周质蛋白酶Prc底物的亲合蛋白质组学鉴定及其调控黄单胞菌致病过程的分子机制

蛋白酶催化的底物受控水解是一种典型的不可逆代谢过程，也是病原细菌－寄主互作的前沿领域。系统鉴定蛋白酶在细胞内的生理学底物，阐明底物水解的精细过程和调控意义是相关研究的难点。在前期分析黄单胞菌周质空间蛋白酶Prc功能及其互作蛋白的基础上，拟利用亲合蛋白质组学方法，采取串联亲合纯化技术（TAP）规模化地筛选Prc的互作蛋白；研究病菌侵染寄主甘蓝和胁迫反应过程中Prc对底物的识别过程、酶切位点、切割步骤以及切割产物的功能。其中，重点分析Prc水解致病相关组氨酸激酶XhsK、细胞分裂蛋白DivD等的调控机制，阐明不可逆蛋白水解途径与可逆蛋白磷酸化途径、细胞有丝分裂调控等主要信号通路之间的交叉控制过程。Prc在革兰氏阴性细菌中比较保守，是目前植物病原细菌中唯一被实验研究的编码PDZ结构域的蛋白酶，项目成果将会为理解病原细菌周质空间蛋白酶控制致病过程的机理提供具有普遍意义的证据。

The regulated proteolysis is a typical irreversible metabolic process, and it is also a major mode of signal transduction. In this study area, systematic identification of physiological substrates of protease is critical to investigate the role of proteolysis in cellular regulation. Based on our previous study that found Prc is a PDZ-domain containing protease that controls virulence of Xanthomonas oryzae pv. oryzae, this project aimed to systematically screen physiological substrates of Prc by the method of tandem affinity purification (TAP), a highly efficient technology of affinity proteomics. After verification of Prc-substrate interactions by both in vitro and in vivo experiments, the recognition mechanism, primary proteolytic site, cleavage process, and the function of proteolytic products will be investigated to understand the regulatory role of Prc in bacterial physiology. Among the substrates, we will focus on the XhsK, a virulence regulating histidine kinase, and DivD, a cell-division related protein. The two proteins have been confirmed to be the proteolytic substrates of Prc in X. campestris pv. campestris. The project will decipher the cross-talk between irreversible signal transduction mediated by Prc and the others, such as reversible protein phosphorylation and cytogenesis regulation. Since Prc is relatively conserved in Gram-negative bacteria and it is the first PDZ-domain containing protease that was experimentally studied in plant bacterial pathogens, we believe that the results of this project will have a broad interesting to scientists whose works are focused on bacterial pathogenesis, proteolysis, and signal transduction of cells.