Estudis estructurals dels canvis conformacionals de les E3 ubiquitin lligases per RMN

L'estudi a nivell molecular de la ruta de senyalització activada per TGF-B té un impacte notable en el panorama actual donada la seva implicació en processos autoimmunitaris i carcinogènics. D'altra banda, l'elucidació a nivell estructural dels mecanismes moleculars que permeten a les ubiquitin lligases de tipus E3 marcar específicament llurs dianes per a la degradació proteosòmica - entre d'altres - resulta fonamental donada la seva importància pel que fa al control sobre el turnover proteic a nivell intracel•lular. En aquest projecte es pretén elucidar els mecanismes d'activació i catlàlisi de les ubiquitin lligases E3 tipus HECT Smurf1 i Nedd4L al mateix temps que se n'estudia la implicació en la regulació dels agents missatgers de la ruta del TGF-B. Així, la tasca es divideix en tres sub-projectes els quals se centren en a) l'estudi de la interacció d'aquestes lligases amb llurs dianes; b) l'elucidació del mecanisme d'activació i c) del de catàlisi d'aquests enzims. Per tal d'assolir aquests objectius ens servim principalment dels avantatges que ens aporta la Ressonància Magnètica Nuclear i altres tècniques biofísiques, principalment la ITC. Durant el temps que he gaudit de la beca FI, m'he centrat principalment en la preparació de pèptids per SPPS, llur purificació per HPLC i caracterització per MS i RMN. Aquests pèptids representen diferents patrons de fosforilació de certes dianes de les lligases esmentades, de manera que han estat emprats per a l'estudi d'interaccions proteïna-proteïna per ITC i RMN. M'he iniciat doncs en l'ús d'aquestes tècniques. D'altra banda, també he preparat mostres protèiques mitjançant l'ús de sistemes d'expressió bacterians basats en E.coli, incloent l'amplificació i clonació del gen que codifica per la proteïna d'interés així com la seva expressió, purificació i caracterització per MS i RMN.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Muscle transcriptomic profiles in pigs with divergent phenotypes for fatness traits

BACKGROUND: Selection for increasing intramuscular fat content would definitively improve the palatability and juiciness of pig meat as well as the sensorial and organoleptic properties of cured products. However, evidences obtained in human and model organisms suggest that high levels of intramuscular fat might alter muscle lipid and carbohydrate metabolism. We have analysed this issue by determining the transcriptomic profiles of Duroc pigs with divergent phenotypes for 13 fatness traits. The strong aptitude of Duroc pigs to have high levels of intramuscular fat makes them a valuable model to analyse the mechanisms that regulate muscle lipid metabolism, an issue with evident implications in the elucidation of the genetic basis of human metabolic diseases such as obesity and insulin resistance. RESULTS: Muscle gene expression profiles of 68 Duroc pigs belonging to two groups (HIGH and LOW) with extreme phenotypes for lipid deposition and composition traits have been analysed. Microarray and quantitative PCR analysis showed that genes related to fatty acid uptake, lipogenesis and triacylglycerol synthesis were upregulated in the muscle tissue of HIGH pigs, which are fatter and have higher amounts of intramuscular fat than their LOW counterparts. Paradoxically, lipolytic genes also showed increased mRNA levels in the HIGH group suggesting the existence of a cycle where triacylglycerols are continuously synthesized and degraded. Several genes related to the insulin-signalling pathway, that is usually impaired in obese humans, were also upregulated. Finally, genes related to antigen-processing and presentation were downregulated in the HIGH group. CONCLUSION: Our data suggest that selection for increasing intramuscular fat content in pigs would lead to a shift but not a disruption of the metabolic homeostasis of muscle cells. Future studies on the post-translational changes affecting protein activity or expression as well as information about protein location within the cell would be needed to to elucidate the effects of lipid deposition on muscle metabolism in pigs.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

From SNPs to pathways: integration of functional effect of sequence variations on models of cell signalling pathways

