| Synapses are the hubs of information exchange between neurons,and their structure and function are the basis for higher-level behaviors such as cognition,memory,and thinking in the brain.Neurotransmitter-loaded synaptic vesicles undergo trafficking,tethering,docking,and priming processes,getting closer to the presynaptic membrane.SNAREs proteins are the core proteins that mediate membrane fusion,including Syntaxin-1 and SNAP-25 located on the plasma membrane,and Synaptobrevin-2 located on synaptic vesicles.The three SNARE proteins assemble in an antiphase parallel manner to form a four-stranded helical SNARE complex,which acts as a zipper to bring the synaptic vesicle closer to the plasma membrane and ultimately mediates membrane fusion.Many regulatory proteins are involved in the assembly of the SNARE complex,and Munc18-1and Munc13-1 are two of the core components that regulate the assembly of the SNARE complex.Munc13-1 opens the closed Munc18-1/Syntaxin-1 complex and eventually converts to the SNARE complex in the presence of Synaptobrevin-2 and SNAP-25.The transformation of the Munc18-1/Syntaxin-1 complex into the SNARE complex assembly involves two core conformational changes: conformational opening of the Syntaxin-1linker region and stretching of the Munc18-1 domain 3a.Munc18-1 and Munc13-1 play a template role in the assembly of the SNARE complex.However,the specific mechanism of interaction between the Munc18-1/Syntaxin-1 complex and Munc13-1 during SNARE complex assembly is not clear.In order to further elucidate the specific mechanism of interaction between Munc18-1and Munc13-1 during SNARE complex assembly,the following studies were carried out using a combination of biophysical and molecular biology experiments.The specific studies and main findings are as follows.(1)The interaction of the Munc13-1 MUN domain with the Munc18-1/Syntaxin-1complex was verified,the involvement of Q301/K308 in the was confirmed by singlemolecule FRET and GST pull-down experiments during the interaction between Munc18-1/Syntaxin-1 complex and MUN domain.(2)Opening of the Syntaxin-1 linker region leads to Munc18-1 domain 3a binds to Synaptobrevin 2,but domain 3a stretching cannot lead to Syntaxin-1 linker region opening.(3)A more open Munc18-1/Synatxin-1 complex: Munc18-1/H3/Habc complex was reconstituted,and K332/K333 was identified as the key site involved in the binding of Munc18-1 to Syntaxin-1 H3.With the help of this complex,MUN domain was found to be have the function of deviating Habc from Munc18-1.(4)Munc18-1/Syntaxin-1/Synaptobrevin-2/MUN intermediate state was found during the MUN-catalyzed conversion of the Munc18-1/Syntaxin-1 complex into the SNARE complex.Syntaxin-1 L165A/E166 A and Munc18-1 P335 A mutations could enhance the stability of the intermediate state complex,The samples of the intermediate complex were prepared by Gra Fix method,and the preliminary exploration of the structure and function was carried out.This thesis elucidates the molecular mechanism of Munc13-1 activation of the Munc18-1/Syntaxin-1 complex from multiple perspectives;clarifies the relationship between the opening of the Syntaxin-1 linker region and the conformational change of the Munc18-1 structural domain 3a stretch: the conformational change of the Syntaxin-1linker region is an earlier event independent of the stretch of the Munc18-1 domain 3a;demonstrates a critical role of the activated state Munc18-1 domain 3a in synaptic secretion;and provides a basis for Munc18-1 to better exercise its template function during subsequent SNARE complex assembly. |
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