Dolors Serra2, Núria Casals3
1
Basic Sciences Department, Faculty of Medicine and Health
Sciences, Universitat Internacional de Catalunya, Sant Cugat del
Vallès, ES, 2CIBER Fisiopatología de la Obesidad y la Nutrición
(CIBEROBN), Instituto de Salud Carlos III, Madrid, ES. Department
of Biochemistry and Molecular Biology, Institut de Biomedicina
de la Universitat de Barcelona (IBUB), Universitat de Barcelona,
Barcelona, ES, 3CIBER Fisiopatología de la Obesidad y la Nutrición
(CIBEROBN), Instituto de Salud Carlos III, Madrid, ES; Basic
Sciences Department, Faculty of Medicine and Health Sciences,
Universitat Internacional de Catalunya, Sant Cugat del Vallès, ES
Carnitine palmitoyltransferase 1 (CPT1) C is a brain specific isoform
of the CPT enzymes family, which is localized in neuronal endoplasmic
reticulum. Despite its minimal CPT1 catalytic activity, CPT1C is able to bind
malonyl-CoA and long-chain acyl-CoAs. CPT1C KO mice present impaired
spatial learning and cognition, motor deficits and deregulation of food
intake and energy homeostasis. However, the exact molecular mechanisms
mediating these functions and if they depend on malonyl-CoA are still
unknown. Therefore, our aim was to evaluate whether CPT1C function in
neurons is regulated by malonyl-CoA. To carry out this study we constructed
the mutant CPT1CM589S by site directed mutagenesis to alter its binding site to
malonyl-CoA. Methionine 589 had been previously described to be crucial for
malonyl-CoA binding to CPT1A. We first determined the loss of sensitivity
to malonyl-CoA by performing binding assays between CPT1CM589S and
malonyl-CoA in microsome samples. Secondly, we studied the involvement
of malonyl-CoA in the interaction of CPT1C with the subunit 1 of glutamate
AMPA receptors (GluA1), a recently well-known interaction, and in the
regulation of GluA1 expression levels. For this purpose, CPT1C and
CPT1CM589S were overexpressed in cortical neurons and immunoprecipitation
and western blot assays were performed. We found that CPT1CM589S
maintained the interaction with GluA1 and increased GluA1 protein levels
similarly to CPT1C overexpression. These results suggest that the CPT1C
role in GluA1 receptor expression regulation is malonyl-CoA-independent,
and hence confirming the functionality of CPT1CM589S. In conclusion, we
have developed a tool to study whether CPT1C is acting as a malonyl-CoA
sensor in neurons and how this interaction further regulates its binding with
other neuronal proteins.
P17-14
Detecting lipid variance in cellular models
of Parkinson’s disease
Aoife Bannon1, Massimo Valoti2
University of Siena, Siena, IT, 2Supervising Associate Professor,
Univeristy of Siena, IT
1
Background: Maintaining lipid homeostasis is essential for neuronal
function and evidence suggests that lipid metabolism alterations are
involved in Parkinson’s disease (PD). Lipid profiling can be used to
investigate lipid pathways that are essential in cell biology and in specific
disease processes.
Aim: To develop a method in which SHSY5Y cells can be used to detect
lipid variance in PD.
Methods: The lipid profile of SHSY5Y cells was obtained using a newly
developed one-phase lipid extraction method and Q-TOF LC/MS analysis.
Cells were differentiated either by retanoic acid(RA) or RA and phorbol
ester (PE), the latter pushing further towards a dopaminergic phenotype.
Lipid extraction procedures are being optimised to compare the lipid
profiles of undifferentiated and differentiated cells. Neurotoxin toxicity is
being investigated on different cell types.
131
Pósters / Posters
Results: Preliminary trials suggest the new lipid extraction method
provides satisfactory recovery of lipids from cell samples compared to the
gold standard methods. Western blot analysis of dopaminergic proteins
confirmed differentiation. Analysis of undifferentiated, RA-differentiated
and RA/PE differentiated cells suggests there is variance in lipid profiles
of the cell types, though further experiments must be carried out to confirm
this. This can provide a profile against which cells treated with toxic/
protective agents can be compared.
Conclusions: These experiments could provide a method by which the
SHSY5Y cell line can be used to detect lipid variance caused by toxins/
neuroprotective compounds, allowing identification of lipid biomarkers for
specific diseases and development of neuroprotective drugs.
Acknowledgements: The presented research is funded by TINTIN, a Marie
Curie ITN funded project 2013-17(GA no:608381).
P17r-15
Effect of cell penetrating peptides based
on connexin43 on the rate of glucose uptake
in glioma stem cells
Sara Gutiérrez-Pelaz, Elvira Criado-Moronati, José María Medina,
Arantxa Tabernero
Instituto de Neurociencias de Castilla y León (INCYL), Universidad
de Salamanca, Salamanca, ES
Connexin43 (Cx43), the main gap junction channel-forming protein
in astrocytes, is downregulated in malignant gliomas. These tumors
are composed of a heterogeneous population of cells, some of which,
termed glioma stem cells (GSCs), display stem-cell-like properties
and high tumorigenic potential. Restoring Cx43 in GSCs reverses their
phenotype through the inhibition of c-Src and consequently reduces
GSCs tumorigenicity. We have developed a cell-penetrating peptide
(TAT-Cx43266-283) containing the region of Cx43 that interacts with
c-Src that mimics the effect of Cx43 on GSC phenotype. In terms of
catabolism, GSC phenotype is characterized by the Warburg effect - part
of the metabolic reprogramming considered a cancer hallmark - in which
mitochondrial OXPHOS is decreased in favor of aerobic glycolysis and
glucose uptake is increased. In the tumor microenvironment, GSCs can
reprogram their metabolism to compete for glucose resources through
expression of GLUT-3, the high affinity glucose transporter. Expression
of GLUT-3 and other glucose metabolism proteins is regulated by
transcription factor HIF-1-alpha, which can in turn be regulated by c-Src.
In this study we have analysed the effect of TAT-Cx43266-283 on the rate
of glucose uptake in human GSCs and in non-tumoral cells. Our results
showed that glucose uptake in human GSCs treated with TAT-Cx43266-283
is decreased compared to that of control human GSCs. Furthermore, we
have determined that glucose uptake in astrocytes from rat brain primary
cultures treated with TAT-Cx43266-283 shows no differences compared to that
of control astrocytes. In conclusion, our results suggest that TAT-Cx43266-283
reduces the rate of glucose uptake specifically on GSCs.
P17m-16
Adipose tissue expansion in obesity. Is oxidative
stress friend or foe?
Martín Alcalá Díaz-Mor1, Dolors Serra Cucurull2, Laura Herrero
Rodríguez2, María del Pilar Ramos Álvarez1, Marta Viana Arribas1
1
Departamento de Química y Bioquímica, Facultad de Farmacia,
Universidad San Pablo CEU, Madrid, ES, 2Departamento
de Bioquímica y Biología Molecular, Facultad de Farmacia,
Universidad de Barcelona, Barcelona, ES
Adipose tissue expansion plays a key role in the physiopathology of
obesity. Changes in adipocytes size and number, macrophage infiltration
132
XXXIX Congreso SEBBM
and extracellular matrix fibrosis are key features that define the expansion
of the adipose tissue. A healthy expansion may prevent the lipotoxicity
in other tissues. Thus, reducing oxidative stress, which is present in the
molecular basis of all the mechanisms of adipose tissue remodeling, may
improve the pathological profile of obesity.
Objectives: To determine, in an animal model, the role of oxidative stress
in the adipose tissue expansion.
Methods: CBL57/6J mice were divided into 3 groups: control group (C), fed
on standard diet; an obese group (O), fed on high-fat diet (45% energy from
fat) and a third group, fed on high-fat diet and a vitamin E supplementation
(OE). Animals were sacrificed after 14 and 28 weeks of treatment. Adipose
tissue structure, macrophage infiltration and extracellular matrix were
analyzed and related to oxidative stress parameters.
Results: Vitamin E supplementation for 14 weeks in obesity caused adipose
tissue fibrosis, adipocyte hypertrophy and macrophage accumulation,
which may account for the insulin resistance observed in these animals.
However, after 28 weeks, when obesity caused a marked oxidative stress,
vitamin E reduced adipose tissue fibrosis, improving the metabolic,
oxidative and inflammatory profile.
Conclusions: The preventive use of vitamin E in short-term obesity
causes adipose tissue remodeling that leads to lipotoxicity, probably due
to a reduction in the physiological levels of ROS. However, in mid-term
obesity, vitamin E reduced the pre-existing oxidative stress, enhancing the
expansion of the adipose tissue, preventing lipotoxicity and improving the
metabolic profile.
P17r-17
G protein-coupled receptor kinase 2 plays
a role in the development of non-alcoholic
steatohepatitis
Marta Cruces Sande1, Rocío Vila-Bedmar1, Elisa Lucas1,
Ángela M. Valverde2, Águeda González-Rodríguez2, Cristina Murga1,
Federico Mayor Menéndez1
1
Centro de Biologia Molecular Severo Ochoa UAM-CSIC, Madrid,
ES, 2Instituto de Investigaciones Biomédicas Alberto Sols
(CSIC/UAM), Madrid, ES
Introduction: Insulin resistance (IR) and obesity are major health
problems and important risk factors for the development of non-alcoholic
fatty liver disease, a disease spectrum that may include hepatic steatosis,
non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. G
protein-coupled receptor kinase 2 (GRK2), first identified as a regulator
of G protein-coupled receptors (GPCRs), has been described to play a
relevant role in the development of IR and obesity in vivo. However, the
effect of GRK2 in the development of NASH had not been addressed so
far. Since the deletion of GRK2 prevents excessive body weight gain, we
fed WT and GRK2 global hemizygous mice (GRK2+/-) with a methionine
and choline-deficient diet (MCD), a well stablished model of NASH that is
independent of fat mass accretion.
Results: Even though the MCD diet induced similar metabolic alterations
and a comparable elevation in plasma transaminase activity in WT and
GRK2+/- mice, other negative effects of the MCD were partially alleviated
in GRK2 +/- animals. The increase in hepatic triglyceride content caused
by this diet was significantly lower in GRK2+/- mice and, interestingly,
MCD feeding induced an increase in GRK2 protein levels in WT but not
in GRK2+/- livers. Moreover, GRK2+/- mice presented protection from
some deleterious effects of MCD in the liver as indicated by reduced
markers of endoplasmic reticulum stress, and conversely maintained some
hepatic protective mechanisms such as autophagy or mitochondrial fusion.
Accordingly, these results provide a link between GRK2 levels and hepatic
lipid homeostasis leading to NASH.
Conclusion: Taken together, these results suggest a role for GRK2 in the
establishment and/or development of NASH.
Salamanca 2016
P17m-18
Papel dual de APC/C-Cdh1 en obesidad
Rubén Rodríguez, Irene García-Higuera, Sergio Moreno
Instituto de Biología Funcional y Genómica (IBFG),
CSIC-Universidad de Salamanca/Instituto de Investigación
Biomédica de Salamanca (IBSAL), Salamanca, ES
Nuestro grupo lleva años estudiando el papel biológico de Cdh1, una
proteína activadora del Complejo Promotor de la Anafase, o ciclosoma
(APC/C), utilizando para ello modelos murinos de inactivación génica.
El complejo APC/C-Cdh1 dirige, durante la fase G1 del ciclo de división
celular, la poliubiquitinación de ciclinas y otros reguladores de ciclo, para
su posterior degradación por el proteasoma. Nuestro trabajo ha desvelado
que APC/C-Cdh1 promueve diversos procesos de diferenciación, como
neurogénesis o eritropoyesis. Sin embargo, nada se sabe acerca de su
posible papel en adipogénesis, un proceso de diferenciación complejo e
inusual, del que aún queda mucho por descifrar.
Realizando ensayos de diferenciación in vitro, con MEF y preadipocitos
primarios, hemos comprobado que la ausencia de Cdh1 provoca un
aumento significativo de la adipogénesis, y estimula tanto la expansión
clonal de los preadipocitos, como su diferenciación terminal. Por otro lado,
también hemos observado que los adipocitos diferenciados carentes de
Cdh1 presentan ciertas características de grasa marrón y expresan niveles
incrementados de UCP1 tras la inducción del proceso de browning. In vivo,
la deleción de Cdh1 restringida a tejido adiposo da lugar a ratones con
depósitos de grasa blanca y marrón de mayor tamaño, lo que confirma que
APC/C-Cdh1 ejerce una sorprendente y novedosa actividad inhibitoria en
la formación del tejido adiposo.
En conjunto nuestros resultados apuntan a un posible papel dual del
complejo APC/C-Cdh1 en obesidad, modulando la formación de los
depósitos de grasa blanca y marrón.
P17-19
La falta de osteopontina está vinculada
a la esteatosis precoz durante el envejecimiento
Beatriz Gómez-Santos1, Maitane Núñez-García1, Diego Sáenz
de Urturi-Indart1, Larraitz Fernández-Ares2, Daniela Mestre
Congregado2, Juan L. García-Rodríguez1, Igor Aurrekoetxea2, Xabier
Buqué2, Patricia Aspichueta2
1
Departamento de Fisiología, Facultad de Medicina y Enfermería,
Universidad del País Vasco, Bilbao, ES, 2BioCruces Health
Research Institute, Hospital Universitario Cruces y Departamento de
Fisiología, Facultad de Medicina y Enfermería, Universidad del País
Vasco, Bilbao, ES
El envejecimiento se caracteriza por la pérdida progresiva de la integridad
fisiológica. La osteopontina (OPN) es una glicoproteína multifuncional
que muestra expresión incrementada en patologías derivadas del síndrome
metabólico y varios tipos de cáncer, siendo la prevalencia de estas
patologías mayor con la edad. Nuestro objetivo fue determinar si OPN
ejerce algún papel en la modulación del metabolismo hepático durante el
envejecimiento y en tal caso definir el mecanismo implicado. Se usaron
ratones deficientes en OPN (OPN-KO) y sus controles de 3, 10 y 20 meses.
Parte de los ratones de 10 y 20 meses fueron alimentados 16 semanas con
dieta rica en grasa. Se estudió el contenido lipídico hepático y su síntesis de
novo. Se analizaron parámetros séricos y de señalización ligadas a procesos
de síntesis y oxidación. Los resultados mostraron que en animales control
los niveles séricos de OPN aumentaban de 3 a 10 meses y se mantenían
de 10 a 20. El mismo patrón se observó en la expresión proteica de OPN
en hígado. Los ratones de 20 meses con dieta rica en grasa presentaban
niveles séricos de OPN incrementados respecto a los de dieta control. Sin
embargo, el incremento inducido por la dieta no se produjo en animales
de edad intermedia. En animales de 10 meses la falta en OPN resultó
en el aumento del índice hepático y el contenido hepático en colesterol
esterificado (CE), triglicérido (TG) y distintos glicerofosfolípidos (PL) que
Pósters / Posters
se mantenía constante a los 20 meses. Sin embargo, los animales salvajes
mostraron un aumento paulatino del contenido de TG y CE a lo largo del
envejecimiento, produciéndose el mayor incremento a los 20 meses. La
síntesis de novo de PL, DG, TG y CE en hígado estaba aumentada en los
animales OPN-KO de 10 meses que también mostraron mayor expresión
de la acetil-CoA carboxilasa, enzima limitante en la síntesis de novo de
ácidos grasos, compatible con la mayor síntesis de novo de ácidos grasos.
Como conclusión, la falta de osteopontina a lo largo del envejecimiento
está asociada al incremento precoz del almacén lipídico hepático,
principalmente debido a la mayor la síntesis de novo de lípidos.
P17r-20
The autophagic protein DOR/TP53INP2
is a novel regulator of brown adipose tissue
metabolism
Alba Sabaté Pérez1, Montserrat Romero de Pablos1, Xavier Testar
Ymbert2, Antonio Zorzano Olarte1
1
Institute for Research in Biomedicine (IRB Barcelona); CIBER
in Diabetes and Associated Metabolic Diseases (CIBERDEM);
Departament of Biochemistry and Molecular Biomedicine, Faculty
of Biology, University of Barcelona, Barcelona, ES, 2CIBER in
Diabetes and Associated Metabolic Diseases (CIBERDEM);
Departament of Biochemistry and Molecular Biomedicine, Faculty
of Biology, University of Barcelona, Barcelona, ES
Brown adipose tissue-mediated thermogenesis is an important effector
on energy dissipation and can have an impact on total energy balance.
Due to the recent discovery that adult humans have active brown adipose
tissue depots, this tissue has emerged as a novel target for the treatment
of metabolic disorders. Diabetes and obesity-regulated protein (DOR),
also called TP53INP2, is a nuclear cofactor that exits to the cytosol and
stimulates autophagy in cellular models, in Drosophila melanogaster and
in skeletal muscle under in vivo conditions. Moreover, we found that DOR
mRNA levels are markedly induced in brown adipose tissue under situations
characterized by enhanced thermogenic activity such as cold exposure.
DOR is also a negative modulator of white adipogenesis and its ablation
causes augmented adiposity in mice. In this regard, tamoxifen-inducible
total DOR knockout (DOR KO) mice presented increased brown adipose
tissue weight and lipid droplet size, downregulation of key genes involved
in brown adipogenesis and thermogenesis, and cold intolerance. Using
brown preadipocytes, we also found that DOR is required for brown fat
differentiation. DOR loss-of-function in these cells resulted in reduced lipid
accumulation, mitochondrial content and expression of brown adipocyte
marker genes, as UCP1 and PGC1α. Therefore, we generated a Myf5-DOR
knockout mice to study the impact on tissue homeostasis of DOR ablation
in brown adipose precursor cells. Preliminary data on brown adipose
tissue from Myf5-DOR knockout mice showed a gene expression profile
comparable to the global DOR KO mice model. All these results suggest
that DOR has a crucial role controlling whole body energy homeostasis by
modulating brown adipose tissue metabolism.
P17-21
Desregulación de la colesterogénesis por
sobreexpresión de SND1 en células de hepatoma
Hiart Navarro Imaz, Yuri Rueda Estévez, Olatz Fresnedo Aranguren
UPV/EHU, Leioa, ES
SND1 es una proteína muy conservada cuya función aún no ha sido
esclarecida. Se considera una proteína multifuncional, que a través de
interacciones muy diversas con RNA y proteínas parece que participa en
procesos dispares. Nuestros estudios funcionales en torno al metabolismo
lipídico han mostrado que podría jugar un papel relevante en la homeostasis
celular del colesterol. En este trabajo hemos profundizado en los mecanismos
133
Pósters / Posters
implicados en la sobreacumulación de ésteres de colesterol (CE) en células
de hepatoma debida a la sobreexpresión de SND1. Para ello se han empleado
células McA-RH7777 que sobreexpresan SND1 de forma estable (McA-S).
Estas células no muestran la respuesta homeostática de las células control
(transformadas con el cDNA de la β-galactosidasa; McA-L) tras el suministro
exógeno de colesterol, acumulando mayores cantidades de CE. Esta alteración
es debida a una activación constitutiva de la proteína de unión al elemento de
respuesta a esteroles tipo 2 (SREBP2), causada por un menor contenido en
colesterol libre (CL) de las membranas del retículo endoplasmático (RE). El
tratamiento de los cultivos con un inhibidor de la esterificación del colesterol
y el consecuente aumento del contenido en CL del RE revierten la activación
de SREBP2 y moderan la velocidad de síntesis de novo del colesterol. Los
resultados de este trabajo sugieren que el papel de SND1 en la homeostasis
del colesterol está vinculado a la modulación de su distribución celular, en
particular al mantenimiento del contenido de CL del RE.
Subvencionado por el GV (SA-2010/00050, IT-336-10 and AE-2012-1-69),
la UPV/EHU (UFI11/20) y la Fundación Jesús de Gangoiti Barrera.
P17-22
Characterization of Endothelial Cells metabolism
in normal and pathological conditions
Marta Cascante Serratosa1, Erika Zodda1, Anusha Jayaraman1,
Pedro de Atauri1, Ramon Messeguer2, Olga Tura-Ceide3, Timothy
Thomson4
1
Department of Biochemistry and Molecular Biomedicine, Faculty of
Biology, Universitat de Barcelona, Barcelona, ES, 2Biomed Division,
Leitat Technological Center, Parc Científic Barcelona, Barcelona,
ES, 3Department of Pulmonary Medicine, Hospital Clínic-Institut
d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),
University of Barcelona, Barcelona, ES / Centro de Investigación
Biomédica en Red de Enfermedades Respiratorias (CIBERES),
Madrid, ES, 4Department of Cell Biology, Molecular Biology
Institute, National Research Council (IBMB-CSIC), Barcelona, ES
Atherosclerosis is considered a disease associated with premature biological
aging, as atherosclerotic plaques show evidence of cellular senescence.
Endothelial dysfunction is a primary factor in the onset and progression
of atherosclerosis and other vascular related diseases as well as in the
thrombus formation and stabilization. Not just the size but rather the
stability of atherosclerotic plaques is a determinant for acute clinical
implications.
Emerging evidences indicate that pathological blood vessel responses and
dysfunctionality of Endothelial Cells (ECs) are associated with metabolic
alterations in ECs. Preliminary data from our group have suggested that
ECs derived from patients show an altered hyperproliferative phenotype
and a resistance to apoptosis when compared to controls.
In this study we aim to establish an in vitro model of endothelial pathology,
using patient-derived endothelial cell lines which are subjected to a systematic
evaluation against control cells in order to determine the metabolic profile
and to understand if endothelial dysfunctionality is a consequence of an
abnormal endothelial cell metabolism. The endothelial cell lines are studied
in terms of morphology, proliferation and metabolic profiling.
P17-23
La deficiencia en MAT1A confiere resistencia
al desarrollo de obesidad y a la resistencia a
insulina en ratones
Diego Sáenz de Urturi-Indart1, Juan L. García-Rodríguez1,
Igor Aurreoketxea2, Daniela Mestre2, Beatriz Gómez-Santos1,
Maitane Núñez-García1, Larraitz Fernández-Ares2, Virginia
Gutiérrez-de Juan3, M. Luz Martínez-Chantar3, José María Mato3,
Xabier Buqué2, Patricia Aspichueta2
134
XXXIX Congreso SEBBM
1
Departamento de Fisiología, Facultad de Medicina y Enfermería,
Universidad del País Vasco, Bilbao, San Sebastián, ES,
2
Departamento de Fisiología, Facultad de Medicina y Enfermería,
Universidad del País Vasco, Bilbao y BioCruces Health Research
Institute, Hospital Universitario Cruces, Barakaldo, ES, 3CIC
bioGUNE, Centro de Investigación Biomédica en Red de
Enfermedades Hepáticas y Digestivas (CIBERehd), Parque
Tecnológico de Bizkaia, Elexalde Derio, ES
La S-adenosilmetionina (SAMe) es sintetizada en la primera reacción del
metabolismo de metionina a través del enzima Metionina adenosiltransferasa
(MAT), producto del gen MAT1A. El descenso hepático de SAMe, provocado
por la falta de MAT1A, induce el desarrollo de la enfermedad de hígado graso
no alcohólica (NAFLD). Sin embargo, poco se sabe sobre la implicación
de MAT1A en el desarrollo de obesidad. El objetivo fue investigar si la
falta del gen MAT1A podría conferir resistencia al desarrollo de obesidad
y de resistencia a insulina así como a la desregulación metabólica de tejido
adiposo blanco (WAT). Se utilizaron animales control (WT) y deficientes
en MAT1A (MAT1A-KO). Los cuales recibieron dieta control (CD) o dieta
rica en grasa (HFD) durante 10 semanas. Sorprendentemente, la HFD
provocó obesidad solo en los WT. Los animales WT y MAT1A-KO con
dieta control presentaban una tolerancia a la glucosa similar sin resistencia a
insulina. La HFD provocó únicamente la resistencia a insulina esperada en
animales WT. Además, los MAT1A-KO, presentaron una mayor respuesta
en la señalización de insulina aumentando la actividad de AKT en WAT. El
tratamiento con HFD provocó el incremento en los niveles de triglicéridos
(TG) y ácidos grasos (AG) en WAT de los ratones WT. Sin embargo, no lo
logró en los MAT1A-KO, que por su parte mostraron un aumento de cuerpos
cetónicos (KB) séricos y un descenso en los de AG con respecto a los WT.
Además, los MAT1A-KO alimentados con CD tenían una menor lipólisis
que sus respectivos controles. Esto no se observó en animales alimentados
con HFD. La síntesis de novo de TG no cambiaba en los animales
MAT1A-KO alimentados con HFD con respecto a sus WT. Sin embargo,
la esterificación y la oxidación lipídica estaban aumentadas, junto con una
mayor fosforilación de AMPK. Como conclusión, la resistencia al desarrollo
de obesidad y resistencia a insulina en los ratones MAT1A-KO está ligada
al incremento en la oxidación lipídica de WAT más que a cambios en su
lipolisis o síntesis de novo de lípidos.
P17-24
TGF-β enhances glucose metabolism through
PFKFB3 gene expression in T98G glioblastoma
cell line
Ana Rodríguez1, Paula Samsó1, Pere Fontova1, Anna Manzano1,
Helga Simon1, Esther Castaño2, Francesc Ventura1, Àurea
Navarro-Sabaté1, Ramon Bartrons1
1
Departament de Ciències Fisiològiques, Universitat de Barcelona,
Barcelona, ES, 2Centres Científics i Tecnològics, Universitat de
Barcelona, Barcelona, ES
The aim of this study was to investigate the mechanisms connecting
TGF-β, glucose metabolism and PFKFB3 in a glioblastoma cell line
(T98G). It is known that TGF-β expression in malignant gliomas renders
survival advantages to the tumor cells by enhancing cell growth, migration,
invasion, angiogenesis, immune suppression and stem cell properties.
PFKFB3 is an homodimeric bifunctional enzyme, belonging to the family
of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, that controls the
conversion of fructose-6-phosphate (Fru-6-P) to fructose-2,6-bisphosphate
(Fru-2,6-P2). This metabolite is important for the dynamic regulation of
glycolytic flux by allosterically activating the rate-limiting enzyme of
glycolysis phosphofructokinase-1 (PFK-1). It has been shown that PFKFB3
serves as an essential regulator of glycolysis during cell cycle progression and
growth. Its activity is regulated by HIF-1α, Akt, p38 and PTEN, and required
for the survival and growth of multiple cancer types. On the other hand, in
human cancers, TGF-β plays a dual role by acting both as a tumor suppressor
Salamanca 2016
and as a promoter of tumor metastasis. Also, TGF-β overexpression has
been related to the Warburg effect induction. However, little is known about
the molecular mechanisms connecting TGF-β with enhanced glycolysis.
Here, we demonstrate that TGF-β upregulates PFKFB3 mRNA and protein
expression resulting in an increase in Fru-2,6-P2 concentration. Transcription
and translation-blockage experiments confirm that PFKFB3 is regulated
at the transcriptional level by TGF-β treatment. Accordingly, Smad3 and
Smad4 overexpression also show an increase in endogenous PFKFB3
protein expression. In contrast, PFKFB3 protein expression is reduced in
presence of siRNA transfection against Smad2/3 and Smad4 after TGF-β
treatment. In conclusion, this study demonstrates that PFKFB3 gene is a
direct downstream target of TGF-β and, that Smad signaling pathway would
be the link between TGF-β and PFKFB3 induction in T98G cancer cells.
This work was supported by Instituto de Salud Carlos III-FIS (PI13/0096)
and Fondo Europeo de Desarrollo Regional (FEDER).
P17-25
Efectos de la realimentación sobre la translocación
de la glucógeno sintasa hepática y el metabolismo
del glucógeno en ratas sanas ayunadas
Josep M. Fernández Novell1, Carmen López Iglesias2, Joan J.
Guinovart Cirera3
1
Universitat de Barcelona, Barcelona, ES, 2Serveis
cientifico-tècnics. Parc Científic de Barcelona, Barcelona, ES,
3
Universitat de Barcelona. Institut de Recerca Biomèdica. Parc
Científic de Barcelona, Barcelona, ES
Se ha descrito que la incubación de hepatocitos aislados de ratas sanas
ayunadas 24 h en presencia de glucosa induce la síntesis de glucógeno de
una forma espacialmente ordenada. Comienza en la periferia celular, desde
la membrana celular hacia el interior del hepatocito. Esta síntesis se produce
de forma simultánea con el movimiento, la translocación, de la glucógeno
sintasa hepática (GS), desde el citoplasma hacia la periferia celular. El
presente estudio se ha llevado a cabo para determinar si la concomitancia
entre ambos procesos se produce en situaciones fisiológicas. Para ello
se utilizaron ratas adultas sanas que se mantuvieron en ayuno durante
las 24 horas anteriores al experimento de realimentación, se sacrificaron
a las 0h, 1h, 2h y 4h de realimentación y se obtuvo de cada animal un
trozo de hígado, una muestra se procesó inmediatamente para obtener
los parámetros bioquímicos. Otra muestra más, se fijo inmediatamente
para posterior procesamiento y análisis mediante microscopía confocal
y, una tercera, también se fijó inmediatamente, para su análisis mediante
microscopía electrónica. En ambos análisis por microscopía se utilizó
el mismo anticuerpo primario contra la GS. Así, en la microscopía
confocal se localizó la GS mediante un anticuerpo secundario marcado
con un fluorocromo, mientras que en la microscopía electrónica se
localizó mediante la técnica de inmunogold. Los análisis bioquímicos y
microscópicos muestran que la realimentación de ratas sanas ayunadas 24
h aumenta la síntesis de glucógeno hepático y, al mismo tiempo, produce
una acumulación progresiva de la GS hepática en la membrana celular.
Nuestros resultados indican que el fenómeno de la translocación de la
glucógeno sintasa es, en las ratas estudiadas, un proceso fisiológicamente
activo y necesario para la síntesis del glucógeno hepático.
P17-26
Análisis del efecto de inhibidores de enzimas
metabólicas sobre la proliferación y viabilidad
de una línea celular modelo de leucemia
mieloide aguda
Carla Ijurko Valeta, Marta Romo González, Ángel Hernández
Hernández
Pósters / Posters
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca (USAL). Instituto de Investigación Biomédica de
Salamanca (IBSAL), Salamanca, ES
Desde hace décadas se conoce que la mayoría de las células cancerosas
y, en particular, las células leucémicas, presentan una incrementada
dependencia de la glucólisis como fuente principal de obtención de
ATP, incluso en presencia de suficiente oxígeno (mecanismo conocido
como “efecto Warburg”). En esencia, este hecho no es más que un
fundamento bioquímico exclusivo de dichas células; por tanto, resultaría
interesante utilizar, como tratamiento anticáncer, inhibidores de este
mecanismo. En este trabajo se ha empleado una línea celular modelo de
leucemia mieloide aguda (LMA), MV4-11, para estudiar la importancia
del metabolismo glucídico en las células leucémicas. Se ha analizado
la proliferación y viabilidad de estas células frente a inhibidores del
transportador de glucosa (phloretin), la hexoquinasa (2-DG), o el
complejo piruvato deshidrogenasa (DCA). Los tres inhibidores han
demostrado frenar la proliferación celular, con IC50 de 85μM, 2,5mM y
30mM, respectivamente, e inducir la apoptosis de las células MV4-11.
Estos datos demuestran la importancia de la vía glucolítica para la
supervivencia de esta línea celular, que ha resultado ser más sensible a
los tratamientos que otras líneas celulares de origen leucémico como por
ejemplo K562. De entre las tres estrategias probadas, phloretin ha sido
la más eficaz. DCA, sin embargo, ha resultado ser el tratamiento menos
eficaz, probablemente por no ser sustrato de una enzima directamente
implicada en la glucólisis. A pesar de que estos inhibidores aún están
lejos de ser los fármacos de elección para el tratamiento del cáncer, parece
claro que la particular adaptación metabólica de las células cancerosas
ofrecerá nuevas posibilidades terapéuticas en el futuro.
P17-27
La UCP2 inhibe la cadena respiratoria
y el consumo de oxígeno en adenocarcinoma
pancreático
Margalida Torrens-Mas1, Jorge Sastre-Serra1, Massimo Donadelli2,
Ilaria Dando2, Jordi Oliver1, Pilar Roca1
1
Grupo Multidisciplinar de Oncología Traslacional, Institut
Universitari d’Investigació en Ciències de la Salut (IUNICS),
Universitat de les Illes Balears. Centro de Investigación Biomédica
en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn,
CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma
(IdISPa), Palma de Mallorca, ES, 2Department of Neuroscience,
Biomedicine and Movement, Biochemistry Section, University of
Verona, Verona, IT
La proteína desacoplante UCP2 está sobreexpresada en el cáncer de
páncreas. Estudios recientes sugieren que la UCP2 puede proteger a las
células cancerosas del estrés oxidativo y participar en la reprogramación
metabólica hacia la glucólisis, siendo de esta manera importante para la
promoción del tumor y el desarrollo de la quimioresistencia.
