Publicação
Development of new cellular models for step-by-step analysis of Astrogliogenic pathways
| Resumo: | The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an evolutionarily conserved mechanism for signalling transduction and involves four steps regulated by protein-protein interactions. Cytokines bind to the common receptor glycoprotein 130 (gp130) to activate JAKs, which then phosphorylate STATs leading to its dimerization and translocation to the nucleus to induce target genes. The activation of this pathway is essential for astroglia production during brain development and upon central nervous system (CNS) damage. Astrocytes are crucial to neuronal survival and determine the ability of the CNS to repair damage and regenerate. Multiple brain insults elicit a reactive response from astrocytes called astrogliosis, which can impair axonal regeneration and the reestablishment of the neural circuits. Due to the pleiotropic effects of the JAK/STAT pathway several attempts have been made to target this signalling pathway. However, further investigation on protein interactions and dynamics are required for an efficient determination of molecular targets. In this regard, bimolecular fluorescence complementation (BiFC) assays offer a direct method to identify potential modulators of protein interactions. In this study, we developed two Venus-based BiFC systems to screen for modulators of key protein-protein interactions in the JAK/STAT pathway, a gp130-Venus and a Venus-STAT3 BiFC system. Both systems worked in living cells and displayed a similar behaviour to endogenous proteins sustaining novel information regarding the dimerization of these proteins. The Venus-STAT3 BiFC system was further validated by a specific inhibitor of STAT3. These results show the potential of the BiFC systems as tools for drug and genetic screenings, as well as step-by-step analysis of the JAK/STAT pathway in diverse biological contexts. |
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| Autores principais: | Rocha, Ana Catarina Maia |
| Assunto: | via JAK/STAT gp130 STAT3 astrócito sistema BiFC Teses de mestrado - 2015 |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an evolutionarily conserved mechanism for signalling transduction and involves four steps regulated by protein-protein interactions. Cytokines bind to the common receptor glycoprotein 130 (gp130) to activate JAKs, which then phosphorylate STATs leading to its dimerization and translocation to the nucleus to induce target genes. The activation of this pathway is essential for astroglia production during brain development and upon central nervous system (CNS) damage. Astrocytes are crucial to neuronal survival and determine the ability of the CNS to repair damage and regenerate. Multiple brain insults elicit a reactive response from astrocytes called astrogliosis, which can impair axonal regeneration and the reestablishment of the neural circuits. Due to the pleiotropic effects of the JAK/STAT pathway several attempts have been made to target this signalling pathway. However, further investigation on protein interactions and dynamics are required for an efficient determination of molecular targets. In this regard, bimolecular fluorescence complementation (BiFC) assays offer a direct method to identify potential modulators of protein interactions. In this study, we developed two Venus-based BiFC systems to screen for modulators of key protein-protein interactions in the JAK/STAT pathway, a gp130-Venus and a Venus-STAT3 BiFC system. Both systems worked in living cells and displayed a similar behaviour to endogenous proteins sustaining novel information regarding the dimerization of these proteins. The Venus-STAT3 BiFC system was further validated by a specific inhibitor of STAT3. These results show the potential of the BiFC systems as tools for drug and genetic screenings, as well as step-by-step analysis of the JAK/STAT pathway in diverse biological contexts. |
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