26S蛋白酶体识别底物的分子机制

26S proteasome is the major molecular machine that is responsible for protein degradation in eukaryotic cells. By specifically eliminating target proteins, it is involved in almost every life activities of organisms. 26S proteasome consists of 47 protein members, and can be divided into 19S regulatory particle and 20S core particle. The target protein substrate with polyubiquitin-tag was firstly recognized by 19S regulatory particle, and then unfolded and translocated into 20S core particle for degradation. The high resolution structures of 26S proteasome were determined with CryoEM techniques. However, molecular mechanism of substrate recognition and processing by 26S proteasome remains unclear, because the substrates with homogeneous ubiquitin chains have not been prepared successfully until now. In this project, the PIMUL (Premade IsoUb-Mediated Ubiquitin Ligation) will be applied to synthesize the substrates with defined homogeneous ubiquitin chains, which has already been proved to be feasible by our unpublished data. And the prepared samples of 26S proteasome bound substrate with defined polyubiquitin chain in different states will be used for CryoEM structure determination. These structures will serve as a molecular basis for mechanistic understanding for substrate recognition by 26S proteasome.

26S 蛋白酶体是真核细胞内负责蛋白质降解的主要分子机器, 通过特异性降解目的蛋白质, 几乎参与了生物体的绝大多数生命活动。在结构上，26S 蛋白酶体可分为19S 调节颗粒和20S 核心颗粒两部分。19S 调节颗粒负责识别带有泛素链标记的蛋白质底物及对其进行去折叠, 并最终将去折叠的蛋白质底物传送至20S 核心颗粒中进行降解。经过多年努力，本项目组已经解析了两个不同构象的26S蛋白酶体的高分辨冷冻电镜结构。然而，由于缺少化学均一性的蛋白质底物，26S蛋白酶体结合蛋白质底物的三维结构尚未获得解析，严重阻碍了对其分子机制的了解。本项目拟采用PIMUL (Premade IsoUb-Mediated Ubiquitin Ligation)方法制备泛素修饰的均一性底物，并制备底物结合的26S蛋白酶体复合物，采用冷冻电镜学的方法进行解析，揭示26S蛋白酶体识别底物的分子机制。

泛素-蛋白酶体系统（Ubiquitin-Proteasome System, UPS）控制着哺乳动物细胞内至少80%的蛋白质降解。26S蛋白酶体是一个大约2.5 MDa的多亚基复合物，由20S核心颗粒（20S core particle, CP）和19S调节颗粒（regulatory particle, RP）两个亚复合物组成。特定泛素化修饰的蛋白质会被蛋白酶体RP识别，并在ATP水解供能下经过去泛素化、去折叠和易位进入蛋白酶体CP中被降解。近年来低温电子显微镜技术和方法的突飞猛进使得酵母与人源26S蛋白酶体全酶高分辨率结构被解析，这使得部分蛋白酶体工作原理的模型被搭建出来。但是目前人源26S蛋白酶体与K48链接四泛素底物（K48-Ub4）尚未有任何结构报道，阻碍了人源26S蛋白酶体识别底物的分子机制研究。本项目利用高分辨的单颗粒冷冻电镜技术首次解析了人源26S蛋白酶体与K48-Ub4底物的近原子分辨率的三维结构（3.86Å）。在结构中我们首次发现3个泛素密度，其中一个近端泛素分子Ub1结合在去泛素化酶Rpn11附近，而两个远端泛素分子Ub2和Ub3结合在泛素受体Rpn10附近。此外，我们首次在蛋白酶体全酶结构中发现了Rpn13的部分密度。Rpn13 N端靠近Rpn2 P927位氨基酸残基，暗示二者之间可能存在相互作用。综上所述，本项目为揭示人源26S蛋白酶体识别K48-Ub4底物分子机制奠定了结构基础。