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Analysis of syntaxin-1 chaperones during synaptic activity
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Orientador(es)
Resumo(s)
Neuronal communication is based on synaptic vesicle exocytosis, which is strongly regulated. The release of neurotransmitters from presynaptic nerve terminals requires cycles of protein-protein interactions. SNARE and SM proteins are universally involved in all intracellular membrane fusion reactions, and reside either on the target membrane (syntaxin-1 and synaptosome-associated protein of 25kDa (SNAP-25) or on the synaptic vesicle (synaptobrevin-2). Recent studies have identified chaperones for two SNARE proteins: synaptobrevin-2 and SNAP-25 . Since these SNARE chaperones seem essential for the long-term functioning of synapses, the question arises which molecule(s) may chaperone syntaxin-1. Previous studies have suggested that Munc-18 and SNAP-25 may chaperone syntaxin-1. Furthermore, it has been shown that chemical modification of syntaxin-1 or mutation on cysteine residue 145 increases its stability. To investigate a possible chaperone function of Munc-18 and SNAP-25 for syntaxin-1, I aimed to clarify whether this chemical modification inhibits syntaxin-1 degradation, whether the C145S mutation reproduces this modification, and whether this cysteine is normally involved in ubiquitination and degradation of syntaxin-1. To approach these aims, HEK-293T cells and neuronal cultures from wild-type mice were used in combination with overexpression of syntaxin-1 full-length, several truncations and its mutant C145S. Chemical agents were used to monitor syntaxin-1 levels. These experiments were analyzed by immunoprecipitation, immunoblotting or immunocytochemistry. Results suggest that munc-18 chaperones syntaxin-1, based on the following observations: 1) it increases syntaxin-1 levels and inhibits syntaxin-1 degradation in cotransfected HEK cells; 2) C145S mutation significantly stabilizes syntaxin-1 levels and results in less degradation products. C145S also dramatically reduces ubiquitination of syntaxin-1; 3) syntaxin-1 may be degraded via the lysosome. Lysosomal inhibitors revealed a trend towards stabilization of syntaxin-1 whereas proteasomal inhibitors showed no change. Yet, further experiments are needed to understand the precise role of C145S in the degradation mechanism of syntaxin-1.
Descrição
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
Palavras-chave
SNARE complex Syntaxin-1 Munc-18 SNAP-25 Chaperones Teses de mestrado - 2011