泛素连接酶E4B介导POLDIP2的泛素化及其降解  

E3 ligase E4B mediates the ubiquitination and degradation of POLDIP2

作　　者：李莎莎 赵博[1] LI Shasha;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）》2021年第4期496-502,共7页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(31971187,31770921);上海市科委基础研究领域项目(20JC1411200)。

摘　　要：目的旨在细胞外和HEK293细胞中鉴定聚合酶δ相互作用蛋白2(POLDIP2)是否为泛素连接酶E4B的底物,并探究E4B能否介导POLDIP2经由26S蛋白酶体降解。方法利用BL21表达并纯化POLDIP2蛋白,设置体外E1-E2-E3泛素传递反应,通过western blot检测POLDIP2的泛素化;同时,构建pcDNA-Flag-POLDIP2重组质粒并转染进HEK293细胞、E4B敲减稳转株(shE4B)、E4B过表达细胞株(E4B OE)中,通过免疫共沉淀检测E4B表达量的变化是否影响POLDIP2的泛素化水平;其次,进一步通过CHX Chase实验,以及在HEK293细胞中转染依次增量的pLVX-E4B质粒,通过western blot检测POLDIP2蛋白水平的变化。结论胞外泛素化反应和胞内泛素化鉴定均证实泛素连接酶E4B可以将POLDIP2泛素化,并且能够介导其通过26S蛋白酶体进行降解。Objective We aim to identify whether POLDIP2(polymerase delta-interacting protein 2)is the substrate of E3 ligase E4B,and further explore whether E4B mediates the degradation of POLDIP2 by 26S proteasome.Methods POLDIP2 protein was expressed and purified in BL21,then ubiquitination transfer reaction was set up in vitro in the presence of E1,E2 and E3.The ubiquitination of POLDIP2 was detected by western blot.HEK293 cells were transfected with different pcDNA-Flag-POLDIP2 recombinant plasmids to generate E4B knockdown stable cell lines(shE4B)and E4B overexpression cell lines(E4B OE),then we performed co-immunoprecipitation to determine whether E4B expressions affects the ubiquitination of POLDIP2.Furthermore,we carried out CHX Chase assay,and detected the protein levels of POLDIP2 by western blot with different amounts of pLVX E4B plasmid in the cells.Conclusions Both in vitro and in vivo assays confirmed that ubiquitin E3 ligase E4B could mediate the ubiquitination of POLDIP2 and induce its degradation by 26S proteasome.

关 键 词：泛素连接酶 E4B 聚合酶δ相互作用蛋白2 泛素化 降解 

分 类 号：Q291[生物学—细胞生物学] Q55