DegS protease and initiation of the envelope-stress response

描述（由申请人提供）：DegS通过切割RseA的周质结构域（sigmaE活性的主要调节因子）启动革兰氏阴性菌的细菌应激反应。通过OMP-肽与DegS的PDZ结构域的结合激活的该初始事件触发RseA的一系列蛋白水解并导致σ E应激反应的诱导。与DegS和RseA在sigmaE途径中的既定生理意义相反，对这两种蛋白质之间的分子相互作用知之甚少。我已经开发了一种定量分析来详细监测切割反应。我还结晶了一个缺乏调节PDZ结构域的DegS变体。将首先检查由OMP-肽对DegS的变构活化和PDZ结构域的作用。将使用遗传互补和lacZ报告基因试验在体内检测体外结果。DegS和RseA之间的相互作用区域将通过晶体学和丙氨酸扫描来鉴定。我还将测试二聚体或三聚体RseA是否与DegS更好地相互作用。最后，我将研究DegS如何影响RseP对RseA的下一次切割。这项研究将增强我们对蛋白水解信号转导途径的理解，并可能为新的抗微生物和抗癌治疗打开大门。DegS对E.大肠杆菌中表达，是sigmaE应激反应中不可缺少的组成部分。值得注意的是，sigmaE途径是许多病原菌的毒力所必需的。因此，了解DegS如何识别激活剂和底物可以为开发新型抗菌剂打开大门。此外，由于DegS的哺乳动物直系同源物HtraA 2/Omi与细胞凋亡和肿瘤发生相关，因此我的研究可能对癌症有影响。

DESCRIPTION (provided by applicant): DegS initiates the envelop-stress response of gram-negative bacteria by cleaving the periplasmic domain of RseA, the major regulator of sigmaE activity. This initial event, which is activated by the binding of OMP- peptides to the PDZ domain of DegS, triggers a series of proteolysis of RseA and leads to induction of the sigmaE stress-response. In contrast to the established physiological significance of DegS and RseA in the sigmaE pathway, little is known for the molecular interactions between these two proteins. I have developed a quantitative assay to monitor the cleavage reaction in detail. I have also crystallized a DegS variant lacking the regulatory PDZ domain. The allosteric activation of DegS by OMP-peptide and the role of the PDZ domain will be first examined. The in vitro findings will be tested in vivo using genetic complementation and lacZ reporter assays. The interaction regions between DegS and RseA will be identified by crystallography and alanine scanning. I will also test whether dimeric- or trimeric RseA interacts better with DegS. Finally, I will investigate how DegS influences the next cleavage of RseA by RseP. This research will enhance our understanding of proteolytic signal transduction pathways and potentially open doors for novel anti-microbial and anti-cancer treatments. DegS is essential for the survival of E. Coli, and is an indispensible component of the sigmaE stress response. Notably, the sigmaE pathway is required for the virulence of many pathogenic bacteria. Thus, understanding how DegS recognizes activators and substrates could open doors for developing novel anti-microbial agents. Moreover, since the mammalian ortholog of DegS, HtraA2/Omi, is associated with apoptosis and oncogenesis, my studies may have implications for cancer.