泛素E3连接酶CHIP和E4B互换U-box结构域突变体的构建及泛素化活性验证

doi:10.13560/j.cnki.biotech.bull.1985.2021-0230

摘要/Abstract

摘要：

旨在构建互换U-box结构域的泛素E3连接酶CHIP和E4B的突变体,用以研究U-box结构域对两种U-box家族泛素E3连接酶CHIP和E4B活性的影响。通过overlap PCR的方法构建CHIP U-box替换为E4B U-box的突变体C+E,以及E4B U-box替换为CHIP U-box的突变体E+C。将突变体质粒分别与CHIP底物RCC2、E4B底物NIPSNAP1、CHIP和E4B共同底物p53、CDC37质粒共转染至HEK-293T细胞。Western Blot检测底物和CHIP、E4B及其突变体表达情况,免疫共沉淀法检测底物的泛素化水平。结果显示,成功构建了两种泛素连接酶互换U-box结构域的突变体,且两种突变体在HEK-293T细胞中均能表达。两种突变体均部分保留了泛素化各自底物的活性,C+E能够泛素化共同底物p53和CDC37。

关键词: 泛素E3连接酶CHIP, 泛素E3连接酶E4B, 突变体, U-box结构域, 底物

Abstract:

This work aims to construct mutants of ubiquitin E3 ligases CHIP and E4B that exchange U-box domains and to study the effect of U-box domain on the activities of 2 U-box family ubiquitin ligases CHIP and E4B. By employing overlap PCR,a mutant CHIP named C+E that contained the U-box domain derived from E4B,and a mutant E4B named E+C whose U-box was replaced by the U-box domain from CHIP,were constructed respectively. These mutants’ plasmids and the substrate of CHIP named RCC2,the substrate of E4B named NIPSNAP1,and their common substrates p53 and CDC37 respectively were co-transfected into HEK-293T cells. Western Blot were adapted to detect the expressions of these substrates,CHIP,E4B and their mutants. Immunoprecipitation was to detect the ubiquitination of the substrates. The results showed that two mutants swapping the U-box domain were successfully constructed,and both of them were expressed in HEK-293T cells. Both mutants partially retained the activity on the ubiquitination of their respective substrates and C+E could have common substrates p53 and CDC37 in the ubiquitination.

Key words: ubiquitin E3 ligase CHIP, ubiquitin E3 ligase E4B, mutant, U-box domain, substrate

范宇宸, 陆瑶, 刘香男, 赵博. 泛素E3连接酶CHIP和E4B互换U-box结构域突变体的构建及泛素化活性验证[J]. 生物技术通报, 2021, 37(12): 191-197.

FAN Yu-chen, LU Yao, LIU Xiang-nan, ZHAO Bo. Construction of Mutants Swapping Ubiquitin E3 Ligase CHIP and E4B U-box Domain and Verification of Ubiquitination Activity[J]. Biotechnology Bulletin, 2021, 37(12): 191-197.

图/表 4

图1 RCC2、p53底物质粒克隆及构建 M:DNA分子量标准;A:RCC2目的基因PCR扩增产物;B:p53目的基因PCR扩增产物;C:pLVX空载体质粒及阳性克隆质粒电泳条带

Fig.1 Plasmid cloning and construction of substrate RCC2 and p53 M:DNA molecular weight standard. A:PCR products of RCC2 gene. B:PCR products of p53 gene. C:Electrophoresis band of pLVX empty vector plasmid and positive cloned plasmid

图2 融合基因C+E及E+C的PCR结果 M:DNA分子量标准;A:CA、CB、EA、EB片段PCR扩增产物;B:C+E基因PCR扩增产物;C:E+C基因PCR扩增产物

Fig.2 PCR results of fusion genes C+E and E+C M:DNA molecular weight standard. A:PCR products of CA,CB,EA and EB fragments. B:PCR products of C+E gene. C:PCR products of E+C gene

图3 底物、CHIP和E4B及其突变体的细胞内表达结果 M:蛋白分子量标准;A、B、C、D:细胞裂解液中底物、CHIP和E4B及其突变体的表达

Fig.3 Intracellular expression results of substrates,CHIP,E4B and their mutants M:Protein molecular weight standard. A,B,C and D:Expression levels of substrates,CHIP,E4B and their mutants in cell lysates

图4 底物RCC2、NIPSNAP1、p53、CDC37的免疫共沉淀实验结果 A、B、C、D:免疫共沉淀检测细胞内CHIP和E4B及其突变体对底物的泛素化活性水平

Fig.4 Co-immunoprecipitation experiment results of sub-strate RCC2,NIPSNAP1,p53 and CDC37 A,B,C and D:Detection of the ubiquitination activity of CHIP,E4B and their mutants on the substrates in the cell by co-immunoprecipitation

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