Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011Neuronal 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