Nuestro objetivo fue determinar el efecto de silenciar mediante un siRNA
la UCP2 sobre la funcionalidad mitocondrial y el consumo de oxígeno en
las células de cáncer de páncreas PANC-1. Para ello, se determinaron los
niveles proteicos de los complejos de la cadena respiratoria OXPHOS por
Western Blot, así como las actividades enzimáticas de los complejos IV
y V, la cantidad de DNA mitocondrial por PCR y el consumo de oxígeno
mediante un electrodo de Clark.
El silenciamiento de la UCP2 provocó un incremento general en todos los
complejos de la cadena respiratoria mitocondrial. Además, las actividades
enzimáticas de los complejos IV y V incrementaron un 27% y un 25%
respectivamente, sin alterar el número de copias del DNA mitocondrial,
sugiriendo una mayor funcionalidad manteniendo el número de
mitocondrias. Por otra parte, se observó que la tasa de consumo de oxígeno
basal fue significativamente mayor en las células con el siRNA para UCP2,
así como la capacidad máxima y la producción de ATP.
135
Pósters / Posters
Estos resultados parecen indicar que la UCP2 podría mediar un cambio
energético en las células cancerosas, inhibiendo la respiración mitocondrial
y favoreciendo así el efecto Warburg.
Financiado: beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y
FEDER-Unión Europea “Una manera de hacer Europa”.
P17m-28
Analyzing the etiology of metabolic disorders
by means of lipidomic analysis
Virginia López Gómez-Carreño1, Sandra Fernández Gallego2,
Nilda Gallardo Alpizar1, Christer S. Ejsing2, Antonio Andrés Hueva1
1
Facultad de Ciencias y Tecnologías Químicas, (UCLM), Ciudad
Real, ES, 2University of Southern Denmark (SDU), Odense M, DK
There are substantial evidences suggesting that hypothalamic inflammation
due to excessive accumulation of long-chain fatty acids and/or ceramides
exacerbates the development of central and systemic insulin resistance.
Wistar rats like humans, increase body weight and adiposity, and become
hypertriglyceridemic and hyperleptinemic with aging. Our previous results
point out aging associated alterations are responsable of this situation and
could be implicated in the development of insulin and leptin resistance.
Besides, based on recents publications and in our results we hypothesized
that hypothalamus is the first tissue affected. Thus, we aim to analyze
whether ceramide levels involved on the sphingolipid metabolism in
hypothalamus is altered with aging, and whether food restriction modifies
ceramide content. To this end, lipidomic enables to analyze the lipidome,
the entire collection of chemically distinct lipid species, in a cell, an organ
or a biologycal system. For this reason lipidomic plays an essential role
in defining the biochemical mechanisms underlying lipid-related disease
process through the determination of alterations in celular lipid signaling,
metabolism, trafficking and homeostasis.
Hypothalamic lipidomic analysis were carried out using shotgun lipidomics
in 3, 8 and 24 months old Wistar rats in order to analyze the effect of aging
in the hypothalamus of these groups of rats. Moreover we have analyzed
the hypothalamic lipidome of 8 and 24 months old Wistar rats subjected to
a moderate food restriction during 3 months.
Unexpectally, the total amount of Cer and GM3 decrease with aging. On
the other hand, we found that SHexCer, HexCer and Hex2Cer are increased
with aging, while food restriction reduced significantly their levels but
only in 8 months old rats.This comprehensive analysis provide information
about the changes in total content and relative abundance of ceramides and
its derivatives (amongst other lipid species) in the hypothalamus during
aging and food restriction.
With aging and food restriction there are changes not only in ceramides,
also in its derivatives which shows that the complex sphingolipid pathway
can not be only focused in ceramides. Extend the analysis to all its
derivatives would provide more information that will allow us for a better
understanding in the mechanisms involved in the development of central
and peripheral insulin and leptin resistance.
P17r-29
NADPH oxidasas como reguladoras del metabolismo
intermediario en leucemia mieloide crónica
Marta Romo González, Carla Ijurko, Rodrigo Prieto Bermejo,
Alejandro Pérez Fernández, Ángel Hernández Henández
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca (USAL). Instituto de Investigación Biomédica de
Salamanca (IBSAL), Salamanca, ES
Tanto la sobreproducción de especies reactivas del oxígeno (ROS) como
la desregulación del metabolismo (efecto Warburg) son características
propias del cáncer, que están íntimamente ligadas a su transformación
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XXXIX Congreso SEBBM
oncogénica. Por una parte, la excesiva producción de ROS en el cáncer
se ha relacionado de un modo directo con los procesos de promoción de
la supervivencia, metástasis, proliferación e incluso con la resistencia
al tratamiento. En la leucemia mieloide crónica (LMC) se ha descrito
que la activación constitutiva de la quinasa BCR-ABL desencadena la
producción de ROS vía NADPH oxidasas, una familia de enzimas cuya
única función conocida es la producción de ROS. Por otra parte, las
células cancerígenas al estar sujetas a un crecimiento y proliferación
descontrolada, se ven forzadas a readaptar su metabolismo, recurriendo
a la fermentación de la glucosa a lactato como fuente para obtener de
energía. Además, es importante destacar, que son las mismas rutas
de señalización las que inducen la producción de ROS vía NADPH
oxidasas y las que provocan un cambio metabólico en la célula
tumoral. Por todo ello, en este trabajo nos hemos propuesto estudiar
el efecto que ejercen las ROS producidas vía NADPH oxidasas en la
particular adaptación metabólica de las células tumorales. Para ello,
empleamos dos aproximaciones: el uso del inhibidor químico DPI y
el silenciamiento mediante ARN de interferencia de la subunidad
p22phox (proteína común a cuatro de los siete miembros de la familia
NADPH oxidasa) o del miembro NOX2 en la línea celular K562 de
LMC. Nuestros resultados muestran que las NADPH oxidasas afectan
a parámetros clave del metabolismo tumoral como son la captación de
glucosa, la actividad lactato deshidrogenasa y los niveles de ATP/ADP,
NADH/NAD+ y NADPH/NADP+.
P17-30
El resveratrol promueve la respiración
mitocondrial y la oxidación de ácidos grasos
en la línea de cáncer de colon SW620
María de Mar Blanquer-Rosselló, Jordi Oliver Oliver, Pilar Roca
Salom, Ádamo Valle Gómez
Grupo Multidisciplinar de Oncología Traslacional, Institut
Universitari d’Investigació en Ciències de la Salut (IUNICS),
Universitat de les Illes Balears. Centro de Investigación Biomédica
en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn,
CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma
(IdISPa), Palma de Mallorca, ES
El resveratrol (RSV) es un polifenol presente en la piel de la uva conocido
por sus propiedades anticancerígenas. Varios estudios han descrito que el
RSV actúa en las etapas de iniciación, promoción y progresión del cáncer.
Aunque su biodisponibilidad es baja debido a su rápida metabolización, su
efecto preventivo en el cáncer de colon merece especial atención puesto
que se trata de un tejido altamente expuesto a los componentes de la dieta,
incluido el resveratrol. Una de las principales características metabólicas
de las células tumorales es su voraz consumo de glucosa mediante la vía
glucolítica en detrimento de la fosforilación oxidativa (efecto Warburg).
Aunque el RSV ha sido descrito como un mimético de la restricción
calórica, hay controversia en cuanto a su efecto sobre el metabolismo de las
células tumorales. El objetivo del estudio fue analizar los efectos del RSV
sobre el metabolismo energético de la línea de cáncer de colon SW620.
Para ello se analizó el efecto sobre el consumo de oxígeno en respuesta a
inhibidores específicos, los niveles de ATP, la producción de lactato y los
niveles de proteínas clave en la regulación del metabolismo energético.
El RSV aumentó la biogénesis mitocondrial y en consecuencia la tasa de
consumo de oxígeno en las células SW620. La mayor respiración se asoció
a un mayor uso de los ácidos grasos como combustible metabólico. No
hubo diferencias en la tasa de producción de lactato ni en los niveles de
enzimas clave del metabolismo del piruvato. Estos datos sugieren que el
efecto citotóxico del RSV en las células de cáncer de colon puede estar
asociado a un shift metabólico, revertiendo parcialmente el efecto Warburg.
Financiado: beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y
FEDER-Unión Europea “Una manera de hacer Europa”.
Salamanca 2016
P17-31
El 17β-estradiol ejerce un efecto antiinflamatorio
en el hígado de ratas ovariectomizadas
Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco
Bauzá-Thorbrügge, Francisco J. García-Palmer, Ana M. Proenza,
Isabel Lladó, Magdalena Gianotti
Grup de Metabolisme Energètic i Nutrició, Dept. Biologia
Fonamental i Ciències de la Salut, IUNICS, Universitat de les
Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación
Biomédica en Red Fisiopatología de la Obesidad y la Nutrición
(CIBERobn, CB06/03/0043), Instituto de Salud Carlos III,
Palma de Mallorca, ES
Los estrógenos protegen de la acumulación hepática de triacilglicéridos
(TAG) y son importantes agentes antioxidantes. Su deficiencia favorece
la esteatosis hepática y el aumento del estrés oxidativo, alteraciones que
pueden desembocar en inflamación hepática.
El objetivo de este estudio fue determinar la modulación que ejerce el
17β-estradiol (E2) en la respuesta inflamatoria hepática, así como elucidar
los mecanismos implicados en la mejora del perfil lipídico hepático y en la
atenuación del estrés oxidativo.
Se realizó un estudio in vivo en ratas Wistar de 14 semanas divididas en tres
grupos: hembras control, ovariectomizadas (OVX) y OVX suplementadas
con E2 (OVX+E2). Se determinaron la acumulación de TAG hepáticos y
los niveles de los principales marcadores de las vías de oxidación y síntesis
de ácidos grasos, así como la actividad de la superóxido dismutasa (SOD),
el daño oxidativo y los niveles de proteínas involucradas en la respuesta
inflamatoria.
En las ratas OVX, se puso de manifiesto un desajuste entre la oxidación
y la síntesis de ácidos grasos que provocó la acumulación hepática de
TAG. Además, se incrementó el estrés oxidativo y, paralelamente, se
activaron las vías inflamatorias. La suplementación con E2 revirtió la
acumulación de TAG y aumentó las defensas antioxidantes, lo que
supuso una disminución de las proteína quinasas activadas por mitógenos
(MAPK) JNK y P38.
En conclusión, el E2 ejercería efectos antiinflamatorios en el hígado a
través de la mejora del perfil lipídico y de la atenuación del estrés oxidativo.
Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato
predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y
beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”.
P17-32
La inflamación del tejido adiposo generada por
una dieta hiperlipídica responde al tratamiento
con rosiglitazona de manera dependiente del
sexo en ratas
Marco Bauzá-Thorbrügge, Miquel Sbert-Roig, Bel M.
Galmés-Pascual, Francisco J. García-Palmer, Magdalena Gianotti,
Isabel Lladó, Ana M. Proenza
Grup de Metabolisme Energètic i Nutrició, Dept. Biologia
Fonamental i Ciències de la Salut, IUNICS, Universitat de les
Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación
Biomédica en Red Fisiopatología de la Obesidad y la Nutrición
(CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma
de Mallorca, ES
Existe un dimorfismo sexual en la funcionalidad mitocondrial del tejido
adiposo blanco (TAB) en ratas. Sin embargo, estas diferencias se atenúan
en respuesta a una dieta hiperlipídica (HL).
El objetivo ha sido determinar si la rosiglitazona (Rsg), un fármaco que
mejora la sensibilidad a la insulina y aumenta la funcionalidad mitocondrial
de los adipocitos, ejerce efectos antiinflamatorios también dependientes
del sexo en el TAB.
Pósters / Posters
Se utilizaron ratas Wistar de ambos sexos alimentadas durante 16 semanas
con dieta control o dieta HL. Durante dos semanas previas al sacrificio,
la mitad de los animales HL fueron tratados con Rsg (100 mg/kg dieta).
En el TAB retroperitoneal se determinaron parámetros marcadores de
inflamación, apoptosis, hipoxia y movilización de lípidos, así como PPARg
y PGC1a.
El aumento de PPARg y de lipasa sensible a hormonas en las hembras HL
es indicativo de una mayor movilización y oxidación de lípidos. En los
machos, esta respuesta solo se observa con el tratamiento con Rsg. Los
marcadores de hipoxia, inflamación y apoptosis fueron más elevados en
los machos HL y disminuyeron de forma más acusada por efecto de la Rsg.
En conclusión, la mayor capacidad de movilizar y oxidar combustibles
lipídicos del TAB de las hembras las protege de la apoptosis e inflamación
inducidas por la dieta HL. Al ser la Rsg un agonista del PPARg, el
dimorfismo sexual observado en los niveles de PPARg con la dieta,
condiciona una mayor respuesta de los machos al tratamiento con
este fármaco, mejorando el perfil inflamatorio generado por la dieta y
reforzando el papel modulador de las hormonas sexuales.
Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato
predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y
beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”.
P17-33
El 17β-estradiol mejora la función y biogénesis
mitocondrial hepática a través de PGC-1β
Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco
Bauzá-Thorbrügge, Francisco J. García-Palmer, Ana M. Proenza,
Isabel Lladó, Magdalena Gianotti
Grup de Metabolisme Energètic i Nutrició, Dept. Biologia
Fonamental i Ciències de la Salut, IUNICS, Universitat de les
Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación
Biomédica en Red Fisiopatología de la Obesidad y la Nutrición
(CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma
de Mallorca, ES
Los estrógenos, y en concreto el 17β-estradiol (E2), se han postulado
como moduladores de la función y biogénesis mitocondrial en el hígado.
La familia de coactivadores 1 del receptor activado por proliferadores
peroxisomales gamma (PGC-1) juega un papel central en la regulación
de estos procesos en diferentes tejidos, aunque la contribución de sus
principales miembros, PGC-1α y PGC-1β, no ha sido elucidada en el
hígado. El objetivo de este estudio fue investigar el efecto del 17β-estradiol
(E2) sobre la función y la biogénesis mitocondrial en el hígado, así
como evaluar la implicación de PGC-1α y PGC-1β como principales
moduladores de estos efectos. Se realizó un estudio in vitro en hepatocitos
HepG2 tratados con E2, en combinación con el silenciamiento de PGC-1α
o PGC-1β mediante un siRNA específico, y se evaluaron marcadores
de función y biogénesis mitocondrial. El tratamiento de HepG2 con E2
incrementó la función y biogénesis mitocondrial. El silenciamiento de
PGC-1β disminuyó los niveles de TFAM y de ATPasa, tanto en situación
basal como con el tratamiento con E2. No obstante, el silenciamiento de
PGC-1α no supuso la disminución de estos marcadores en ninguna de las
condiciones estudiadas, sino que se incrementaron con la administración de
E2. Nuestros resultados aportan evidencias de que los efectos beneficiosos
del E2 en la función y biogénesis mitocondrial en el hígado están mediados
por el PGC-1β. Por tanto, la activación de PGC-1β podría constituir una
diana terapéutica contra los desórdenes mitocondriales hepáticos.
Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato
predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y
beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”.
137
Pósters / Posters
P17-34
El 17β-estradiol mejora la resistencia hepática a
la insulina asociada a la inflamación. Papel de la
vía de JNK
Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco Bauzá-Thorbrügge,
Melanie Raquel Martínez-Cignoni, Francisco J. García-Palmer,
Ana M. Proenza, Magdalena Gianotti, Isabel Lladó
Grup de Metabolisme Energètic i Nutrició, Dept. Biologia
Fonamental i Ciències de la Salut, IUNICS, Universitat de les
Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación
Biomédica en Red Fisiopatología de la Obesidad y la Nutrición
(CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma
de Mallorca, ES
La ingesta de dietas hiperlipídicas (HL) conduce a alteraciones metabólicas
que se asocian al desarrollo de numerosas patologías. En el hígado,
la acumulación de triacilglicéridos (TAG) se relaciona con un estado
de resistencia a la insulina, potenciada por la activación de las vías
inflamatorias. Destaca el papel de c-Jun-N-terminal kinase (JNK), cuya
activación produce una disminución de la respuesta a la insulina.
El objetivo de este estudio fue determinar las diferencias entre sexos en la
resistencia hepática a la insulina y el estado de inflamación inducidos por
una dieta HL y, en particular, el papel modulador del 17β-estradiol (E2).
Se realizó un estudio in vivo en ratas Wistar de ambos sexos alimentadas
durante 16 semanas con una dieta HL (22,5% materia grasa), paralelamente
con el desarrollo de experimentos in vivo en hepatocitos HepG2 tratados
con palmitato (PA) y E2. En ambos modelos se cuantificaron elementos de
las vías de señalización de la insulina y de las proteína quinasas activadas
por mitógenos (MAPK).
En respuesta a la dieta HL, las ratas macho mostraron, en comparación con
las hembras, una mayor resistencia sistémica y hepática a la insulina, que
correlacionó con una mayor activación de JNK. La implicación del E2 en
estos efectos fue confirmada en los estudios realizados en los hepatocitos
HepG2, en los que esta hormona atenuó los efectos lipotóxicos del PA,
disminuyendo la activación de JNK.
En conclusión, el E2 protege frente al desarrollo de resistencia hepática a la
insulina disminuyendo la inflamación, a través de JNK.
Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato
predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y
beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”.
P17-35
Los efectos antinflamatorios del estradiol
en la lipotoxicidad cardíaca no dependen
de la activación de AMPK
Miquel Sbert-Roig, Marco Bauzá-Thorbrügge, Bel M.
Galmés-Pascual, Agustí González-Vicens, Francisco J.
García-Palmer, Isabel Lladó, Magdalena Gianotti, Ana M. Proenza
Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental
i Ciències de la Salut, IUNICS, Universitat de les Illes Balears.
IdISPa, Palma de Mallorca. Centro de Investigación Biomédica
en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn,
CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES
La lipotoxicidad cardíaca supone una disfunción metabólica que conduce
a la activación de las vías inflamatorias en los cardiomiocitos, entre las
cuales cabe destacar las MAPK. Se ha visto que los estrógenos atenúan
la activación de las MAPK en el corazón y, además, pueden activar la
proteína AMPK. Esta proteína ejerce un efecto beneficioso sobre el
corazón lipotóxico, entre otros mecanismos, a través de la disminución de
la respuesta inflamatoria.
El objetivo de este estudio fue analizar si los efectos antinflamatorios del
17β-estradiol (E2) en cardiomiocitos sometidos a un estrés lipotóxico
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XXXIX Congreso SEBBM
dependían de la activación de la AMPK por parte de esta hormona. Para
ello se realizó un estudio in vitro con cardiomiocitos H9c2 diferenciados
y se activaron las vías inflamatorias mediante el tratamiento con palmitato
(PA). Además, las células fueron tratadas con A769662 (activador de
AMPK), E2 y E2 en combinación con el compuesto c (inhibidor de
AMPK). Finalmente, se determinó el nivel de activación de las quinasas
AMPK, ACC, ERK, JNK y p38 por western blot.
Se observó una activación de las MAPK ERK, JNK y p38 en respuesta al
tratamiento con PA, la cual disminuyó mediante la activación de AMPK
por parte de A769662. Este efecto inflamatorio del PA disminuyó también
en presencia de E2 y no se vio afectado por el compuesto c.
En conclusión, el E2 atenúa la activación de las vías inflamatorias de las
MAPK en cardiomiocitos H9c2 en una situación lipotóxica, pero estos
efectos no implican a la AMPK pese a los efectos antiinflamatorios de esta
proteína.
Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato
predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y
beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”.
P17-36
Efectos de la ovariectomía y del tratamiento
con 17β-estradiol sobre la función y biogénesis
mitocondrial hepática
Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco
Bauzá-Thorbrügge, Francisco J. García-Palmer, Ana M. Proenza,
Magdalena Gianotti, Isabel Lladó
Grup de Metabolisme Energètic i Nutrició, Dept. Biologia
Fonamental i Ciències de la Salut, IUNICS, Universitat de les
Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación
Biomédica en Red Fisiopatología de la Obesidad y la Nutrición
(CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma
de Mallorca, ES
Estudios previos han puesto de manifiesto la existencia de un dimorfismo
sexual en la función y biogénesis mitocondrial en hígado de rata, apuntando
a las hembras como poseedoras de mitocondrias más diferenciadas y con
mayor capacidad oxidativa.
El objetivo de este estudio fue profundizar en estas diferencias de sexo,
investigando el efecto de la ovariectomía y del reemplazamiento con
17β-estradiol (E2) en la biogénesis y función mitocondrial en el hígado.
Se usaron ratas Wistar hembra de 14 semanas de edad divididas en
tres grupos experimentales: control, ovariectomizadas (OVX) y OVX
suplementadas con E2 (OVX+E2). En el hígado, se determinaron las
actividades citocromo c oxidasa (COX) y citrato sintasa (CS), así como los
niveles de PGC-1α, PGC-1β, TFAM, NRF1, COX I, ATPasa y del ADN
mitocondrial (ADNmt).
En respuesta a la ovariectomía, la expresión de TFAM disminuyó,
acompañado de un descenso del ADNmt y el consecuente empeoramiento
de la capacidad oxidativa, reflejado por una disminución de la actividad CS
y de los niveles de PGC-1β y ATPasa. El tratamiento con E2 revirtió estos
efectos a través de un incremento del ADNmt,de la actividad COX y de los
niveles de PGC-1β, NRF1, COX I y ATPasa.
En conclusión, el tratamiento con E2 revierte los efectos deletéreos que la
ovariectomía provoca en la función y biogénesis mitocondrial en el hígado,
aumentando el ADNmt y la capacidad oxidativa. Estos resultados están
de acuerdo con el dimorfismo sexual previamente descrito en este tejido,
y atribuyen un papel clave en estas diferencias a las hormonas ováricas, y
en concreto al E2.
Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato
predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y
beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”.
Salamanca 2016
P17r-37
Subcellular distribution of α and β adrenergic
receptors in liver of Wistar rats: effects of central
leptin and caloric restriction
Lorena Mazuecos Fernández-Pacheco1, Virginia López
Gómez-Carreño1, Alejandro Fernández Briones1, Carmen Arribas
Mocoroa2, Nilda Gallardo Alpízar1, Antonio Andrés Hueva1
1
Facultad de Ciencias y Tecnologías Químicas, Ciudad Real
(UCLM), Ciudad Real, ES, 2Facultad de Ciencias Ambientales
y Bioquímica (UCLM), Toledo, ES
Leptin convey information to the hypothalamus regarding to long-term
energy stores. The brain delivers signals to peripheral organs, including
the liver, to keep homeostasis. Sympathetic hepatic nerves can modulate
hepatocyte function by direct action of their neurotransmitter noradrenaline
on α- and β- adrenergic receptors. Alterations in the connections between
brain and liver through the adrenergic system could favor the accumulation
of hepatic fat and the development of diseases such as fatty liver.
Focus on anti-steatotic and anti-diabetic functions of leptin, the indirect
effects of this hormone on the hepatic adrenergic receptors were analyzed
in order to reveal the involvement of the autonomic nervous system
in the central leptin regulation of liver function. To carry out this study,
osmotic pumps were implanted, by stereotaxic surgery, in the brain of male
8-month-old Wistar rats fed ad libitum and rats under a moderate caloric
restriction. Animals were infused with leptin (0.2 μg/d) or its vehicle (PBS)
for 7 days. After that, rats were sacrificed, metabolites and hormones
were measured in serum and adrenergic receptor levels were analyzed by
Western blot in total liver extract and sub-cellular fractions, in plasma and
inner membranes.
Our results show that an intra-cerebroventricular leptin administration alters
subcellular distribution of α- and β- adrenergic receptors in the liver. Leptin
tends to increase α1A adrenergic receptors levels in plasma membrane,
which could be associated with increased hepatic glycogenolysis, fatty
acid oxidation and ketogenesis. Furthermore, leptin decreases the presence
of α2A receptor in the plasma membrane, which could improve lipolysis
and attenuate the de novo synthesis of fatty acids. In parallel, central leptin
downregulates the hepatic β-adrenergic receptors (β1 and β2) at the plasma
membrane and increased their levels in the internal membranes, suggesting
that the β-adrenergic receptors play a crucial role in limiting hepatic lipid
accumulation. These findings suggest that brain leptin contribute to the
prevention of hepatic steatosis throughout activation of the neuronal brain–
liver connection.
P17-38
La microbiota intestinal como factor
determinante del dimorfismo sexual de
enfermedades metabólicas
José Antonio Santos Marcos1, Oriol Alberto Rangel Zúñiga1,
Sonia García Carpintero1, Juan Francisco Alcalá Díaz1, J
osé Andrés Morales Martínez1, Manuel Tena Sempere2, Blanca
Landa del Castillo3, José López Miranda1, Francisco Pérez Jiménez1,
Antonio Camargo García1
1
Unidad de Lípidos y Aterosclerosis. IMIBIC/Hospital Universitario
Reina Sofía /Universidad de Córdoba; CIBER Fisiopatología
Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos
III, Córdoba, ES, 2Departamento de Biología Celular, Fisiología
e Inmunología, IMIBIC/Hospital Universitario Reina Sofía /
Universidad de Córdoba; CIBER Fisiopatología Obesidad y
Nutrición (CIBEROBN), Instituto de Salud Carlos III, Córdoba, ES,
3
Instituto de Agricultura Sostenible (IAS), Consejo Superior de
Investigaciones Científicas (CSIC), Córdoba, ES
En los últimos años, el estudio de la microbiota intestinal ha cobrado
especial interés tras descubrirse que la transferencia a modelos animales
Pósters / Posters
carentes de microbiota intestinal, de un sistema bacteriano relativamente
pobre en Bacteroidetes y rico en Firmicutes, conllevaba un aumento de
peso en estos animales. De hecho, actualmente se considera a la microbiota
intestinal como un órgano totalmente integrado en la fisiología del
organismo hospedador que interviene en procesos como la regulación de
la inmunidad innata y adaptativa, la extracción de la energía, y el control
del balance energético. Estudios en modelos animales han identificado una
relación directa entre microbiota intestinal, hormonas sexuales y desarrollo
de enfermedad. Este hecho sugiere que las diferencias en la composición de
la microbiota entre géneros podría estar relacionada con las diferencia que
existe en la incidencia de enfermedades metabólicas y sus comorbilidades,
las cuales son sexualmente dimórficas y en mujeres, aumentan después
de la menopausia. En este estudio comparamos la microbiota intestinal
de mujeres pre- y post-menopáusicas, y de 2 grupos control de hombres
con la misma edad e índice de masa corporal que los grupos de mujeres.
Nuestro estudio mostró que los géneros Prevotella, Ruminococcus y
Oscillospira y las familias Erysipelotrichaceae y Desulfovibrionaceae
son más abundantes en mujeres pre-menopaúsicas que en mujeres
post-menopaúsicas y en ambos grupos de hombres. Nuestros resultados
sugieren que diferencias entre la microbiota intestinal de hombres y
mujeres podría ser un factor determinante en la prevalencia en el desarrollo
de enfermedades metabólicas como diabetes, síndrome metabólico y
enfermedades cardiovasculares.
P17r-39
Metabolic adaptations of neurons and astrocytes
to physiological oxygen levels
Moussa Wardé, Juan P. Bolaños
Institute of Functional Biology and Genomics (IBFG), University
of Salamanca-CSIC, Salamanca, ES
Brain energy metabolism is highly dependent on the oxygen levels. The
vast majority of biochemical studies aimed to understand the metabolic
interactions between astrocytes and neurons, using cultured systems,
have been performed at atmospheric, 21% oxygen concentrations.
However, it has been estimated that neural cells in vivo are exposed to
oxygen concentrations ranging from 1 to 5%, depending on the distance
from the brain vessels. Accordingly, it is unknown whether our current
knowledge on brain cells energy metabolism is the result of an adaptation
to cultured cells to non-physiological high oxygen levels conditions.
To understand this issue, here we have established a long-term primary
culture of cortical neurons and astrocytes grown on 2.0% oxygen levels
using an oxygen-controlled incubator chamber. We found that these cells,
when chronically incubated under a 2.0% oxygen concentration, adapt
their mitochondrial energy metabolism giving rise to significant changes
in glucose metabolism and mitochondrial reactive oxygen species (mROS)
production when compared with cells incubated under atmospheric oxygen
conditions. We believe that these observations may be of critical relevance
when understanding different pathophysiological features of the brain and
the astrocyte-neuronal metabolic interactions.
P17r-40
Impact of mitochondrial ROS on brain cells
energy metabolism in vivo
Carlos Vicente-Gutiérrez, Nicoló Bonora, Juan Pedro Bolaños
Universidad de Salamanca, Salamanca, ES
A substantial body of evidence suggests that mitochondrial reactive
oxygen species (mROS) generation is a hallmark of certain neural diseases.
However, the underlying molecular mechanisms involved are not fully
understood, and whether they play physiological roles is still controversial.
We are interested in studying how mROS orchestrate alternative signaling
pathways dictating specific changes in cell metabolism. To explore this, we
139
Pósters / Posters
have generated an inducible, floxed knock-in mouse model that expresses
a version of catalase fused to a canonical mitochondrial localization signal
(mCat). We believe that the adequate use of mCat mice would allow us
to probe tissue- or cell-specific functions of mROS in vivo. Here, we
present data showing the efficacy of mCat at reducing mROS levels in
a cell-specific manner, and that this is sufficient to induce changes in
astrocyte gene expression as well as their metabolism in vitro. We are
currently characterizing the changes at the protein level with the aim to
elucidate the impact of mROS in different energy metabolism-related
pathways both in astrocytes and in neurons in vivo. We expect to reveal the
functional physiological roles for mROS in the brain and to contribute to
ascertain novel mechanisms whereby astrocytes and neurons cooperate in
the brain energy homeostasis.
P17-41
Modelo de células quiescentes para el estudio
del papel de DCTPP1 en la homeostasis
mitocondrial de nucleótidos
Blanca Martínez Arribas, Antonio E. Vidal, Dolores
González-Pacanowska
Instituto de Parasitología y Biomedicina López Neyra (CSIC),
Granada, ES
En mamíferos, el pool de desoxirribonucleótidos trifosfato (dNTP) se
encuentra físicamente separado en el citosol y la mitocondria. En células en
división, el pool citosólico, producido principalmente por la ribonucleótido
reductasa (RNR), proporciona los precursores necesarios para la síntesis
tanto del DNA nuclear como mitocondrial (mtDNA). A diferencia de lo
que ocurre en células proliferativas, en células post-mitóticas de tejidos
diferenciados no hay replicación del DNA nuclear pero sí existe síntesis y
reparación de DNA mitocondrial por lo que el aporte de nucleótidos debe ser
constante. En estas células, el aporte de precursores mitocondriales proviene
fundamentalmente de la ruta de salvamento mitocondrial ya que la síntesis
de novo y las vías de salvamento citosólico están apagadas. Así, mutaciones
en los genes que intervienen en la ruta de salvamento mitocondrial se han
asociado con diversas patologías humanas denominadas en conjunto como
síndromes de depleción de DNA mitocondrial (MDS) y caracterizadas por la
disminución del número de copias de mtDNA y disfunción mitocondrial en
los tejidos afectados. En este trabajo se describe un modelo de fibroblastos
de pulmón en quiescencia como herramienta para el estudio del papel de
DCTPP1 en la homeostasis de nucleótidos mitocondrial. DCTPP1 es una
NTP pirofosfatasa de la familia de las todo-α encargada de catabolizar el
dCTP produciendo dCMP y pirofosfato. En células en división DCTPP1 se
expresa de forma ubicua en núcleo, citosol y mitocondria, mientras que en
células quiescentes la expresión disminuye pronunciadamente en citosol y
núcleo, y se mantiene constante en mitocondria. Proponemos que DCTPP1
tiene un papel significativo en el mantenimiento de la homeostasis de
nucleótidos pirimidínicos mitocondrial proceso que es esencial para la
correcta replicación del mtDNA.
