DegS protease and initiation of the envelope-stress response

DegS 蛋白酶和包膜应激反应的启动

• 批准号: 7777378
• 负责人: JUNGSAN SOHN
• 金额: $ 5.01万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777378
• 关键词: Affect; Alanine; Amino Acids; Apoptosis; Binding; Biological Assay; C-terminal; Catalytic Domain; Cleaved cell; Crystallography; Cytoplasmic Tail; DNA Sequence Rearrangement; Diffuse; Escherichia coli; Event; Genetic; Gram-Negative Bacteria; In Vitro; LacZ Genes; Malignant Neoplasms; Membrane; Modeling; Molecular; Monitor; Orthologous Gene; Pathway interactions; Peptide Hydrolases; Peptides; Physiological; Play; Process; Property; Proteins; Proteolysis; Reaction; Reporter; Research; Role; Scanning; Series; Signal Transduction; Signal Transduction Pathway; Site-Directed Mutagenesis; Testing; Triad Acrylic Resin; Variant; Virulence; Work; antimicrobial; antimicrobial drug; base; biological adaptation to stress; cancer therapy; design; in vivo; monomer; mutant; novel; pathogenic bacteria; periplasm; porin; protein aminoacid sequence; tumorigenesis

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.

描述（由申请人提供）：DEGS通过裂解Sigmae活性的主要调节剂RSEA的周质结构域来启动革兰氏阴性细菌的信封应力反应。这种初始事件是通过抗肽与DEGS的PDZ结构域的结合而激活的，触发了一系列RSEA蛋白水解，并导致诱导Sigmae应力 - 响应。与Sigmae途径中DEG和RSEA建立的生理意义相反，这两种蛋白质之间的分子相互作用鲜为人知。我已经开发了一种定量测定，以详细监测切割反应。我还结晶了缺乏调节PDZ域的DEG变体。首先检查由OMP肽对DEG的变构激活和PDZ结构域的作用。体外发现将在体内使用遗传互补和LACZ报告基因测定。 DEG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与DEG的相互作用更好。最后，我将研究DEG如何影响RSEP下一次RSEA裂解。这项研究将增强我们对蛋白水解信号转导途径的理解，并可能打开新型抗微生物和抗癌处理的门。 DEG对于大肠杆菌的存活至关重要，并且是Sigmae应力反应的不可或缺的组成部分。值得注意的是，许多致病细菌的毒力需要Sigmae途径。因此，了解DEG如何识别激活剂和底物可以为开发新型抗微生物剂打开门。此外，由于DEG的哺乳动物直系同源物HtraA2/OMI与凋亡和肿瘤发生有关，因此我的研究可能对癌症有影响。