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

Construction of Mutants Swapping Ubiquitin E3 Ligase CHIP and E4B U-box Domain and Verification of Ubiquitination Activity

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。

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.