Characterization of YjbH: Insights into its role in oxidative stress response

YjbH 的表征：深入了解其在氧化应激反应中的作用

• 批准号: 8313033
• 负责人: Chio Mui Chan
• 金额: $ 5.22万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8313033
• 关键词: ATP-Dependent Proteases; Affect; Affinity; Aftercare; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Alkylation; Alleles; Award; Bacillus (bacterium); Bacillus subtilis; Bacteria; Binding; Binding Proteins; Boron; Cell physiology; Cells; Code; Complex; Crystallography; DNA-Directed RNA Polymerase; Data; Developmental Process; Devices; Diamide; Disulfides; Environment; Escherichia coli; Galactosidase; Genes; Genetic Screening; Goals; Gram-Positive Bacteria; Homeostasis; Homologous Gene; Immune system; In Vitro; Knowledge; LacZ Genes; Mediating; Methods; Modeling; Molecular; Mutagenesis; Oxidants; Oxidation-Reduction; Oxidative Stress; Peptide Hydrolases; Peptides; Phenotype; Plasmids; Play; Process; Proteins; Proteolysis; Quality Control; Reaction; Reactive Oxygen Species; Recovery; Reducing Agents; Reporter; Reproduction spores; Role; Sampling; Screening procedure; Series; Stress; Structure; Sulfhydryl Compounds; System; Testing; Transcriptional Regulation; Work; bacterial genetics; biological adaptation to stress; c-myc Genes; design; growth promoting activity; in vivo; insight; loss of function; microorganism; mutant; operation; oxidative damage; prevent; research study; response; skills; vector

DESCRIPTION (provided by applicant): Using Bacillus subtilis as a gram-positive model, we propose to investigate the mechanism of proteolytic control that is applied in response to oxidative stress. Proteolytic control is essential for protein homeostasis and also plays a key role in modulating the levels of specific regulatory factors. One such factor, Spx, is a global transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Its concentration is elevated when cells undergo oxidative stress. When stress is alleviated, Spx is degraded by the ATP-dependent protease, ClpXP, a process that requires the substrate recognition factor, YjbH. Spx interacts directly with YjbH, which promotes ClpXP-catalyzed Spx proteolysis. Our recent studies show that a small peptide YirB binds to YjbH thereby releasing Spx from proteolytic control. One of our goals is to study the interactions between these four components, Spx/YjbH/ClpX/YirB, both in vivo and in vitro during oxidative stress and as stress subsides. This will be determined using active, affinity-tagged versions of the binding partners for pull down interaction experiments to be conducted with in vivo samples and in vitro reactions. In addition, to gaining in-depth understanding of the interactions, I will undertake structural studies of the key player YjbH and its complexes with Spx and YirB. During this award period, I intend to gain skills in bacterial genetics and knowledge to design approaches to elucidate the complexities of regulated proteolysis, while utilizing my advanced expertise in crystallography to define in detail the interactions between the protein components at the molecular level. PUBLIC HEALTH RELEVANCE: Infectious microorganisms must defend themselves from stress brought about by toxic agents such as oxidants produced by the host immune system. Bacteria can regulate their response to oxidants by precisely and rapidly changing the levels of specific factors that govern the processes that prevent and/or alleviate stress. Our application is to characterize the system of control that determines the concentration of an important regulator of the bacterial stress response, the protein called Spx. I will define in detail the structure and function of the factors that control the degradation of Spx before and during recovery from oxidant-induced stress.

描述（由申请人提供）：使用枯草芽孢杆菌作为革兰氏阳性模型，我们建议研究用于响应氧化应激的蛋白水解控制机制。蛋白水解控制对于蛋白质稳态至关重要，并且在调节特定调节因子的水平方面也发挥着关键作用。其中一个因子 Spx 是革兰氏阳性菌氧化应激反应的全局转录调节因子。当细胞经历氧化应激时，其浓度会升高。当压力减轻时，Spx 会被 ATP 依赖性蛋白酶 ClpXP 降解，该过程需要底物识别因子 YjbH。 Spx 直接与 YjbH 相互作用，从而促进 ClpXP 催化的 Spx 蛋白水解。我们最近的研究表明，小肽 YirB 与 YjbH 结合，从而将 Spx 从蛋白水解控制中释放出来。我们的目标之一是研究氧化应激期间和应激消退期间体内和体外这四种成分 Spx/YjbH/ClpX/YirB 之间的相互作用。这将使用活性的、亲和力标记的结合配偶体进行确定，以进行体内样品和体外反应的下拉相互作用实验。此外，为了深入了解相互作用，我将对关键参与者 YjbH 及其与 Spx 和 YirB 的复合物进行结构研究。在此奖励期间，我打算获得细菌遗传学方面的技能和知识，以设计方法来阐明调节蛋白水解的复杂性，同时利用我在晶体学方面的先进专业知识在分子水平上详细定义蛋白质成分之间的相互作用。 公共卫生相关性：传染性微生物必须保护自己免受有毒物质（例如宿主免疫系统产生的氧化剂）带来的压力。细菌可以通过精确而快速地改变控制预防和/或减轻压力过程的特定因子的水平来调节它们对氧化剂的反应。我们的应用程序是 表征控制系统，该系统决定细菌应激反应的重要调节剂（称为 Spx 的蛋白质）的浓度。我将详细定义结构和 在从氧化剂诱导的应激恢复之前和恢复过程中控制 Spx 降解的因素的功能。