P17-42
Liver and serum lipidomic analysis of mice
overexpressing carnitine palmitoyltransferase
1AM in liver
Sandra Recalde Luna1, Minéia Weber1, Fina Casas2, Gemma
Fabrias2, Joan Francesc Mir Bonnín1, María Calderón Domínguez3,
Eduviges Bustos1, Núria Casals4, Josefa Badia1, Laura Herrero3,
Dolors Serra3
1
Department of Biochemistry and Physiology, School of Pharmacy,
Institut de Biomedicina de la Universitat de Barcelona (IBUB),
Universitat de Barcelona, Molins de Rei, ES, 2Research Unit on
Bioactive Molecules, Department of Biomedicinal Chemistry,
Institute for Advanced Chemistry of Catalonia (IQAC-CSIC),
140
XXXIX Congreso SEBBM
Barcelona, ES, 3Department of Biochemistry and Physiology, School
of Pharmacy, Institut de Biomedicina de la Universitat de Barcelona
(IBUB), Universitat de Barcelona, Barcelona, ES/ Centro de
Investigación Biomédica en Red de Fisiopatología de la Obesidad
y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid,
ES, 4Centro de Investigación Biomédica en Red de Fisiopatología de
la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos
III, Madrid, ES/ Department of Basic Sciences, Faculty of Medicine
and Health Sciences, Universitat Internacional de Catalunya,
Barcelona, ES
Obesity is strongly associated with several metabolic disturbances,
including insulin resistance and type 2 diabetes. Excessive lipid storage
in several tissues leads to dysregulation of intracellular lipid metabolism.
In liver, abnormally large accumulations of triacylglycerides produce
hepatic steatosis and insulin resistance. Previous studies from our
group have shown that an enhanced fatty acid oxidation (FAO) by
overexpressing a permanent activated CPT1A isoform (CPT1AM) in
liver of high-fat diet (HFD) mice reduces hepatic steatosis, blood glucose
and insulin levels. In an attempt to identify lipid species that could
be involved in the improvement of HFD-treated mice we performed
a lipidomic analysis in liver and serum. Our results show that liver of
HFD CPT1AM-expressing mice have a decrease in the major part of
triacylglycerol (TAG) and diacylglycerol (DAG) species compared to
HFD control mice, which agrees with the reduced steatosis observed
in the HFD CPT1AM-treated mice. However, this massive TAGs and
DAGs decrease is not observed in serum, only C54:5, C54:4, and C54:3
TAGs were reduced in CPT1AM-treated mice respect to HFD control
mice. Among the phosphatidylcholines (PC)s, PC 36:2 and PC 38:3
in liver and PC 36:3 and PC 38:3 in serumwere reduced compared to
normal chow diet mice. Lysophosphatidylcholine (LPC)s 16:0 and 18:0
were also reducedin liver but no changes were observed in serum of HFD
CPT1AM-expressing mice. Finally, the analysis of sphingolipids showed
a significant reduction of liver ceramides 16:0, 18:0, 20:0, 22:0 and 24:2
and dihidrocerimide 22:0. However, ceramide 18:1 was the only one
reduced in the serum. All these results show that the enhancement of FAO
in liver of HFD mice reduces mainly neutral lipids such as TAGs and
DAGs in liver. In contrast, few lipid species showed changes in serum.
These serum metabolites may serve as valuable “biomarker candidates”
for the steatosis reversion and insulin resistance improvement in future
studies.
P18. Señalización celular /
Cellular signalling
P18r-1
Gαq interactions with PB1 domain-containing
proteins open new avenues for Gq-coupled GPCR
signaling
Sofía Cabezudo Violero, Guzmán Sánchez Fernández, Federico
Mayor Menéndez, Catalina Ribas Núñez
Centro de Biología Molecular Severo Ochoa CBMSO, Universidad
Autónoma de Madrid, Madrid, ES
Heterotrimeric G proteins play an essential role in the initiation of G
protein-coupled receptor (GPCR) signaling through specific interactions
with a variety of cellular effectors. The Gαq/11 family canonically
activates PLCβ. However, additional effectors may underlie other Gαq
functions. We have reported that GPCR activation promotes a direct
interaction between Gαq and protein kinase C  (PKC) through a novel
acidic region in Gαq, different from the classical effector-binding region,
Salamanca 2016
and the PB1 domain of PKC. This Gαq/PKCinteraction leads to the
stimulation of the ERK5 pathway and to the induction of apoptotic
cell death independently of PLCβ. G protein-coupled receptor kinase 2
(GRK2), a known Gαq negative modulator, abrogates ERK5 stimulation
by disrupting the complex.
Interestingly, we have uncovered that Gαq interacts with another PB1
domain-containing protein, the key p62/SQSTM1 signaling hub, and the
implication of this complex in the modulation of autophagy. We find that
Gαq and p62 associate in cells and are able to directly interact. Furthermore,
their interaction increases upon Gαq activation by GPCR or in the presence
of constitutively active Gαq mutants, and is disrupted by GRK2, consistent
with p62 being a functional Gαq effector. Gαq/p62 association involves the
PB1 domain of p62 and the acidic region of Gαq described to be essential
for PKCbinding, although PKCis not required for the formation of the
complex. On the other hand, we uncover that Gαq controls autophagy by a
mechanism dependent on the modulation of the mTORC1 pathway under
different nutrient stress conditions. Given the key reported role of p62 in
these processes, we postulate a central role of this novel Gαq/p62 pathway
in the control of autophagy.
Overall, our studies indicate that PKCand p62 act as functional effectors
of Gαq through the engagement of a PB1-type I-like binding region in this
subunit. Our data also point out a relevant role for Gαq/11 as a link between
nutrient-sensing GPCRs and autophagy modulation. Finally, we suggest
that the interaction of Gαq with additional PB1 domain-containing proteins
may participate in the control of cellular functions by Gq-coupled GPCR.
P18r-2
Role of C3G in hepatocarcinoma tumor growth
and progression. Regulation of stemness capacity
Celia Sequera1, Sara Manzano1, María Arechederra1, Cristina
Baquero1, Carmen Guerrero2, Almudena Porras1
1
Departamento de Bioquímica y Biología Molecular II, Facultad de
Farmacia, UCM, IdISSC, Madrid, ES, 2Centro de Investigación del
Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES
C3G is a guanine nucleotide exchange factor for Rap1 and R-Ras. It is
essential for embryonic development and it regulates several cellular
functions such as cytoskeletal remodeling, differentiation and cell death.
However, its role in cancer is controversial acting either as a tumor
promoter or suppressor. Using different hepatocarcinoma (HCC) cell
models with different degrees of epithelial phenotypes (Hep3B and HLE),
our group found that C3G knock-down increased invasion and migration
through induction of an epithelial-mesenchymal transition (EMT)
process. Accordingly, C3G knock-down cells expressed higher levels of
mesenchymal markers (Vimentin, N-Cadherin) and EMT transcription
factors (Snail, Zeb1). All these changes induced by C3G silencing were
similar to those elicited by TGF-β, a well known EMT inducer. Based on
this pro-migratory effect of C3G knock-down, we have evaluated the role of
C3G in tumorigenesis in HCC cells. Anchorage-dependent assays showed
reduced number of foci and greater dispersion of cells within a focus in
C3G knock-down HCC cells, as compared to parental cells. This correlates
with the low adhesion of C3G silenced Hep3B cells. In contrast, anchorage
independent assays in soft agar revealed an increase in the number and
size of foci in C3G-silenced Hep3B cells, as compared to parental cells.
However, each individual focus was composed of few and scattered cells.
To further understand the function of C3G in tumor growth, we studied its
role on cell survival and found that C3G knock-down decreased cell death.
Moreover, our preliminary studies on the role of C3G on stemness capacity
of HCC cells showed that C3G knock-down favors the induction of a stem
cell like phenotype in Hep3B cells. Thus, C3G silenced cells form more
spheres and increase the expression of some stemness markers. All these
data support a role for C3G as an inhibitor of migration and invasion, while
its function in tumor growth remains unclear. We are currently evaluating
in vivo the role of C3G in HCC tumor growth.
Pósters / Posters
P18r-3
Concomitant deletion of H-Ras and N-Ras leads
to pulmonary immaturity, respiratory failure
and neonatal death in mice
Rocío Fuentes Mateos1, David Jimeno2, Carmela Gómez2,
Eugenio Santos de Dios2, Alberto Fernández Medarde2
1
Laboratorio 1, Centro de Investigación del Cáncer. Campus Miguel
de Unamuno, Fundación de Investigación del Cáncer Universidad
de Salamanca, Salamanca, ES, 2Laboratorio 1, Centro de
Investigación del Cáncer. Campus Miguel de Unamuno, Instituto
de Biología Molecular y Celular del Cáncer. Universidad de
Salamanca/CSIC, Salamanca, ES
The canonical members of the Ras gene family (H-ras, N-ras, and K-ras)
encode four protein isoforms (H-Ras, N-Ras, K-Ras4A and 4B) regulating
cell proliferation, differentiation or death/survival. Whereas genomic
disruption of K-ras4B causes embryonic lethality, H-ras, N-ras and
K-ras4A knockout mice are viable. H-ras and N-ras double KO (DKO)
mice are also viable but show increased perinatal mortality suggesting
critical physiological roles of those proteins around birth.
Our characterization of DKO (Hras-/-;Nras-/-) mice uncovered a significant
mortality rate due to respiratory failure during the first two postnatal days.
Although DKO mice develop normal lung branching, they show delayed
pulmonary maturation, a defect affecting both bronchiolar (Ciliated and
Clara Cells) and alveolar lineages (type I and type II Pneumocytes), plus
a reduced alveolar space and thicker septa. Clara Cells showed abnormal
morphology and lack of proteoglycans production, whereas Ciliated Cells
showed incorrect cilia structure. Additionally, these cells were not evenly
juxtaposed in the bronchiolar space but formed a continuous layer in which
markers for both cell types merged. Alveolar cells showed also abnormal
phenotypes. Whereas type II Pneumocytes were wrongly localized inside a
cell mass instead of surrounding the alveolar spaces, the type I cells did not
exhibit typical flat shape, pointing to a defect in differentiation. The delay
in lung maturation was further supported by the observation of increased
number of precursors cells of alveolar lineage (more RCA/SftpC+ cells) as
well as by higher proliferation rates.
Our data uncover important, specific functional roles of H-Ras and N-Ras
for lung maturation and neonatal survival that cannot be substituted by
K-Ras action.
P18-4
The Rac-GAP β2-chimaerin has a dual function
in breast carcinogenesis
Victoria Casado-Medrano, María José Caloca Roldán
CSIC/IBGM, Valladolid, ES
β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor
suppressor in breast cancer. Despite the relevance of this function, the
precise contribution of β2-chimaerin to this pathology has been largely
unknown. We show evidence of a differential role of β2-chimaerin in
the migration and invasion of breast cancer cells depending on whether
they are epithelial or mesenchymal. Expression of β2-chimaerin in
LM2 cells, a mesenchymal-like breast cancer cell line, significantly
reduced cell migration (analyzed by wound healing assays) and invasion
(analyzed by trasnswell assays). However, expression of β2-chimaerin in
the epithelial breast cancer cell line MCF7 had no effect in the migratory
and invasive properties of these cells. This cell context dependency of the
effects of β2-chimaerin raised an important question about the exact role
of β2-chimaerin in breast cancer, a pathology that arises from epithelial
cells that switch to a mesenchymal phenotype during cancer progression.
To answer this question we analyzed the effect of the genetic ablation of
β2-chimaerin in the MMTV-Neu/ErbB2 mouse model of breast cancer.
We demonstrate that loss of β2-chimaerin accelerates tumor onset and,
importantly, delays tumor progression, most likely by delaying the
141
Pósters / Posters
epithelial to mesenchymal transition of breast cancer cells. Finally,
we show evidence that our findings can be extrapolated to the human
pathology, since analysis of breast cancer databases shows that patients
with low β2-chimaerin expression have reduced relapse free survival but
develop metastasis at similar times.
Financial support: JCYL (BIO/VA34/15).
P18-5
Mitochondrial dynamics: blasting off
to pluripotency
Javier Prieto1, Marian León1, Xavier Ponsoda1, Carlos López1,
José Manuel Torres Ibáñez2
1
UVEG, Burjassot, ES, 2Universitat de Valencia, Meliana, ES
During the process of reprogramming to induced-Pluripotent Stem (iPS)
cells, somatic cells switch from oxidative to glycolytic metabolism, a
transition associated with profound mitochondrial reorganisation. Neither
the importance of mitochondrial remodelling for cell reprogramming nor
the molecular mechanisms controlling this process are well understood.
Here, we show that an early wave of mitochondrial fragmentation occurs
upon expression of reprogramming factors. Reprogramming-induced
mitochondrial fission is not associated with mitophagy but rather with an
increase in organelle biogenesis. The pro-fission factor Drp1 is phosphorylated
early in reprogramming, and its knockdown impairs both mitochondrial
fragmentation and generation of iPS cell colonies. Drp1 phosphorylation
depends on Erk activation in early reprogramming, which occurs, at least
in part, due to down-regulation of the MAP kinase phosphatase Dusp6.
Taken together, our data indicate that mitochondrial fission controlled by an
Erk-Drp1 axis constitutes an early and necessary step in the reprogramming
process to pluripotency. The role of the reprogramming factors in triggering
mitochondrial reorganisation and function will be discussed.
P18r-6
C3G promotes angiogenesis through
the regulation of the differential release
of pro-/anti-angiogenic factors from
thrombin-activated platelets
Víctor Martín-Granado1, Sara Ortiz-Rivero1, Sara Gutiérrez-Herrero1,
Celia Sequera2, José Ramón González-Porras3, Francisco
Martín-Herrero4, Almudena Porras2, Carmen Guerrero1
1
Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL,
Salamanca, ES, 2Departamento de Bioquímica y Biología Molecular
II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES, 3Departamento
de Hematología, IBSAL, Hospital Universitario de Salamanca,
Salamanca, ES, 4Departamento de Cardiología, IBSAL, Hospital
Universitario de Salamanca, Salamanca, ES
C3G is a guanine nucleotide exchange factor (GEF) of several proteins
within the Ras superfamily, including Rap1a/b and R-Ras. Rap1 is known
to be involved in critical aspects of platelet function, including aggregation,
adhesion and spreading, through the activation of the platelet integrin
αIIbβ3. Using transgenic mouse models expressing C3G or C3G∆Cat (a
mutant lacking the catalytic domain) in megakaryocytes and platelets, our
group has identified C3G as a mediator of Rap1 actions leading to platelet
activation and aggregation. Furthermore, C3G promotes α-granule release,
evidenced by the increase in P-selectin exposure on the platelet surface,
following its activation. In addition, preliminary immunofluorescence
analysis suggests that C3G modulates the release of VEGF and endostatin
from thrombin-activated platelets.
The goal of the present study was to further characterize the underlying
mechanisms that regulate the function of C3G in the differential release of
pro- and anti-angiogenic factors upon platelet activation, and to evaluate
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XXXIX Congreso SEBBM
the angiogenic potential of the platelet releasate. We have found that VEGF
release was up-regulated in thrombin-activated mouse platelets from both,
C3G and C3GΔCat, transgenic mice. Additionally, thrombospondin release
was also up-regulated in the latter. The releasate of TgC3G platelets had an
overall pro-angiogenic effect as evidenced in a capillary-tube formation
assay with HUVECs and in heterotopic tumor mouse models using B16
and 3LL cells. On the other hand, platelet spreading analysis revealed an
up-regulation of this function in both transgenic mouse platelets upon
thrombin activation, with TgC3G platelets showing the highest extension
area. A preliminary co-IP analysis revealed an interaction between C3G
and VAMP-7 upon activation, which could explain this result.
Taken together, our data indicate the co-existence of RapGEF-dependent
and independent mechanism through which C3G is regulating platelet
secretion, and suggest an overall pro-angiogenic effect of platelet C3G.
P18r-7
C3G: a new player in the control of invasiveness,
stemness and tumorigenesis in glioblastoma
Sara Manzano1, Celia Sequera1, María Arechederra1, Cristina
Baquero1, Neibla Priego1, Carmen Guerrero2, Almudena Porras1
1
Departamento de Bioquímica y Biología Molecular II, Facultad
de Farmacia, UCM, IdISSC, Madrid, ES, 2Centro de Investigación
del Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES
C3G is a guanine-nucleotide exchange factor (GEF) for some Ras family
members, although it can act through GEF independent mechanisms. It
is essential for embryonic development due to its role in adhesion and
regulates different cellular functions. The role of C3G in human cancer is
controversial, acting as either a tumour suppressor or mediator. Although
the function of C3G in migration depends on the context, C3G was shown
to inhibit migration in various cell types. We also found that C3G acts as
a migratory and invasive repressor in MEFs and HCT116 cells through
down-regulation of p38α MAPK activity.
Based on this, we have studied the role played by C3G in human
glioblastoma through gene silencing, using the U87 cell line as an in vitro
model. We found that C3G knock-down enhances invasion and reduces
cell adhesion. In fact, upon C3G silencing, U87 cells re-organize actin
cytoskeleton, leading to neurite-like extensions. These functional and
morphological changes correlate with the expression of transcription
factors and proteins involved in the epithelial-mesenchymal transition
(EMT). Moreover, C3G knock-down induces anchorage-dependent and
-independent growth of U87 cells, suggesting that C3G could inhibit
tumour growth. C3G may also play a role in the control of stemness and the
acquisition of a glioblastoma initiating cell-like phenotype, as its silencing
favours spheres formation. C3G silencing also induces changes in different
signalling pathways, such as p38 MAPKs, ERKs, STAT3 or c-Met, which
might contribute to regulate the observed functional changes.
These data indicate that C3G is a new player in the regulation of
glioblastoma progression, as it would inhibit tumour growth and invasion.
Therefore, its down-regulation might promote tumour growth and the
generation of metastasis through a mechanism involving an EMT process
and stemness induction. However, more studies are required to fully
characterize the function of C3G in glioblastoma, as well as to determine
the molecular basis of this regulation and its relevance in clinic.
P18-8
PPAR-α-FGF21 signaling is induced by a
combined docosahexaenoic acid and thyroid
hormone protocol
Luis Videla Cabrera, Romina Vargas Villagrán, Virginia Fernández
Arancibia
Universidad de Chile, Facultad de Medicina, ICBM, Programa de
Farmacología, Santiago, CL
Salamanca 2016
Combined omega-3 and thyroid hormone (T3) treatment prevents
ischemia-reperfusion liver injury. Considering that energy requirements
of this protection may entail peroxisome-proliferator activated
receptor-α (PPAR-α)-fibroblast growth factor 21 (FGF21) signaling, a
combined protocol of docosahexaenoic acid (DHA) (300 mg/kg for 3
days) plus T3 (0.05 mg/kg, single dose) was administered to fed rats.
Enhanced liver DHA content and serum T3 levels were paralleled
by increased PPAR-α mRNA and its DNA binding. Higher PPAR-α
target genes mRNA expression (carnitine-palmitoyl transferase 1α,
acyl-CoA oxidase, and 3-hydroxyl-3-methylglutaryl-CoA synthase
2) were achieved, effects that were mimicked by 0.1 mgT3/kg or the
PPAR-α agonist WY-14632. As increased mRNA expression of retinoic
X receptor-α (RXR-α), accompanied by augmented liver mRNA and
protein FGF21 levels and those of serum FGF21, were also achieved,
it is concluded that PPAR-α-FGF21 induction by DHA combined with
T3 may involve ligand activation of PPAR-α by DHA and enhanced
expression of PPAR-α by T3, with consequent upregulation of the
FGF21 that is controlled by PPAR-α.
Supported by FONDECYT 1150104.
P18r-9
Función de PKCδ en el tráfico polarizado
de cuerpos multivesiculares y secreción de
exosomas por los linfocitos T en la sinapsis
inmune
Gonzalo Herranz Gómez, David Fernández Moreno, Sergio Dávila
Martínez, Laura Márquez Expósito, Álvaro Merchán Martín, Raúl de
Martín Esteban, Jesús Gómez Alonso, Ioana Bianca Stancu, Teresa
Fernández Caballero, Mario Quintanilla Álvarez, Víctor Calvo López,
Manuel Izquierdo Pastor
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM,
Madrid, ES
La formación de la sinapsis inmune (SI) es crucial para garantizar
una respuesta inmune eficaz. La SI entre una célula presentadora de
antígeno y un linfocito T, induce en este último el tráfico de los cuerpos
multivesiculares (MVB), que se fusionan a la membrana plasmática en
la SI. Tras la fusión, las vesículas intraluminales contenidas en los MVB
se liberan al exterior celular en forma de exosomas. Los exosomas son
vesículas de doble membrana lipídica que intervienen en la comunicación
intercelular, ya que contienen diversas moléculas bioactivas. En
linfocitos T, los exosomas contienen proteínas proapoptóticas que
median la citoxicidad de linfocitos T citotóxicos y la muerte celular
inducida por reactivación (AICD) de linfocitos T. Por tanto, dilucidar
los mecanismos que controlan su secreción permitiría modificar la
función y la homeostasis en el sistema inmune. Se ha documentado que
el tráfico polarizado de MVB hacia la SI se controla de forma directa por
reorganización del citoesqueleto de actina, e indirectamente por el enzima
diacilglicerol quinasa α (DGKα) y diacilglicerol (DAG), su sustrato.
Sin embargo, las bases moleculares de este fenómeno están sin aclarar.
Hemos observado que la proteína quinasa C delta (PKCδ), un isotipo
de PKC activado por DAG, se acumula cerca de los MVB. Cuando se
interfiere la expresión de PKCδ en linfocitos T disminuye la eficiencia de
la polarización de los MVB hacia la SI, y también se reduce la secreción
de exosomas y la AICD. La reorganización del citoesqueleto de actina
cortical que ocurre en la SI está afectada en los clones interferidos en
PKCδ, tanto espacial como temporalmente. Esto sugiere que PKCδ y
su control del citoesqueleto de actina median el efecto de DGKα en el
tráfico secretor polarizado del linfocito T.
Pósters / Posters
P18-10
Papel de la AMPK (proteína quinasa activada por
AMP) como promotor tumoral en cáncer de colon
Ana Chocarro-Calvo1, José Manuel García-Martínez1, María
Gutiérrez-Salmerón1, Antonio de la Vieja2, Custodia García-Jiménez1
1
Universidad Rey Juan Carlos, Alcorcón, ES, 2Instituto de Salud
Carlos III, Majadahonda, ES
Estudios epidemiológicos asocian una mayor incidencia de cáncer de
colon con la diabetes. La AMPK permite a la célula adaptarse al estrés
metabólico y es una diana molecular importante en diabetes. El papel de
AMPK en la progresión tumoral y su potencial como diana terapéutica es
controvertido ya que puede actuar como un supresor tumoral condicional o
como un oncogen contextual.
Objetivo: Caracterización del papel de la AMPK durante la adaptación al
estrés metabólico impuesto por la captura facilitada de glucosa en cáncer
de colon.
Metodología: Usamos células de colon tumorales (HCT 116) y sanas
(CCD-18Co) en normo o hiper-glucemia. Los tratamientos incluyen
inhibidores/activadores y siRNA específicos de AMPK. Las alteraciones
en las proteínas se miden por western Blot e inmunoprecipitaciones.
Resultados: La hiperglucemia aumenta la actividad de AMPK en células
tumorales de colon induciendo su fosforilación en la T172 e inhibiéndola
en la S485. La activación de AMPK correlaciona con la fosforilación de
p300 y su activación y con autofagia aumentada, pero no con la inhibición
de la vía de mTORC1. La inhibición o depleción de AMPK bloquean estos
efectos pero la Metformina no los reproduce.
Conclusiones: La fosforilación de AMPK en la S485 puede determinar su
papel como oncogén o supresor tumoral en células de colon. Los resultados
sugieren un nuevo mecanismo de adaptación de algunas células tumorales
al estrés metabólico.
P18-11
Analysis of the function of Sp6 and Sp8
transcription factors in the limb ectoderm
Rocío Pérez Gómez1, Marc Fernández-Guerrero1, Sandra
Zunzunegui2, Juan F. López-Giménez1, Marian Ros1
1
Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC),
Consejo Superior de Investigaciones Científicas (CSIC), Santander,
ES, 2Instituto de Biomedicina y Biotecnología de Cantabria
(IBBTEC), Universidad de Cantabria (UC), Santander, ES
We have recently identified the crucial role played by Sp6 and Sp8, two
transcription factors members of the Sp family, during limb development.
In their absence or substantial reduction no limbs form and their progressive
dose reduction associates a spectrum of limb malformations ranking from
syndactyly through Split-hand/split-foot malformation, to olygodactyly.
When digits form, they exhibit bidorsal tips. We have postulated that Sp6
and Sp8 are necessary, downstream of Wnt/B-catenin, to activate Fgf8
and downstream of Bmp signaling, possible cooperating with Smads, to
activate En1, the marker of the ventral ectoderm.
Currently we are exploring these suspected protein-protein interactions
by co-immunoprecipitation (CoIP) and by bimolecular fluorescence
complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged
with Myc or FLAG epitopes to their N-terminal end and with YFP full
length or YFP moieties to their C-terminal end. We show by CoIP and
BiFC that Sp8 homodimerizes. To determine the region involved in Sp8
interaction, we have generated different truncated versions of Sp8. Our
results show that Sp8 homodimerizes at its C-terminal domain. Our study
underscores the relevance of Sp6/8 in mediating B-catenin and Bmp
signaling pathways in the limb ectoderm.
143
Pósters / Posters
P18-12
El avance del ciclo celular en G1/S está regulado
por la estabilidad de PLK1 vía CDK1/βTrCP
María Galindo-Moreno1, Servando Giráldez1, M. Cristina
Limón-Mortés1, Joaquín Herrero-Ruiz1, Mar Mora-Santos1, Carmen
Sáez2, Miguel Á. Japón2, María Tortolero1, Francisco Romero1
1
Departamento de Microbiología, Facultad de Biología, Universidad
de Sevilla, Sevilla, ES, 2Instituto de Biomedicina de Sevilla (IBIS)
y Departamento de Anatomía Patológica del Hospital Universitario
Virgen del Rocío de Sevilla, Sevilla, ES
Polo-like kinase 1 (PLK1) es una quinasa de serina/treonina implicada en
diversas etapas del ciclo celular, entre las que destacan la entrada y salida de
mitosis y la citocinesis. Además, tiene un papel esencial en la regulación de
la replicación y, junto con ciclina A/CDK, fosforila a la subunidad CDH1
del APC/C, induciendo su ubicuitilación y degradación por el proteasoma.
Esto permite la acumulación de los sustratos correspondientes y el avance
del ciclo celular.
En muestras de tumores de pacientes de muy diversos orígenes se han
detectado niveles elevados de PLK1, comparados con el tejido normal,
lo que ha llevado a proponer a PLK1 como un marcador pronóstico
de los cánceres humanos. De hecho, se usa como diana de fármacos
anticancerígenos y ya se han descrito una serie de inhibidores que están
siendo probados en ensayos clínicos. Todos estos datos nos han llevado
a estudiar la regulación de la estabilidad de esta quinasa y a profundizar
en su papel en el ciclo celular. En resultados previos de nuestro grupo
determinamos que la proteína PLK1 nuclear es ubicuitilada por SCFFBXW7
y degradada por el proteasoma en respuesta a los daños en el ADN para
impedir la formación de los complejos de pre-replicación y evitar la
proliferación celular. En el presente trabajo mostramos que la proteína
PLK1 citoplasmática es ubicuitilada y degradada por SCFβTrCP/proteasoma
cuando no está correctamente plegada, poniéndose esto de manifiesto al
tratar las células con geldanamicina, un inhibidor de la chaperona HSP90.
Además, identificamos a CDK1 como la principal quinasa implicada en
este proceso. Finalmente demostramos que la inhibición de HSP90 detiene
la progresión del ciclo en la transición G1/S a través de la degradación de
PLK1 que, a su vez, evita la destrucción de CDH1. En estas condiciones,
los sustratos del APC/CCDH1 no se acumulan y el ciclo se detiene. Las
consecuencias de estos resultados serán discutidas.
María Galindo-Moreno y Servando Giráldez han contribuido igualmente
a este trabajo.
P18r-13
Characterization of VRK1 and Atxn1 interaction
and its functional implications
Elena Martín Doncel, Lara Cantarero, Pedro A. Lazo
Experimental Therapeutics and Translational Oncology Program,
Instituto de Biología Molecular y Celular del Cáncer, Consejo
Superior de Investigaciones Científicas (CSIC), Universidad de
Salamanca; Instituto de Investigación Biomédica de Salamanca
(IBSAL), Hospital Universitario de Salamanca, Salamanca, ES
Atxn1 is the protein involved in the inherited neurodegenerative disease
SCA1, an autosomal dominant disorder caused by the expansion of a
triplet CAG, encoding a polyglutamine tract (polyQ) which confers toxic
properties to the protein. Nevertheless, polyQ is necessary but not sufficient
to cause pathology. Indeed, is also important the nuclear localization
signal, the AXH domain and a serine 776 residue. VRK1 is a Ser-Thr
kinase associated with multiple processes, including the regulation of cell
cycle progression, chromatin remodeling or Cajal Bodies dynamics. Also
VRK1 is mutated in neurodegenerative diseases. Previously we identified
Atxn1 like a potential target for VRK1, through the use of linear peptide
chips. Our aim is to characterize the interaction VRK1-Atxn1 and its
144
XXXIX Congreso SEBBM
potential functional implication. Through immunoprecipitation and nuclei
isolation assays, we detected an interaction between both proteins. Besides,
by HPLC fractionation is proved that Atxn1 and VRK1 elute in the same
high molecular size fractions. By immunofluorescence, we did not detect
any colocalization, which may indicate a transient interaction between
both proteins. In a kinase assay, we observed distinct phosphorylation
levels between Atxn1 with a physiological polyQ and the protein with an
expanded polyQ. Only wild-type Atxn1 can be phosphorylated by VRK1.
Atxn1 is degraded via ubiquitin proteasome system. Here, we showed
that different mutants of Atxn1 have a similar protein accumulation in the
nuclear inclusions when proteasome is inhibited with MG-132. Yet, we
have evidence that VRK1 downregulation enhances the degradation of
Atxn1. We believe other proteins, like VCP, which colocalized with Atxn1
nuclear inclusions with expanded polyQ, might be involved in this process.
P18-14
Chromatin dynamics in response to DNA Damage
Ignacio Campillo-Marcos1, Marcella Salzano1,
Raúl García-González2, Pedro A. Lazo1
1
Experimental Therapeutics and Translational Oncology Program,
Instituto de Biología Molecular y Celular del Cáncer, Consejo
Superior de Investigaciones Científicas (CSIC), Universidad de
Salamanca; Instituto de Investigación Biomédica de Salamanca
(IBSAL), Hospital Universitario de Salamanca, Salamanca, ES,
2
Experimental Therapeutics and Translational Oncology Program,
Instituto de Biología Molecular y Celular del Cáncer, Consejo
Superior de Investigaciones Científicas (CSIC), Universidad de
Salamanca, Salamanca, ES
Introduction: Genome integrity is continuously challenged by exogenous
and endogenous agents causing DNA damage, both in euchromatin and
heterochromatin. The repair of these lesions involves changes in the histone
organization, such as recruitment and/or incorporation of histone variants
or covalent modifications of histones, in order to promote the formation
of open, relaxed chromatin structures. Recently, a growing number of
chromatin components, remodelers and modifications has been identificated
as key players in DNA repair, emphasizing the complex role of chromatin
in this process. On the other hand, defects in DNA repair pathways enable
cancer cells to survive DNA damage. For this reason, the combination of
DNA-damaging agents with specific inhibitors of these pathways could be
used in cancer treatment. Currently, specific inhibitors against chromatin
remodelers have been developed, but it is still unclear how they act in tumor
cells. Our aim is to detemine the effect of histone acetylation/deacetylation
and methylation/demethylation inhibition on DNA repair foci.
Results and conclusion: We use pharmacological inhibitors of HATs
(C646 and MG149); HDACs (Entinostat and SAHA); KMTs (Chaetocin);
and KDMs (JMJD2 inhibitor). Some of them are being employed in
preclinical regulatory studies. In this analysis, we can observe that closed
chromatin induced by HATs inhibitors seems to affect the formation of
γH2AX and 53BP1 foci, and the dynamics of foci formation corresponds
with the increase of fluorescence level of H4 acetylation after inducing
DNA damage. Based on these results, we conclude that chromatin
relaxation is an essential early step in DNA repair, which can be blocked
by specific inhibitors against HATs, sensibilizing cells to treatments.
