基于冷冻电镜的真核细胞分子伴侣素TRiC的结构与功能研究

The health and integrity of the cellular proteome depend on molecular chaperones, which maintain protein homeostasis through their distinct substrate specificities and modes of action. Protein misfolding and aggregation are associated with a growing number of human diseases, ranging from cancer to aggregation-associated neurodegenerative diseases such as Alzheimer’s and Huntington’s. The eukaryotic chaperonin TRiC (also known as CCT) serves as an essential protein-folding nanomachine, assisting the folding of 10% of the cytosolic proteins, including many key cytoskeletal components (i.e. actin, tubulin) and cell cycle regulators (i.e. VHL, p53 and CDC20). TRiC also appears important for the prevention of protein aggregation and toxicity. TRiC is hetero-oligomeric, consisting two stacked rings of eight different subunits each, forming the most complex chaperonin. Its functionality is closely related to the ATP-driven conformational cycle of TRiC. However, due to lacking of high-resolution structural information, our understanding on the mechanism of TRiC allosteric cooperativity, and on how the distinct TRiC subunits coordinate to recognize and fold the diversified substrates remains limited. Through this project, by utilizing the cutting-edge cryoelectron microscopy (cryo-EM) technique, we aim to resolve high-resolution cryo-EM structures of TRiC and TRiC-substrate complexes in different nucleotide states. Our goal is to systematically reveal the mechanism of how TRiC recognizes, binds and assists the productive folding of substrate, as well as to understand how the structural asymmetry of TRiC is related to its functional specificity. Our work will facilitate developments in diagnosis and treatment of related human diseases including cancer and neurodegenerative disease.

细胞中蛋白质的正确折叠对其发挥正常的生物学功能、维持细胞内环境的稳态具有重要作用。蛋白质错误折叠及聚集可引起癌症和神经退行性疾病等人类重大疾病。真核细胞中的分子伴侣素TRiC可以协助～10％的胞质蛋白正确折叠，其底物包括许多重要的结构性和调节性蛋白。TRiC 拥有8个不同的亚基，是最为复杂的分子伴侣素，其功能的实施与其在ATP 驱动下的构象变化密切相关。然而由于缺乏高分辨率结构信息，限制了人们对TRiC高度协同性及其特异性识别并折叠底物机制的认知。本项目将应用冷冻电镜技术，解析不同ATP状态下TRiC与代表性底物形成复合体的高分辨率三维结构，推进部分结构至近原子分辨率，以期系统性揭示TRiC识别、结合及协助底物正确折叠的结构机制，理解TRiC各亚基的结构及ATP结合能力的非对称性与其功能特异性之间的关系，进而为相关疾病的诊治提供结构和理论基础。

真核细胞中的分子伴侣素TRiC可以协助～10％的胞质蛋白正确折叠, 其发挥正常的生物学功能对于维持细胞内环境的稳态具有重要作用。TRiC底物包括许多重要的结构性和调节性蛋白，如细胞骨架蛋白actin、tubulin, 细胞周期调节蛋白CDC20等。TRiC功能的异常与癌症和神经退行性疾病等密切相关。TRiC 有8个不同的亚基，是最为复杂的分子伴侣素，其功能的实施与其在ATP 驱动下的构象变化密切相关。由于缺乏高分辨率结构信息，TRiC的高度协同性及其特异性识别并协助底物折叠的分子机制亟待研究。本项目资助的主要研究进展如下：（1）首次捕捉到真核生物分子伴侣素TRiC逐步关环的完整构象变化空间，揭示了TRiC各亚基在ATP消耗和关环过程中的特异性机制（PNAS, 2019）；（2）发展了eGFP标签定位（YISEL）方法及亚基中位PA-NZ-1标签-抗体定位（YISPANL）方法，并应用上述方法，首次明确了TRiC各亚基在其开环结构中的定位，解决了一直以来的争议（Sci Rep, 2018a；J Mol Biol, 2018），也为其他超大分子复合体的亚基准确定位提供了新策略（Mol Cell, 2019；Sci Rep, 2018b；PNAS，accepted）；（3）首次解析了TRiC结合底物tubulin形成的复合体在ATP驱动下的系列高分辨率冷冻电镜结构，揭示了TRiC辅助底物tubulin正确折叠的完整过程和结构机制，为相关疾病的诊疗提供理论基础；（4）首次捕捉到辅助伴侣Plp2在TRiC关环过程中的动态位置迁移，观测到辅助伴侣PFD可以同时结合在TRiC的双环外入口处，为全面揭示TRiC在辅助伴侣参与下协助底物折叠的分子机制奠定结构基础；（5）系统研究了酵母以及人源TRiC单亚基同源寡聚物的成环特性及其结构和功能，揭示了TRiC的组装机制（已投稿）；（6）应邀发表两篇TRiC体系相关的研究专评及综述，并提出了本领域未来需要回答的关键科学问题（Nat Struct Mol Biol, 2017; Macromolecular Protein Complexes II, 2019）。在PNAS、Mol Cell、Sci Adv、Cell Research、Nat Comm、Cell Discovery等高水平期刊发表受本项目资助的研究论文13篇。