膜融合介导的细胞器互作关键分子机器20S的工作机制研究

Membrane fusion is one of the important ways of organelle interaction. As the core molecular machine mediating membrane fusion, SNARE complex is of vital importance for organelle interaction. The SNARE complex is highly stable and must be disassembled into the individual protein for recycling, which is performed in cells through the ATPase enzyme NSF and the attachment protein SNAP. SNAP and NSF sequentially bind to the SNARE complex to form a 20S complex in which NSF, with the help of SNAP, upon using the energy from ATP hydrolysis, disassembles the SNARE complex to recycle the subunits. When the function of 20S machine is inhibited, the membrane fusion events in cells are also inhibited rapidly, which indicates that 20S machine is very important to maintain membrane fusion. Therefore, the working mechanism of 20S has always been a hot issue for scientists to study. In this project, we will use the nanodisc, which can mimic the lipid bilayer, to reconstitute the 20S complex. Then the high-resolution structures of 20S complex in the close physiological state will be resolved by using advanced cryo-electron microscopy combined with the optimized biochemical sample preparation technology. And on the basis of the obtained structures, we will study the mechanism underlying the SNARE disassembly by the 20S complex combined with mutation analysis, in vitro disassembly assay, single-molecule fluorescence assay, electrophysiological analysis and so on. Our research will provide important insights into the understanding of the formation and maintenance mechanisms of the organelle interaction mediated by the membrane fusion.

膜融合是细胞器互作的重要方式之一，作为介导膜融合的核心分子机器，SNARE复合体在细胞器互作中起着至关重要的作用。SNARE复合体高度稳定，它必须被解聚成单体以便循环使用。NSF与SNAP结合到SNARE复合体上形成20S分子机器，进而NSF在SNAP的帮助下利用水解ATP产生的能量将SNARE复合体解开成单体。当20S分子机器的功能被抑制时，细胞内的膜融合事件也被迅速抑制，说明20S分子机器对于维持膜融合至关重要。因此20S的工作机制一直是科学家们研究的热点问题。在本项目中，我们将使用能模拟脂质双分子层的纳米盘重组20S复合体，利用先进的冷冻电镜技术结合优化的生化样品制备技术解析20S复合体在接近生理状态下的高分辨率结构，并在结构的基础上结合突变分析、体外解聚实验、单分子荧光实验、电生理实验等，揭示20S复合体的工作机制，从而深入理解以膜融合为互作方式的细胞器互作网络的形成和维持机制。

本项目旨在利用先进的冷冻电镜技术结合优化的生化样品制备技术解析20S复合体在接近生理状态下的高分辨率结构，并在结构的基础上结合突变分析、体外解聚实验、单分子荧光实验、电生理实验等，揭示20S复合体的工作机制，从而深入理解以膜融合为互作方式的细胞器互作网络的形成和维持机制。我们按原计划基本完成了研究计划。我们获得了20S复合体中α-SNAP-SNARE组装体的高分辨结构，根据结构对关键氨基酸进行了突变，对突变体进行了体外解聚和电生理实验，并提出了一个详细的α-SNAP介导的SNARE复合体解聚的旋转模型。我们还进行了三色单分子荧光实验，并捕捉到了与早先报道不同的信息。我们观察到SNARE复合体的解聚存在两条主要路径。一条路径是Syntaxin首先以可观察到的瞬时状态在N端与复合物的其余部分分离，然后解离开。另一条主要途径是VAMP首先与Syntaxin和SNAP25解离。我们的实验结果澄清了领域内的一个主要问题，即SNARE的解聚主要是一个依次解离的过程，而不是以前报道的同时解离的方式。在本项目的资助下，我们还对膜融合过程中的拴系蛋白TRAPPII进行了研究，我们解析了酵母完整TRAPPII的多种状态的高分辨率结构，并进一步结合酵母遗传学以及生物化学等手段揭示了TRAPPII复合体的组装方式和作用机制。