Background: Single nucleotide polymorphisms (SNPs) are the most frequent type of sequence variation between individuals, and represent a promising tool for finding genetic determinants of complex diseases and understanding the differences in drug response. In this regard, it is of particular interest to study the effect of non-synonymous SNPs in the context of biological networks such as cell signalling pathways. UniProt provides curated information about the functional and phenotypic effects of sequence variation, including SNPs, as well as on mutations of protein sequences. However, no strategy has been developed to integrate this information with biological networks, with the ultimate goal of studying the impact of the functional effect of SNPs in the structure and dynamics of biological networks. Results: First, we identified the different challenges posed by the integration of the phenotypic effect of sequence variants and mutations with biological networks. Second, we developed a strategy for the combination of data extracted from public resources, such as UniProt, NCBI dbSNP, Reactome and BioModels. We generated attribute files containing phenotypic and genotypic annotations to the nodes of biological networks, which can be imported into network visualization tools such as Cytoscape. These resources allow the mapping and visualization of mutations and natural variations of human proteins and their phenotypic effect on biological networks (e.g. signalling pathways, protein-protein interaction networks, dynamic models). Finally, an example on the use of the sequence variation data in the dynamics of a network model is presented. Conclusion: In this paper we present a general strategy for the integration of pathway and sequence variation data for visualization, analysis and modelling purposes, including the study of the functional impact of protein sequence variations on the dynamics of signalling pathways. This is of particular interest when the SNP or mutation is known to be associated to disease. We expect that this approach will help in the study of the functional impact of disease-associated SNPs on the behaviour of cell signalling pathways, which ultimately will lead to a better understanding of the mechanisms underlying complex diseases.

Pathway databases and tools for their exploitation: benefits, current limitations and challenges

In past years, comprehensive representations of cell signalling pathways have been developed by manual curation from literature, which requires huge effort and would benefit from information stored in databases and from automatic retrieval and integration methods. Once a reconstruction of the network of interactions is achieved, analysis of its structural features and its dynamic behaviour can take place. Mathematical modelling techniques are used to simulate the complex behaviour of cell signalling networks, which ultimately sheds light on the mechanisms leading to complex diseases or helps in the identification of drug targets. A variety of databases containing information on cell signalling pathways have been developed in conjunction with methodologies to access and analyse the data. In principle, the scenario is prepared to make the most of this information for the analysis of the dynamics of signalling pathways. However, are the knowledge repositories of signalling pathways ready to realize the systems biology promise? In this article we aim to initiate this discussion and to provide some insights on this issue.

Neutralizing antibodies in Multiple Sclerosis a model-based analysis of Interferon beta signaling pathway in macrophages

Multiple Sclerosis is the most common non-traumatic cause of neurologicaldisability in young people. There is no cure yet, and until recently, few long-termtherapies existed. Interferon beta (IFNβ) was the first treatment, and remains the mostcommonly prescribed. One of the most significant problems of IFNβ therapy is theproduction of drug specific antibodies. Up to 45% of patients develop neutralizingantibodies (NAbs) to IFNβ products. The neutralizing antibody binds to the biologicalagent preventing its interaction with its receptor, inhibiting the biological action of theprotein, which abrogates the clinical efficacy of IFNβ treatment. Interferon-betamediates its response by binding to its high affinity cell surface receptor and initiatingthe JAK/STAT signalling cascade. In this project we have analyzed the IFNβ signalingpathway in macrophages when neutralizing antibodies are present. The response tothis pathway after IFNβ stimulation shows a transient oscillatory rhythm of STAT1phosphorylation, which varies as NAbs concentration increases. To improve ourunderstanding of that behavior, we extended an existing mathematical model based onnonlinear ordinary differential equations of JAK/STAT pathway by including IFN-NAbassociation and IFN-activation receptor. Combining our theoretical model withexperimental data we could study the role of neutralizing antibodies on the molecularresponse and determine its lifetime after cytokine stimulation.

β-catenin signalling in Glioblastoma multiforme and Glioma-initiating cells

Glioblastoma multiforme (GBM) is a commonly occurring brain tumor with a poor prognosis. GBM can develop both “de novo” or evolve from a previous astrocytoma and is characterized by high proliferation and infiltration into the surrounding tissue. Following treatment (surgery, radiotherapy, and chemotherapy), tumors often reappear. Glioma-initiating cells (GICs) have been identified in GBM and are thought to be responsible for tumors initiation, their continued growth, and recurrence. β-catenin, a component of the cell-cell adhesion complex and of the canonical Wnt pathway, regulates proliferation, adhesion, and migration in different cell types. β-catenin and components of the Wnt canonical pathway are commonly overexpressed in GBM. Here, we review previous work on the role of Wnt/β-catenin signalling in glioma initiation, proliferation, and invasion. Understanding the molecular mechanisms regulating GIC biology and glioma progression may help in identifying novel therapeutic targets for GBM treatment.

Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential β-catenin phosphorylation

Tyrosine phosphorylation of ß-catenin, a component of adhesion complexes and the Wnt pathway, affects cell adhesion, migration and gene transcription. By reducing ßcatenin availability using shRNA-mediated gene silencing or expression of intracellular N-cadherin, we show that ß-catenin is required for axon growth downstream of Brain Derived Neurotrophic Factor (BDNF) and Hepatocyte Growth Factor (HGF) signalling. We demonstrate that receptor tyrosine kinases (RTK) Trk and Met interact with and phosphorylate ß-catenin. Neurotrophins (NT) stimulation of Trk receptors results in phosphorylation of ß-catenin at residue Y654 and increased axon growth and branching. Conversely, pharmacological inhibition of Trk or a Y654F mutant blocks these effects. ß-catenin phospho(P)-Y654 colocalizes with the cytoskeleton at growth cones. However, HGF that also increases axon growth and branching, induces ß-catenin phosphorylation at Y142 and a nuclear localization. Interestingly, dominant negative ΔN-TCF4 abolishes the effects of HGF in axon growth and branching, but not of NT. We conclude that NT and HGF signalling differentially phosphorylate ß-catenin, targeting ß-catenin to distinct compartments to regulate axon morphogenesis by TCF4-transcription-dependent and independent mechanisms. These results place ß-catenin downstream of growth factor/RTK signalling in axon differentiation.

BAW notch filter for WiMAX applications

Als darrers anys la necessitat de connectar-se a internet des de qualsevol lloc s’ha incrementat exponencialment sobretot de manera inalàmbrica. Degut al finit espectre radioelèctric es tendeix a aprofitar totes les franges freqüencials d’aquest convivint diferents sistemes en franges properes podent induir-se interferències mútuament. Per evitar aquestes interferències es requereix de filtres a tots els dispositius els quals aïllin un sistema del adjacent. En aquest projecte es dóna una solució al cas concret de la convivència entre els sistemes Wi-Fi y WiMAX eliminant la banda Wi-Fi interferent en sistemes WiMAX. Aquesta solució consisteix en el disseny d’un filtre banda eliminada d’ordre 3 implementat mitjançant tecnologia BAW a partir de l’estructura y especificacions d’un filtre comercial. A més també es fa un petit estudi per veure si seria interessant una millora en els processos de fabricació del filtre per part del fabricant.

La millor comprensió del desenvolupament cardíac i el dany cel•lular en els miòcits ajudarà a definir dianes terapèutiques

En el cor embrional, la senyalització de mort cel•lular apoptòtica iniciada per receptors de mort i les caspases executores 3 i 7 exerceixen un paper important durant el desenvolupament cardíac no relacionat amb la mort cel•lular, i posteriorment són silenciats en l’adult, on les vies independents de caspases estan implicades en la mort cel•lular patològica. Resultats previs del nostre grup han contribuït a entendre com es regula i silencia en el cor l’expressió dels gens de la via apoptòtica depenent de caspases durant el desenvolupament; a més, resultats no publicats demostren que les caspases regulen el procés d’expressió de gens en el cor i, contràriament a la maquinària depenent de caspases, TatD, una nucleasa, ’expressa abundantment al cor postnatal. Es desconeixen les funcions de la senyalització apoptòtica durant el desenvolupament cardíac, tot i que són essencials per al desenvolupament, a més, la senyalització independent de caspases implicada al dany cel•lular en els miòcits només es coneix parcialment, el nostre objectiu és contribuir al coneixement d’ambdós fenòmens. Creiem que les caspases podrien processar proteïnes reguladores de l’expressió de gens musculars alterant la seva activitat, mentre que TatD té un paper rellevant en el dany cel•lular però també en la funció cardíaca normal. Volem caracteritzar la contribució de les caspases 3 i 7 en el desenvolupament cardíac, utilitzant models in vivo (estem finalitzant els creuaments necessaris per a disposar dels animals amb el genotip desitjat) i in vitro (pràcticament hem preparat tot el material i hem optimitzat els protocols per a tirar-ho endavant). També volem caracteritzar la funció de TatD durant el desenvolupament i fisiologia del cor i conèixer-ne la seva funció utilitzant models in vitro i in vivo.