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，我们建议研究响应于氧化应激而应用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，在调节特定调节因素水平中也起着关键作用。 SPX的一个因素之一是革兰氏阳性细菌中氧化应激反应的全球转录调节剂。当细胞承受氧化应激时，其浓度升高。当压力减轻时，SPX会被ATP依赖性蛋白酶CLPXP降解，CLPXP是需要底物识别因子YJBH的过程。 SPX与YJBH直接相互作用，YJBH促进了CLPXP催化的SPX蛋白水解。我们最近的研究表明，较小的肽YIRB与YJBH结合，从而从蛋白水解对照中释放SPX。我们的目标之一是研究这四个组件之间的相互作用，SPX/YJBH/CLPX/YIRB，无论是在氧化应激和应力下降过程中体内和体外的体外与体外的相互作用。这将使用与体内样品和体外反应进行下拉相互作用实验的活跃的，亲和力的版本的结合伙伴的活跃版本确定。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。在此奖项期间，我打算在细菌遗传学和知识方面获得技能，以设计方法来阐明受调节的蛋白水解的复杂性，同时利用我的先进的晶体学专业知识来详细定义分子水平上蛋白质成分之间的相互作用。 公共卫生相关性：传染性微生物必须捍卫自己免受有毒药物所带来的压力，例如宿主免疫系统产生的氧化剂。细菌可以通过精确，快速地改变控制预防和/或减轻压力的过程的特定因素的水平来调节其对氧化剂的反应。我们的申请是 为了表征确定细菌应力反应的重要调节剂浓度的控制系统，该蛋白称为SPX。我将详细定义结构和 控制氧化剂诱导的应激之前和期间控制SPX降解的因素的功能。