P18r-15
Functional interaction between Met and TGF-β
pathways during EMT in mouse liver progenitor
cells
Laura Almalé1, María García-Álvaro1, Adoración Martínez-Palacián1,
Nerea Lazcanoiturburu1, Annalisa Addante1, César Roncero1,
Margarita Fernández1, Eduardo Rial2, Isabel Fabregat3, Blanca
Herrera1, Aránzazu Sánchez1
Salamanca 2016
1
Dep. Bioquímica y Biología Molecular II, Facultad de Farmacia,
Universidad Complutense de Madrid; Instituto de Investigación
Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid,
ES, 2Departamento de Medicina Molecular y Celular, Centro de
Investigaciones Biológicas, CSIC, Madrid, ES, 3Laboratori d´Oncologia
Molecular, Universitat de Barcelona, Institut d´Investigació
Biomèdica de Bellvitge, L´Hospitalet de Llobregat, ES
Adult progenitor liver cells (oval cells, OC) constitute a bipotential cell
population that contribute to liver regeneration in chronic liver disease
(CLD). However, recent evidence supports a pro-fibrogenic role of OC in
the context of the CLD. They are also prone to malignant transformation
having been proposed as the cell of origin of a hepatocellular carcinoma
subgroup. To better understand the mechanisms behind OC fate decisions,
we have analyzed the effect of TGF-β-driven EMT (Epithelial-Mesenchymal
Transition) in OC function and the potential interaction between TGF-β and
HGF/Met signalling pathways in this context. For that, we have used an
in vitro model of OC lines harbouring an active (Metflx/flx cells) or inactive
(Met-/-cells) Met tyrosine kinase. After a chronic TGF-β treatment, we
obtained cells with a stable mesenchymal phenotype (TβT-OC, TGF-β-treated
oval cells). Interestingly, TGF-β-induced EMT did not result in up-regulation
of cancer stem cell markers (Epcam, CD44 and CD133) or in acquisition
of anchorage-independent growth capacity. Nevertheless, TβT-OC show
profound changes in their proliferative and invasive properties and acquire
resistance to apoptosis. Importantly, Met signaling seems to be necessary for
OC expansion after EMT, since Met-/- cells suffer an irreversible senescence
process associated with the induction of cell cycle inhibitors. We are now
exploring whether the effect of the exacerbated oxidative stress detected in
TβT-Met-/- cells could be the key mechanism for senescence induction. On the
other hand, TGF-β-OC no longer respond to HGF in terms of mitogenic or
pro-invasive activities and an altered signaling response to HGF is observed
post-EMT. These changes could be partly explained by amplification of the
HGF autocrine loop detected in OC. In conclusion, our results evidence
profound changes in OC phenotypic and functional properties after EMT
and an interesting crosstalk between HGF and TGF-β pathways during
and post-EMT induction. The molecular mechanisms involved and the
consequences for oval cell fate are under study.
P18-16
MCI inhibition and acute O2 sensing by
peripheral chemoreceptors: Effect of regeneration
of electron acceptors
Ignacio Arias Mayenco, Hortensia Torres Torrelo, Patricia González
Rodríguez, Lin Gao, Patricia Ortega Sáenz, José López-Barneo
López Barneo
Instituto de Biomedicina de Sevilla, Sevilla, ES
The carotid body contains chemoreceptor O2-sensitive glomus cells,
which in response to hypoxia release transmitters to elicit compensatory
cardiorespiratory reflexes. Understanding the mechanisms underlying the
detection of acute changes in O2 tension is of broad biomedical interest.We
have recently reported the involvement of mitochondrial complex I (MCI)
in acute O2 sensing (Fernández-Agüera et al., Cell Metab. 2015). Knockout
mice lacking Ndufs2 (a MCI subunit required for ubiquinone reduction)
in catecholaminergic cells (TH-NDUFS2) failed to hyperventilate
under hypoxic conditions. Hypoxia-induced increases in NADH and
reactive oxygen species (ROS), inhibition of K+ channels and increase
in cytosolic Ca2+ were also selectively abolished in glomus cells from
these mice. In Ndufs2-deficient glomus cells NADH was accumulated,
which could inhibit Kreb’s cycle dehydrogenase activity. Therefore,
to determine whether these phenotypes result from a developmental
generalized metabolic disarrangement or they are a direct consequence of
MCI dysfunction, we have generated the ESR-NDUFS2 mice, in which
the Ndufs2 gene was ablated during adulthood. A time-dependent loss
of hypoxic ventilatory response with a parallel decrease in MCI activity
Pósters / Posters
were observed in ESR-NDUFS2 mice. The effects of hypoxia on glomus
cell parameters (NADH, ROS, and cytosolic Ca2+) were also abolished.
Addition of electron acceptors (alpha ketobutyrate or pyruvate), to
regenerate NAD+ and activate Kreb’s cycle, in combination with succinate
treatment, to increase mitochondrial complex II activity, failed to recover
responsiveness to hypoxia. These results further demonstrate the essential
role of MCI in acute O2 sensing and the involvement of NADH and ROS
in the signalling pathway leading to K+ channel inhibition, glomus cell
depolarization and the trigger of cardiorespiratory reflexes.
P18-17
Hypoxia reduces mitochondrial complex IV
activity via post-transcriptional control
of NDUFA4 by IRP1
Eduardo Balsa1, Bárbara Acosta-Iborra1, Pablo Hernansanz-Agustín2,
Daniel Tello1, Adela Guarás3, Nora Cascante-Estepa1, Ángel
Ordóñez-Navadijo1, Elia López-Bernardo4, Susana Cadenas4, José
Antonio Enríquez3, Manuel O. Landázuri5, Antonio Martínez-Ruiz6
1
Unidad de Investigación, Hospital Universitario Santa Cristina,
Universidad Autónoma de Madrid, Instituto de Investigación
Sanitaria Princesa (IIS-IP), Madrid, ES, 2Servicio de Inmunología,
Hospital Universitario de La Princesa, Instituto de Investigación
Sanitaria Princesa (IIS-IP). Departamento de Bioquímica,
Universidad Autónoma de Madrid, Madrid, ES, 3Centro Nacional
de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, ES,
4
Servicio de Inmunología, Hospital Universitario de La Princesa,
Instituto de Investigación Sanitaria Princesa (IIS-IP). Departamento
de Biología Molecular, Centro de Biología Molecular Severo Ochoa
(CSIC-UAM), Universidad Autónoma de Madrid, Madrid, ES,
5
Unidad de Investigación, Hospital Universitario Santa Cristina,
Universidad Autónoma de Madrid, Instituto de Investigación
Sanitaria Princesa (IIS-IP) Servicio de Inmunología, Hospital
Universitario de La Princesa, Instituto de Investigación Sanitaria
Princesa (IIS-IP), Madrid, ES, 6Servicio de Inmunología, Hospital
Universitario de La Princesa, Instituto de Investigación Sanitaria
Princesa (IIS-IP), Madrid, ES
Cytochrome c oxidase or complex IV (CIV) of the mitochondrial oxidative
phosphorylation system (OXPHOS) is the principal oxygen-consuming
enzyme in eukaryotic cells. Although global OXPHOS and CIV activity
are diminished in hypoxia, a hypoxia inducible factor-1 (HIF-1)-dependent
exchange of critical CIV subunit isoforms optimizes CIV efficiency.
Herein, we demonstrate that CIV abundance is progressively reduced when
oxygen supply becomes limiting. Hypoxia does not alter the mRNA levels
of CIV subunits, but instead induces degradation of the entire complex,
diminishing CIV-containing supercomplexes and reducing oxygen
consumption. We show that CIV stability depends on the abundance of the
CIV subunit NDUFA4, which is in turn regulated by iron regulatory protein
1 (IRP1) activity. Hypoxia decreases IRP1 binding to an iron response
element (IRE) within the 3’-UTR mRNA of NDUFA4, reducing NDUFA4
protein translation. Thus HIF-dependent and independent mechanisms
cooperate to fully adapt CIV to fluctuating oxygen availability.
- Hypoxia triggers a series of adaptive responses in cell metabolism,
including the enhancement of glycolysis and inhibition of different steps
of mitochondrial oxidative phosphorylation (OXPHOS).
- In higher organisms, the only mechanism described for direct regulation
of mitochondrial complex IV (CIV), the main oxygen-consuming system,
is a switch of CIV subunits that increases CIV efficiency in hypoxia, driven
by the hypoxia-inducible factor 1 (HIF-1).
- Several reports have shown that CIV activity decreases in hypoxia, a
plausible adaptation to reduce oxygen consumption.
- We describe a novel mechanism by which hypoxia reduces CIV activity,
mediated by iron regulatory protein-1 (IRP1) binding to the mRNA of
NDUFA4, a protein that has been recently described to be a CIV subunit.
145
Pósters / Posters
XXXIX Congreso SEBBM
P18-18
P18r-20
Activation of arterial chemoreceptor cells by
extracellular lactate; role in acute oxygen sensing
Regulation of mitochondrial respiration
in astrocytes
Hortensia Torres-Torrelo, Lin Gao, Patricia González-Rodríguez, José
López-Barneo, Patricia Ortega-Sáenz
Instituto de Biomedicina de Sevilla, Sevilla, ES
Ines Juaristi1, Irene Llorente-Folch1, Araceli del Arco2, Jorgina
Satrústegui1
1
Departamento de Biología Molecular, Centro de Biología
Molecular Severo Ochoa, Consejo Superior de Departamento
de Investigaciones Científicas-Universidad Autónoma de Madrid
(CSIC-UAM); Centro de Investigación Biomédica en Red de
Enfermedades Raras (CIBERER); Instituto de Investigación
Sanitaria Fundación Jiménez Díaz (IIS-FJD), ISCIII, Madrid, ES,
2
Centro regional de Investigaciones Biomédicas, Facultad de
Ciencias Ambientales y Bioquímica, Universidad de Castilla
La Mancha, Madrid, ES
The carotid body (CB) mediates rapid adaptive responses to hypoxia.
Glomus cellsin the CB contain K+ channels, which are inhibited by
intracellular ROS and NADH generated in mitochondria during hypoxia
leading to cell depolarization, transmitters secretion and activation of
afferent fibers impinging upon respiratory centers. Recently, it has been
reported that glomus cells contain lactate receptors and that lactate released
from these cells during hypoxia activate neighboring cells, in an auto/
paracrine phenomenon, that has been suggested to be the mechanism
responsible for acute responsiveness to lowering O2 tension. We have
investigated the effects of lactate on single glomus cells dispersed or in
CB slices. Extracellular lactate increased glomus cells secretion rate,
evaluated by amperometry, with a half-maximal activation at 15 mM,
without occluding the secretory response to hypoxia; both stimuli (hypoxia
and lactate) had additive effects. In glomus cells, studied with perforated
patch clamp, lactate had no effect on resting K+ conductance and produced
a small but significant increase in voltage-dependent K+ current. This
contrasts with the effects of hypoxia, which normally inhibits both resting
and voltage-dependent K+ channels. In transgenic TH-NDUFS2 mice,
with mitochondrial complex I function almost completely abolished and
selective suppression of hypoxic response, lactate induced normal secretory
activity. These data indicate that although extracellular lactate can exert a
significant stimulatory effect on CB glomus cells it does not seem to play a
major role in acute O2 sensing by arterial chemoreceptors. Ongoing studies
will clarify the role of extracellular lactate, mainly generated in anaerobic
skeletal muscle fibers during exercise, in CB-mediated regulation of
respiration.
P18-19
Macrophages lacking lipin-2 Enhance IL-1β
production through the overactivation of NLRP3
inflammasome
Gema Lordén Losada1, Jesús Balsinde2, María Balboa2
1
Universidad de Valladolid, Valladolid, ES, 2CSIC, Valladolid, ES
Lipin-2 is a member of the lipin family of proteins, which catalyze the
enzymatic conversion of phosphatidic acid to diacylglycerol. Mutations
found in the human LPIN2 gene are known to cause Majeed Syndrome,
an autoinflammatory disorder in which IL-1βis involved. Since NLRP3
inflammasome orchestrate innate immune responses through activation
of caspase-1 leading to the maturation of pro-inflammatory cytokines
pro-IL-1β and pro-IL-18, it was hypothesized that lipin-2 could modulate
its production and thereby be involved in the regulation of inflammasome
activity. We found out that both, classic (LPS and ATP) and metabolic
(LPS and palmitic acid) activation of NLRP3 inflammasome, promote an
increased production of the proinflammatory cytokine IL-1β in macrophages
lacking lipin-2, which depends on the overactivation of NF-κB by LPS.
Besides, depletion of lipin-2 in macrophages alter cellular responses to
ATP, what leads to overstimulation of the NLRP3 pathway triggering the
IL-1β and IL-18 production in an ASC and caspase-1 dependent manner.
These effects may be due to the fact that absence of lipin-2 modifies
lipid membrane levels, leading to an increase of P2X7 receptor activity,
triggering potassium efflux from the cell, and increasing the assembly
of the inflammasome and its activity. Collectively, these studies confirm
a protective role for lipin-2 in the pathology of inflammatory diseases
mediated by activation of inflamasome NLRP3.
146
In the brain, calcium regulates mitochondrial respiration both by activation
of ATP consumption and by Ca signaling. Ca signaling in mitochondria may
regulate respiration: i) after Ca entering through mitochondrial uniporter
(MCU), activating mitochondrial dehydrogenases and F0F1-ATPsynthase;
or ii) activating Ca-binding mitochondrial carriers, ARALAR and SCaMCs
from the outer face of the inner mitochondrial membrane, which involves
metabolite supply. In neurons, ARALAR contributes significantly to workload
and Ca-induced respiration (1). In brain astrocytes, ARALAR levels are
undetectable and this work aims to study the metabolic response of cultured
astrocytes to different workloads and the possible role of Ca in response,
using different ligands, glutamate (L-Glu) and ATP. L-Glu is transported
into the astrocytes resulting in increased Na+ uptake, which drives ATP
breakdown, and also acts on metabotropic L-Glu receptors. L-Glu (200 μM –
500 μM) produced a small rise in Cai in cultured astrocytes, increased glucose
utilization, lactate production and stimulated oxygen consumption in an acute
and stable way (OCR, by Seahorse XF24 Extracellular Flux Analyzer) and
maximal uncoupled respiration (MUR) in the presence of 2.5 mM glucose.
A transportable but non-metabolizable glutamate analog, D-aspartate (D-Asp)
(500 μM), which also increases Cai, stimulated respiration in a way similar
to L-Glu, but failed to increase MUR, clearly showing that L-Glu is used as
respiratory substrate by cultured astrocytes. L-Glu and D-Asp-stimulation of
respiration was only slightly reduced in the absence of Cao, consistent with
a major role of ATP demand in stimulation of OCR.ATP (100 μM - 1 mM)
activates metabotropic and ionotropic purinergic receptors and induces a larger
Cai rise than L-Glu in cultured astrocytes. ATP caused an increase in glucose
utilization and lactate production, and also an acute and transient rise in OCR,
which was dependent on Cao. Whether this is due to increased Ca-dependent
workload or to the effects of Ca on upregulation of respiration is part of
the ongoing work. Remarkably, MUR was also Ca-dependent, especially
in astrocytes exposed to high ATP concentrations. It is likely that glycogen
breakdown via Ca-dependent adenylate cyclase and PKA (2) plays a role in
increasing MUR in the presence of Ca. Further studies are required to dissect
the role of MCU and SCaMC-3 and Ca-dependency with each stimulus.
References
[1] Llorente-Folch et al., J Neurosci (2013) 33 (35).
[2] Müller MS et al., Glia (2014) 62(4).
P18-21
Cross-regulation between GPCRs and glutamate
NMDA receptors: Role of HINT1 proteins and
of σ 1 receptors
Elsa Cortés Montero, María Rodríguez Muñoz, Pilar Sánchez
Blázquez, Javier Garzón Niño
Laboratorio Neurofarmacología, Departamento de Neurobiología
Molecular, Celular y de Desarrollo, Instituto Cajal, CSIC, Madrid, ES
The ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) is a
ligand-gated cation channel highly permeable to Ca2+. This receptor plays an
essential role in neuronal plasticity, development, differentiation, learning and
Salamanca 2016
memory consolidation. The activity of NMDARs is under the influence of G
protein-coupled receptors (GPCRs) which can dampen (as cannabinoid 1
receptor CB1R) or enhance (as mu-opioid receptor MOR) NMDAR function.
The cross-regulation between these GPCRs and NMDARs requires of the
physical interaction of NMDAR NR1 subunits with the C termini of the GPCRs
(1, 2). The present study shows that the interaction between NMDARs and
MOR/CB1R reaches physiological relevance in the presence of the HINT1
protein and of the σ1R and in the absence of these proteins, the GPCR-NMDAR
cross-regulation is disrupted. Within σ1R-MOR-HINT1 complex, the cytosolic
segment C1 of the NMDAR NR1 subunit binds simultaneously to HINT1 and
to σ1R, but σ1R weakens HINT1-NR1 association and impedes HINT1 transfer
to the NMDAR. MOR activation leads to PKC-mediated phosphorylation of
the NR1 C1 reducing the affinity of the coupled NMDAR to the MOR-HINT1
complex, and consequently the active NMDAR-σ1R tandem separates. To avoid
hiperactivation of NMDARs by MORs, the antagonists of σ1R prevents σ1R
binding to NR1 subunits and thus promote the swapping of HINT1 from the
MOR to the NMDAR. Alternatively, within CB1R-HINT1-σ1R-NR1 complex
the cannabinoids restrain NMDAR activity and could provoke its hypofunction,
thus when necessary NMDARs must be disconnected from the negative
influence of the CB1R by transfer of HINT1 from the CB1R to the NMDAR
NR1 C1 subunit. The σ1R and its endogenous regulators (neurosteroids) play
an essential role in this regulatory process, which serves to maintain NMDAR
excitatory activity within physiological limits. Therefore, HINT1 and σ1R form
a molecular switch that connect and disconnect the activity or GPCRs such as the
MOR and CB1 with that of NMDARs.
References
[1] Rodríguez-Muñoz, M., et al., (2012). Neuropsychopharmacology, 37,
338.
[2] Sánchez-Blazquez, P., et al., (2013). Antioxidants & Redox Signaling,
19, 1766.
Pósters / Posters
Universitario de Salamanca, Salamanca, ES, 3Departamento de
Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM,
IdISSC, Madrid, ES
C3G is guanine nucleotide exchange factor for Rap1b, a protein of the
Ras family involved in critical aspects of platelet function through the
activation of integrin aIIbb3. Using transgenic mouse models, with specific
expression in platelets, of full length C3G (tgC3G) or mutant lacking
the GEF domain (tgC3GDCat), our group has unveiled an important
role of C3G in primary hemostasis. Thus, C3G-Rap1 participates in
thrombin-triggered platelet activation, aggregation and clotting through
a PKC-mediated pathway. In addition, C3G-overexpressing platelets also
show a stronger response to ADP, as compared to wt platelets. Based on
these results, we have deepened into the role of C3G in platelet signaling
by using several inhibitors of different platelets signaling pathways.
This includes: clopidogrel and 2MeSAMP (for P2Y12 ADP receptor),
MRS2179 (for P2Y1 ADP receptor), SB203580 (for p38a/b MAPK),
U0126 (for ERK1/2), aspirin (for TXA2 synthesis) and wortmannin (for
PI3K). We have performed activation, aggregation, and Rap1 activity
assays in platelets stimulated with thrombin or ADP/fibrinogen in the
presence or absence of the above referred inhibitors. Our results indicate
that thrombin- or ADP-stimulated tgC3G platelets are more sensitive to
the action of clopidogrel, SB203580, U0126 and aspirin, as compared to
wild type platelets. In contrast, tgC3GDCat platelets are more resistant to
these inhibitors than their corresponding control platelets. No differences
were observed with the rest of inhibitors. These results suggest that C3G
may participate in the activation of Rap1 mediated by ADP-P2Y12, but
independent of PI3K. Furthermore, our results support a role for C3G in
the thromboxane synthesis pathway, probably through modulating ERKs
and p38a MAPK activities.
P18r-24
P18-22
Pharmaco-phosphoproteomics disection of
isoform-selective PI3K signalling in a cell model
of acute myeloid leukaemia
Maruan Hijazi, Pedro R. Cutillas
Cell Signalling & Proteomics Group, Centre for Haemato-Oncology,
Barts Cancer Institute, Queen Mary University of London, London, UK
Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases that
coordinate intracellular signalling in response to extracellular stimuli.
Alterations in PI3K signalling cascades are common events in human cancers,
making this class of enzymes a prime drug target for anti-cancer therapy.
PI3K isoforms have distinct roles in mediating signalling in physiological
and oncogenic contexts. However, the precise roles of specific PI3K isoforms
in terms of biological responses remain poorly defined. In order to assemble
a database of phosphorylation sites annotated with the signalling pathways
they belong to, here we classified phosphorylation sites by their patterns
of modulation by a panel of class Ia isoform-selective PI3K inhibitors in
HL-60 cell line (human myeloid leukaemia cells). Their phosphoproteomes
were analysed relative to DMSO control. We derive kinase activity by using
label-free phosphoproteomics and bioinformatics tools. We show different
patterns of activity modulated in an isoform-dependent manner, suggesting
the presence of isoform-specific functions.
P18-23
Contribution of the Rap-GEF C3G to signaling
pathways in platelets
Sara Gutiérrez-Herrero1, Víctor Martín-Granado1, Sara Ortiz-Rivero1,
José Ramón González-Porras2, Almudena Porras3, Carmen Guerrero1
1
Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL,
Salamanca, ES, 2Departamento de Hematología, IBSAL-Hospital
C3G regulates megakaryocytic differentiation
and pro-platelet formation in mice
Sara Ortiz-Rivero1, Sara Gutiérrez-Herrero1, Víctor Martín-Granado1,
José Ramón González-Porras2, Almudena Porras3, Carmen Guerrero1
1
Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL,
Salamanca, ES, 2Departamento de Hematología, IBSAL-Hospital
Universitario de Salamanca, Salamanca, ES, 3Departamento de
Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM,
IdISSC, Madrid, ES
C3G is an activator of members of the Rap subfamily of Ras proteins.
Several studies suggest that C3G plays a role in different processes of
differentiation acting through its main target Rap1. C3G is induced during
neural differentiation and monocytic differentiation to macrophages.
Similarly, an increase in C3G protein levels and its association with Crk is
required for adipocyte differentiation. There are also evidences supporting
the involvement of C3G in the differentiation of megakaryocytes to
platelets through Rap1 activation, although its function in early stages
of megakaryocytic differentiation has not yet been addressed. Using
transgenic mouse models for C3G and C3GΔCat (C3G mutant lacking the
GEF domain), where the transgenes are expressed under the control of the
megakaryocyte and platelet specific PF4 gene promoter, we have found
that C3G plays a role in the differentiation of immature hematopoietic
cells to megakaryocytes. Bone marrow cells from transgenic C3G, but
not those from transgenic C3GΔCat mice, showed increased CD41 and
CD61 expression upon thrombopoietin (TPO) treatment. Polyploidization
analysis supports the hypothesis of a positive role of C3G in megakaryocytic
differentiation. Thus, C3G overexpression increased the levels of CD41+
megakaryocytes with 16N and 32N ploidy. In addition, preliminary
studies using fresh bone marrow explants, incubated with Tyrode´s Buffer
containing 5% mouse serum, showed an increased migration from the
osteoblastic niche to the vascular niche and an enhanced ability to form
pro-platelets in C3G transgenic megakaryocytes, as compared to wild-type
147
Pósters / Posters
ones. These results suggest the participation of C3G in different key aspects
of megakaryopoiesis by a mechanism mediated by its GEF activity.
XXXIX Congreso SEBBM
son determinantes para controlar la quiescencia y características de célula
madre de las HSC. Nuestros resultados preliminares apuntan a que esto
sucede efectivamente, y que la HSC es más quiescente cuando los niveles
de dichas proteínas se reducen en las células mesenquimales.
P18r-25
Targeted expression of a Spry1 Y53A mutant
precludes replicative senescence
Carlos Anerillas Aljama1, Marta Vaquero Susagna1, Sara Cuesta
Sancho2, Joaquim Egea Navarro2, Mario Encinas Martín3
1
Universitat de Lleida, Quicena, ES, 2IRB Lleida, Lleida, ES,
3
Universitat de Lleida/ IRB Lleida, Lleida, ES
Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor
tyrosine kinase (RTK) signaling. As such, they restrain proliferation
of many cell types and have been proposed as tumor suppressor genes.
Data from our group indicate that Spry1 behaves as a tumor suppressor
independently of ERK activity by promoting cellular senescence, a fail-safe
mechanism opposing cellular transformation. However, the structural
domains critical for such function remain poorly understood. Different
studies relying on ectopic overexpression point to an important role of
a conserved tyrosine in the N-terminus of Spry family members (Tyr53
in Spry1). To confirm the role of this tyrosine in a more physiological
setting we have analyzed skin fibroblasts from knockin mice bearing a
Tyrosine to Alanine mutation in residue 53 of Spry1. When placed in a 3T3
scheme, skin fibroblasts from wild type but not those from Y53A knockin
mice undergo replicative senescence as judged by cell counting, 5’
Bromo-deoxyuridine uptake and senescence-associated beta galactosidase
staining. Interestingly, skin fibroblasts from Spry1 null mice do not
escape replicative senescence, suggesting that Spry1 Y53A functions in
a dominant negative fashion. Finally, in line with our previous data, no
differences on ERK phosphorylation were found between wild type and
knockin fibroblasts, indicating that induction of senescence by Spry1 is
independent of this pathway.
P18r-26
β-catenina y PTP-BL están implicadas en la
regulación de la quiescencia de las HSC por
parte de las células mesenquimales del estroma
Alejandro Pérez Fernández, Rodrigo Prieto Bermejo, Marta Romo
González, Carla Ijurko, Ángel Hernández Hernández
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca (USAL). Instituto de Investigación Biomédica de
Salamanca (IBSAL), Salamanca, ES
La hematopoyesis en el adulto se desarrolla en la médula ósea, en
un entorno compuesto por distintos tipos celulares y condiciones
fisico-químicas que en su conjunto se denomina nicho hematopoyético.
Durante los últimos años se ha establecido de forma sólida que las
células de linaje distinto al hematopoyético influyen en la biología de
las células madre hematopoyéticas (HSC) tanto a través de factores
solubles como por contacto directo. Una de las vías de señalización más
estudiadas en el contexto de la hematopoyesis, entre otras razones por los
resultados contradictorios que arrojan los distintos estudios realizados,
es la denominada ruta canónica de Wnt, cuyo efector final es el factor
de transcripción y molécula de adhesión β-catenina. Nuestro grupo de
investigación ha demostrado recientemente que esta ruta es importante en
la diferenciación megacariocítica, y que existe un control de la actividad
transcripcional y estabilidad de β-catenina mediado por la fosfatasa
PTP-Bas/PTP-BL. Además, estos estudios ponen de manifiesto que los
niveles de estas dos proteínas en las HSC son un factor determinante a
la hora de regular la quiescencia de estas células, fenómeno que parece
ser mediado por la adhesión de las HSC a las células mesenquimales del
nicho. Con los anteriores antecedentes, nos propusimos estudiar si los
niveles de β-catenina y PTP-Bas/PTP-BL a nivel de células mesenquimales
148
P18-27
USP29 a novel upstream HIF-α activator
Amelie Schober, Teresa Martín-Mateos, Encarnación Pérez-Andrés,
Onintza Carlevaris, Sara Pozo, Edurne Berra
CIC bioGUNE, Derio, ES
The transcription factor HIF is the central regulator of the adaptive
cellular program in response to hypoxia. In healthy cells, HIF signalling
is tightly controlled via the availability of its α-subunit. HIF-α is degraded
by the ubiquitin-proteasome system (UPS) in well-oxygenated cells but
the protein is stable in hypoxia, dimerises with HIF-β, binds to RCGTG
motives of target genes, and transcriptionally drives their expression.
HIF-targets involve among many others, genes that enhance glycolysis
and metabolic rewiring, angiogenesis and resistance to apoptosis. HIF-α
is also stabilised in a variety of cancer cells of different origin due to
genetic alterations independently of the present oxygen level. Accordingly,
sustained expression of HIF-α in tumours has been associated with higher
aggressiveness, migratory and metastasis-initiating potential and therefore
worse prognosis.
To better understand which proteins might be responsible for pathological
activation of HIF signalling, the family of deubiquitinating enzymes
(DUBs) was silenced in an RNAi screen. We identified a new non-canonical
positive regulator of HIF-α stability that we further characterised using
standard biochemical methods and confocal fluorescence live cell imaging
techniques. USP29 belongs to the family of ubiquitin-specific proteases
(USPs) and efficiently stabilised HIF-α in an O2/PHD/pVHL-independent
and a proteasome-dependent way. Furthermore, USP29 expression
correlated with disease progression in prostate cancer, suggesting that the
maternally imprinted USP29 gene might act as an oncogene. In the light of
DUBs being a class of highly druggable proteins, our results might open up
possibilities for new therapeutic approaches.
P18r-28
A new non-canonical pathway of Gα protein
regulating Alex3 in the mitochondria
Ismael Izquierdo Villalba1, Serena Mirra2, Cristiane Benincá1,
José Antonio Izquierdo3, Eduardo Soriano2, Anna Aragay Combas1
1
IBMB-CSIC, Barcelona, ES, 2Department of Cell Biology, University
of Barcelona, Barcelona, ES, 3CNIC, Madrid, ES
A novel localization of heterotrimeric G proteins at the mitochondria and
their implications on the physiology of the organelle has been recently
reported (Beninca et al., 2014; Zang et al., 2010;). In particular, the Gq
subfamily is required to keep the proper balance between mitochondria
fusion and fission acting at both outer and inner membrane dynamics
(Beninca et al., 2014; Sánchez-Fernández G et al., 2014). Together with
Gβ (that binds to Mfn1), Gαq stabilizes elongated mitochondria and cristae
structure. Gqis also necessary for the maintenance mitochondrial membrane
potential and the activity of the respiratory chain and mitochondrial
ATP synthesis. Surprisingly, Gαq is necessary for the supercomplex
assembly at the inner membrane. The molecular mechanism of action of
heterotrimeric G proteins at the mitochondria is still unknown. A recent
MS-proteomic analysis has helped us to decipher the Gq-interactome
(“Gq-mitoproteome”). We have utilized mitochondrial enriched fractions
from four different cell lines, among them the Gq/11-MEF knockout, the
recover Gαq-MEF-knockout, MEFs wild type and NIH3T3 cells, as well
as, two different anti-Gq antibodies. The new candidate binding proteins
are being analyzed by their capacity to interact to Gαq.
Salamanca 2016
Among the candidates we found Armadillo-like-domain containing protein
Armcx3, also known as Alex3. This mitochondrial outer-membrane protein
is involved in the regulation of mitochondrial aggregation and trafficking,
among other processes (López-Doménech et. al., 2012). IP studies showed
that the constutitive-active mutant of Gq, GqR183C, interacts specifically
with Alex3 as well as the mitochondrial Rho-GTPase Miro1, one of the main
regulators of mitochondrial transport in neurons. In summary, our group
postulates a new non-canonical mitochondrial-function of heterotrimeric
G proteins that involves their translocation to the mitochondria and the
interaction with several mitochondrial partners.
P18-29
La tetraspanina TSPAN33 controla la activación
de los macrófagos por los receptores Toll a través
de la modulación de Notch
Susana López López, Almudena Ruíz-García, José Javier
García-Ramírez, Victoriano Baladrón, Laura López-Sanz, María
José Ruíz-Hidalgo, Ángela Ballesteros, Jorge Laborda, Eva María
Monsalve, María José M. Díaz-Guerra
Facultad de Medicina, UCLM, Albacete, ES
Los receptores Notch modulan la activación de los macrófagos por
los receptors Toll, sin embargo los diferentes componentes y vías que
controlan la señalización de estas proteínas todavía no se conocen en
detalle. Nuestro grupo ha estudiado la expresión y la función de la
proteína Tspan33, una tetraspanina implicada en la maduración de la
proteasa ADAM10, en la activación proinflamatoria de los macrófagos.
La expresion de Tspan33 aumenta en los macrófagos tras la activación de
diferentes receptores Toll, como TLR4, TLR3 y TLR2, siendo aun mayor
con un coestímulo de INFγ. La inducción de Tspan33 mediante TLR/
INFγ depende de la activación de Notch, disminuyendo su expresión
en macrófagos deficientes en Notch1 y Notch2 y aumentando tras la
sobreexpresión de la forma intracelular activa de Notch1. Tspan33 es la
tetraspanina de la familia TspanC8 que más se induce durante la activación
de los macrófagos, y su sobrexpresión favorece el procesamiento de
ADAM10 y su activación en la membrana. De esta forma, Tspan33
incrementa la señalización de Notch en los macrófagos activados.
Además, Tspan33 modula la expresión de genes proinflamatorios
inducidos por los receptores Toll mediante el aumento de la actividad
transcripcional de NF-kB. Nuestros resultados sugieren que Tspan33 es
un nuevo punto de control en el desarrollo de la inflamación que podría
ser utilizado como diana farmacológica.
