Cpx信号系统中YihE蛋白激酶与Rho转录终止因子相互作用及调控机理研究

The Cpx signalling system of Gram-negative bacteria, such as Escherichia coli, senses extracytoplasmic stress and controls expression of factors that allow the bacterium to adapt to these stressors and thereby enhance survival. Gene yihE in E.coli was activated by the Cpx pathway. Encoded protein YihE is cell survival regulating enzyme and probably acts early in the stress response to restrict cell PCD. Thus, inhibition of The Cpx stress response system can sense cell envelope stress, such as misfolded proteins in the periplasm, and responds by upregulating the expression of folding factors and proteases to modify or degrade the damaged protein. YihE is a known protein kinase implicated in Cpx stress response, although it remains elusive whether its kinase activity is directly involved. We have through pull-down experiments identified Rho, a transcription termination factor, interacts with YihE. Using size exclusion chromatography, we have obtained YihE and Rho complex which is likely in 1:6 ratio as shown by SDS and multi-angle light scattering analyses. In this project ,we will go through various truncation constructs, the C-terminal tail of YihE is critical to the complex formation. Morever, using SEM and 3D-SIM we show that YihE is able to lessen the effects of Rho overexpression, including both cell morphology and Rho distribution in the cell. Real-time PCR results further revealed that the expression of both yihe and rho were significantly increasingly under stress conditions. YihE seems to act as a Rho antagonist to reduce the “side” effects in Rho-mediated Cpx stress. We will also study the protein structure of YihE/Rho complex to elucidate the mechanism the regualtion of YihE on Rho-depeneded transcription termination.

细菌通过Cpx信号通路感知细胞外应力，控制应激反应因子的表达来适应环境。Cpx通路中YihE蛋白是应激环境下细胞生存的调控酶，在应激反应早期发挥作用并抑制细胞程序化凋亡PCD。YihE是一个维持细胞稳态生命动力学中未知的新激酶。因此找到抑制YihE方式可能成为一种提高抗菌药物杀伤力且减弱毒性的方法。本项目最新发现了YihE作用于细胞重要的Rho转录终止因子，Rho转录终止子调控着细胞中多数基因mRNA合成，然而其调控机制尚不清楚。申请人将结合生化生理技术和结构生物学技术，研究YihE激酶调控细胞转录重要因子Rho功能的方式，探讨YihE在细胞应激调控机制中的作用，并通过解析YihE/Rho复合物结构及理论计算研究YihE与Rho的相互作用方式，有可能在原核生物中发现全新蛋白磷酸化修饰信号调控通路。申请人首次发现并证明YihE可能参与依赖Rho细胞转录调控，因而该项目具有科研创新和应用价值。

大肠杆菌Cpx应激反应系统可以感知细胞膜周边环境变化以应对细胞膜间质中折叠损伤的蛋白质。细菌通过上调蛋白修复及降解酶的表达起到修复或降解受损伤蛋白质的作用。YihE是一种与Cpx应激反应相关的蛋白激酶而Rho是原核生物中的转录终止因子，它们之间没有联系却在Cpx系统中一起起作用。本项目我们成功发现了YihE激酶可以与Rho转录终止子间的相互作用，能够形成一个YihE：Rho比为1：6的复合物。我们成功解析了低分辨率的YihE-Rho晶体结构，并分析YihE和Rho之间的相互作用具体位置和氨基酸作用位点，并通过实验证明了一个YihE分子是通过静电作用于Rho六聚体中的F分子互作，这与Rho结合蛋白NusA的所在位置相似。我们观察到细菌在环境变化的压力条件下，如在H2O2应激或抗生素存在的情况下，YihE和Rho在细菌体内表达显着增加并且严重影响细胞的形态和生长。我们还发现YihE过表达可以帮助减轻Rho过表达引起的细菌形态和生理的异常表现，包括细菌在恶劣环境下Rho蛋白介导的细胞伸长表型。因而，通过本项目我们成功确定了YihE是一种新的Rho结合蛋白，充当Rho的拮抗剂以响应细菌Cpx应激反应。YihE与Rho的结合会从RNA分子上释放与RNA分子结合的Rho因子，从而在转录水平上调控细菌Cpx应激系统中的基因表达。这将有助于我们理解细菌如何通过调节Rho依赖性转录终止来应对细菌环境条件的变化，为药物设计提供了一个新的靶点。