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

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.

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。

作为一种单细胞生物，革兰氏阴性细菌在感知外界刺激的过程中，主要利用细胞内膜上的受体监测环境信号。其中，受体组氨酸激酶以蛋白可逆磷酸化方式（磷酸化－脱磷酸化）完成环境信号的跨膜传递和信号转导，发挥着类似高等动物中枢神经系统的作用，因而被科学家们形象地称为细菌的“智商（IQ）”。受体组氨酸激酶主要以可逆的磷酸化-脱磷酸化方式控制细菌生理代谢活动。然而，细菌细胞内不可逆的调控方式，如蛋白质的水解是否与可逆磷酸化之间存在信号交叉，仍然是未知的领域，值得深入进行探讨。.在病原细菌－野油菜黄单胞菌感染植物的过程中，有一个名为VgrS的受体组氨酸激酶发挥着关键作用。VgrS感知寄主体内由固有免疫反应产生的缺铁环境，调节病原细菌从寄主中争抢和吸收铁的过程。因此，VgrS突变或失活后会导致细菌铁代谢紊乱，致病力严重下降。然而，VgrS的功能具有两面性。这项研究发现：当细菌面对高渗胁迫时，VgrS信号通路的激活会严重阻碍细菌的生长。为了迅速淬灭该通路，细菌周质空间内的蛋白酶Prc结合到VgrS位于氨基端的信号感知区域，随即特异性切割前9位氨基酸并且抑制VgrS的激酶活性。VgrS被Prc特异性切割失活是细菌对抗高渗胁迫的前提条件。该研究从遗传学、酶学和生物物理学方面提出了充分的证据，证明上述过程对于细菌适应胁迫环境的重要意义。同时，研究结果也提示：以病原细菌的重要受体作为分子作用药靶时，在特定生存环境中反而有可能提高细菌的适应能力，对此必须进行更全面、深入的研究。.项目结题时，共在包括Nature Communications, PLoS Pathogens, Genetics, Mol Plant-Microbe Interactions，Molecular Plant Pathology等期刊上发表研究论文6篇，培养博士生4人，硕士生1人。完成了任务目标。