P18m-30
Papel de Sam68 en las vías de señalización
de insulina y leptina en células de la granulosa
Teresa Vilariño García1, Antonio Pérez-Pérez2, Flora
Sánchez-Jiménez2, Esther Santamaría-López3, Nicolás
Prados-Dodd3, Manuel Fernández-Sánchez3, Víctor
Sánchez-Margalet2
1
Fundación Pública Andaluza para la Gestión de la Investigación
en Salud de Sevilla (FISEVI), Sevilla, ES, 2Bioquímica Médica y
Biología Molecular e Inmunología, Facultad de Medicina, Hospital
Universitario Virgen Macarena, Sevilla, ES, 3Instituto Valenciano de
Infertilidad de Sevilla (IVI), Sevilla, ES
Antecedentes y objetivo: La obesidad es un problema médico, no
solo por estar asociada a un aumento del riesgo de padecer Diabetes
tipo 2 o enfermedades cardiovasculares, sino porque también se asocia
con problemas de reproducción cuando afecta a mujeres en edad fértil;
de hecho, se asocia con el Síndrome de ovario poliquístico (PCOS),
resistencia a la insulina durante el embarazo y disminución de la fertilidad.
Se ha demostrado la expresión de Sam68, una proteína de unión a RNA,
Pósters / Posters
en ratones hembras y se ha observado que los ratones hembra knockout
para dicha proteína son subfértiles con problemas de ovulación. Dado
que previamente hemos encontrado que Sam68 interviene en las vías de
señalización de los receptores de insulina y leptina, planteamos estudiar
la posibilidad de participación de Sam68 en dichas vías de señalización en
las células de la granulosa. Además pretendemos estudiar la expresión de
Sam68 en respuesta a leptina e insulina in vitro.
Material y métodos: La señalización fue estudiada por inmunoprecipitación
e immunoblot de proteínas fosforiladas. La expresión de Sam68 fue
inhibida mediante la estrategia antisentido. Los niveles de expresión de
Sam68 y de receptores de insulina y leptina, fueron cuantificados mediante
qPCR e immunoblot.
Resultados: Hemos encontrado que Sam68 es fosforilado en tyrosina por
estimulación con insulina y leptina en células de la granulosa, reclutando
Sam68 para complejos de señalización y disminuyendo así su capacidad
de unión a RNA. Por otra parte, tanto insulina como leptina incrementan
la expresión de Sam68 en las células de la granulosa. Finalmente se
evidencia que se requiere la total expresión de Sam68 para que insulina
y leptina activen las vías de señalización PI3K y MAPK en células de la
granulosa.
Conclusión: Sam68 es reclutado para las vías de señalización activadas
por insulina y leptina, su expresión es inducida por ambas hormonas y es
necesaria para la completa activación de la transducción de la señal de los
receptores de insulina y leptina en células de la granulosa. Sam68 podría
ser un nuevo elemento importante en la resistencia a la insulina en le ovario
y descenso de la fertilidad encontrado en mujeres obesas.
P18r-31
Análisis de la función de NADPH oxidadas
en hematopoyesis in vivo mediante ARNi frente
a p22phox
Rodrigo Prieto Bermejo, Guillermo López Ruano, Alejandro Pérez
Fernández, Marta Romo González, Carla Ijurko Valeta, Ángel
Hernández Hernández
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca (USAL); Instituto de Investigación Biomédica de
Salamanca (IBSAL), Salamanca, ES
Las especies reactivas de oxígeno (ROS) son compuestos que se producen
de manera natural en las células como consecuencia de su metabolismo
aerobio, y debido a su gran reactividad siempre se han considerado como
agentes nocivos. En las últimas décadas se ha demostrado que esto no
es así, y que las ROS están relacionadas con el control de múltiples
procesos celulares (como la señalización celular o la expresión génica)
en los que actuarían como segundos mensajeros. Esto es debido en gran
parte a su capacidad de oxidación reversible de proteínas señalizadoras y
reguladoras, y a la capacidad de la familia NADPH oxidasa de generar ROS
de forma específica y en respuesta a diferentes estímulos. Actualmente
se conoce que la señalización redox interviene en la diferenciación de
múltiples tipos de células (cardíacas, neuronales,…). Nuestro laboratorio
también ha demostrado la importancia de la producción de ROS por
medio de NADPH oxidasas para un correcto proceso de diferenciación
megacariocítica. Para esto utilizamos experimentos de ARNi frente a
p22phox, proteína necesaria para la activación de varios miembros de la
familia, tanto en líneas celulares como en progenitores hematopoyéticos.
En este trabajo, hemos querido profundizar aún más en el papel de
estas enzimas en el proceso de diferenciación, de manera que hemos
analizado el efecto del silenciamiento de p22phox en la hematopoyesis in
vivo mediante trasplantes de medula ósea en ratón. Los resultados nos
demuestran que el silenciamiento de p22phox en células Lin- de ratón
provoca un descenso en las poblaciones celulares de la rama mieloide, en
favor de un aumento de las células linfoides. Además, el número de HSC
no se ve alterado, pero sí hay un aumento de células LT-HSC compensado
por un descenso en células ST-HSC.
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Pósters / Posters
P18-32
AIRAPL: a novel tumor suppressor that regulates
IGF1R proteostasis
José María Pérez Freije
Departamento de Bioquímica, Universidad de Oviedo, Oviedo, ES
Protein homeostasis is essential for cell function and involves
complex regulatory mechanisms of protein quality control. AIRAPL
(arsenite-inducible regulatory particle-associated protein-like, encoded
by ZFAND2B) is a highly conserved endoplasmic reticulum protein whose
deficiency had been previously reported to cause shortened lifespan in
Caenorhabditis elegans as a consequence of proteostasis impairment. We
have recently generated AIRAPL-deficient mice, which do not exhibit any
obvious development or fertility alterations. However, we have observed that
these mice suffer a fully-penetrant myeloproliferative neoplastic process,
characterized by early hypercellularity, progressive myeloid skewing and,
at later stages, trilineage dysplasia and myelofibrosis. Proteomic analyses of
AIRAPL-deficient mouse cells revealed that AIRAPL exerts its antineoplastic
function by interacting with newly synthesized IGF1R isoforms, promoting
their ubiquitination and degradation in the proteasome. In agreement with
this fact, genetic and pharmacological targeting of IGF1R prevented the
hematological disease developed not only by AIRAPL-deficient animals,
but also by Jak2V617F mutation carriers, demonstrating the relevance of this
signaling pathway in the pathogenesis of myeloproliferative neoplasms
(MPN). Moreover, we have found that AIRAPL expression is suppressed
in human MPN and we have identified mir-125a-3p as the main factor
responsible for AIRAPL suppression in these disorders. Based on these
findings, we propose that the identification of this novel pathway involving
AIRAPL, IGF1R and miR-125a-3p offers new therapeutic opportunities for
the management of myeloproliferative malignancies.
P18r-33
Regulación del receptor de insulina
por la sirtuína 6
María Gutiérrez-Salmerón1, Ana Chocarro-Calvo1, José Manuel
García-Martínez1, Antonio De la Vieja2, Custodia García-Jiménez1
1
Facultad Ciencias de la Salud. Universidad Rey Juan Carlos,
Alcorcón, ES, 2Instituto de Salud Carlos III, Majadahonda, ES
Introducción: La hiperglucemia favorece el desarrollo tumoral a través
de mecanismos sistémicos y directos sobre la célula tumoral, potenciando
la acetilación de cromatina y la proliferación y migración tumoral. La
sirtuína SIRT6 es nuclear y tiene actividades enzimáticas de desacetilación,
mono-ADP-ribosilación y demiristoylación. El silenciamiento de SIRT6 en
modelos animales permite concluir que es indispensable para la regulación
de la homeostasis de la glucosa y como supresor tumoral. Por ello su
estudio despierta interés en las áreas de diabetes y cáncer y las posibles
relaciones entre ambos.
Objetivo: Evaluar la influencia de la hiperglucemia sobre SIRT6 en células
tumorales (HCT 116) y no tumorales humanas (CCD18-Co) de colon y en
tejido tumoral y no tumoral humano y analizar las consecuencias sobre la
señalización por el receptor de insulina (IR).
Metodología: Células humanas estimuladas con glucosa en condiciones
control o tras depleción o sobre-expresión de SIRT6. Se evalúan cambios
en expresión génica (qPCR), exhibición del IR (citometría de flujo),
señalización y niveles de SIRT6 e IR (Western-Blot e inmunofluorescencia).
Resultados: La hiperglucemia aumenta los niveles de SIRT6 e IR, pero
atenúa la señalización de la vía a través de PI3K/Akt. La sobre-expresión
de SIRT6 aumenta los niveles totales de IR y la depleción (siRNA)
los disminuye. SIRT6 media la regulación por glucosa IR, ya que
el silenciamiento de la sirtuína disminuye tanto los niveles como la
funcionalidad del receptor.
Conclusiones: SIRT6 podría representar un sensor metabólico que media
los cambios inducidos por la hiperglucemia sobre el receptor de insulina,
150
XXXIX Congreso SEBBM
posiblemente para mantener la homeostasis de la glucosa. Por ello, SIRT6
podría representar una buena diana antitumoral.
P18r-34
Impact of mitochondrial ROS on astrocyte
antioxidant defense in vivo
Nicolò Bonora, Carlos Vicente Gutiérrez, Juan Pedro Bolaños
University of Salamanca, Salamanca, ES
A substantial body of evidence suggests that mitochondrial reactive
oxygen species (mROS) generation is a hallmark of certain neural diseases.
However, the underlying molecular mechanisms involved are not fully
understood, and whether they play physiological roles is still controversial.
Our group revealed that the astrocytes robustly express a plethora of
antioxidant enzymes compared to neurons, as a consequence of the major
activity of the Nrf2 pathway, the master transcriptional regulator of the
antioxidant response, in glial cells. However, the molecular mechanism
responsible for the up-regulation of the antioxidant defenses in astrocytes
is unknown.
We hypothesized that astrocytic mROS would play an important role in
neuronal antioxidant protection through the Nrf2 pathway. Nowadays, the
vast majority of studies on ROS-mediated cell signaling have used a strategy
based on increasing ROS, either by exogenous or endogenously-supplied
ROS. We believe that this approach might mask the beneficial roles of
physiological mROS. Hence we plan an alternative approach aimed to
down-modulate endogenous mROS.
To explore this, we have designed and generated an inducible, floxed
knock-in mouse model that expresses a mitochondrial version of catalase
(mCat). We believe that the analysis of mCat mice would allow us to probe
the astrocyte-specific functions of mROS at regulating the antioxidant
defense in vivo. We present results showing the efficacy of mCat at
reducing mROS levels in an astrocyte-specific manner. Now we have
found data that suggest a role of astrocytic mROS maintaining active
the Nrf2 pathway. We think that these results may reveal the functional
physiological relevance of mROS in astrocytes at contributing to the brain
defense against oxidative stress.
P18-35
Papel de la ruta de señalización PI3K/AKT
en la infección del virus de la enfermedad
de Newcastle
Javier Blanco1, Cristina Cameirão1, M. Carmen López-Cuesta2,
Isabel Muñoz-Barroso1
1
Departamento de Bioquímica y Biología Molecular, Universidad
de Salamanca, Edificio Departamental Lab.106, Salamanca,
ES, 2Departamento de Microbiología Genética, Universidad de
Salamanca, Edificio Departamental Lab.236, Salamanca, ES
La ruta de señalización fosfatidilinositol-3-quinasa-Akt (PI3K/Akt) regula
varios procesos celulares importantes como la síntesis de proteínas,
el crecimiento celular, el metabolismo de la glucosa y la inflamación.
Muchos tipos celulares utilizan esta ruta para inhibir la apoptosis celular
e incrementar su supervivencia. Existen numerosos ejemplos de virus que
han desarrollado mecanismos para modular la ruta de señalización PI3K/
Akt con objeto de incrementar su replicación. El virus de la enfermedad
de Newcastle (NDV) es un virus patógeno aviar con RNA de polaridad
negativa cuyo ciclo se desarrolla completamente en el citoplasma de la
célula infectada. En este trabajo, hemos estudiado el efecto del inhibidor
de quinasas LY294002 en la infección del NDV en células de cultivo y
el efecto del NDV sobre la ruta de señalización PI3K/Akt. Observamos
un aumento del efecto citopático en células pretratadas con el inhibidor
e infectadas con NDV. Asimismo, la infectividad del virus disminuyó en
presencia el inhibidor. La infección del NDV de células HeLa determinó la
Salamanca 2016
Pósters / Posters
activación de la ruta de señalización PI3K/Akt, detectándose un incremento
los niveles de fosforilación de Akt respecto a Akt de hasta cuatro veces a las
6 y 12 h postinfección. Nuestros datos sugieren la implicación de la ruta de
señalización PI3K/Akt en la infección del NDV.
player in cellular ROS control. The identification of these novel binding
partners for Gαq will help to understand the molecular mechanisms of
ROS-dependent GPCR signaling and provide new therapeutic targets in
cardiovascular diseases.
P18-36
P18-38
Cell cycle regulation of Whi7, a new insight
in Start progression
Mastl (Greatwall)-PP2A: a new cell cycle pathway
with therapeutic implications in cancer
Mercè Gomar Alba, Ester Méndez Belinchón, Inma Quilis Bayarri,
M. Carmen Bañó Aracil, Juan Carlos Igual García
Departament de Bioquímica i Biologia Molecular, ERI Biotecnologia
i Biomedicina, Universitat de València, Burjassot, ES
Mónica Álvarez-Fernández, Belén Sanz-Castillo, María Sanz-Flores,
María Salazar-Roa, Miguel Ángel Quintela-Fandino, Marcos
Malumbres
Spanish National Cancer Research Centre (CNIO), Madrid, ES
Start is a key point of cell cycle control in eukaryotic cells. Executing Start
is an irreversible decision that implies accurate molecular mechanisms
of control. In S. cerevisae the process begins when the Cln3-Cdc28
cyclin-CDK complex is released from its association to the endoplasmic
reticulum (ER) in late G1 and accumulates in the nucleus in order
to activate a transcriptional program mediated by the SBF and MBF
activators and Whi5 and Stb1 repressors. As a result, Cln1/Cln2-Cdc28
complexes appear and initiate a positive feedback on the transcriptional
program that gives coherence to the process. The Whi5 paralog Whi7 is a
negative regulator of Start controlling cell cycle entry by retaining Cln3 in
the ER in a phosphorylation-dependent manner (Yahya et al., 2012). In this
work, we describe a new function of Whi7 in Start regulation. Whi7 is a
cell cycle regulated protein whose protein level and phosphorylation state
increases from late G1 to G2-M. It is an unstable protein which is degraded
by the SCFGrr1 ubiquitin-ligase. Whi7 degradation requires phosphorylation
by any Cyclin-CDK complex, remaining the unphosphorylated protein
stable only in early G1. More important, Whi7 is found associated to CLN2
and RNR1 promoters, mostly in a SBF dependent manner. The interplay
with the main effectors of Start suggests a double repressive function of
Whi7 through Cln3 retention in the ER and SBF transcriptional program
repression, and confirms the necessity of overlapping security mechanisms
to ensure a model of robust control in cell division.
Protein phosphorylation is an essential mechanism to control progression
throughout the different phases of the cell cycle. During the last years, the
Greatwall-PP2A axis has emerged as a new pathway implicated in control
of mitosis. Greatwall kinase, also known as Mastl in mammals, specifically
inhibits PP2A complexes containing the B55 family of regulatory subunits
(PPP2R2A-D). Inhibition of PP2A-B55 complexes by Greatwall occurs
in a kinase-dependent manner through the phosphorylation of Ensa and
ARPP19, the only two Greatwall substrates identified to date. Depletion
of Greatwall results in chromosome segregation and condensation defects,
which are partially rescued by concomitant depletion of B55 regulatory
subunits. Whereas the relevance of Greatwall in cancer is mostly unknown,
the phosphatase PP2A is a well-known tumor suppressor, frequently
inactivated in cancer. Since PP2A reactivation has been proposed as a
therapeutic strategy for cancer treatment, inhibition of Greatwall may
have therapeutic benefits through the reactivation of PP2A-dependent
pathways. Interestingly, PPP2R2A has been found to be deleted in breast
tumors, suggesting that the Greatwall-PP2A pathway may be relevant
for breast tumor progression. Indeed, we have found that Greatwall is
overexpressed in breast tumors, and its expression level correlates with
poor prognosis, both in hormone-positive as well as triple-negative breast
tumors. Moreover, Greatwall depletion results in defective proliferation
of specific breast tumor cell lines. By using a Mastl conditional knockout
model, we have found that Greatwall deletion also impairs the growth of
breast tumors in vivo. Altogether, these data suggest Greatwall as a new
therapeutic target for breast cancer.
P18-37
Gq-interactome and oxidative stress signaling
pathways
P18-39
Álvaro Caballero, Federico Mayor Menéndez, Catalina Ribas Núñez
Centro de Biologia Molecular Severo Ochoa (CSIC-UAM) and
Instituto de Investigación Sanitaria La Princesa, Madrid, ES
Caracterización de NLRC5 y su relación
con la respuesta inmune antiviral en branquias
de salmónidos
Activation of G protein-coupled receptors (GPCR) coupled to the
Gq/11 family of heterotrimeric G proteins plays a paramount role in
cardiovascular physiopathology. In addition to the canonical PLCß effector,
additional proteins are being identified to underlie some of the functions of
Gq. Recently, we have reported that GPCR activation promotes a direct
interaction between Gαq and protein kinase C ζ (PKCζ) through a novel
acidic region in Gαq, different from the classical effector-binding region,
and the PB1 domain of PKCζ. This Gαq/PKCζ interaction leads to the
stimulation of the ERK5 pathway and is required for angiotensin-induced
cardiac hypertrophy in mice, and is also able to trigger the induction of
apoptotic cell death independently of PLCß. We hypothesize that the
interaction of Gαq with additional PB1 domain-containing proteins may
participate in the control of cellular functions by Gq-coupled GPCR.
GPCR activation can lead to increased production of reactive oxygen
species (ROS) in a way depending on specific agonists and cell type.
Gq-GPCR are known to promote an excess of ROS during hypertension,
ischemia-reperfusion injury and heart failure, thus contributing to cell
death and apoptosis. In this work, we have uncovered that Gαq associates
with another PB1 domain-containing protein, the Nox2 activator subunit
p67PHOX. Moreover, Gαq functionally interacts with Keap1, also a relevant
Felipe Ramírez, Claudio Álvarez, Daniel Oyanedel, Paula Santana,
Nicole Canto, Byron Morales-Lange, Jimena Cortés, Luis Mercado
Vianco
Pontificia Universidad Católica de Valparaíso, Valparaíso, CL
La creciente investigación acerca de receptores de reconocimiento de
patrón (PRR), activados por patrones moleculares asociados a patógenos
(PAMP), no solo ha concitado el interés en vertebrados superiores, sino
que también en invertebrados y vertebrados inferiores. De hecho en estos
grupos animales han venido a explicar la fortaleza de la inmunidad en
sistemas sin una inmunidad adaptativa robusta como sucede en aves y
mamíferos. Entre los PRR, en peces se ha identificado un número mayor de
receptores tipo Toll (TLR) en comparación a mamíferos, y para la mayoría
de ellos no se ha establecido con claridad cuál es su ligando. Con respecto a
los PRR solubles o citosólicos, la información es mucho menos abundante
para peces. Con la idea de conocer la capacidad de respuesta inmune no
relacionada con el tejido linfático asociado a branquias (GiALT), iniciamos
la caracterización de PRR citosólicos en epitelio branquial, identificando
su potencial ligando, así como también la respuesta inmune que es capaz de
desarrollar. Cuatro nuevas secuencias de receptores tipo NOD (NLR) fueron
151
Pósters / Posters
caracterizadas en trucha arcoíris, y se pudo establecer la sobreexpresión de
NLRC5 en respuesta a Poly I:C, en la línea celular de epitelio branquial
RT-Gill de O. mykiss. Inicialmente además se pudo demostrar que Poly
I:C induce la sobreexpresión de TNF-alpha e IFN de tipo I en este mismo
tipo celular. Mediante la técnica de siRNA, se inhibió la transcripción de
NLRC5 en células RT-Gill, lo que provocó que en presencia de Poly I:C
disminuyó la sobreexpresión de TNF-alpha e IFN tipo I. Paralelamente
y mediante IF pudimos establecer que Poly I:C induce el incremento de
NLRC5 e IFN tipo I en RT-Gill, a nivel de proteína. Para confirmar estos
resultados in vitro, un grupo de peces salmónidos fueron desafiados con el
virus ISA, en éstos se detectó una sobreexpresión de NLRC5, seguida de
un incremento de IFN de tipo I. Estos resultados confirman lo observado
in vitro y constituyen las primeras evidencias de respuesta inmune antiviral
vía NLR en branquias de salmónidos.
P18-40
Dual role of the pseudokinaseTRIB3
in the development of breast cancer
Alba Orea-Soufi1, María del Mar Lorente1, Sonia Castillo1,
María Salazar-Roa1, Elena García-Taboada1, José Miguel Lizcano2,
Endre Kiss-Toth3, Guillermo Velasco1
1
Complutense University; Department of Biochemistry and
Molecular Biology. School of Biology, Madrid, ES, 2Institut
de Neurociències and Departament de Bioquímica i Biologia
Molecular, Universitat Autònoma de Barcelona, Bellaterra, ES,
3
Department of Cardiovascular Science, University of Sheffield,
Medical School, Beech Hill Road, Sheffield, UK
Tribbles pseudokinase 3 (TRIB3) participates in the regulation of multiple
signaling pathways that are involved in the regulation of cell survival,
Emerging evidences obtained during the last few years suggest that
TRIB3 is a crucial modulator of tumorigenesis. Recent observations by
our research group have shown that TRIB3 plays a tumor suppressor
role in several models of cancer through a mechanism that relies on the
regulation of the MTORC2/AKT axis Although there is limited and often
conflicting literature concerning to the role of TRIB3 in breast cancer, the
existing data suggest that this pseudokinase could be deregulated during
the development of this pathology. In this work, we have investigated the
effect of the genetic inactivation of TRIB3 in the malignant properties of
several human breast cancer cell lines. Our results show that TRIB3 genetic
inhibition increases proliferation and invasiveness of BT474 (ER+, PR+,
HER2+) whereas it decreases both parameters in MDA-MB-231 (ER-,
PR-, HER2-), and MCF7 (ER+, PR+, HER2-) cells. Moreover, Trib3
silencing accelerated the growth of tumor xenografts generated with BT474
and produced the opposite effect in tumour xenografts generated from
MDA-MB-231 and cells. Our data also show that Trib3 genetic inhibition
enhances Akt and FOXO phosphorylation in BT474 and decreases the
phosphorylation of these proteins in MDA-MB-231 cells. suggesting that
the ability of Trib3 to modulate the AKT/FOXO pathway determines its
tumor suppressor or oncogenic role in breast cancer cells. Ongoing work
is currently investigating the role of postranslational modifications and
its influence on the suncellular location of TRIB3 on the dual role of this
pseudokinase in breast cancer.
P18-41
El plasma de pacientes con tumor pulmonar
activa la expresión de VCAM e ICAM-1 en la
superficie de células endoteliales en cultivo
Alejandra Romero, Andrea Veiga Barrientos, Ana Dione Ibáñez,
Isabel Vaquero, Juan-Carlos Murciano, Fátima Esquivel
Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS),
Madrid, ES
152
XXXIX Congreso SEBBM
El cáncer es una patología muy asociada con múltiples procesos que
benefician el desarrollo de la enfermedad y la aparición de otras patologías
de riesgo elevado. Los procesos inflamatorios pueden asociarse a metástasis,
mediante la cual pueden aumentar aquellos receptores o moléculas
involucradas en la traslocación celular. El paciente con tumor pulmonar
posee un cáncer de muy mal pronóstico y evolución pronunciada, hecho
que se ve potenciado por su virulencia, pero también, por su diagnosis en
periodos del cáncer con estadiaje avanzado. Nuestro grupo viene realizando
una colección de muestras sanguíneas de pacientes diagnosticados de un
tumor en sus primeros periodos tras el diagnóstico. Estos pacientes aun no
han comenzado un tratamiento antitumoral activo. Junto con las muestras,
la base de datos recoge datos analíticos, epidemiológicos y de laboratorio.
Los datos generales representativos de los pacientes muestran una edad
próxima a los 70 años y un porcentaje de pacientes masculinos cercanos
al 70 %, datos muy similares en cuanto a la distribución y diagnóstico
de este tipo de tumores independientemente de su localización geográfica.
Es interesante resaltar que cuando exponemos células endoteliales de
cordón umbilical en cultivo a la presencia del plasma de estos pacientes, se
produce un aumento de la expresión de VCAM e ICAM-1 en su superficie,
obteniendo valores superiores a aquellas células expuestas únicamente al
plasma de controles sanos. Esta expresión se encuentra más elevada tras 10
horas de exposición, siguiendo cinéticas de expresión de dichas moléculas
muy similares a aquellas obtenidas con inductores proinflamatorios como
el LPS. Estos resultados parecen incluso asociarse al tipo de histología
del tumor, viéndose más incrementada la expresión de estas moléculas en
los adenocarcinomas, que en los epidermoides o en los microcíticos. Este
estudio trata de establecer la relación del citado incremento de moléculas
proinflamatorias que podrían ir asociadas a un mayor riesgo metastático
del tumor, o cuando menos de peor pronóstico.
Agradecimientos: Este estudio ha sido realizado con cargo al proyecto
PI11/0668 donde J-C.M es el IP. Este proyecto recibe financiación del
Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013-2016) y ha sido
cofinanciado por el Fondo Europeo de Desarrollo Regional «Una manera
de hacer Europa», FEDER. La Dra. FE recibe su salario a cargo de este
proyecto.
P19. Transgénesis en mamíferos /
Transgenesis in mammals
P19-1
Implicación de Igf1r en la respuesta inflamatoria
pulmonar aguda a la bleomicina y en la
hiperreactividad de las vías respiratorias tras
inducción de asma con extracto de ácaros del
polvo doméstico
Sergio Piñeiro Hermida1, Icíar P. López1, Raquel Torrens1, Joshua A.
Gregory2, Mikael Adner2, José G. Pichel1
1
Centro de Investigación Biomédica de La Rioja (CIBIR), Fundación
Rioja Salud, Logroño, ES, 2Karolinska Institute, Institute of
Environmental Medicine (IMM), Unit of Experimental Asthma and
Allergy Research, Estocolmo, SE
IGF1R (Insulin-like Growth Factor type 1 Receptor) es un receptor
transmembrana de expresión ubicua con funciones biológicas esenciales
como la supervivencia, proliferación, diferenciación y adhesión celular.
En el pulmón, participa tanto en el desarrollo embrionario como en el
mantenimiento de la homeostasis en el adulto. También se ha relacionado con
el estrés oxidativo asociado al proceso inflamatorio y con el remodelado de
Salamanca 2016
las vías respiratorias. Con el objeto de determinar su función en la respuesta
al daño pulmonar, los ratones mutantesUBC-CreERT2; Igf1rfl/fl con deleción
de Igf1r inducida posnatalmente, se sometieron a dos tipos de challenges
respiratorios. i) Tras la administración intratraqueal de bleomicina, los
mutantes presentaron mejor tasa de supervivencia que los controles.
A los tres días sus pulmones mostraron menos congestión pulmonar y
contenido de células inflamatorias, así como un descenso de marcadores
inflamatorios (TNF, Il1b, Il6, Ly6g, Cxcl1 y Adgre1), de adhesión (Icam,
Pecam1) e hipoxia (Hif1a), y un incremento del marcador de antiestrés
oxidativo Gpx8. ii) Después de la administración intranasal de extracto de
ácaros durante un mes, los mutantes revelaron mejor función pulmonar y
menos eosinófilos en lavados bronquioalveolares. Sus pulmones estaban
menos inflamados y contenían menos células mucosecretoras, colágeno,
eosinófilos y niveles reducidos de marcadores de producción de moco
(Spdef y Muc5ac). La menor hiperreactividad bronquiolar en el mutante
se reflejó en la reducción del grosor del músculo liso y en la alteración
morfológica del epitelio bronquiolar distal. Estos resultados demostraron
que Igf1r está implicado en la respuesta inflamatoria y en la hiperreactivad
respiratoria tras el daño pulmonar tanto agudo como sostenido.
P19-2
The role of fibronectin synergic site in cutaneous
wound healing
Irene Gimeno Lluch, María Benito Jardón, Mercedes Costell
Universidad de Valencia, Almacera, ES
In humans, there are several pathological conditions where wound healing is
delayed, such as in diabetes, or excessive healing as occurs in the hypertrophic
and keloid scars. Fibronectin (FN) is thought to be one of the most important
extracellular matrix proteins involved in cutaneous wound repair. FN is an
extracellular matrix glycoprotein formed by two identical subunits joined at
the C-terminus by two disulphide bonds. Its structure includes three different
types of repeating units named type I, type II and type III. FN binds several
heterodimeric transmembrane receptors called integrins. The main integrin
binding site is located at the FN type III10 module. In this region, an Arg-Gly-Asp
(RGD) motif joins α5β1, αIIbβ3, α8β1 and the αv family of integrins. Adjacent
to the RGD motif there is a Pro-His-Ser-Arg-Asn (PHSRN) sequence which
has been described to enhance FN adhesion to α5β1and αIIbβ3 integrins and
so-called synergic site (FNsyn). The in vitro role of FNsyn has been intensively
studied and is thought to have a crucial role in situations where the FN-integrin
bond is tensioned. We studied wound healing capacity in mice carrying point
mutations in the FN gene that impair the synergy site function (FNsyn/syn) and
compared with wild type littermates (FN+/+). Our results show a delay in the
gap closure in the FNsyn/syn mice at early times. During re-epithelialization, the
keratinocytes, confined in the epidermis, start to migrate from both sides of
the wound through a FN-fibrin provisional matrix. We analysed in vitro the
keratinocyte migration on substrates of either purified wild type FN or FN-syn.
We observed that keratinocytes migrated at lower speeds on substrates of
FN-syn. These results confirm the mechanistic role of synergic site in the
integrin-FN engagement. This is the first time that the synergic site is studied
in vivo and we will discuss its implication during cutaneous wound healing.
P19r-3
Destrucción in vitro del oncogen BCR-ABL
p210 mediante nucleasas de edición genómica
CRISPR/Cas9
Ignacio García-Tuñón1, Lucia Méndez-Sanchez2, Luis
Hernández-Cano2, María Hernández-Sánchez1, Miguel Quijada1,
José Luis Ordóñez1, Verónica Alonso-Pérez1, Rocio Benito1, Carmen
Guerrero3, Jesús María Hernández-Rivas1, Manuel Sánchez-Martín2
1
Centro de Investigación del Cáncer (CIC), IBMCC, Instituto de
Investigación Biomédica de Salamanca (IBSAL), Servicio de
Hematología, Hospital Universitario, Salamanca, ES, 2Servicio
Pósters / Posters
de Transgénesis, Nucleus. Universidad de Salamanca, Salamanca,
ES, 3Departamento de Medicina. Facultad de Medicina. Universidad
de Salamanca, Salamanca, ES
La caracterización molecular de las alteraciones génicas implicadas en
la transformación celular así como el desarrollo de nuevas herramientas
de edición genómica cambiarán radicalmente el panorama terapéutico de
algunas neoplasias en los próximos años, especialmente el de aquellas
asociadas a mutaciones “drivers” bien caracterizadas, como sucede en la
leucemia mieloide crónica (LMC). Esta enfermedad se caracteriza por
la presencia de la translocación t(9;22)(q34;q11) que genera el oncogén
de fusión BCR/ABLp210 y que produce una proteína con actividad
tirosinquinasa (TK) constitutiva. Esta actividad TK es crucial y se considera
indispensable en el mecanismo de transformación celular al que conduce.
El diseño de fármacos inhibidores de TK específicas ha constituido uno
de los mayores logros terapéuticos en la medicina actual. Sin embargo, su
administración es continua y prolongada en el tiempo, la acción terapéutica
se limita a la anulación del efecto a nivel proteico y en ningún caso hay
reparación o anulación del daño a nivel genético, lo que en ocasiones
conlleva a la aparición de resistencias. Afortunadamente, la aparición de las
nuevas herramientas de edición genómica, tipo CRSIPR-Cas9, solventaría
todos estos inconvenientes se podrían corregir/eliminar mutaciones a nivel
genómico lo que abre un nuevo panorama terapéutico. Este estudio explora
el uso de esta tecnología CRSIPR-Cas9 en un modelo celular tumoral
específico del oncogén p210 y demuestra su eficacia y utilidad como
posible nueva herramienta terapéutica para la LMC.
Financiación: HUS272413, PI15/01471 y Junta de Castilla y León.
