Study And Application Of Reconstituted Human Neuronal SNARE Fusion Mechanism In Saccharomyces Cerevisiae

In human neural networks,signal transmission between neurons is mediated by neurotransmitters,and release of neurotransmitters is regulated by synaptic vesicle fusion.During synaptic vesicle fusion,syntaxin-1A,VAMP2 and SNAP-25 form a SNARE complex through their SNARE domains,synaptic vesicles fuse with the presynaptic membrane under the joint control of many proteins and factors,eventually leading to the release of neurotransmitters.The regulation of membrane fusion is extremely complicated,and there are still some mechanisms for regulating proteins that have not yet been explained completely.In eukaryotic cells,the membrane fusion mechanism is highly conserved,human and yeast share the same process.Sso1/2,Snc1/2 and Sec9,which are orthologous proteins of the human SNARE proteins syntaxin-1A,VAMP2 and SNAP-25,form a SNARE complex in the cell secretory process of yeast.The destruction of human neurotransmitter release causes various neurological diseases,and the destruction of the extracellular secretion process of yeast affects the growth of yeast.Based on the advantages of simple gene operation and short growth cycle of yeast,the human neuronal membrane fusion process can be reconstituted in yeast,and the neuronal SNARE membrane fusion mechanism can be studied in yeast.Botulinum neurotoxins inhibit neurotransmitter release by cleaving the SNARE protein and syntaxin-1 binding protein tomosyn inhibits neurotransmitter release by forming a SNARE complex with SNAP-25 and syntaxin-1A.On the basis of the previous study,the model of humanized yeast was further improved,and it could be used to study the SNARE membrane fusion mechanism and construct the activity analysis system of botulinum neurotoxin C and function analysis system in yeast.The main research results of this thesis are as follows:（1）Analyse the reasons why Sso1/STX1A could not replace the function of Sso1and Sso2.Sso1/STX1A is a chimera of Sso1 and syntaxin-1A in which the SNARE domain in Sso1 was replaced with that of syntaxin-1A,was not functional in yeast.Through a screening we found that when introducing mutations in the Habc domain,the Sso1/STX1A chimeric protein was functional,which was similar to the introduction of mutations in SNARE region in previous study.Protein localization analysis indicated that the functional incompatibility of Sso1/STX1A in yeast cells may be attributable to its accumulation in ER（endoplasmic reticulum）.Based on this hypothesis,it was further verified that the functional Sso1/STX1A mutant（D133V and A220E）could be released from ER.（2）Characteristic analysis of SNARE proteins in syntaxin family.Firstly,the ER localization of Sso1/STX1A and Sso1/STX1A^A220E/D133V under the condition of overexpression of Sec9 or Snc1 was caused by the formation of ectopic SNARE complex in ER.Then,through analyzing the characteristic differences of the SNARE region of Sso1 and syntaxin-1A by fluorescence localization and IP experiment,we found that the SNARE region of syntaxin-1A was more likely to form ectopic SNARE complex in ER.The self-interaction of Sso1 and syntaxin-1A was analyzed through IP experiment,and it was found that syntaxin-1A could self-interact through its SNARE region,while Sso1 could not,which further indicated that Sso1 and syntaxin-1A proteins had different properties.Finally,fluorescence localization and IP results indicate that syntaxin-1A maintains a tight closed state by binding to munc-18,which further indicates that the SNARE regions of syntaxin-1A and Sso1 have different characteristics.（3）Obtaining a yeast mutant strain in which Sso1/STX1A could replace the function of Sso1 and Sso2 through 5-FOA screening.Firstly,a viable sso1?sso2?harbouring Sso1/STX1A was obtained by 5-FOA drug screening;Then the effective mutant gene in the strain was isolated by yeast tetrad disecssion.The G373D mutation was found on the candidate gene GFA1 by yeast whole genome sequencing and database alignment.Subsequently,by analyzing the effect of GFA1^G373D on the localization of Sso1/STX1A recombinant protein and the study of GFA1 metabolic pathway,the compensatory mechanism of G373D mutation was proposed.It was further verified that the mutation of the human orthologous protein GFPT1 corresponding site has the same function as the G373D mutation of GFA1 in yeast,which provides the foundation for a study of GFPT1 in human neurological diseases and other related diseases.（4）BoNT/C-LC activity was analyzed by using a humanized yeast model.Expression of BoNT/C-LC did not interfere with the growth of sso1?sso2?cells harboring Sso1;Next,expression of BoNT/C-LC interfered with the growth of sso1?sso2?cells harboring Sso1/STX1A^D133V by degrading the Sso1/STX1A^D133V protein.Therefore,the activity of BoNT/C-LC can be analyzed by monitoring the growth of yeast.Finally,the sensitivity of different Sso1/STX1A recombinant proteins to BoNT/C were analyzed by the difference of yeast growth phenotype,and substrate recognition by BoNTs is mediated by H213,D214 and M215 in the exosites remote from the active site and and the degradation recognition site K253 and V254.（5）Establishing a yeast model for the analysis of tomosyn function.Tomosyn overexpression did not affect the growth of wild-type yeast,while inhibited the growth of sso1Δsso2Δcells harbouring Sso1/STX1A^D133V.Further analysed different Sso1/STX1A mutants in combination with tomosyn in yeast by monitoring yeast growth phenotypic differences,we found that the 12 amino acids in front of the SNARE region contribute to the binding of syntaxin-1A to tomosyn,also said the entire alpha helix region of syntaxin-1A is required for binding to tomosyn.Thus,the function of tomosyn could be characterized by monitoring the growth of humanized yeast.