26S蛋白酶体在DNA损伤应答中的底物识别机制研究

26S蛋白酶体负责真核细胞中绝大多数蛋白质的降解，对于几乎所有细胞活动都不可或缺。然而，人们对蛋白酶体自身如何被调控却知之甚少。我们发现蛋白酶体的泛素识别亚基Rpn10在多种DNA损伤条件下会发生磷酸化，且该修饰对受损细胞的存活有重要调控作用。我们推测Rpn10磷酸化可以改变其对不同泛素链的识别能力，从而影响蛋白酶体对损伤修复相关蛋白的降解。尽管DNA损伤应答中有多种蛋白会发生不同形式的泛素化，也有多项研究证明蛋白酶体活性对DNA修复至关重要，但是蛋白酶体如何识别、区分这些泛素化的蛋白尚不完全清楚。本项目将运用生物化学、细胞生物学、基因编辑、化学生物学、定量质谱、小鼠模型等多种手段研究Rpn10磷酸化与蛋白酶体底物选择性的关系及其在DNA损伤修复中的生物学意义，明确DNA损伤应答过程中蛋白酶体的底物范围，为相关抗癌药物与蛋白酶体抑制剂的合理联用提供理论和实验依据。

The 26S proteasome is responsible for degradation of the majority of cellular proteins in eukaryotes. Despite its indispensable roles in almost every cellular activity, how the proteasome itself is regulated remains unclear. Current research of my laboratory is focused on proteasome regulation by chemical modifications. Our preliminary study has established that Rpn10/PSMD4, an essential ubiquitin receptor subunit of the 26S proteasome, becomes phosphorylated in response to a variety of DNA damages. It is well known that numerous DNA repair-related proteins are modified by mono- or poly-ubiquitination, and that the proteolytic activity of the proteasome is essential for several types of DNA repair. However, how the 26S proteasome recognizes and distinguishes the variety of ubiquitinated proteins involved in DNA damage responses (DDR) remains poorly understood. We hypothesize that DNA damage-induced Rpn10 phosphorylation represents a novel substrate selection mechanism of the 26S proteasome that may be critical in regulating the turnover of DDR proteins and the outcomes of DNA damage repair. Here we propose a series of strategies including biochemistry, cell biology, gene editing, chemical biology, quantitative proteomics and mouse models to investigate the role of Rpn10 phosphorylation in determining the substrate selectivity of the proteasome, to elucidate its relevance to DNA damage repair and to define the spectrum of proteasome substrates involved in DDR. This work will provide important clues for the potential co-administration of DNA-damaging drugs and proteasome inhibitors as a combinatorial therapy for cancer treatment.