P19-4
Shortage of dNTPs underlies replication defects
and genome instability in the absence of the
APC/C cofactor Cdh1
Javier Garzón Hidalgo, Sergio Moreno Pérez, Irene García-Higuera
Instituto de Biología Funcional y Genómica (IBFG), CSIC /
Universidad de Salamanca. Instituto de Investigación Biomédica de
Salamanca (IBSAL), Salamanca, ES
The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin
ligase that promotes proteasomal degradation of mitotic cyclins and other
critical cell-cycle regulators. Its activity is limited to the cell cycle period
comprised between anaphase and the end of the G1 phase, and is controlled
by the regulated binding of two co-factors: Cdc20 or Cdh1. APC/C-Cdc20
is active during anaphase, while APC/C-Cdh1 is active from anaphase until
the end of G1.Over the last few years, our group has been addressing the
biological function of the APC/C-Cdh1 complex using mouse models of
Cdh1 deficiency.
Cells derived from mutant embryos (MEFs) accumulate DNA breaks
and show increased levels of RPA foci and phospho-Chk1, suggestive
of replication stress. Direct analysis of the DNA replication process
on stretched DNA fibers (DNA fiber assays) revealed reduced rates of
replication fork progression and increased frequency of origin firing
in the absence of Cdh1. Interestingly, addition of exogenous dNTPs
or nucleosides, normalized replication dynamics in mutant cells, and
prevented DNA breakage. Consistently, quantification of intracellular
dNTP pools confirmed decreased levels of all four dNTPs in Cdh1-deficient
MEFs. Moreover, overexpression of RRM2, the regulatory subunit of
Ribonucleotide Reductase (the rate-limiting enzyme for dNTP synthesis),
restored efficient replication and genome stability in cells lacking Cdh1
expression.
These results highlight the importance of APC/C activity during the G1
phase to prevent replication defects, and provide evidence that, in the
absence of Cdh1, insufficient supply of dNTPs to the replisome leads to
replication stress, which, in turn, causes chromosomal breaks.
153
Pósters / Posters
P20. Transportadores de membrana /
Membrane transporters
P20-1
Deciphering the role of the exomer along
the evolution
Carlos Antón Plágaro, Irene Arcones Ríos, Rosario Valle Vicente,
César Roncero Maíllo
Universidad de Salamanca, Salamanca, ES
In order to reach the plasma membrane (PM) receptors, channels and
enzymes use the secretory pathway. Along this route, the main sorting
station is the Trans-Golgi-Network (TGN). Here, proteins can be
sent to multiple destinations: vacuole/lysosomes, PM or earlier Golgi
compartments. To address these purposes, cells use the lipid language,
the cytoskeleton, several small GTPases, adaptors and coats, although the
precise mechanisms involved in the process are not still fully understood.
One of the best studied platform for cargo sorting at the TGN is the yeast
exomer, originally described in S. cerevisiae as an effector of Arf1
necessary for the transport of the chitin synthase Chs3 to the PM. It is
formed by two molecules of Chs5 bound to a dimer of any of the four
ChAPs: Chs6, Bud7, Bch1 and Bch2. The ChAP proteins will act not
only as adaptors for the three cargoes described: Chs3, Fus1 and Pin2, but
they will play additional roles in exomer function. Here we will show that
exomer is involved in the intracellular transport of several other proteins
not previously identified as cargoes. These include several PM transporters
as Ena1, Gap1 or Tat2, whose transport is only impaired in the absence of
exomer. Moreover, we will show that the absolute requirement of exomer
for the bona fide cargos seems linked to their interaction to AP-1 complex.
This interaction has been probably acquired later in the fungal evolution,
being a rather unique characteristic of the Saccharomyces genus. Our
results thus suggest that exomer has a general and evolutionary conserved
function in protein sorting at the TGN which has later evolved to more
specialized function in cargo selection in some yeast.
P20-2
Bases moleculares de la quimiorresistencia
en el cáncer gástrico: papel del transportoma
Ruba Al-Abdulla1, Rocío I.R. Macías1, Elisa Lozano1, Oscar Briz1,
Marta Alonso1, Beatriz Castaño1, Francisco González San-Martín1,
Jesús M. Bañales2, Luis Bujanda2, José J. G. Marín1
1
Hepatología Experimental y Vectorización de Fármacos
(HEVEFARM), IBSAL, Universidad de Salamanca. Centro de
Investigación Biomédica En Red para el Estudio de Enfermedades
Hepáticas y Digestivas (CIBERehd), Salamanca, ES, 2Biodonostia,
Hospital Universitario de San Sebastián, Universidad del País
Vasco, Ikerbasque; Centro de Investigación Biomédica En Red para
el Estudio de Enfermedades Hepáticas y Digestivas (CIBERehd),
San Sebastian, ES
XXXIX Congreso SEBBM
de confirmación, se determinó el nivel de expresión de proteínas capaces de
transportar 5-FU o cisplatino. Tanto en tumores como en tejido no tumoral
la expresión de transportadores de la familia SLC22 fue baja, mientras que
las de la familia SLC28 y del transportador SLC31A1 fue elevada.
Resultados: Las bombas de la superfamilia ABC con mayor expresión en
ACG fueron MRP5>MRP3>BCRP. No se observaron diferencias según la
localización y estadio del tumor. El análisis inmunohistoquímico confirmó
una elevada expresión de MRP5 en ACG, mientras que la de BCRP fue
muy variable. En células AGS derivadas de ACG humano, la inhibición de
las bombas MRP con probenecid incrementó la sensibilidad de las células
al efecto citotóxico del 5-FU.
Conclusión: El transportoma, y en particular algunas bombas exportadoras
de la familia MRP, está implicado en la quimiorresistencia del ACG, lo que
permitiría identificar potenciales dianas moleculares para el desarrollo de
nuevas estrategias sensibilizantes a la quimioterapia del ACG.
P20r-3
Structural determinants of glycine
neurotransporter 2 (GlyT2) selective inhibitor
ALX1393
Cristina Benito-Muñoz1, Almudena Perona2, Enrique Núñez1,
Carmen Aragón3, Beatriz López Corcuera3
1
Departamento de Biología Molecular and Centro de Biología
Molecular Severo Ochoa, Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid, Madrid, ES,
2
Smartligs, Parque Científico de Madrid, Campus de Cantoblanco,
Madrid, ES, 3Departamento de Biología Molecular and Centro
de Biología Molecular Severo Ochoa, Consejo Superior de
Investigaciones Científicas-Universidad Autónoma de Madrid;
IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de
Madrid, Madrid, ES
Glycine transporters are plasma membrane proteins that remove
the neurotransmitter glycine from the synaptic cleft promoting the
termination of the glycinergic signal (GlyT1) and the pre-synaptic
terminal reloading (GlyT2). GlyT1 and GlyT2 have been associated to
the pathogenesis of nervous system disorders, including schizophrenia
and related affective and cognitive disturbances, alcohol dependence,
pain, epilepsy, breathing disorders and hyperekplexia or startle disease.
GlyTs belong to the solute carrier 6 (SLC6) family of sodium and
chloride-dependent transporters, and their three dimensional structure
has been recently modeled by homology with the crystal structure of
the Drosophila melanogaster dopamine transporter. Since the study of
the molecular determinants of the binding of selective GlyT inhibitors
may have therapeutic consequences, in this report our aim is to localize
the binding site of the GlyT2-specific inhibitor ALX1393. By using
computational and biochemical docking on GlyT2 models subjected to
dynamic simulation, we have identified the key residues involved in
the binding of the inhibitor. The effect of the N-glycosylation of the
transporter has also been evaluated
P20-4
Antecedentes: El adenocarcinoma gástrico (ACG) es un tipo de tumor
muy resistente a la quimioterapia, generalmente basada en la combinación
de 5-fluorouracilo (5-FU) y cisplatino, lo que justifica que el tratamiento
farmacológico fracase en >95% de los pacientes con ACG no operable.
Objetivo: Investigar el papel de las proteínas transportadoras que median
la capacidad de las células tumorales de captar fármacos antitumorales o el
aumento de su expulsión como mecanismos que contribuyen a la falta de
respuesta del ACG a la quimioterapia.
Métodos: Se obtuvieron muestras quirúrgicas pareadas (n=24x2) de tejido
tumoral y adyacente no tumoral de pacientes con ACG en estadios II,
III y IV, que no habían recibido previamente quimioterapia antitumoral.
Mediante Taqman Low Density Arrays (TLDA) y RT-QPCR convencional
154
GLUT12: functional mechanism
and pathophysiological implications
Eva Gil-Iturbe, Jonai Pujol-Giménez, Carla Valriveras,
María Pilar Lostao
Universidad de Navarra, Pamplona, ES
GLUT12 belongs to the class III of the facilitative glucose transporter
family SLC2A. It was cloned from the mammary cancer cell line
MCF-7, and its expression was found in human crude membranes of
adipose tissue, small intestine and skeletal muscle, where it responds
to insulin. GLUT12 was also found in mammary gland and prostate
Salamanca 2016
tumors, where it may act as a key in the energy supply to malignant
cells. Studies from our group, expressing GLUT12 in Xenopus laevis
oocytes, have characterized some of its functional properties: GLUT12
is able to transport a wide variety of sugars, including α-methyl-glucose
(αMG), a characteristic substrate of the SGLT family. Sugar transport
is increased in the presence of extracellular Na+, is voltage dependent
and up-regulated by PKA. Strikingly, GLUT12 is electrogenic and the
glucose-induced currents are driven by a chloride conductance, which
is uncoupled to the substrate transport. In small intestine, GLUT12 is
located in the apical cytoplasm, below the brush border membrane of rat
and human enterocytes; in the perinuclear region of human enterocytes
and in rat basolateral cytoplasm. Interestingly, GLUT12 is expressed in
brush border membrane vesicles of Caco-2 cells where it is upregulated
by its substrates. The pro-inflammatory cytokine TNFα increases αMG
uptake by GLU12 through the translocation of the transporter to the
apical membrane. GLUT12 protein is also expressed in retroperitoneal
fat from mouse and rat and 3T3-L1 cells. Immunohistochemistry
assays localize GLUT12 around the nuclei of the adipocytes, nerves
and arterial myocytes. Uptake of αMG by GLUT12 in 3T3-L1 cells is
increased by insulin. Finally, GLUT12 is overexpressed in the frontal
cortex of Alzheimer disease patients and in several distinct cancer cell
lines, supporting GLUT12 role in the glycolytic metabolism of cancer
cells. Overall, the functional features found for GLUT12 are very
different from those of the other GLUT isoforms, indicating that it must
have a specific physiological role within glucose homeostasis, still to
be discovered.
P20r-5
Tráfico intracelular y regulación de variantes
del transportador neuronal de glicina GlyT2
asociadas a hiperplexia humana
Andrés de la Rocha Muñoz1, Enrique Núñez1, Carmen Aragón2,
Beatriz López-Corcuera2
1
Departamento de Biología Molecular and Centro de Biología
Molecular Severo Ochoa, Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid, Madrid, ES,
2
Departamento de Biología Molecular and Centro de Biología
Molecular Severo Ochoa, Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid; IdiPAZ-Hospital
Universitario La Paz, Universidad Autónoma de Madrid, Madrid, ES
La hiperplexia o enfermedad de sobresalto es un síndrome clínico
raro caracterizado por reacciones motoras exageradas frente a
estímulos triviales auditivos o táctiles. Se origina por disfunción de la
neurotransmisión glicinérgica inhibidora, pudiendo producir la muerte
del neonato por problemas cardiorrespiratorios. El transportador
neuronal de glicina GlyT2 aporta neurotransmisor a la presinapsis desde
la hendidura sináptica para el rellenado de vesículas y su posterior
reutilización. Por ello, algunas mutaciones en este gen humano
(SLC6A5) producen una forma presináptica de la enfermedad. Se han
asociado a hiperplexia unas 30 mutaciones en GlyT2. La mayoría son
autosómicas recesivas y causan la inactividad del transportador o su
ausencia de la membrana. En este trabajo se estudian las propiedades
de tráfico intracelular y regulación de cuatro mutantes puntuales
de GlyT2 presentes en pacientes de hiperplexia humana que son
parcialmente activos. Mediante ensayos de acumulación de glicina
tritiada y biotinilación de superficie de los transportadores expresados
en cultivos celulares (COS7, MDCK II, neuronas primarias), hemos
demostrado que las variantes difieren en la expresión en membrana y en
la velocidad máxima del transporte, lo que sugiere una alteración de su
tráfico intracelular. Utilizando técnicas inmunoquímicas se estudiarán
los puntos de retención intracelular y su capacidad de ubiquitinación,
así como las alteraciones en la unión a proteínas del interactoma de
GlyT2.
Pósters / Posters
P20-6
Participation of brain aquaporins in adult
hydrocephalus associated to hypoxia and aging
Miriam Echevarría, José Luis Trillo-Contreras, Reposo
Ramírez-Lorca, Ismael Sánchez-Gomar, Ana Galán-Cobo,
Nela Suárez-Luna, Magdalena Olivares Blanco, María Oliver,
Juan José Toledo-Aral, Javier Villadiego
Instituto de Biomedicina de Sevilla (IBiS), Campus Hospital
Universitario Virgen del Rocío, Sevilla, ES
Aquaporins (AQPs) are integral membrane proteins that allow transport
of water across cell membranes. The demonstration that AQP1 and AQP4,
expressed in choroid plexus (CP) epithelial cells (AQP1) and ependymal
cells bordering the intraventricular compartments (AQP4) play an important
role in the cerebrospinal fluid (CSF) production, make us to think that both
proteins may have an important role on the onset of adult hydrocephalus.
The description by our group that AQP1 expression is up-regulated in the
CP of animals exposed to low oxygen tension (10% O2 for 48h), led us to
propose that overexpression of brain AQPs during hypoxia may yield to
an increment in CSF production an thus may participate in the origin of
adult hydrocephalus. We explored in mice exposed to hypoxia for 2-5 days
whether expression levels of AQP1 and AQP4 in different regions of the
brain were affected. Cortex, striatum, ependyma and CP, were analyzed
separately both for mRNA and protein levels under control conditions
and after hypoxia. Magnetic resonance images (MRI) and intracranial
pressure were evaluated in both conditions using young and aged
animals. Moreover, in patients with hydrocephalus of different origins, we
evaluated by ELISA assays the amount of AQP1 and AQP4 shed to the
CSF to establish a correlation between amount of AQPs and hydrocephalus
condition in humans. A general increaseof mRNA and protein for brain
AQPs was observed with the hypoxic treatment, evaluated by RT-qPCR,
western blot and immunocytochemistry assays. In animals exposed to
hypoxia ventricular dilatation was observed by MRI being clearly larger
the ventricle volume in aged animals. AQPs levels in human CSF also
varied upon the hydrocephalus condition. Our results indicate a likely
correlation between AQPs and hydrocephalus in humans and animals. As
a working hypothesis both conditions, hypoxia and aging, acting together
could be crucial to explain the developing of the hydrocephalus state in
adult patients. However, more experiments are needed to strength this
association and further explore the role of AQPs on adult hydrocephalus.
P20-7
An assay to quantify, at the cellular scale,
the membrane binding profile of protein
assemblies involved in vesicle transport
Irene Pazos Capell, Ana García, Carla Belmonte, Nereida Jiménez,
Oriol Gallego
IRB Barcelona, Barcelona, ES
Vesicle trafficking is fundamental to distribute correctly lipids and proteins
among cellular compartments. A complex network of interconnected
pathways transports vesicles between organelles according to the cellular
demand. However, the study of vesicle trafficking is often limited to local
routes between two organelles and ignores its contribution at the global
cellular scale. PICT (Protein interactions from Imaging of Complexes after
Translocation) is a live-cell imaging technique to study the interaction
between two proteins. Here we have used an assay based on PICT to
quantify the membrane binding profile of protein assemblies in yeast. We
have further developed the method to increase the sensitivity two orders
of magnitude and to quantify the association between two proteins bound
to the same membrane. MTCs (Multisubunit Tethering Complexes) are
a group of 9 protein assemblies conserved from yeast to human that work
as long distance connectors between the vesicle and the acceptor organelle,
providing specificity and directionality to vesicle trafficking. We used a set
155
Pósters / Posters
of 19 proteins that localize in specific cellular compartments as markers to
quantify the binding of MTCs to each of these membrane compartments of
the cell. We then quantified the effect on MTCs membrane binding profile at
the cellular scale upon perturbing locally the trafficking in the trans-Golgi–
endosomes transport. Overall, these results shed light on the physiological
role of MTCs and the dependency among distant vesicle transport routes.
This assay could find applications in the study of other protein assemblies
and to understand the interplay between different trafficking pathways.
P20r-8
Papel del transportoma en la sensibilidad
al sorafenib en líneas celulares de leucemia
mieloide aguda
Anabel Sánchez-Martín1, Óscar Briz2, Gabriela Rodríguez-Macías3,
Luis I. Sánchez-Abarca4, María D. Odero5, José J.G. Marín2,
Rocío I.R. Macías2
1
IBSAL, Salamanca, ES, 2Laboratorio de Hepatología Experimental
y Vectorización de Fármacos, Universidad de Salamanca, IBSAL.
CIBERehd , Salamanca, ES, 3Servicio de Hematología, Hospital
Gregorio Marañón, Madrid, ES, 4Servicio de Hematología, Hospital
Universitario de Salamanca, IBSAL, Salamanca, ES, 5Departamento
de Bioquímica y Genética, Universidad de Navarra, Pamplona, ES
Introducción: Las mutaciones en el receptor FLT3 justifican la aparición
de resistencia al sorafenib en pacientes con leucemia mieloide aguda
(LMA), aunque otros mecanismos aún desconocidos contribuyen a la falta
de respuesta. El objetivo fue investigar si los niveles de expresión de las
proteínas implicadas en la captación y el eflujo de sorafenib se relacionan
con la sensibilidad de líneas celulares al fármaco.
Métodos: El efecto sobre la viabilidad celular se determinó mediante el test de
formazán y con el kit anexina V/7-AAD tras 72 h de exposición a concentraciones
crecientes de sorafenib. La expresión de los genes de interés se determinó por
RT-PCR y la funcionalidad de las proteínas por citometría de flujo.
Resultados: La sensibilidad de las líneas celulares al sorafenib fue
MOLM-13>>HL-60>HEL≈K-562. Los niveles del transportador OCT1
fueron mayores en células MOLM-13 que en HL-60 y no se detectaron
en HEL y K-562. En cuanto a las bombas de eflujo, la MDR1
estaba sobre-expresada en células HEL>K-562≈MOLM-13 y no se
detectó en HL-60. La expresión de BCRP fue moderada en células
HEL>K-562>HL-60>MOLM-13, los niveles de MRP3 fueron bajos en
las células MOLM-13 y K-562 y no se detectaron en HL-60 y HEL y
todas las líneas presentaron una elevada expresión de MRP4. Las células
MOLM-13, además de la mutación FLT3-ITD presentaron niveles 1000
veces superiores de FLT3 al resto de líneas. Los resultados de funcionalidad
de los transportadores confirmaron la relación entre la mayor capacidad de
las células de retener sorafenib y su sensibilidad a este fármaco.
Conclusión: Junto con los niveles de las dianas del fármaco y la presencia
de mutaciones, la expresión de proteínas de captación y eflujo de sorafenib
contribuye a la sensibilidad de las células de LMA a este fármaco.
P21. Grupo invitado: Biología química /
Invited group: chemical biology
P21-1
Generation of ubiquitinated p15 protein
for structural studies
Amaia González Magaña1, Nekane Merino1, Alfredo De Biasio2,
Francisco J. Blanco3
1
CIC bioGUNE, Derio, ES, 2Elettra Sincrotrone, Trieste, IT, 3CIC
bioGUNE; IKERBASQUE Basque Foundation for Science, Bilbao, ES
156
XXXIX Congreso SEBBM
p15PAF (proliferating-cell-nuclear-antigen-associated factor, hereafter
p15) is an intrinsically disordered protein of 111 amino acids with
increased expression in tumors(1). It binds PCNA through its conserved
PCNA-interacting protein motif, the PIP-box, located in the central
region. It also binds DNA, mainly through its N-terminal region. The
binding to PCNA and DNA occurs simultaneously and independently
with similar affinity (Kd = 1.1 μM at 25 ºC). This mode of binding
suggests that p15 acts as a flexible regulator of the velocity of PCNA
sliding on the DNA(2). Recent studies have shown that p15 facilitates
the switch from replicative to translesion synthesis polymerases at
stalled replication forks. This role is related with the existence in the
cell of a fraction of PCNA-bound p15 ubiquitinated at Lys 15 and Lys
24 during normal S-phase progression(3). Monoubiquitination at these
two Lys residues should not affect PCNA binding but would reduce
the affinity of p15 for the DNA as it removes two positive charges
and introduces steric hindrance. To confirm this hypothesis we are
preparing milligram amounts of ubiquitinated p15. We are following
a non-enzymatic strategy that results in a disulfide directed ligation
mimicking the isopeptide bond between the p15 Lys side chain and
the carboxyl group at ubiquitin C-terminus(4). We have produced a
p15 mutant with Lys 15 and 24 mutated to Cys and two native Cys
residues mutated to Ser. An ubiquitin with a C-terminal reactive thiol
group is prepared from a fusion protein with an intein that is cleaved
with cysteamine. The ubiquitinated p15 protein has been generated and
purified with high homogeneity but low yields. Structural analysis by
circular dichroism and multiangle light scattering indicate that the p15
chain remains disordered and monomeric while the bound ubiquitin is
cooperatively folded.
P21r-2
Alteraciones en la estructura y función
de la Conexina 43 en melanoma
Adrián Varela-Vázquez1, Paula Carpintero-Fernández1,
María L. Díaz2, María C. Vázquez-Blanco1, Ángel Fernández-Flores3,
Carmen Rivas2, Eduardo Fonseca1, María D. Mayán1
1
Grupo de Investigación CellCOM. Instituto de Investigación
Biomédica de A Coruña (INIBIC). Complexo Hospitalario
Universitario de A Coruña (CHUAC-XXIAC). Universidade da Coruña.
Sergas, ES, 2Grupo Virus and Cancer. CIMUS, USC, Santiago de
Compostela, ES, 3Servicio de Anatomía Patológica. Hospital del
Bierzo, Ponferrada, ES
Las conexinas (Cx) son proteínas transmembrana que forman canales
que permiten la comunicación directa célula-célula a través de uniones
comunicantes (UC) y célula-matriz a través de hemicanales. En
las distintas capas de la epidermis se han detectado diferentes Cxs,
siendo la Cx43 la más ampliamente expresada. La Cx43 es esencial
para la función de barrera de la piel, y ha sido descrita como factor
condicional supresor de tumores presentando diferente actividad
durante el desarrollo del tumor y el proceso de metástasis. Resultados
de co-IP, inmunofluorescencia y western-blot (líneas celulares y tejidos
de pacientes) evidenciaron que la Cx43 en melanoma se encuentra
sumoilada, junto con disminución en los niveles de la proteína y
alteraciones en la localización subcelular. Ensayos de scrape loading
demostraron una pérdida total de comunicación celular a través de
UC. La restauración de los niveles de la Cx43 en líneas celulares de
melanoma rescató la comunicación célula-célula reduciendo los niveles
de migración y proliferación celular. Los resultados obtenidos sugieren
que alteraciones en la estructura y niveles de la Cx43 asociados con
cambios en la localización celular de la proteína podrían impedir la
comunicación directa célula-célula a través de UC, alterar proliferación
y migración celular y por tanto estar jugando un papel importante en el
desarrollo o malignidad del melanoma.
Salamanca 2016
P21r-3
The invasion of glioblastoma stem cells
is reduced by the sequence of the connexin43
that interacts with c-Src
Myriam Jaraíz Rodríguez1, Mª Dolores Tabernero2, María
González-Tablas3, Álvaro Otero4, Alberto Orfao3, José M. Medina1,
Arantxa Tabernero1
1
Instituto de Neurociencias de Castilla y León, Universidad de
Salamanca, Salamanca, ES, 2Instituto de Estudios de Ciencias
de la Salud de Castilla y León (IECSCYL); Research Laboratory
of the University Hospital of Salamanca, Instituto de Investigación
Biomedicina de Salamanca (IBSAL); Centre for Cancer Research
(CIC-IBMCC; CSIC/USAL; IBSAL), Salamanca, ES, 3Centre for
Cancer Research (CIC-IBMCC; CSIC/USAL; IBSAL); Department
of Medicine, Universidad de Salamanca, Salamanca, ES,
4
Neurosurgery Service of the University Hospital of Salamanca
and IBSAL, Salamanca, ES
Glioblastoma stem cells (GSC) constitute a niche of cells with self-renewal
ability and resistance to conventional therapies. Thus, they are responsible
for relapse in these aggressive brain tumors when their resection is not
complete. The expression of connexin43 (Cx43), the main gap junction
channel-forming protein, and c-Src, a non receptor tyrosine kinase, is
inversely related in glioblastoma cells. Interestingly, restoring Cx43 in
GSC reverses GSC phenotype. The sequence responsible for this effect
is the region that interacts with c-Src (amino acids 266-283). The Focal
Adhesion Kinase (FAK) requires the c-Src-mediated phosphorylation in
tyrosines 576 and 577 in order to be fully active and promote cell migration.
In this study, fresh human tumour biopsies were disaggregated to obtain
human primary GSCs. These cells were treated with a cell-penetrating
peptide containing the Cx43-Src interacting sequence (Tat-Cx43266-283). The
migration was analyzed by tracking individual cell trajectories in cultures
recorded by Time-Lapse Live-cell Imaging. Matrigel-treated transwell
inserts were used to analyze the invasion and the levels of phosphorylation
of FAK tyrosines 576 and 577 were evaluated by western Blot. Our results
showed that Tat-Cx43266-283 reduced the migration and invasion of human
primary GSC. The analysis of c-Src and FAK proteins suggests that the
mechanism by which this reduction takes place includes the inhibition of
c-Src/FAK axis.
In conclusion, Tat-Cx43266-283 inhibits c-Src with the subsequent reduction
in FAK phosphorylation required to establish appropriate focal adhesions
for migration. All together, these results indicate an important antivasive
role of the sequence of Cx43 that interacts with c-Src in glioma stem cells.
P21-4
Whole body imaging and therapeutic targeting
of aggressive cancers: focus on premetastatic
niches
Maria Soengas, David Olmeda
Melanoma Laboratory, Molecular Oncology Programme, Spanish
National Cancer research Centre (CNIO), Madrid, ES
Cutaneous melanoma is the deadliest form of skin cancer. Although the
presence of tumor cells in lymph nodes is a poor prognosis indicator, the
specific contribution of lymphangiogenesis to the mortality associated
with melanoma metastasis remains unclear. This is mainly due to the lack
of tumor markers and experimental models for non-invasive imaging of
lymphangiogenesis in vivo. To address molecular mechanism(s) regulating
metastatic spread of melanoma, we developed a series of genetically
engineered mouse strains derived from a “lymphoreporter model” in
which a luciferase cassette is expressed as a reflection of the endogenous
Flt4 (VEGFR3) promoter. These “lymphoreporter” melanoma models
allowed tracing of metastatic dissemination from very early stages of
melanoma development. In particular, we have identified distinct patterns
Pósters / Posters
of melanoma-driven neo-lymphangiogenesis activated at distal sites before
the actual colonization by tumor cells. Proteomic analysis of the melanoma
secretome revealed new pro-lymphangiogenic factors that promote tumor
metastasis. Here we will present the prognostic value of these metastatic
genes and discuss the use of the lymphoreporter mice for the screening
of anti-metastatic agents. Together, these results support the VEGFR3-luc
mouse melanoma models as a cost-effective strategy for gene discovery
in the context of otherwise aggressive and intrinsically metastatic tumors.
P21-5
IFN-γ receptor signaling is critically dependent
on its location in lipid nanodomains
Ornella Morana1, Cedric Blouin2, Dalila Ciceri1, Maier Lorizate1,
Christophe Lamaze2, Xabier Contreras3
1
Instituto Biofisika (CSIC-UPV/EHU), and Departamento de
Bioquímica, Universidad del País Vasco, Bilbao, ES, 2Institut Curie
- Centre de Recherche, Membrane Dynamics and Mechanics of
Intracellular Signaling Laboratory. Centre National de la Recherche
Scientifique (CNRS), UMR 3666, París, FR, 3Instituto Biofisika
(CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad
del País Vasco. IKERBASQUE, Basque Foundation for Science,
Leioa, ES
Interferon-γ (IFN-γ) is a cytokine that orchestrates many critical cell
functions and signaling processes through transcriptional activation and
regulation of a vast number of genes. Among others, INF-γ plays crucial
roles in controlling host defense, immunopathological processes, and
fighting tumors. IFNγ mediates its pleiotropic effects on cells binding to
a receptor (IFNGR) expressed on the membrane surface of a large variety
of cells. However, understanding how membrane nanoscale organization
controls transmembrane receptors signaling activity remains a challenge
mainly due to a lack of accurate methodology. Here, we show that
IFNGR localizes, in vivo, in lipid nanodomains and that IFN-γ addition
induces a conformational change in IFNGR that leads to a specific
IFNGR-sphingolipid interaction that could play a role as a docking site
for receptor phosphorylation and JAK-STAT signaling cascade activation.
This experiment of nature established the critical importance of dynamic
interactions with lipid nanodomains in IFN-γR signaling.
P21-6
NMR, molecular recognition and chemical
glycobiology
Jesús Jiménez-Barbero
CIC BIOGUNE, Derio, ES
Molecular recognition by specific targets is at the heart of the life
processes. In recent years, it has been shown that the interactions between
proteins (lectins, enzymes, antibodies) and carbohydrates mediate a
broad range of biological activities, from fertilization, embryogenesis,
and tissue maturation, to pathological processes. The elucidation of
the mechanisms that govern how glycans are accommodated in the
binding sites of these receptors is currently a topic of interest. Thus,
the determination of the structural and conformational factors and the
physicochemical features that govern the molecular recognition of these
molecules is of paramount importance. This presentation is focused on
the application of NMR methods both from the ligand and receptor’s
perspective (especially HSQC-based chemical shift perturbation analysis
and 19F-based Saturation Transfer Difference) to the study of molecular
recognition processes between a variety of polypeptides of biomedical
interest and carbohydrate-based molecules, drugs and inhibitors. The use
of novel lanthanide-binding tags[1, 2] and 19F-containing glycomimetics [3]
as probes for monitoring interactions from the ligand’s point of view will
be highlighted. Different glycan receptors, both wild type and mutants,
157
Pósters / Posters
have been used as receptors with the final aim to know and to evaluate the
relative importance of polar (hydrogen bonding, electrostatic interactions)
and non polar (van der Waals, CH-π) forces in the molecular recognition
processes and to design novel molecules with improved binding properties.
As examples, the structural and conformational details of the effect of
branching in the glycan chain and how this affects its recognition by
lectins, enzymes, and antibodies will be shown.
References
[1] Á. Canales, Á. Mallagaray, M.Á. Berbís, A. Navarro-Vázquez, G.
Domínguez, F.J. Cañada, S. André, H.J. Gabius, J. Pérez-Castells, J.
Jiménez-Barbero, J Am Chem Soc. 136, 8011-7 (2014).
[2] A. Canales, A. Mallagaray, J. Pérez-Castells, I. Boos, C. Unverzagt, S.
André, H.J. Gabius, F.J. Cañada, J. Jiménez-Barbero, Angew Chem Int Ed
Engl. 52, 13789-93 (2013).
[3] L.P. Calle, B. Echeverria, A. Franconetti, S. Serna, M.C.
Fernández-Alonso, T. Diercks, F.J. Cañada, A. Ardá, N.C. Reichardt, J.
Jiménez-Barbero, Chem Eur J. 21, 11408-16 (2015).
P21-7
Toll-like receptors modulation:
computational studies and drug repositioning
Sonsoles Martín-Santamaría, Lucía Pérez-Regidor, Joan
Guzmán-Caldentey, Jean-Marc Billod, Laura Ortega, Malik Zarioh,
Alessandra Lacetera
Centro de Investigaciones Biológicas, CIB-CSIC, Madrid, ES
Innate immunity is the first defensive wall a pathogen needs to beat to
flourish in our body and, due to the enormous diversity of microorganisms
present in our environment, it is composed by a number of agents able
to respond in a highly effective way. Toll-like receptors (TLR) family
can recognize a wide variety of pathogens, making them an interesting
target to help our body fight the disease. Each TLR is specialized in the
recognition of a particular pathogen-associated molecular pattern arisen
from a bacteria or virus. TLR modulators have the potential to be used
with different biomedical applications, especially in the field of infection,
inflammation and autoimmune diseases, but also in central nervous system
disorders such as Alzheimer´s disease, and cancer. However, just a few of
them are currently under clinical development. Therefore, it is imperative
to find new chemical entities as TLR modulators with drug-like properties
in order to facilitate their development as drugs. Our group has directed
its efforts towards the finding of novel TLRs modulators (in particular,
TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2 systems) by virtual screening
and drug repositioning approaches, leading to the identification of novel
TLR4 antagonists with advantageous drug-like properties. We have also
applied different computational and molecular modeling tools to the study
of the mechanism of the TLR4/MD-2 system and the molecular recognition
by different ligands: we have studied the dynamics of the MD-2 binding
pocket and its ability to accommodate lipid A,(1) and we have also provided
a molecular model for activation of the human TLR4/MD-2 complex by
penta-acylated lipid A from B. cenocepacia LPS.(2) Our computational
approaches have also assisted to the rational design of glycolipid-based
TLR4 modulators and fluorescent probes.(3) Finally, we also here report
the computational study of the dynamics of the full TLR4/MD-2 system
(extracellular + intracellular + transmembrane domains) inserted in the
membrane environment by MD simulations aiming to understand the
dimerization mechanism at atomic detail.
