Shen Wang,Ucheor B Choi,Jihong Gong,Xiaoyu Yang,Yun Li,Austin L Wang,Xiaofei Yang,Axel T Brunger,Cong Ma
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1 adopts a closed conformation when bound to Munc18-1, preventing binding to synaptobrevin-2 and SNAP-25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13-1 catalyzes the transition from the Munc18-1/syntaxin-1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin-1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18-1/syntaxin-1/MUN complex, in which syntaxin-1 still adopts a closed conformation tightly bound to Munc18-1, whereas the syntaxin-1 linker region changes its conformation, similar to that of the LE mutant of syntaxin-1 when bound to Munc18-1. We suggest that the conformational change of the syntaxin-1 linker region induced by Munc13-1 initiates ternary SNARE complex formation in the neuronal system.
Background : Radix Bupleuri (RB) and acetaminophen (APAP) are two popular medications having potential hepatotoxicity and substantial risks of irrational co-administration and excessive use, posing an overlooked danger of drug-induced liver injury (DILI). Autophagy is a protective mechanism against APAP-induced DILI, yet, saikosaponin d (SSd) in RB has been characterized to regulate autophagy, although the current findings are controversial. Purpose : We aim to elucidate whether SSd promoted APAP-induced liver injury by regulating autophagy. Methods : UPLC-MS analysis was employed to measure the hepatic abundance of APAP-cysteine protein adducts. Multiple techniques such as fluorescence probe, proteinase K protection assay, immunoprecipitation-coupled proteomic analysis, surface plasmon resonance, molecular docking and et.al were applied to evaluate the SSd on autophagy flux. Results : We discovered that, by inhibiting autophagy, SSd impaired the removal of APAP-cysteine protein adducts and delayed the compensation of damaged mitochondria. This ultimately potentiated the development of severe liver toxicity induced by subtoxic APAP. The use of autophagy probes, transmission electron microscopy, membrane curative assay, and protein K assay collectively revealed that SSd predominately disrupted autophagosome-lysosome fusion, without affecting other stages of autophagic flux. Immunoprecipitation-coupled proteomic analysis and surface plasmon resonance further found that SSd directly bound to GABARAP, thus preventing the recruitment and autoactivation of STX17 and the following assembly of STX17-SNAP29-VAMP8 complex. Conclusion : In conclusion, our findings not only highlight the significant risk of drug-induced liver injury associated with the co-administration of RB and APAP in clinical practice but also unveils that GABARAP-SNARE complex is a novel druggable target for the treatment of autophagy-related diseases.
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