泛素E3连接酶CHIP介导RCC2的泛素化及降解  

E3 ligase CHIP mediates the ubiquitination and degradation of RCC2

作　　者：李贝 范宇宸 赵博[1] LI Bei;FAN Yuchen;ZHAO Bo(School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学药学院,上海200240

出　　处：《石河子大学学报（自然科学版）》2023年第3期327-334,共8页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(31971187);上海市科委基础研究领域项目(20JC1411200)。

摘　　要：目的 本课题组前期构建了一种正交泛素传递方法,并发现染色体浓缩调节蛋白2(RCC2)是泛素连接酶CHIP的潜在底物。本实验旨在验证CHIP是否能介导RCC2的泛素化及降解,以期通过CHIP来泛素化调控RCC2的表达和功能。方法 首先构建CHIP及其突变体质粒,验证CHIP与RCC2是否有相互作用;然后构建pLVX-RCC2-FLAG重组质粒并转染进CHIP敲减细胞株(shCHIP)、HEK293细胞株、CHIP过表达细胞株(OE CHIP),通过免疫共沉淀下拉富集RCC2-FLAG并检测其泛素化水平;接下来在HEK293细胞中转染不同量的CHIP,以及设置CHX chase蛋白稳定性实验,检测RCC2的蛋白水平变化;最后利用泛素突变体K48R-Ub及K11R-Ub进行CHIP介导RCC2上形成泛素链的实验检测。结果 在HEK293细胞中,CHIP能够介导RCC2的泛素化,并在RCC2上形成K48和K11多聚泛素链,促进其通过蛋白酶体发生降解。结论 泛素连接酶CHIP能够介导RCC2的泛素化及降解,为实现通过CHIP来泛素化调控RCC2提供了理论基础。Objective Through the OUT(orthogonal ubiquitin transfer) pathway,we identified RCC2(regulatory of chromosome condensation 2) as a potential substrate of E3 ubiquitin ligase CHIP.Here,we verified that CHIP can mediate the ubiquitination and degradation of RCC2 and provided a new direction for pharmacology research targeting RCC2.Methods We verified the interaction between CHIP and RCC2 by co-immunoprecipitation.We then examined RCC2 ubiquitination levels in HEK293 cells with knockdown or overexpression of CHIP.To determine if CHIP regulates the degradation of RCC2 by ubiquitination,we transiently transfected CHIP plasmids with titration into HEK293 cells and measured the steady-state levels of RCC2.We also conducted a CHX chase assay to investigate whether CHIP could induce the degradation of RCC2.Furthermore,we validated that CHIP can form K48-and K11-linked polyubiquitin chains on RCC2 by Ub mutants K48R-Ub and K11R-Ub.Results In HEK293 cells,CHIP can mediate the ubiquitination of RCC2.CHIP can form K48-and K11-linked polyubiquitin chains on RCC2 and promote its degradation.Conclusions Overall,our work uncovers an important mechanism underlying the regulation of RCC2 through CHIP-mediated ubiquitination and degradation.

关 键 词：泛素化 泛素连接酶CHIP 染色体浓缩调节蛋白2 降解 

分 类 号：R346[医药卫生—基础医学]