References
[1] J. Vasl; A. Oblak; T. T. Peternelj; J. Klett; S. Martín-Santamaría; T. L.
Gioannini; J. P. Weiss; J. Jerala. J. Immunol. 2016, 196, 2309-18.
[2] F. Di Lorenzo; L. Kubik; A. Oblak; N. I. Lore; C. Cigana; R. Lanzetta;
M. Parrilli; M. A. Hamad; A. De Soyza; A. Silipo; R. Jerala; A. Bragonzi;
M. A. Valvano; S. Martin-Santamaria; A. Molinaro. J. Biol. Chem. 2015,
290, 21305-19.
158
XXXIX Congreso SEBBM
[3] C. Ciaramelli; V. Calabrese; S. E. Sestito; L. Pérez-Regidor; J. Klett; A.
Oblak; R. Jerala; S. Martín-Santamaría; F. Peri. Chem. Biol. & Drug Des.,
2016, doi: 10.1111/cbdd.12749.
P21-8
Synthetic probes and tools for biological
intervention
José Luis Mascareñas Cid
Universidade de Santiago de Compostela, Santiago de Compostela, ES
The ability to synthesize molecules is at the heart of the progress in
many branches of science. Therefore there is a continuum need to devise
practical and efficient synthetic methods that allow to construct relevant
products from readily available starting materials in a sustainable and
reliable manner. In this context, one of our research lines is focused to
the development of new and efficient synthetic processes.(1) As molecular
makers we are also highly interested in the design and synthesis of
molecules that can lead to new type of biomolecular recognition processes,
or promote specific biological and cellular responses. An important part
of our work has been focused to devise molecular agents and strategies
for the interaction with specific DNAs. Some of these derivatives take
advantage of the coordination capability of late transition metals to attain
their recognition properties.
We have also developed variations that not only bind the designed targets
but are also capable of emitting a signal upon that interaction, therefore
working as selective biosensors.(2)
References
[1] F. López, J.L. Mascareñas Chem. Soc. Rev. 2014, 2904. M. Gulías, J.
L. Mascareñas Angew. Chem. Int. Ed. 2016, 10.1002/anie.201511567R1.
[2] M. I. Sánchez, J. Martínez-Costas, F. González, F., M. A. Bermúdez,
M.E. Vázquez, J. L Mascareñas ACS Chem. Biol. 2012, 7, 1276. C. Penas,
E. Pazos, J. L. Mascareñas, M. E. Vázquez J. Am. Chem. Soc. 2013, 135,
3812. M. I. Sánchez, C. Penas, M. E. Vázquez, J. L. Mascareñas Chem.
Sci. 2014, 5, 1901. J. Rodríguez, J. Mosquera, J. R. Couceiro, M. Eugenio
Vázquez, J. L. Mascareñas Chem. Sci. 2015, 6, 4767.
P21-9
Protein-targeted dynamic combinatorial
chemistry in drug discovery
Ruth Pérez-Fernández1, L. Martínez-González1,
M.J. Sánchez-Barrena2, A. Martínez1
1
Centro de Investigaciones Biológicas, CIB-CSIC, Madrid, ES,
2
Instituto de Química Física Rocasolano, IQFRS-CSIC, Madrid, ES
The development of synthetic molecules that mimics the precision of nature
in molecular recognition represents a huge challenge to chemists. Dynamic
combinatorial chemistry (DCC) holds enormous potential for drug
discovery speeding the identification and optimization of novel ligands for
biological targets. In DCC, building blocks react with one another using
reversible chemical reactions giving mixtures of oligomers (Dynamic
Combinatorial Libraries, DCLs). If a protein is added to the system and one
or more molecules show affinity to it, these building blocks will, according
to the Le Chatelier’s principle, be amplified on the expense of the other
non-bonding constituents. We present the discovery of novel ligands of
Frequenine-2 (Frq2), a high-affinity Ca2+-binding protein conserved from
yeast to humans (named Neuronal Calcium Sensor-1). Frq2 is involved in
pathologies that result from an abnormal synapse number such as Fragile X
syndrome (FXS). Fragile X syndrome is the most common inherited cause
of intellectual disability and a common known single gene cause of autism
spectrum disorders.
Salamanca 2016
P21r-10
pH-labile PGA-Doxorubicin based combination
conjugates with enhanced antitumor activity
in breast cancer models
Juan José Arroyo Crespo, David Charbonnier, Ana Armiñán,
María Jesús Vicent Docón
Centro de Investigación Príncipe Felipe, Valencia, ES
Introduction: It has been already demonstrated that coupling of small
drugs in a synergistic ratio to the same polymer ensures the arrival of both
drugs to the same cell at the same time, yielding to an improved therapeutic
index, and allowing to control the drug release kinetics profile and reduce
side effects1,2. We have previously reported a novel PGA-AGM-DOX
conjugate family, bearing the combination of Aminoglutethimide (AGM)
with Doxorubicin (Dox), in which both drugs were attached to the polymer
through different protease-cleavable linkers, such as G, GG or GFLG,
being PGA-GAGM10-DOX5 the conjugate showing the greatest antitumor
activity in an orthotopic 4T1 breast cancer model3.We now propose the use
pH-labile hydrazone linkers as a strategy to delivery DOX in a new family
of PGA-AGM-DOX conjugates.
Pósters / Posters
Results
and
discussion:
PGA-GAGM10-hyd-DOX5
and
PGA-GAGM10-EMCH-DOX5 families have been developed bearing
pH-sensitive hydrazone linkers. Looking for P.Q. descriptors, a deep
characterization of the conjugates has been carried out. Conjugates have a
concentration-dependent behavior, showing the capacity to self-assemble
in aggregates up to 200 nm in aqueous solution, with differences in either
their critical aggregation concentration and/or size, depending on the
linker and on the presence of one or both drugs. CD analysis revealed
a clear trend for some of the conjugates to self-assemble as an alpha
helix with greater rigidity, data in agreement with the release kinetics
observed. Cell viability in a representative model is coherent with such
release profiles. Conjugates showing the greatest in vitro activity were
chosen for further in vivo evaluation in an orthotopic and metastatic 4T1
murine breast tumor model to analyze, not only the antitumor activity, but
also their capacity to reduce metastasis progression.
References
[1] Duncan R. J Control Release. 2014, 190:371-80.
[2] Greco, F, Vicent M.J, Adv. Drug. Deliv. Rev. 2009, 61(13):1203-13.
[3] C. Deladriere, J.J. Arroyo, E. Masiá, V.J. Nebot, A. Armiñán, M.J.
Vicent. Submitted.
PW1. Enseñanza de la bioquímica / Teaching biochemistry
PW1-1
PW1-2
El origen de la vida en la Tierra y fuera de ella:
una perspectiva bioquímica
Implementación del modelo flipped classroom
y videotutoriales como herramientas para la
mejora del autoaprendizaje del alumno en el
grado de Bioquímica y estudios de posgrado
relacionados
Juan Antonio Aguilera Mochón
Dpto. Bioquímica y Biología Molecular I, Universidad de
Granada, Granada, ES
Como dijo Günter Wächtershäuser, “Una teoría de la evolución
temprana debe tener como propósito principal el problema de explicar la
Bioquímica”. Lo mismo cabe decir, por supuesto, respecto a las teorías
sobre el origen de la vida. No cabe, pues, eludir los aspectos bioquímicos
en estas materias, ni siquiera cuando se trate de divulgarlas.
Con estas ideas de principio, el autor ha intentado trasladar, en un
lenguaje lo más asequible posible, su experiencia de más de una
década de enseñanza de “Bioquímica evolutiva” en un libro divulgativo
acerca de “El origen de la vida en la Tierra” (RBA, 2016). ¿Cuándo
ocurrió, y en qué escenario?, ¿cómo se pudo lograr la complejidad que
alcanzó el último pasado común universal (LUCA)?, ¿cómo se gestó
el código genético y se desarrolló el metabolismo?, ¿cómo aparecieron
los eucariotas?… Carecemos de respuestas firmes, pero las hipótesis —
avaladas en lo posible por datos experimentales y filogenéticos— son
fascinantes.
Este análisis sobre la biogénesis en nuestro planeta es imprescindible de
cara a especular con alguna base sobre las posibilidades de vida en otros.
¿Qué condiciones se necesitan para el desarrollo de una bioquímica
similar a la nuestra en otros mundos? Y, además, ¿son posibles otras
bioquímicas, basadas en la química del carbono o no? Todas estas
preguntas las ha abordado el autor en otro libro de divulgación científica,
“La vida no terrestre” (RBA, 2016), en el que intenta acercarse con rigor
a unos temas fácilmente propensos a la pseudociencia.
Los dos textos, aunque parten de una base sencilla, podrían (sobre
todo merced a recuadros más técnicos) servir de recurso al profesorado
de Bioquímica para ofrecer a los alumnos un enfoque evolutivo,
imprescindible (recordemos a Dobzhansky) en cualquier explicación
biológica.
Joan Ribot, Paula Oliver, Juana Sánchez
Laboratorio de Biología Molecular, Nutrición y Biotecnología
(Nutrigenómica y Obesidad), Universidad de las Islas Baleares
(UIB) y CIBER Fisiopatología de la Obesidad y Nutrición
(CIBEROBN), Palma de Mallorca, ES
Hemos incorporado el uso del modelo de flipped classroom y
videotutoriales tratando de potenciar el interés del estudiante en la
resolución y ejecución de actividades propuestas y favorecer un ambiente
de aprendizaje colaborativo e interactivo en estudios de grado y posgrado.
En la asignatura “Bioinfomática de Sistemas Aplicada”, los alumnos
debían analizar los resultados de un microarray (actividad obligatoria),
para lo que disponían de bibliografía y de vídeo tutoriales preparados por
el profesor. En la asignatura “Bioquímica Analítica y Clínica”, los alumnos
(actividad grupal y optativa) debían preparar un vídeo-presentación sobre
“nuevos biomarcadores” de una enfermedad. Finalmente, en la asignatura
de posgrado “Nutrición Molecular”, los alumnos (actividad grupal y
obligatoria) debían preparar un proyecto de investigación y defenderlo
delante de sus compañeros, que los evaluaron.
Se realizó una encuesta de satisfacción a los alumnos de grado y se valoró
el impacto de las actividades propuestas sobre sus notas. Participaron
19 alumnos (actividad obligatoria) y 7 de 45 (optativa). Contestaron la
encuesta un 75% de los alumnos participantes.
La aceptación de los videotutoriales por parte de los alumnos fue
excelente. Los alumnos consideraron las actividades de dificultad media
invirtiendo las mismas o menos horas que en otras asignaturas con carga
lectiva similar. Las actividades resultaron muy interesantes, motivadoras
e útiles para los alumnos que no dudarían en repetir (9 de cada 10), siendo
159
Pósters / Posters
el grado de satisfacción global muy alto (8,5 sobre 10). La realización de
estas actividades les supuso un incremento significativo en la nota final.
Destaca que las evaluaciones de los propios alumnos a sus compañeros
fueron muy homogéneas y más exigentes que las del profesor.
Estos resultados reflejan la buena aceptación entre los alumnos y
nos anima a continuar con el uso del modelo de flipped classroom y
videotutoriales como herramientas para la mejora del autoaprendizaje
en los estudios de Bioquímica.
Financiado por Institut de Recerca i Innovació Educativa de la UIB.
XXXIX Congreso SEBBM
docentes, investigadoras y de gestión de los profesores universitarios
como requisito previo para la contratación de éstos en alguna de las
figuras establecidas en la mencionada Ley. En particular, establece que
la contratación de Profesores Ayudantes Doctores (PAD) y Profesores
Contratados Doctores (PCD) se celebrará entre doctores que hayan
obtenido la evaluación positiva por parte de la ANECA o del órgano de
evaluación externo que la Ley de la Comunidad Autónoma determine.
La ponencia pretende aportar información práctica sobre la estructura
de los programas, los criterios de evaluación establecidos en las
correspondientes resoluciones de la Dirección General de Universidades
para los campos de las Ciencias Experimentales y de las Ciencias de la
Salud y el funcionamiento de los Comités de Evaluación.
PW1-3
Uso de YouTube como recurso didáctico
en el aprendizaje de la Bioquímica
Inmaculada Ballesteros-Yáñez1, Beatriz Rodríguez-Martín2,
Carlos Alberto Castillo Sarmiento2
1
Facultad de Medicina, Universidad de Castilla-La Mancha,
Ciudad Real, ES, 2Facultad de Terapia Ocupacional, Logopedia
y Enfermería, Universidad de Castilla-La Mancha, Talavera
de la Reina, ES
Introducción: Desde la implantación del Espacio Europeo de Educación
Superior, la formación integral del estudiante se basa en la adquisición
de competencias, actitudes y habilidades desde el inicio de sus estudios
de Grado y no solo en conocimientos teóricos.
Objetivo del profesor: Fomentar la adquisición de competencias
potenciando el uso de herramientas gratuitas de publicación en red, que
permitan a nuestros alumnos un contacto más directo con la sociedad, en
este caso la red social YouTube.
Objetivo de los alumnos: Realizar un vídeo divulgativo de la
resolución de un caso práctico con los medios de grabación y edición
que consideraran convenientes.
Metodología: Como trabajo grupal se propuso resolver un caso práctico
a elegir entre un listado de 33 problemas complejos que incluían 1 o
varios temas de la asignatura. Tras la entrega del caso, se debía grabar
y editar en formato audiovisual un material que explicara de forma
sencilla el caso resuelto. La única restricción era que el vídeo sería
público (soportado en YouTube) y no debía durar más de 10 minutos.
Resultados y conclusiones: Se presentan fragmentos de vídeos realizados
por los alumnos que demuestran que, además de competencias específicas
de la asignatura, se trabajaron de forma satisfactoria competencias básicas
(ej: aplicar conocimientos a su vocación), transversales (ej: dominio de
las TIC) y generales (ej: demostrar dotes de innovación), algunas de las
cuales no estaban contempladas en la guía de la asignatura. Estos trabajos
ponen de relevancia el valor a explotar de este tipo de herramientas no
solo como potenciadores del aprendizaje sino también como medios de
información para orientar y cubrir las necesidades de la comunidad en la
que los alumnos desarrollarán sus trabajos.
PW1-4
Orientaciones para superar la evaluación
de Profesor Ayudante Doctor y Profesor
Contratado Doctor
Begoña Ochoa
Departamento de Fisiología, Facultad de Medicina y Enfermería,
Universidad del País Vasco UPV/EHU, Leioa, ES
La Ley Orgánica 4/2007, de 12 de abril, por la que se modifica la Ley
Orgánica 6/2001, de 21 de Diciembre, de Universidades, atribuye
a la Agencia Nacional de Evaluación de la Calidad y Acreditación
(ANECA) funciones relacionadas con la evaluación de las actividades
160
PW1-5
El contrato de aprendizaje en la enseñanza
de la Bioquímica. ¿Un elemento motivador
para el alumnado universitario de Ciencias?
Ángel Luis García-Ponce1, Ángel Blanco-López1, Ana Mª Rodríguez
Quesada2, Francisco J. Alonso Carrión2, María Fernanda Suárez2,
Miguel Ángel Medina Torres2
1
Universidad de Málaga, Andalucía Tech, Departamento de
Didáctica de la Matemática, de las Ciencias Sociales y de
las Ciencias Experimentales, Facultad de Ciencias de la
Educación, Málaga, ES, 2Universidad de Málaga, Andalucía Tech,
Departamento de Biología Molecular y Bioquímica, Facultad de
Ciencias, Málaga, ES
La Bioquímica es una materia que, organizada en sus múltiples
asignaturas, se imparte a lo largo del currículo de algunos grados de
Ciencias (Biología, Química, Bioquímica). Es habitual que la primera
aproximación a esta disciplina que el alumnado afronta sea cursando
una asignatura de Fundamentos de Bioquímica o Bioquímica General,
que a su vez puede estar organizada semestralmente en forma de dos
asignaturas independientes y sucesivas, Bioquímica I y II.
En bastantes casos, los contenidos de esta disciplina, y en concreto los
correspondientes a Bioquímica II, habitualmente dedicada al estudio
del metabolismo, su regulación e integración, es percibida por los
estudiantes como una disciplina muy exigente y de notable dificultad.
Ello hace que, ya casi desde el inicio de curso, muchos estudiantes le
muestren un cierto desapego inicial que se traduce en un retardo en
afrontar su estudio, llegando incluso al abandono temporal de la misma.
En los últimos años, es conocido que, en algunas de nuestras
universidades españolas, las tasas de éxito y rendimiento para esta
asignatura son sensiblemente menores comparadas con las de otras
cursadas en los mismos cursos.
La propuesta pedagógica que se presenta se ha desarrollado en el marco
de un Proyecto de Innovación Educativa de la Universidad de Málaga
(PIE15-163) que se centra en el empleo de una estrategia pedagógica,
el contrato de aprendizaje. Se considera que –sin ser extremadamente
novedosa– esta estrategia puede constituir un elemento motivador para
ese sector del alumnado que encuentra una especial dificultad en el
aprendizaje y asimilación de los contenidos de los fundamentos de la
Bioquímica en general, y del metabolismo y su regulación en particular.
PW1-6
Medida de glucosa en refrescos: una útil
herramienta para ilustrar el aprendizaje
de los fundamentos del análisis enzimático
Beatriz Martínez-Poveda1, Ángel Luis García-Ponce2, Ángel
Blanco-López2, Miguel Ángel Medina Torres1, Ana Mª Rodríguez
Quesada1
Salamanca 2016
1
Universidad de Málaga, Andalucía Tech, Departamento de
Biología Molecular y Bioquímica, Facultad de Ciencias, Málaga,
ES, 2Universidad de Málaga, Andalucía Tech, Departamento de
Didáctica de la Matemática, de las Ciencias Sociales y de las
Ciencias Experimentales, Facultad de Ciencias de la Educación,
Málaga, ES
Es indudable la relevancia que el análisis enzimático tiene en el ámbito
de la Bioquímica, la Química Clínica y la Química de la Alimentación,
entre otras. En la asignatura “Bioquímica Aplicada” de cuarto curso
del grado en Química de nuestra Universidad, los alumnos aprenden
los fundamentos teóricos del análisis enzimático, que luego ponen
en práctica en el laboratorio, en un contexto cotidiano, mediante la
determinación de glucosa en diferentes refrescos carbonatados.
Aunque existen diversos métodos enzimáticos para la valoración de
la concentración de glucosa, hemos elegido para la sesión práctica
el que se basa en las reacciones acopladas de la glucosa oxidasa
(EC 1.1.3.4.) y la peroxidasa (EC 1.11.1.7.) por ser especialmente
ilustrativo a la hora de afianzar los conceptos teóricos desarrollados
en clase, así como para despertar su espíritu crítico al enfrentarles
a la resolución de problemas “del mundo real” y hacerles elegir qué
método analítico puede ser mejor para un caso concreto. Los alumnos
usan dos protocolos distintos (métodos cinético y a punto final) para
determinar la concentración de glucosa, inicialmente en una muestra de
glucosa en agua, familiarizándose con ambos métodos y permitiéndoles
construir las rectas de calibrado, mediante las que deberán establecer el
rango de aplicación para cada método. La medida de las variaciones de
absorbancia con el tiempo, que en el método cinético son proporcionales
a la concentración de glucosa, permite ilustrar aspectos metodológicos
de la estimación de la velocidad inicial de una reacción enzimática.
Posteriormente los alumnos se enfrentan al problema de la medida de la
concentración de glucosa en una serie de bebidas que incluyen refrescos
de cola y tónicas, con o sin azúcar (refrescos “cero”), encontrándose con
nuevas dificultades como son la necesidad de diluir en varios órdenes de
magnitud las muestras (lo que les familiariza con el uso de las diluciones
seriadas), y la aparición de interferencias debidas al color de la muestra,
decantándose por uno u otro método en función de la naturaleza del
problema analítico planteado.
PW1-7
Memoria de 20 años del curso “¿Y tú?
Yo, Bioquímica?”
Josep M. Fernández Novell1, Joan J. Guinovart2
1
Departamento de Bioquímica y Biomedicina Molecular,
Universitat de Barcelona, Barcelona, ES, 2Departamento de
Bioquímica y Biomedicina Molecular, Universitat de Barcelona;
Institut de Recerca Biomèdica, Parc Científic de Barcelona,
Barcelona, ES
El curso Iniciación a la Bioquímica y Biología Molecular para Alumnos
de Secundaria, conocido con el lema “¿Y tú? Yo, Bioquímica”, cumple
este 2016 su 20 aniversario. Al hacer un balance de estos veinte cursos el
resultado numérico es muy alentador así, se debe remarcar que de las más
de 8000 solicitudes presentadas se han preseleccionado y entrevistado a
más de 2000 de los cuales, finalmente, se han escogido 480 excelentes
alumnos, 24 por curso. Se han realizado más de 140 conferencias con
la participación del profesorado universitario involucrado en dicha área.
Estas charlas, con títulos muy sugerentes para los jóvenes participantes,
les han mostrado los últimos avances de la Bioquímica. Además, las
sesiones de laboratorio han estado preparadas con alto rigor científico
por parte de nuestro alumnado predoctoral y pregraduado, donde les
han mostrado algunas de las técnicas que actualmente utilizan los
investigadores. Cada curso termina con el acto de clausura y entrega
de certificados que otorga la UB. El balance “afectivo” nos dice que
Pósters / Posters
ha sido un trabajo coral, sin la ayuda de tantas personas e instituciones
esta aventura habría fracasado. Pero, debemos reconocer que uno de los
pilares han sido y son los centros de secundaria, en especial su profesorado
y es bueno señalar que se ha producido un aumento constante a lo largo
de estos 20 años de la buena relación entre la educación universitaria y
la secundaria. Hemos sido capaces de convertir en realidad el título del
artículo “Bridging the gap in biochemistry between secondary school
and university”.
PW1-8
La coevaluación como recurso formativo.
Aplicación y resultados en la enseñanza
de la cinética enzimática
Néstor V. Torres, Guido Santos
Systems Biology and Mathematical Modelling Group. Departamento
de Bioquímica, Microbiología, Biología Celular y Genética.
Universidad de La Laguna, San Cristóbal de La Laguna, ES
La coevaluación o evaluación entre pares es reconocida como una
estrategia eficaz para fomentar el papel activo del estudiante en el
proceso de aprendizaje. El alumnado tiene la oportunidad de revisar el
trabajo de sus compañeros/as de clase frente a su propia evaluación y este
ejercicio tiene reflejo en su propio proceso de aprendizaje, provocando la
reorientación de sus propias estrategias de aprendizaje y el reforzamiento
y la mejor comprensión de las materias. En esta comunicación se muestran
los resultados de un ejercicio de coevaluación llevado a cabo con un grupo
de 90 estudiantes del Grado en Biología, en la asignatura de Bioquímica,
en el tema cinética enzimática. Una vez realizada la actividad práctica
cada estudiante redactó un informe de la misma de acuerdo en un
formato previamente determinado. Cada informe (anónimo) fue evaluado
individual y anónimamente por uno, dos o tres compañeros/as de curso y
por el profesorado. Los resultados de las evaluaciones muestran un nivel
alto de convergencia entre las evaluaciones del alumnado y de estas con
las del profesorado. Se muestra la percepción del alumnado, se señalan los
problemas detectados y las propuestas de mejora.
Agradecimientos: Este trabajo ha sido realizado gracias a la
financiación del Proyecto del MINECO (ref. BIO2014-54411-C2-2-R).
PW1-9
Utilizando Jmol para explicar la bioquímica
y biología molecular del cáncer
Jordi Sastre-Serra, Jordi Oliver, Margalida Torrens-Mas, Pilar Roca
Grupo Multidisciplinar de Oncología Traslacional, Institut
Universitari d’Investigació en Ciències de la Salut (IUNICS),
Universitat de les Illes Balears. Centro de Investigación
Biomédica en Red Fisiopatología de la Obesidad y la Nutrición
(CIBERObn, CB06/03), ISCIII, Instituto de Investigación
Sanitaria de Palma (IdISPa), Palma de Mallorca, ES
La Bioquímica y Biología Molecular del Cáncer (3 ECTS) es una
asignatura optativa del grado de Bioquímica en la Universitat de les Illes
Balears. La asignatura tiene el objetivo de introducir al alumno en las
bases moleculares de esta patología, explicando las causas, así como las
consecuencias de las alteraciones de las proteínas claves implicadas en
el desarrollo del proceso carcinogénico. Se presta especial interés a las
alteraciones del control del ciclo celular (con el papel de la proteína del
retinoblastoma y su control por las proteínas quinasas dependientes de
ciclina), las vías de activación de las MAPK quinasas, el papel central
del p53 en el control de la apoptosis, los mecanismos de la apoptosis,
tratamientos del cáncer.
161
Pósters / Posters
Jmol nos proporciona una herramienta que permite acercarnos a
nivel molecular a los cambios que sufren proteínas implicadas en
el desarrollo de esta patología. Así hemos desarrollado una serie de
guiones de diferentes proteínas implicadas en el proceso carcinogénico
para ayudar a los alumnos a comprender los fenómenos moleculares
y los efectos de las mutaciones en las proteínas que intervienen en el
control del ciclo celular, apoptosis y vías de señalización de las MAPK
quinasas: Proteína Retinoblastoma, Proteínas quinasas dependientes
XXXIX Congreso SEBBM
de ciclinas, p53, Caspasas (estructura, activación y mecanismo
enzimático), Scr, Ras y la familia de las proteínas Bcl-2. (Más
información en: http://gmot.uib.es/moleculas/molec_cancer.html.)
Jmol y sus guiones realizados se han demostrado como una herramienta
de gran ayuda para que los alumnos puedan adentrarse en los procesos
moleculares implicados en el cáncer, donde pueden observar los
cambios que se producen al interaccionar varias proteínas, o los cambios
ocasionados por la presencia de mutaciones en proteínas como Ras, p53.
PW2. Workshop: Investigador emergente / Workshop emergent investigator
PW2-1
Bringing light to receptors with LOV
Álvaro Inglés Prieto, Harald Janovjak
Institute of Science and Technology Austria, Viena, AT
Receptor tyrosine kinases (RTKs) are a large family of membrane
receptors that sense growth factors and hormones and regulate a
variety of cell behaviors in health and disease. We engineered RTKs
that can be selectively activated by low-intensity blue light. We
screened light-oxygen-voltage (LOV)-sensing domains for their ability
to activate RTKs by light-activated dimerization. Incorporation of
LOV domains resulted in robust activation of relevant RTKs and the
induction of cellular signaling in human cancer and endothelial cells
with high spatial and temporal precision. Furthermore, light faithfully
mimicked complex mitogenic and morphogenic cell behavior induced
by growth factors. Finally, we applied these light-activated RTKs in an
optogenetics-assisted drug screen. This platform obviates the addition
of chemical activators and reporters, which reduces the number of
operational steps in a cell-based drug screen against human protein
kinases including an orphan RTK.
PW2-2
Ligand engaged TCR is triggered by Lck
not associated with CD8 coreceptor
Javier Casas Requena1, Joanna Brzostek2, Veronika I. Zarnitsyna3,
Jin-sung Hong4, Quianru Wei2, John A.H. Hoerter5, Guo Fu5,
Jeanette Ampudia5, Rose Zamoyska6, Cheng Zhu4, Nicholas R.J.
Gascoigne2
1
Instituto de Biología y Genética Molecular (IBGM),
CSIC-Universidad de Valladolid, Valladolid, ES, 2Department
of Microbiology, Yong Loo Lin School of Medicine, National
University Health System, National University of Singapore,
Singapore, SG, 3Wallace H Coulter Department of Biomedical
Engineering, Georgia Institute of Technology and Emory
University, Atlanta, US, 4George W Woodruff School of
Mechanical Engineering, Georgia Institute of Technology and
Emory University, Atlanta, US, 5Department of Immunology and
Microbial Sciences, The Scripps Research Institute, La Jolla, US,
6
Institute of Immunology and Infection Research, University of
Edinburgh, Edinburgh, UK
Recognition of antigen by the T cell receptor (TCR) is the key event for the
T cell activation. The earliest molecular events in T cell recognition have
not yet been fully described, and the initial TCR triggering mechanism
remains a subject of controversy. By using supported lipid bilayers to
162
present peptide MHC class I complexes to CD8+ cells, we can monitor
the molecular events occurring at the immune synapse. The lipid bilayer
technology mimics the signaling environment for T cell activation as
judged by Ca2+ flux, total phospho-Tyr levels, and IL-2 secretion. Using
TIRF/FRET microscopy, we observe a two-stage interaction between
TCR, CD8, and MHCp. There is an early (within seconds) interaction
between CD3ζ and the coreceptor CD8 that is independent of the binding
of CD8 to MHC, but that requires CD8 association with Lck. Later
(several minutes) CD3ζ-CD8 interactions require CD8-MHC binding.
Lck can be found free or bound to the coreceptor. This data suggests that,
upon antigen recognition, TCR may be initially phosphorylated by Lck
not associated with coreceptor, followed by MHC dependent recruitment
of CD8-Lck complexes. This work indicates that the initial TCR triggering
event is induced by free Lck.
PW2-3
Understanding the role of RAS-PI3K signalling
in lung cancer
Esther Castellano Sánchez1, Clare Sheridan2, Miriam
Molina-Arcas2, Julian Downward2
1
Centre for Cancer and Inflammation, Barts Cancer Institute,
Londres, UK, 2Oncogene Biology, The Francis Crick Institute,
Londres, UK
Non-small cell lung cancer (NSCLC) is the most frequent type of lung
cancer. The prognosis is poor with a 15% overall 5year survival rate
for patients with advanced disease. This is attributable to the presence
of metastasis at the time of diagnosis. KRAS mutations occur in
approximately 25% of NSCLC patients. Unfortunately, understanding
oncogenic changes in NSCLC has not provided effective long-term
therapeutic strategies. Thus, there is a profound need to uncover the
molecular mechanisms of RAS-driven lung tumours to develop new
therapies to target NSCLCs. We have shown in a mouse model of lung
cancer that disruption of RAS signaling through PI3-Kinase (PI3K),
one of its main effectors, is essential for RAS driven tumor initiation
and maintenance. We have also shown that genetically blocking the
interaction of RAS with p110α causes partial regression of established
tumors, followed by long-term tumor stasis. Interestingly, simultaneous
inhibition of p110α together with inhibition of ERK, another major
RAS effector pathway, causes major tumor regression.Proteomic
analysis of the regressing lung tumors lacking RAS-PI3K signaling
showed significant changes in proteins involved in cell adhesion and
actin cytoskeleton reorganization. This correlated with a decrease in
cell migration via regulation of Reelin (Reln) expression. We found that
RAS-PI3K regulation of Reln is essential for proper cell migration and
failure of regulation of this pathway results in loss of Reln expression
Salamanca 2016
and increased migratory phenotype both in in vitro and in vivo settings.
Furthermore, loss of Reelin correlates with decreased survival of lung
and breast cancer patients. Reelin thus plays a role in restraining RAS
and PI3-kinase promotion of cell motility and potentially tumour
metastasis.
PW2-4
Interplay between SUMOylation
and ubiquitination during DNA replication
Emilio Lecona1, Sara Rodríguez-Acebes2, Julia Specks1,
Antonio Galarreta1, Patricia Ubieto1, Sergio Ruiz1, Andrés
López-Contreras3, Isabel Ruppen4, Matilde Murga1, Javier
Muñoz4, Juan Méndez2, Óscar Fernández-Capetillo1
1
Genomic Instability Group, Spanish National Cancer Research
Centre (CNIO), Madrid, ES, 2DNA Replication Group, Spanish
National Cancer Research Centre (CNIO), Madrid, ES, 3Center
for Chromosome Stability, University of Copenhagen ,
Copenhagen, DK, 4Proteomics Unit, Spanish National Cancer
Research Centre (CNIO), Madrid, ES
Post-translational modification of proteins by ubiquitin (Ub) and
Ub-like modifiers regulates DNA replication and genome stability.
