26S蛋白酶体调节亚基组成蛋白Rpn5-Rpn9复合物的晶体结构

26S proteasome is the major ATP-dependent regulated protein degradation machine in eukaryotes . This giant macromolecular complex consists of two parts: 20S core particle(CP) and 19S regulatory particle(RP). The poly-ubiquitinated protein substrates can be recognized, unfolded and translocated by RP into CP for degradation. RP could be further divided into two subcomplexes: base and lid. Base is composed of 4 Non-ATPase subunits and 6 different ATPases(Rpt1-6), which is responsible for substrate unfolding and translocation. And lid contains 9 non-ATPase subunits, which is involved in substrate recognition and poly-ubiquitin chain trimming. Among these 9 subunits, there are 6 PCI domain and 2 MPN domain containing proteins. Interestingly, this subunits organization in lid is similar to those in eIF3 and COP9 signolosome complex. Until now，little has been reported about the exact role of the PCI domain containing proteins in the proteasome lid, eIF3 and COP9 signolosome complex assembly. In this project, we are trying to get the crystal structure of Rpn5-Rpn9 complex. Combined with structure analysis directed biochemical and biophysical investigation, this project will provide the insight into the molecular basis for the interaction between PCI containing proteins and further lid assembly.

26S 蛋白酶体是真核细胞负责ATP依赖的可调控蛋白降解的主要分子机器，由核心亚基和调节亚基构成。泛素化底物首先被调节亚基识别并去折叠，进而传送到核心亚基中降解。调节亚基主要由两部分构成：base和lid。其中base主要由6个ATPase和4个非ATPase蛋白亚基构成，负责底物去折叠及传送；lid包含9个非ATPase蛋白亚基，负责底物识别和泛素链剪切。这9个亚基的组成方式同另两个大分子复合物eIF3起始因子和Cop9 信号小体相似，都是由6个含PCI结构域及1－2个含MPN结构域的同源蛋白构成。关于含PCI结构域的蛋白在这些复合物上的准确组装机制尚未见报道。本项目拟通过解析lid中两个含PCI结构域的蛋白Rpn5和Rpn9的二元复合物晶体结构，并基于结构分析，运用生物化学和生物物理学等手段来阐明含PCI 结构域蛋白之间的相互作用方式，为进一步阐明lid等复合物的组装机制奠定结构基础。

真核26S蛋白酶体是体内主要的蛋白质降解机器，介导泛素链标记的可调控蛋白质降解。本项目利用高分辨的单颗粒冷冻电镜技术首次解析了来自酿酒酵母内源的26S 蛋白酶体的近原子分辨率的三维结构（ 4.6- to 6.3-Å）。基于高分辨的冷冻电镜图谱数据，本项目重建了26S蛋白酶体中调节颗粒中的18个蛋白亚基和催化颗粒中所有28个蛋白质亚基。基于冷冻电镜数据处理，本项目解析了两个构象明显不同的26S蛋白酶体三维结构，分别命名为M1和M2。其中M1和先前报道的ATP-γS 结合的26S蛋白酶体相似，而M2和ATP结合的26S蛋白酶体相似。另外，M1状态和先前报道的结合内源底物的26S蛋白酶体构象相似，M2和未结合底物的26S蛋白酶体构象相似。此外，基于结构分析，我们体外重构了Rpn8-11，lid core 和lid，并表明Rpn11在组装成lid后去泛素化酶活性明显提高。综上所述，本项目为揭示26S蛋白酶体执行功能的分子机制奠定了扎实的结构基础。