线粒体内膜融合蛋白OPA1促进膜重塑的分子机制研究

Mitochondria are dynamic organelles that play important roles in many life processes, such as energy metabolism, mitochondrial autophagy and apoptosis. Those roles largely rely on the continually going mitochondrial fusion and fission. A better understanding of the fusion and fission related molecular mechanism will ultimately lead to improvements in the interpretation of the mitochondrial functions..In mammals, the mitochondrial inner membrane fusion is mediated by OPA1. Under physiological state, OPA1 undergo proteolytic processing to form a long isoform (L-OPA1) that is membrane anchored and a short isoform (S-OPA1) that lacks the membrane anchor but can still interact with membranes. A combination of both long and short isoforms is required for membrane fusion. However, when mitochondria lose membrane potential, have low levels of ATP, or lose some of the other quality-control mechanisms, all L-OPA1 will be cleaved to S-OPA1 followed by inner membrane fusion prevention. The mechanism of OPA1 mediated inner membrane fusion is not well understand, so do the respective function of the long and short form OPA1s. Absence of structural study is one of the restraints. Since the interaction of OPA1 and membrane is the basis of OPA1 mediated membrane remodeling, and S-OPA1, as has been reported, can deform the membrane surface and cause the elaboration of lipid tubules, we will focus on the structure of S-OPA1-membrane complex by using the technique of three-dimensional helical reconstruction of cryo-electron microscopy and three-dimensional reconstruction of cryo-electron tomography. Those structures will tell the S-OPA1-membrane interaction and help understanding the molecular mechanism of OPA1 mediate membrane remodeling. This work will contribute to the interpretation of mitochondrial membrane dynamics.

线粒体融合分裂在多种生命过程中具有重要作用，因此研究与融合分裂密切相关的线粒体膜的动态变化对于阐释线粒体发挥多种生理功能的机制至关重要。哺乳动物线粒体内膜融合过程主要由OPA1介导。生理状态下，体内的OPA1存在长短两种活性形式，分别为L-OPA1和S-OPA1，两种形式的蛋白共同作用保障线粒体融合的正常进行。当细胞处于一定压力条件下时，L-OPA1被全部剪切为S-OPA1，此时线粒体的融合过程被抑制。由于确定OPA1与膜的相互作用方式是研究OPA1促进线粒体内膜融合机制的第一步，而S-OPA1可单独与膜作用形成规则排列的螺旋结构，因此本课题将综合利用冷冻电镜螺旋三维重构技术和冷冻电子断层三维重构技术，对S-OPA1与膜形成的复合物的结构进行解析，获得S-OPA1的结构及并了解其与膜相互作用的方式，以阐释OPA1在促进线粒体内膜融合的过程中发挥作用的分子机制。

视神经萎缩蛋白OPA1是介导线粒体内膜融合的关键蛋白。在生理状态下，OPA1通过蛋白酶水解形成锚定在线粒体内膜上的长型L-OPA1和可溶的短型S-OPA1两种活性形式。L-OPA1与S-OPA1的共同作用促进线粒体内膜的融合。当细胞处于一定压力条件下，L-OPA1被全部剪切为S-OPA1，线粒体的融合过程被抑制。然而，在融合功能发生的过程中，S-OPA1到底承担怎样的角色，S-OPA1促使内膜融合具体分子机制等尚不清楚。. 本项目围绕S-OPA1促进线粒体内膜融合的分子机制，在体外建立S-OPA1的表达纯化和脂质体制备体系，鉴定重组表达的S-OPA1蛋白的生化性质，并制备出用于结构解析的电镜样品；设计构建S-OPA1突变体，解析不同核苷酸结合状体下S-OPA1与脂质体形成的管状结构的三维结构，阐释GTP结合与水解对S-OPA1发挥其生理功能的影响及S-OPA1驱动的膜重塑的分子机制；. 本项目解析了剪切的短型S-OPA1与脂质体形成的螺旋三维结构。结构表明，与经典的Dynamin家族蛋白结构类似，S-OPA1也可分为用于GTP水解的GTPase结构域，介导高聚组装状态形成的Stalk区域和膜结合相关的EMB结构域三部分。螺旋结构的不对称单位之间的作用面对stalk区域的相互作用及成管起至关重要的作用。突变体实验表明EMB结构域中存在促使S-OPA1引起膜形变并进行管状组装的关键位点，且S-OPA1的成管过程与GTP的结合和水解无关，然而膜结合对于S-OPA1的GTP水解活性有显著增强作用，提示GTP水解发生在S-OPA1促进膜融合的晚期过程。本项目进一步利用冷冻电子断层三维重构和体平均技术，解析了GTPγS结合状态下S-OPA1与脂质体形成的管状复合物三维结构。与未结合核苷酸的结构相比，GTPγS结合之后的S-OPA1通过调节自身构象和组装方式，使得其在脂质体管上的排列更加松散，从而引起了脂质体管直径的扩张。. 该项目的研究工作揭示了人源线粒体内膜融合蛋白S-OPA1与线粒体内膜结合组装的模式，发现了核苷酸结合所引起的S-OPA1构象和组装的变化，提出了核苷酸结合所触发的膜形变的分子机理。