泛素连接酶E4B介导PM20D2、PABPC1和TACO1蛋白质的泛素化验证  

Verification of the ubiquitination of PM20D2,PABPC1 and TACO1 proteins by ubiquitin ligase E4B

作　　者：刘香男 范宇宸 王亚楠 陆瑶 赵博[1] 武正华[1] LIU Xiangnan;FAN Yuchen;WANG Yanan;LU Yao;ZHAO Bo;WU Zhenghua(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：《生物学杂志》2021年第3期31-36,共6页Journal of Biology

基　　金：国家自然科学基金项目(31770921;31971187)。

摘　　要：泛素连接酶E4B通过U-box基序可将经泛素活化酶(Ubiquitin-activating enzyme, E1)和泛素结合酶(Ubiquitin-conjugating enzyme, E2)传递的泛素(Ubiquitin, UB)标记至底物蛋白质。探究3个蛋白质:金属蛋白酶M20家族蛋白质2(Peptidase M20 domain-containing protein 2,PM20D2)、多腺苷酸结合蛋白质1(Polyadenylate-binding protein 1,PABPC1)、细胞色素C氧化酶Ⅰ翻译激活剂(Translational activator of cytochrome C oxidase Ⅰ,TACO1)是否可被E4B介导泛素化。从HEK293细胞中提取总RNA,反转录为cDNA,以其为模板调取底物基因并构建重组质粒,利用大肠杆菌表达系统表达并纯化泛素化过程所需的各个相关蛋白质,通过蛋白质体外泛素化实验,对3个蛋白质进行泛素化验证。结果表明3个蛋白质均可以在蛋白质水平被E4B泛素化,证明3个蛋白质都是E4B的底物,为进一步在细胞中研究其泛素化的机理奠定基础。Ubiquitin ligase E4 B can transfer ubiquitin(UB) that comes from ubiquitin-activating enzyme(E1) and ubiquitin-conjugating enzyme(E2) to substrates via U-box motif. Three proteins such as peptidase M20 domain-containing protein 2(PM20 D2), polyadenylate-binding protein 1(PABPC1), and translational activator of cytochrome C oxidase Ⅰ(TACO1) ubiquitinated by E4 B were verified. Total RNA was extracted from HEK293 cells and reverse-transcribed into cDNA. The cDNA was used as template for obtaining the genes of substrates by PCR, and recombinant plasmids were constructed with genes of substrates and vector, respectively. E. coli system was used to express proteins which were required for the ubiquitination of E4 B substrates. Result showed that PM20 D2, PABPC1 and TACO1 could be ubiquitinated by E4 B.The result indicated that all the three proteins were the substrates of E4 B, which would lay a foundation for the further study of the mechanism of ubiquitination in cells.

关 键 词：泛素化 泛素连接酶E4B 金属蛋白酶M20家族蛋白质2 多腺苷酸结合蛋白质1 细胞色素C氧化酶Ⅰ翻译激活剂 

分 类 号：Q78[生物学—分子生物学]