DegS protease and initiation of the envelope-stress response

描述(由申请人提供)：DEGS通过裂解SigmaE活性的主要调节因子RseA的周质结构域，启动革兰氏阴性细菌的包膜应激反应。这一初始事件由OMP多肽与DEGS的PDZ结构域结合而激活，触发了RseA的一系列蛋白分解，并导致SigmaE应激反应的诱导。与SigmaE途径中DEGS和RseA的生理意义相比，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方法来详细监测切割反应。我还明确了一种缺乏调控PDZ结构域的DEGS变体。我们将首先研究OMP多肽对DEGS的变构激活以及PDZ结构域的作用。体外研究结果将使用遗传互补和LacZ报告分析在体内进行测试。通过结晶学和丙氨酸扫描确定DEGS与RseA之间的相互作用区域。我还将测试二聚体或三聚体RseA与DEGS之间的相互作用是否更好。最后，我将研究DEGS如何影响RseP对RseA的下一次切割。这项研究将加深我们对蛋白质分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对于E.Coli的生存是必不可少的，是SigmaE应激反应中不可或缺的组成部分。值得注意的是，SigmaE途径是许多致病细菌毒力所必需的。因此，了解DEGS如何识别激活剂和底物可以为开发新型抗微生物制剂打开大门。此外，由于哺乳动物DEGS的同源基因HtraA2/Omi与细胞凋亡和肿瘤发生有关，我的研究可能对癌症有意义。

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.