TRIM27在RNF169调控的DNA损伤修复响应中的相互作用与功能研究

To safeguard genome integrity, cells utilize a repertoire of genotoxic stress responses, collectively known as the “DNA Damage Response” (DDR), that culminate in DNA repair and cell-cycle arrest. Key to the formation of DSB-associated ubiquitin conjugates are the E3 ubiquitin ligases RNF8 and RNF168. Current evidence suggests that these ubiquitin ligases work in concert to promote lys63-based ubiquitin polymers at the vicinity of DSBs, allowing redistribution of genome-caretaker proteins BRCA1, 53BP1 and RAD18 into cytologically discernible foci that overlap with the DNA damage marker gH2AX. the Ring Finger Protein RNF169 has recently identified as a paralogue of the RIDDLE syndrome RNF168 protein that plays negative roles in ubiquitin-signaling events at DSBs. RNF169 competes for DSB-associated ubiquitin adducts and prevents hyper-accumulation of genome-caretaker proteins BRCA1 and 53BP1 from DSBs. In a research recently accepted by PNAS, we document an unprecedented role of an NLS that supports the nuclear function of RNF169. We show that the RNF169 NLS serves not only to target the Ring finger protein into the nucleus, but also enforces its occupancy at DSBs by mediating its interaction with USP7, a deubiquitylase that protects it from proteolytic degradation. Accordingly, genetic ablation or chemical inhibition of USP7 destabilises RNF169, compromises high-fidelity DSB repair, and hyper-sensitize cells to PARP inhibition. Together, we propose that the USP7-RNF169 axis is integral to the DSB signal transduction cascade, and allows fine-tuning of chromatin responses at the DSB microenvironment. Using TAP-MS, we identified TRIM27 as another RNF169-interacting protein, and TRIM27, USP7, and RNF169 might form a ternary complex in vivo. In this project, we are planning to investigate the interactions among these three proteins, using both structural and cell biology methods, to understand the role of TRIM27 played in the functional regulation of RNF169 and potential influence caused by USP7 for this regulation.

在DNA损伤应答信号传导网络中，细胞精确的使用RNF8/RNF168两个E3泛素连接酶在DNA双链断裂(DSB)位点附近实现泛素化来定位若干重要的信号蛋白(BRCA1和53BP1等)，同时又使用RNF169来和上述蛋白竞争泛素化位点，从而实现了DSB响应的动态平衡，使细胞可以对DNA损伤进行精确修复。我们在前期研究中发现了USP7是RNF169的结合蛋白，通过一系列的结构和细胞生物学研究，我们揭示了USP7利用去泛素化来稳定RNF169，从而提高RNF169在DSB位点附近的水平，促进了细胞利用同源重组HR方式进行修复的机制。我们最近又确认了RNF169的另一个结合蛋白TRIM27，并发现TRIM27、USP7、和RNF169在细胞内会形成一个三元复合物。在本项目研究中，我们计划通过结构生物学方法研究这三个蛋白之间的相互作用关系和细节，并结合细胞实验揭示其在DNA损伤修复中的功能意义。

在DNA损伤应答信号传导网络中，细胞精确的使用RNF8/RNF168两个E3泛素连接酶在DNA双链断裂(DSB)位点附近实现泛素化来定位若干重要的信号蛋白(BRCA1和53BP1等)，同时又使用RNF169 来和上述蛋白竞争泛素化位点，从而实现了DSB响应的动态平衡，使细胞可以对DNA损伤进行精确修复。我们在前期研究中发现了USP7是RNF169的结合蛋白，并揭示了USP7利用去泛素化来稳定RNF169，从而提高RNF169在DSB位点附近的水平，促进了细胞利用同源重组HR方式进行修复的机制。在本项目研究中，我们针对DNA损伤应答信号通路中的两个蛋白RNF168和USP7展开了进一步的研究。通过合作，我们阐明了RNF168是如何准确的定位组蛋白尾巴H2A和H2AX上的13位和15位的lysine残基来实现泛素化，从而引导下游的DNA损伤修复应答的分子机制。另外，我们发现了一个USP7的新的调控蛋白RBMX，它可以通过调控USP7对MDM2的去泛素化过程来影响MDM2的稳定性，潜在进一步影响p53的正常功能。