Our previous results show that the chromatin around replisomes
is rich in SUMO and depleted in Ub, whereas an opposite pattern is
observed in mature chromatin. The mechanism responsible for the
generation and maintenance of this SUMO-rich/Ub-low environment
at sites of DNA replication is not known. We have identified USP7 as
a replisome-enriched SUMO deubiquitinase that is essential for DNA
replication. The chemical inhibition or genetic deletion of USP7 blocks
both new origin firing and the activity of active forks. The analysis of
the targetome of USP7 revealed SUMO2/3 as a new substrate for this
deubiquitinase and USP7 counteracts the ubiquitination of SUMO2/3
and SUMOylated proteins on chromatin. The blockade of USP7 leads
to the ubiquitination of SUMOylated proteins and their displacement
from the replisomes, in cooperation with the VCP/p97 segregase. Our
findings provide a model to explain the accumulation of SUMO and
depletion of Ub at replication forks through a new essential function
of USP7 in DNA replication. Currently, we are exploring the potential
ubiquitin ligases that target SUMOylated proteins on the replisome and
the mechanisms that direct VCP/p97 to proteins modified by SUMO and
ubiquitin. Given the potential of targeting USP7 for cancer treatment,
this new role of USP7 should be taken into account in the application of
USP7 inhibitors as anticancer agents.
PW2-5
Coordinated actin and septin
cytoskeletal rearrangements modulate
mechano-transduction and the emergence
of Cancer-associated fibroblasts
Fernando Calvo1, Romana Ranftl1, Aaron J. Farrugia1, Steven
Hooper2, Erik Sahai2
1
Institute of Cancer Research, London, UK, 2Crick Institute,
Londres, UK
Cancer-associated fibroblasts (CAFs) are non-malignant cells found
in tumours that can favour tumour progression, dissemination and
therapeutic resistance through remodelling of the extracellular matrix
and signalling to cancer, endothelial and immune cells. To investigate
the mechanisms governing the tumour promoting phenotype of CAFs,
we isolated fibroblasts from different stages of breast cancer progression
and normal counterparts, and analysed their phenotype, function and gene
Pósters / Posters
expression. Our analyses reveal that enhanced mechano-transduction
and activation of the transcriptional regulator YAP are signature features
of CAFs. YAP regulates the expression of cytoskeletal genes that are
required for CAFs to remodel the extracellular matrix and promote
cancer cell invasion and angiogenesis. Matrix stiffening further enhances
YAP activation, thus establishing a feed-forward self-reinforcing loop
that helps to maintain the CAF phenotype. In addition, we identify
Cdc42EP3, a previously uncharacterised Cdc42 effector, as a YAP target
gene in CAFs. Using super-resolution microscopy and biochemical
approaches we demonstrate that Cdc42EP3 functions by coordinating
the actin and septin networks, which change dramatically during
CAF emergence. In CAFs, Cdc42EP3 and septin fibres are required
for mechano-transduction, force-mediating matrix remodelling and
promoting cancer cell invasion, angiogenesis and tumour growth. To
conclude, our studies underlie the relevance of mechano-transduction
and coordinated cytoskeletal rearrangements in modulating the
aggressive behaviour of stromal fibroblasts in cancer, and define a novel
molecular and biological role for the septin cytoskeleton.
PW2-6
Early events in ribosome biogenesis
Christina Schmidt1, Fanny Boissier2, Sebastién Fribourg2,
Jorge Pérez-Fernández1
1
University of Regensburg, Regensburg, DE, 2Univ. Bordeaux,
IECB, F-33607, Pessac, FR. INSERM, U1212 & CNRS UMR
5320, F-33077, Pessac, FR
Ribosome synthesis is an essential multistep process which involves the
correct folding of the mature ribosomal (r)RNAs and the incorporation
of the ribosomal proteins. In eukaryotic cells, more than 150 biogenesis
factors associate transiently with the pre-rRNA to promote the correct
formation of ribosomes. During the early events of ribosome biogenesis,
a large set of proteins associates to the nascent rRNA to form the first
pre-ribosomal particle or SSU-processome (small subunit). Formation
of the SSU-processome occurs by the stepwise association of the early
assembly factors to the rRNA in a highly hierarchical process. Some of
these early-acting factors have been isolated as small protein complexes
(UTP complexes) and they are the primary binders of the pre-rRNAs.
However, the molecular mechanism behind the hierarchical relationships
that control the formation of the SSU-processome remains elusive. To
uncover this aspect, we have studied the role of Pwp2, a component of
the UTP-B complex which may play a central role during the formation
of the SSU-processome. The crystal structure of the N-terminal domain
of Pwp2 shows a classical tandem β-propeller folding. Analysis of
different mutants revealed that the N-terminal domain of Pwp2 is not
enough to support the formation of the UTP-B complex. Instead, the
C-terminal domain of Pwp2 establishes interactions with three other
UTP-B components and is required for the stabilization of the full
complex and the stable formation of the SSU-processome. Overall,
our data suggest that the UTP-B complex works as a platform for the
association of some assembly factors.
PW2-7
Carnitine palmitoyltransferase 1 increases
lipolysis, UCP1 protein expression and
mitochondrial activity in brown adipocytes
Laura Herrero1, María Calderón-Domínguez1, David Sebastián2,
Raquel Fucho1, Minéia Weber1, Joan F. Mir1, Esther
García-Casarrubios3, María Jesús Obregón3, Antonio Zorzano2,
Ángela M. Valverde4, Dolors Serra1
1
Department of Biochemistry and Physiology, Institut de
Biomedicina de la Universitat de Barcelona (IBUB), Universitat
163
Pósters / Posters
de Barcelona, Barcelona, ES; Centro de Investigación Biomédica
en Red de Fisiopatología de la Obesidad y la Nutrición
(CIBEROBN), Instituto de Salud Carlos III, Madrid, ES, 2Institute
for Research in Biomedicine (IRB Barcelona) and Departament
de Bioquímica i Biologia Molecular, Facultat de Biologia,
Universitat de Barcelona, Barcelona, ES; Centro de Investigación
Biomédica en Red de Diabetes y Enfermedades Metabólicas
Asociadas (CIBERDEM), Instituto de Salud Carlos III , Madrid,
ES, 3Instituto de Investigaciones Biomédicas Alberto Sols
(CSIC-UAM) and Instituto de Investigación Sanitaria La Paz,
Madrid, ES, 4Instituto de Investigaciones Biomédicas Alberto
Sols (CSIC-UAM) and Instituto de Investigación Sanitaria La
Paz; Centro de Investigación Biomédica en Red de Diabetes y
Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de
Salud Carlos III, Madrid, ES
The discovery of active brown adipose tissue (BAT) in adult humans
and the fact that it is reduced in obese and diabetic patients have put
a spotlight on this tissue as a key player in obesity-induced metabolic
disorders. BAT regulates energy expenditure through thermogenesis;
therefore, harnessing its thermogenic fat-burning power is an attractive
therapeutic approach. We aimed to enhance BAT thermogenesis by
increasing its fatty acid oxidation (FAO) rate. Thus, we expressed
carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active
mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown
adipocyte (rBA) cell line through adenoviral infection. We found that
CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein
levels and mitochondrial activity. Additionally, enhanced FAO reduced
the palmitate-induced increase in triglyceride content and the expression
of obese and inflammatory markers. Thus, CPT1AM-expressing
rBA had enhanced fat-burning capacity and improved lipid-induced
derangements. This indicates that CPT1AM-mediated increase in
brown adipocytes FAO may be a new approach to the treatment of
obesity-induced disorders.
PW2-8
Regulation of human Polλ by post-translational
modification
José F. Ruiz
Universidad de Sevilla, Sevilla, ES
The genome integrity in mammalian cells is constantly threatened
by a wide variety of lesions, the repair of which often require a DNA
synthesis step either to replace damaged nucleotides or DNA strands
or to bypass them provisionally until their subsequent removal. DNA
synthesis during these repair or tolerance processes is mainly performed
by specialized DNA polymerases belonging to the X and Y families.
Among these enzymes DNA polymerase lambda (Polλ) is one of the
most versatile, having relevant roles in the repair of oxidative DNA
damage through Base Excision Repair (BER) as well as in the repair
of DNA double-strand breaks by Non-Homologous End Joining
(NHEJ). The involvement of Polλ in these different processes suggests a
complex regulation, which remains largely unknown so far. In this work
we will discuss on the complex interplay between post-translational
modifications (PTMs) and human Polλ, describing novel modifications
that we have recently identified and showing their relevance in different
aspects like the subcellular localization or its activity during the repair of
IR-induced DSBs by NHEJ. Our work will contribute to the mechanistic
understanding of how DNA polymerases lambda can influence genome
integrity through its participation in different repair pathways, with
an emphasis on the means by which these pathways intersect human
diseases.
164
XXXIX Congreso SEBBM
PW2-9
Clot formation as a potential diagnostic tool
for Alzheimer’s disease
Marta Cortés Canteli, Jesús Ruiz-Cabello, Valentín Fuster
Fundación Centro Nacional de Investigaciones Cardiovasculares
Carlos III, Madrid, ES
Alzheimer’s disease (AD) is a multifactorial disorder with no effective
treatment available. Accumulating evidence shows that AD presents
an important vascular component. Epidemiological studies show a
correlation of AD with vascular risk factors. There are also profound
alterations of cerebrovascular structure and function present in AD,
and deposition of beta-amiloid in the cerebral blood vessels, that could
affect brain circulation. Indeed, a decrease in cerebral blood flow has
been reported in AD patients which could have a very detrimental effect
on neuronal viability and cerebral function in general. Several pieces
of evidence indicate increased thrombosis, decreased fibrinolysis and
increased obstruction of the cerebral blood vessels in the AD brain.
These obstructions in AD cerebral vasculature could initiate and/or
aggravate the brain hypoperfusion and inflammation present in the
AD brain. Cerebral hypoperfusion seems to be a good indicative for
dementia conversion, which suggests that the occlusion of vessels might
be happening at very early stages of the disease, many years before
the clinical manifestation of AD. We are using state-of-the-art imaging
technology to develop an in vivo non-invasive method to identify these
occlusions. This research could serve as a novel biomarker since it will
identify the subpopulation of AD patients with early dysregulation
of cerebral blood flow and might prove to be a useful tool to identify
patients at very early stages of the AD pathology.
PW2-10
Cristae shape determines mitochondrial
bioenergetics under compromised respiration
Rubén Quintana Cabrera, Mauro Corrado, Luca Scorrano
Dpto. Biología. Universidad de Padua; Venetian Institute of
Molecular Medicine (VIMM), Padua, IT
Mitochondrial cristae function as a membrane platform where
respiratory (super)complexes sit to account for respiration. A growing
body of evidence shows that maintaining the architecture of the
cristae is key to assemble respiratory supercomplexes and improve
respiration rates; however, how mitochondrial ultrastructure controls
mitochondrial bioenergetics remains obscure. Capitalizing on genetic
modulation of Opa1 as a master regulator of cristae shape, we show
that cristae determine F0F1-ATP synthase stability and mitochondrial
bioenergenetic parameters such as matrix pH, ATP or membrane
potential. Narrowing of cristae by Opa1 stabilizes F0F1-ATP synthase
to account for sustained mitochondrial function and cell survival under
compromised respiration or metabolic reliance on mitochondrial ATP
production. Electron microscopy analysis of inner membrane and
apoptotic cristae remodeling by Bid confirmed that keeping cristae
tight is essential to preserve F0F1-ATP synthase stability and function.
As a result of improved F0F1-ATP synthase composition and activity,
OPA1 accounts for lower reactive oxygen species production under
compromised mitochondrial respiration. Interestingly, the pro-survival
and bioenergetic advantage from OPA1 overexpression are lost when
F0F1-ATP synthase dimerization is impaired, thus indicating a need for
dimers as a link for structural and functional consequences of cristae
remodeling. In sum, our results prove a connection of mitochondrial
ultrastructure and bioenergetics, offering a proof of principle for
therapies aimed at restoring mitochondrial function in disease.
Salamanca 2016
PW2-11
Caracterización funcional y estructural
del regulador transcripcional pleiotrópico RcsB
de Salmonella typhimurium
Patricia Casino1, Laura Miguel-Romero2, Alberto Marina2
Departamento de Bioquímica, Universitat de València.
Estructura de Recerca Interdisciplinar en Biotecnologia
i Biomedicina (ERI BIOTECMED), Universitat de València ,
Burjassot-Valencia, ES, 2Instituto de Biomedicina de Valencia,
CSIC, Valencia, ES
1
El sistema RcsCDB es un sistema de dos componentes complejo presente
en Enterobacteriacea, que regula la formación de biofilm así como otros
genes implicados en el mantenimiento de la pared celular, la división
celular, el factor sigma σS, motilidad y virulencia. El sistema RcsCDB
está formado por la histidina quinasa híbrida RcsC, la fosfotransferasa
RcsD y el regulador de la respuesta pleiotrópico RcsB que se activan
mediante múltiples pasos de fosforilación, también denominados
como “phosphorelay”. Como respuesta a cambios ambientales, RcsC
se autofosforila en un residuo de His conservado y fosfotransfiere el
grupo fosforilo a un residuo de Asp conservado presente en su misma
cadena polipeptídica. Posteriormente el grupo fosforilo se transfiere de
nuevo desde el Asp a otro residuo de His presente en la fosfotransferasa
RcsD. Finalmente, el grupo fosforilo se transfiere desde RcsD a un
residuo de Asp presente en RcsB, el cual controla la transcripción
de múltiples genes bien de manera independiente o junto con otros
reguladores transcripcionales auxiliares. Así pues, la complejidad en la
respuestaa cambios ambientales generada por el sistema RcsCDB viene
determinada principalmente por la actividad pleiotrópica que presenta el
regulador de la respuesta RcsB para regular la transcripción de múltiples
genes. Con el fin de comprender las bases moleculares del mecanismo
de “phosphorelay” en RcsCDB así como el mecanismo pleiotrópico de
regulación transcripcional, hemos realizado estudios funcionales con el
complejo formado por RcsD y RcsB, así como estudios estructurales
y funcionales con RcsB. Ello nos han permito resolver la estructura
tridimensional de RcsB en dos estados diferentes de activación arrojando
así luz sobre su activación y regulación transcripcional.
Pósters / Posters
a strict cell-cycle regulatory mechanism that depends on changes in its
phosphorylation status by the Cdk kinase and the Cdc14 phosphatase.
Such control implies that Yen1 will display low activity and cytoplasmic
localization in S-phase, while in anaphase it will re-enter the nucleus
and become highly active. Interestingly, expression of a mutant that is
constitutively active and nuclear (Yen1ON) results in detrimental effects
for the cell. These range from increased genome instability when Yen1ON
is expressed at endogenous levels to cell death when it is overexpressed.
Here, we will present the lab strategy and novel data where we are using
Yen1ON as a tool to explore the importance of cell cycle control on SSE
activation as well as the dynamics of early recombination/replication
intermediates under unperturbed conditions.
PW2-13
Understanding the structurally dynamic AMPA
receptors
Javier García Nafria, Beatriz Herguedas, Ingo H. Greger
MRC Laboratory of Molecular Biology, Cambridge, UK
AMPA receptors (AMPARs) are glutamate-gated ion channels localized
at the post-synaptic density. AMPARs are not only involved in synaptic
transmission, but also have a central role in synaptic plasticity, which
is believed to be the basis of human cognitive functions and memory
formation. In the brain, the channel is mainly a hetero-tetramer, formed
by combinations of four different subunits that yield functionally
different channels. Structurally, AMPAR is divided in three domain
layers with a transmembrane region (TMD), acting as the ion channel,
a ligand binding domain (LDB), which binds glutamate, and the
membrane-distal N-terminal domain (NTD). Using a variety of
techniques including Anisotropic Network Model, cysteine crosslinking,
X-ray crystallography and single-particle electron microcopy, we were
able to 1) predict and confirm new conformations of AMPA receptor
homomers, 2) show that, although flexible, all AMPARs subunits can
adopt a common set of discrete conformations, 3) describe two new
conformations using single-particle cryo-electron microscopy, one of
which is the description of the first AMPAR heteromer, and 4) speculate
about the potential of these different conformation in regulating synaptic
protein-protein interactions.
PW2-12
Too early to prune: Premature activation
of structure-selective endonucleases destroys
essential branched DNA structures
PW2-14
Miguel González Blanco, Francisco Javier Aguado Domínguez,
Vanesa Nieves Hurtado
Centro de Investigación en Medicina Molecular y Enfermedades
Crónicas. Universidade de Santiago de Compostela, Santiago de
Compostela, ES
Jorge Alegre-Cebollada
Fundación CNIC, Madrid, ES
The transmission of genetic information from mother to daughter cells
depends on the faithful duplication of their DNA content and its accurate
distribution into two equal genomic packages. Despite conceptually
simple, such a task requires the coordinated actions from different
molecular machineries, like those responsible for DNA replication,
repair and segregation. In most cases, this synchronization relies on the
master cell cycle regulator kinase Cdk, which dictates the main order
of events in the cell. However, in situations of high genotoxic stress,
a signaling cascade known as the DNA damage response (DDR) can
trigger a checkpoint that arrests cell cycle progression, providing enough
time for the cell to repair DNA before committing to the next cell cycle
stage. Yen1 is a structure-selective endonuclease (SSE) involved in the
removal of persistent homologous recombination intermediates. The
activation of this nuclease, rather than depending on the DDR, is under
Single-molecule mechanobiochemistry
laboratory at CNIC
We use biochemistry and biophysics techniques to study the mechanics
of heart tissue and its regulation in health and disease. Our lab takes
a multidisciplinary approach to try to learn more about molecular
determinants of the mechanical properties of the heart. We specialize in
single molecule methods using atomic force microscopy (AFM), which
are able to measure the effects of mechanical forces on cardiac structural
proteins. Our main lines of research are:
1. Regulation of protein elasticity by redox posttranslational modifications
(PTM). Using in vitro experiments, we have recently described that
PTM of buried cysteines in the giant protein titin makes heart tissue
more elastic (Cell 2014, 156:1235). We are using mass spectrometry
techniques to detect native redox PTMs from heart tissue. Once relevant
redox PTMs are identified, we use single-molecule methods by AFM to
determine their effect in protein elasticity. We want to explore how these
mechanical PTMs are differentially present during heart disease.
2. Genotype/phenotype relations in familial cardiomyopathies. The
majority of familial cardiomyopathies are caused by mutations in
165
Pósters / Posters
sarcomeric genes that code for proteins with mechanical roles. We are
exploring the connection between dysregulation of protein mechanics
and development of cardiomyopathy. Using single-molecule AFM, we
measure the mechanical properties of proteins whose mutation cause
cardiomyopathy. We plan to examine several other cellular functions
that may be affected by mutations.
3. Development of engineered muscle-mimicking biomaterials. Using
a directed crosslinking strategy, we are able to polymerize proteins
into hydrogels whose stiffness can be measured in the lab using tensile
testers built by us (Macromolecular Materials and Engineering 2015,
300, 369). We are developing experimental platforms that allow us to
predict the stiffness of the biomaterial from the mechanical properties of
the constituent proteins measured at the single-molecule level.
PW2-15
Microenvironment-induced metabolic switch
promotes metastasis in pancreatic cancer
Sara Trabulo, Andrés Cano, Shanthini Crusz, Petra Jagust,
Andrew Palfreeman, David Barneda, Christopher Heeschen,
Patricia Sancho
Centre for Stem Cells in Cancer & Ageing, Barts Cancer Institute,
Queen Mary University of London, London, UK
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest
cancers worldwide due to its chemoresistance and early onset of
metastases, characteristics that, at least in part, can be attributed to the
presence of cancer stem cells (CSCs). We have established that CSCs
are eminently OXPHOS-dependent, with MYC/PGC1A expression ratio
as the main determinant for their sensitivity to mitochondrial targeting
(i.e. metformin) (Sancho et al., Cell Metabolism 2015). We now show
that, concomitant with the acquisition of migratory and invasive
capacities, microenvironmental factors such as Tumour-Associated
Macrophages (TAMs), modulate the metabolic phenotype of PDAC
cells (Sainz et al., Gut 2015). Specifically, incubation of PDAC cells
with TAM-conditioned medium or direct co-culture with TAMs in either
2D or 3D conditions, increased their MYC/PGC1A ratio, leading to a
metabolic shift towards glycolysis. As expected, cells that underwent
EMT became unresponsive to the mitochondrial inhibitor metformin,
which was attributed to upregulation of MYC, as previously described
(Sancho et al., 2015). Importantly, MYC knockdown blocked these
metabolic and phenotypic changes induced by TAMs, implying a
crucial role for MYC in EMT induction via modulation of metabolism.
Interestingly, single-cell gene expression analysis of circulating-tumour
cells (CTCs) demonstrated an increased MYC/PGC1A ratio in the CTC
population as compared to cell derived from primary tumours. This was
even more pronounced in the subset of circulating CSCs, suggesting that
CSCs evaded from the primary tumour have switched their preferential
metabolism from OXPHOS to glycolysis via upregulation of MYC.
In summary, our results suggest that in PDAC an increased MYC/
PGC1A ratio promotes a pro-metastatic programme through metabolic
reprogramming of CSCs. Inhibition of this process may provide a novel
approach for combating metastasis in PDAC.
PW2-16
Identification of protective cellular pathways
involved in ischemic preconditioning
María Delgado Esteban
Instituto de Investigación Biomédica de Salamanca (IBSAL)/
Hospital Universitario de Salamanca; Instituto de Biología
Funcional y Genómica (IBFG)/Universidad de Salamanca (CSIC/
USAL), Salamanca, ES
166
XXXIX Congreso SEBBM
Ischemic preconditioning (IPC) is one of the most important
endogenous mechanisms responsible for the increased tolerance
of brain and heart after an ischemic insult. Although the molecular
mechanisms underlying IPC-induced cell protection are still
unknown, several studies suggest that IPC promotes the activation
of endogenous antioxidant systems and modulates apoptosis. Then,
IPC is an effective tool for identifying molecular targets involved
in cell survival. In this context, two transcription factors, p53
-the key regulator of apoptotic cell death- and the nuclear factor
(erythroid-derived 2)-like 2 (Nrf2) protein -the key regulator of
antioxidant response-, have been described to play an essential role
in the progression of cerebral ischemia and heart failure. Our work is
focused on the study of the role of p53 and Nrf2 on the IPC-induced
cell protection against an ischemic insult. Indeed, cellular redox
status is directly regulated by p53 through modified expression of
pro- and anti-oxidant proteins. For this purpose, we used two different
models of IPC insults: i) primary cultured cortical neurons were
stimulated with moderate concentrations of N-methyl-D-aspartate
(20 μM NMDA); and ii)human endothelial cells were incubated
upon sublethal oxygen and glucose deprivation (OGD) conditions
for 20 min. After the corresponding IPC insult, cells were incubated
for further 2 h either in the presence (IPC) or absence (IPC+OGD)
of oxygen and glucose (lethal OGD). Our results show that IPC
modulated p53-mediated neuronal apoptosis by interfering with
the caspase-3 pathway. IPC prevents the OGD-induced p53 mRNA
levels abrogating the accumulation of p53 and its target p21 in
cortical neurons.Furthermore, IPC modulated mRNA levels of Nrf2
and its targets hemeoxygenese-1 (HO-1) and NADPH quinona
reductase NQO1 in endothelial cells. Our results demonstrate that
IPC promotes cell survival through the control of the p53 signaling
pathway and the Nrf2-antioxidant pathway. Then, IPC plays a key
role in the control of cellular redox homeostasis and survival.
Funded by grants from the Instituto de Salud Carlos III (Miguel Servet
I CP0014/00010; RD12/0014/0007); FEDER (European regional
development fund).
PW2-17
Iron overload causes endolysosomal deficits
modulated by NAADP regulated two pore
channels and RAB7A
Belén Fernández López1, Elena Fernández Fernández1, Patricia
Gómez Suaga1, Fernando Gil Hernández2, Isabel Molina Villalba2,
Isidro Ferrer3, Sandip Patel4, Grant C. Churchill5, Sabine Hilfiker1
1
Instituto de Parasitología y Biomedicina López-Neyra (CSIC),
Armilla (Granada), ES, 2Universidad de Granada. Facultad
de Medicina. Departamento de Medicina Legal, Toxicología y
Antropología Física, Granada, ES, 3Universidad de Barcelona.
Instituto de Nueropatología. IDIBELL-Hospital Universitario
Bellvitge, L’Hospitalet de Llobregat, ES, 4University College
London. Dept. of Cell and Developmental Biology, Londres, UK,
5
Dept. of Pharmacology, University of Oxford, Oxford, UK
Various neurodegenerative disorders are associated with increased
brain iron content. Iron is known to cause oxidative stress, which
concomitantly promotes cell death.Whereas endolysosomes are known
to serve as intracellular iron storage organelles, the consequences
of increased iron on endolysosomal functioning, and effects on
cellviability upon modulation of endolysosomal iron release remain
largely unknown. Here,we show that increasing intracellular iron
causes endolysosomal alterations associated with impaired autophagic
clearance of intracellular protein aggregates, increased cytosolic
oxidative stress and increased cell death. These effects are subject
Salamanca 2016
to regulation by NAADP, a potent second messenger reported to
target endolysosomalTPCNs (two pore channels). Consistent with
endolysosomal iron storage, cytosolic iron levels are modulated by
NAADP, and increased cytosolic iron is detected when overexpressing
active, but not inactive TPCNs, indicating that these channels
can modulate endolysosomal iron release. Cell death triggered by
altered intralysosomaliron handling is abrogated in the presence of
an NAADP antagonist or when inhibiting RAB7A activity. Taken
together, our results suggest that increased endolysosomal iron causes
cell death associated with increased cytosolic oxidative stress as well
as autophagic impairments, and these effects are subject to modulation
by endolysosomalion channel activity in a RAB7A-dependent
manner. These data highlight alternative therapeutic strategies for
neurodegenerative disorders associated with increased intracellular
iron load.
PW2-18
Immunoregulatory properties of human adipose
derived stem cells (hASCs) in inflammatory
diseases. Differences between Crohn´s disease
and obesity
Carolina Serena, Noelia Keiran, Ana Madeira, Joan Vendrell,
Sonia Fernández-Veledo
Instituto de Investigación Sanitaria Pere Virgili. Hospital Joan
XXIII de Tarragona. CIBERDEM, Tarragona, ES
Background: Inflammation and dysbiosis are common in several
highly prevalent diseases, such as Cohn’s disease (CD), obesity
and type 2 diabetes (T2D). Visceral fat depot plays a decisive role
in the inflammatory environment observed in these pathologies.
Specifically, in CD subjects there is frequently an increase of
mesenteric fat-tissue which is called “creeping fat”. However, CD
patients show an aggressive local inflammatory response whereas
obese and T2D patients develop a subtle chronic inflammatory
status. The main objective of this study was to identify key factors
involved in triggering the immune innate response of adipose
tissue (AT). Specifically, we focus on the potential role that human
adipose-derived stem cells (hASCs) play in local and systemic
inflammation in the setting of CD and obesity.
Methods: We isolated hASCs from subcutaneous (SAT) and visceral
AT (VAT) of a well-characterized cohort including 4 groups: inactive
CD (in remission), active CD, obese, and obese subjects with diabetes
(T2D). hASCs were immunophenotyped by flow cytometry. Gene
expression profile, migration, invasion, as well as phagocytic capacity
were analyzed in hASCs obtained from different donors.
Results: hASCs obtained from CD subjects shown a significantly
greater migratory and invasive capacity than those derived from
obese subjects, regardless of the presence of diabetes. This
distinctive difference in phenotype between CD versus obese hASCs
was observed in both active and inactive CD donors and in both
depots SAT and VAT. The phagocytic capacity was also higher in
CD subjects compare obese subjects but in this occasion in inactive
CD donors showed lower phagocityc activity to compare active CD
donors. Accordingly, ATinflammation markers (IL-6, TNFa, IL-1b,
MCP-1) indicated that both active and inactive CD subjects present
a higher proinflammatory environment compared to obese subjects.
Remarkably, we demonstrate that CD produces a detrimental effect
not only on mesenteric adipose tissue-resident stem cells, but also on
the subcutaneous fat depots.
Conclusions: hASCs may have different immuno-modulatory capacities
depending on the inflammatory event.
Pósters / Posters
PW2-19
Peripheral modification of disease pathogenesis
in skeletal muscle affects systemic
Huntington´s disease
Silvia Corrochano Sánchez1, Debbie Williams1, Gonzalo Blanco2,
Michelle Simon1, Michael R Duchen3, Henning Wackerhage4, David
C Rubinsztein5, Steve D.M. Brown1, Abraham Acevedo-Arozena6
1
MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK,
2
York University, York, UK, 3University College London (UCL),
London, UK, 4University of Aberdeen, Scotland, UK, 5Cambridge
University, Cambridge, UK, 6MRC Mammalian Genetics Unit,
Harwell, Oxfordshire, UK, and Unidad de Investigación, Hospital
Universitario de Canarias, Tenerife, ES
Polyglutamine expansions in huntingtin (HTT) cause Huntington’s
disease (HD). HD is primarily a neurodegenerative condition; however,
HTT is ubiquitously expressed, leading to pathological alterations
also in peripheral organs, as well as general metabolic abnormalities.
To identify new HD modifiers, we performed a genetic screen using
N-ethyl-N-nitrosourea (ENU) mutagenesis in mice, identifying a mutation
in the skeletal muscle voltage-gated sodium channel (Scn4a) that
exacerbated the overall HD phenotype. Huntingtin and Scn4a are only
highly expressed together in skeletal muscle, leading to cell autonomous
effects of their genetic interaction. That in turn causes systemic effects,
including metabolic alterations associated with energy imbalance leading
to further weight loss and a dramatic reduction in survival in double
mutants. These systemic alterations, including further changes in the
kynurenine system, ultimately perturb the expression of key HD genes
in the brain. Our results reveal that local metabolic changes in skeletal
muscle can modulate the systemic nature of HD pathogenesis.
PW2-20
Positioning of centrioles is a conserved readout
of Frizzled planar cell polarity signalling
José María Carvajal González, Sonia Mulero-Navarro
Universidad de Extremadura, Badajoz, ES
Planar cell polarity (PCP) signalling is a well-conserved developmental
pathway regulating cellular orientation during development. An
evolutionarily conserved pathway readout is not established and,
moreover, it is thought that PCP mediated cellular responses are
tissue-specific. A key PCP function in vertebrates is to regulate
coordinated centriole/cilia positioning, a function that has not been
associated with PCP in Drosophila. Here we report instructive input of
Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in
Drosophila wings. We show that centrioles are polarized in pupal wing
cells as a readout of PCP signalling, with both gain and loss-of-function
Fz/PCP signalling affecting centriole polarization. Importantly, loss or
gain of centrioles does not affect Fz/PCP establishment, implicating
centriolar positioning as a conserved PCP-readout, likely downstream
of PCP-regulated actin polymerization. Together with vertebrate data,
these results suggest a unifying model of centriole/cilia positioning as
a common downstream effect of PCP signalling from flies to mammals.
PW2-21
Structural basis of TubZ filament assembly
and dynamic modulation
María A. Oliva Blanco1, María Eugenia Fuentes Pérez2, Rafael Núñez
Ramírez1, Alejandro Martín Gonzalez2, David Juan Rodríguez1, José
Manuel Andreu Morales1, Oscar Llorca1, Fernando Moreno Herrero2
1
CSIC-CIB, Madrid, ES, 2CSIC-CNB, Madrid, ES
167
Pósters / Posters
Accurate DNA segregation is essential for genome transmission during
cell division and partition systems are key on low copy plasmids
maintenance. These systems require only three elements to effectively
produce DNA movement: a cis-acting centromere-like DNA sequence,
a CBP (centromere-binding protein) and a motor protein. Based on the
nature of the motor protein partition systems are classified into type I
(Walker A-like ATPases), II (actin-like ATPase) and III (tubulin-like
GTPase).
In type III systems, the motor protein (TubZ) grows in a helical fashion
following treadmilling or dynamic instability, although the underlying
molecular mechanism is not clear. Using a biochemical approach in
combination with electron and atomic force microscopy we have studied
the structural changes in TubZ filament upon GTP hydrolysis and how
those drive the disassembly process.
We present phage CbTubZ primary assembly into a 4-stranded helical
arrangement, which is just supported by the presence of GTP caps. The
transition from GTP to GDP-bound state propagates along the filament
only when GTP caps are released and involves the stiffening of the
filament. Our results point to the TubZ C-terminal flexible tail as an
unstructured domain directly involves in the filament dynamics because
is essential on the formation of a 4-stranded helical filament and helps
in the conformational change upon hydrolysis for depolymerization.
Further, the C-tail also modulates the interaction with the partner
regulator TubY and can induce t