Wang Zhen, McCloskey Alix, Cheng Sen, Wu Mei, Xue Chenyu, Yu Zhengyou, Fu Jiaqi, Liu Yanhua, Luo Zhao-Qing, Liu Xiaoyun
1Institute of Analytical Chemistry and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China.
2Purdue Institute of Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 USA.
Cell Discov. 2018 Oct 9;4:53. doi: 10.1038/s41421-018-0055-9. eCollection 2018.
Posttranslational modification of key host proteins by virulence factors is an important theme in bacterial pathogenesis. A remarkable example is the reversible modifications of the small GTPase Rab1 by multiple effectors of the bacterial pathogen . Previous studies have shown that the effector SetA, dependent on a functional glucosyltransferase domain, interferes with host secretory pathways. However, the enzymatic substrate(s) of SetA in host cells remains unknown. Here, by using cross-linking mass spectrometry we uncovered Rab1 as the target of SetA during infection. Biochemical studies establish that SetA covalently attaches a glucose moiety to Thr within the switch II region of Rab1, inhibiting its intrinsic GTPase activity. Moreover, we found that SetA preferentially modifies the GDP-bound form of Rab1 over its GTP-associated state and the modification of Rab1 inhibits its interaction with the GDP dissociation inhibitor GDI1, allowing for Rab1 activation. Our results thus add an extra layer of regulation on Rab1 activity and provide a mechanistic understanding of its inhibition of the host secretory pathways as well as cellular toxicity.
毒力因子对关键宿主蛋白进行的翻译后修饰是细菌致病机制中的一个重要主题。一个显著的例子是细菌病原体的多种效应子对小GTP酶Rab1进行的可逆修饰。先前的研究表明，依赖功能性葡糖基转移酶结构域的效应子SetA会干扰宿主分泌途径。然而，SetA在宿主细胞中的酶促底物仍不清楚。在这里，我们通过交联质谱法发现Rab1是感染过程中SetA的靶标。生化研究表明，SetA将一个葡萄糖部分共价连接到Rab1开关II区域内的苏氨酸上，抑制其内在的GTP酶活性。此外，我们发现SetA优先修饰Rab1的GDP结合形式而非其GTP结合状态，并且Rab1的修饰会抑制其与GDP解离抑制剂GDI1的相互作用，从而激活Rab1。因此，我们的结果为Rab1活性增加了一层额外的调控，并提供了对其抑制宿主分泌途径及细胞毒性的机制理解。
