UbcH5B活性位点突变体对野生型E2传递泛素活性的影响

Effects of UbcH5B Active Site Mutant on the Ubiquitin Transfer Activity of Wild-Type E2

蛋白质泛素化需要泛素活化酶E1、泛素结合酶E2和泛素连接酶E3的依次作用,但泛素结合酶E2不仅在泛素传递中起中间者的作用,还可能决定泛素链的特异性以及调控泛素化过程.为了更加深入了解E2参与的泛素传递调控机制,通过将泛素结合酶UbcH5B催化活性位点Cys85突变成Ser,获得可以与Ub结合得到更加稳定复合物的突变体UbcH5B-C85S,探究突变体对野生型E2体外泛素传递的影响.首先构建野生型E2和UbcH5B-C85S的表达质粒,然后通过原核表达和使用Ni-NTA亲和柱层析纯化,获得有活性的人重组E2蛋白;设置体外泛素转移反应,通过Western Blot等方法,分别在E2和E3水平分析UbcH5B-C85S对野生型E2泛素转移反应的作用.结果显示:突变体UbcH5B-C85S可以部分抑制泛素从E1传递到UbcH5B和E2-25K,并且可以抑制UbcH5B将泛素传递到泛素连接酶TRIM23.这些结果有助于更深入了解泛素化的活性调控机制.

Ubiquitination is the attachment of ubiquitin(Ub)to its substrate proteins through sequential transfer by E1,E2 and E3 enzymes.However,E2 not only acts as a middle man in the transfer,but also determines the specificity of ubiquitin chain formation and regulates the ubiquitination process.In order to better understand the mechanism of Ub transfer and the role of involved E2,we mutated the catalytic site Cys85 of UbcH5B to Ser and generated a mutant UbcH5B-C85S that could bind to Ub and form a more stable conjugate.We explored the effect of the mutant on Ub transfer reactions in vitro.Expression plasmids containing the genes of wild type E2s and UbcH5B-C85S were constructed,and these recombinant E2s were expressed in E.coli and purified by Ni-NTA affinity chromatography.Finally,the effects of UbcH5B-C85S on the ubiquitin transfer conducted by wild-type E2 were analyzed in the presence of E2 and E3 enzymes.The results demonstrated that the mutant UbcH5B-C85S could partially inhibit the Ub transfer from E1 to UbcH5B and E2-25K,and could also inhibit the transfer of Ub to E3 ligase TRIM23 from UbcH5B.These results provide deeper understanding of the regulation of ubiquitination by E2.