冷冻电子显微学研究ESCRT-III剪切膜的分子机制

The endosomal sorting complex required for transport (ESCRT) is essential for many biological processes including multivesicular body formation, budding and release of HIV-1, cytokinesis, autophagy and nuclear pore assembly. ESCRT assembles into a conserved multisubunit machinery that performs a topologically unique membrane bending and scission reaction away from the cytoplasm. Among ESCRT subunits, ESCRT-III is the key part to sever membrane neck via assembling into in-plane spiral filaments, which harbor energy in the outer part. The domain reorganization of ESCRT-III subunit can release energy as possible source for membrane abscission. However, the detailed abscission mechanism about how ESCRT-III assembles and dissembles via Vps4 accompanied with energy change remains elusive due to the lack of high-resolution structure of ESCRT-III assemblies. .In this project, we will draw cryo-Electron Microscopy and cryo-Electron Tomography as the heart of our tools, complemented with biochemistry, cell biology, molecular dynamic simulation and optical tweezer aiming to study the detailed mechanism for ESCRT-III to abscise membrane. The whole project will include three correlative parts: 1) determining the high resolution structure of ESCRT-III in plane spiral in the presence of membrane via combining both cryo-EM and cryo-ET techniques; 2) resolving the high resolution structure of wild-type Vps4 with ESCRT-III filament passing through; 3) elucidating the energy recycle during ESCRT-III assembly and disassembly. .This project aims to answer how ESCRT do what they do. All the above work together will clearly show us the detailed membrane abscission mechanism accompanied with ESCRT-III assembly and the exact recovery of ESCRT-III from filaments to individual subunits via Vps4. Obviously, this project is not limited to basic research only, but will yield many promising compounds targeting cancer, neurodegenerative disease and Huntington disease caused by dysfunctional ESCRT components.

转运必需内吞体分选复合物（ESCRT）是目前发现的唯一一类可以使膜表面形成凹陷进而剪切形成独立膜系统的蛋白超分子机器，在细胞分裂，病毒出芽，细胞自噬，核膜重塑等过程中发挥了重要的作用。其中的ESCRT-III复合体组装成平面线状螺旋结构，通过结构的重排完成膜的剪切，是ESCRT系统中最重要的执行膜剪切的分子机器。.本项目将以冷冻电子显微学作为主要技术手段，综合利用生物化学，细胞生物学，分子动态模拟和光镊等技术，从解析膜上ESCRT-III平面螺旋高分辨率结构入手，研究ESCRT-III与下游解螺旋因子Vps4的复合体结构，进而检测ESCRT-III组装与去组装过程中的能量变化，从而深入理解ESCRT-III剪切膜的分子机制。.该项目的完成对解决由ESCRT功能紊乱造成的肿瘤，神经退化性疾病和亨廷氏在内的多种疾病具有重大指导意义，同时也将为胞内诸多蛋白与膜相互作用系统的研究提供借鉴。

转运必需内吞体分选复合物(ESCRT)是目前发现的唯一一类可以使膜表面形成凹陷进而剪切形成独立膜系统的蛋白超分子机器，在细胞分裂、病毒出芽、细胞自噬、核膜重塑等过程中发挥了重要的作用。其中的ESCRT-III复合体组装成平面线状螺旋结构，通过结构的重排完成膜的剪切，是ESCRT系统中最重要的执行膜剪切的分子机器。 .在本项目的资助下，我们以冷冻电镜为主要技术手段，率先发现了ESCRT-III在膜上形成的平面螺旋线状结构，克服了平面螺旋线状结构解析中诸多困难，开发了一种基于螺旋轨迹赋予欧拉角的重构新算法，解析了ESCRT-III膜上两种不同曲率多聚体的结构，明确指出了ESCRT-III亚基以其helix a0与膜相互作用，为全面理解ESCRT-III从平面螺旋线状结构到3D螺旋结构的转变提供了坚实的结构生物学基础。.通过比较不同曲率的ESCRT-III结构，我们发现了ESCRT-III的曲率来自其自身helix a2/3的弯曲，我们的分子动态模拟表明弯曲的helix a2/3具有更低的自由能，也提示ESCRT-III平面螺旋线状结构自内向外随着弯曲程度减少，自由能不断增加。进一步的数学建模证实了ESCRT-III平面螺旋线状结构不断积累的能量足以完成膜的剪切过程，首次提出了ESCRT-III剪切膜的能量来源。.本项目的开展，填补了ESCRT-III剪切膜初始状态的空白，提供了领域内期盼已久的平面螺旋线状结构。另外，本项目创造性把结构研究，功能分析和能量联系在一起，不仅在基础研究研究中起到了很好的例证作用，也将为解决由ESCRT功能紊乱造成的肿瘤，神经退化性疾病和亨廷氏在内的多种 疾病具有重大指导意义。

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