Ruan Hai-Hua, Li Ye, Zhang Xi-Xuan, Liu Qiang, Ren He, Zhang Kun-Sheng, Zhao Hui
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China.
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China.
Biochem Biophys Res Commun. 2014 Apr 25;447(1):172-7. doi: 10.1016/j.bbrc.2014.03.126. Epub 2014 Apr 2.
The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonella typhimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6(-/-) mouse embryonic fibroblasts (MEFs) compared with Traf6(+/+) MEFs. However, the ectopic expression of TRAF6 in Traf6(-/-) MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host's E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.
磷脂酰肌醇磷酸酶SopB是鼠伤寒沙门氏菌（S.typhimurium）注入的效应蛋白之一，它通过泛素依赖性差异定位实现功能多样化。然而，尚不清楚哪种E3泛素连接酶负责SopB的泛素化。基于负责泛素激活并将其转运至底物蛋白的E1-E2-E3酶系，我们构建了SopB泛素化的体外检测方法。利用该检测方法，我们从Henle-407 S100提取物中纯化出一种E3泛素连接酶TRAF6，它可能负责SopB的泛素化。为了研究TRAF6的功能相关性，我们发现重组TRAF6在体外以剂量依赖性方式特异性地使SopB泛素化。感染后，与Traf6(+/+)小鼠胚胎成纤维细胞（MEFs）相比，Traf6(-/-) MEFs中TRAF6缺失导致SopB的泛素化完全受阻。然而，在Traf6(-/-) MEFs中异位表达TRAF6可挽救两种泛素偶联的SopB，这加强了TRAF6在SopB泛素化中的作用。对E2的分析表明，在体外和体内，TRAF6介导的SopB泛素化都需要UbcH5c而非其他E2缀合酶。总之，这些结果表明在鼠伤寒沙门氏菌感染期间UbcH5c/TRAF6与SopB相关，从而意味着鼠伤寒沙门氏菌已进化出一种机制，利用宿主的E3泛素连接酶来修饰和调节其效应蛋白的功能，以确保病原体和宿主细胞的存活。
