Transcriptional control by the global regulator Spx

全球监管机构 Spx 的转录控制

基本信息

批准号：
8325221
负责人：
PETER ZUBER
金额：
$ 5.54万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-02-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8325221
关键词：
ATP-Dependent Proteases Adaptor Signaling Protein Affect Affinity Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Bacillus anthracis Bacillus subtilis Bacteria Base Sequence Binding Binding Proteins Binding Sites Biological Assay C-terminal Cells Chimeric Proteins Co-Immunoprecipitations Code Communicable Diseases Complex Cysteine DNA DNA Binding DNA Sequence DNA Shuffling DNA-Directed RNA Polymerase DNA-protein crosslink DNase-I Footprinting Defect Disease Disulfides Doctor of Medicine Drug Design Electrophoretic Mobility Shift Assay Elements Energy Transfer Enzymes Exhibits Family FeBABE Fluorescence Fluorescence Anisotropy Gel Genes Genetic Transcription Goals Gram-Positive Bacteria Holoenzymes Immune Immunoprecipitation In Vitro Infection Label LacZ Genes Learning Listeria monocytogenes Mass Spectrum Analysis Measures Mediating Methods Modeling Molecular Conformation Mutation N-terminal Nature Orthologous Gene Oxidants Oxidation-Reduction Oxidative Stress Peptide Hydrolases Peptides Positioning Attribute Process Promoter Regions Protein Family Proteins Proteolysis Reaction Recovery Reducing Agents Regulation Reproduction spores Research Resistance Resolution Response Elements Screening procedure Specific qualifier value Staphylococcus aureus Streptococcus pneumoniae Stress Structure Sulfhydryl Compounds Surface System Technology Testing Transcription Initiation Transcriptional Activation Transcriptional Regulation Ursidae Family Virulence arsenate reductase cell envelope cis acting element combat gene function genetic regulatory protein in vitro Assay in vivo member microorganism mutant oxidation pathogen pathogenic bacteria prevent promoter protein protein interaction public health relevance research study response stoichiometry thioredoxin reductase transcription factor zinc-binding protein

项目摘要

DESCRIPTION (provided by applicant): In pathogenic bacteria, complex systems of control have evolved that activate defenses against the various toxic agents used by immune cells to combat infection. These systems regulate important virulence determinants, which are targets of drugs designed to alleviate infectious disease. The low GC Gram-positive bacteria have a unique system of transcriptional control, mediated by the protein Spx, that functions in the response to toxic oxidants, including those produced by phagocytic immune cells. Spx is a transcriptional regulator that we discovered in the spore-forming bacterium, Bacillus subtilis, but is also found in Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, and Bacillus anthracis. Spx has been shown to control the expression of virulence-associated activities in S. aureus, and is produced to high levels in Listeria monocytogenes and B. anthracis during host cell invasion. Spx defines a family of regulators that structurally resemble the enzyme arsenate reductase. It bears an N-terminal CXXC redox disulfide center that regulates its activity. Its mechanism of control is unique in that free Spx does not exhibit DNA-binding activity, but when bound to RNA polymerase (RNAP) in its oxidized disulfide form, it directs the enzyme to transcribe specific genes that function in oxidative stress defense. Spx concentration is reduced to low levels after recovery from stress by the protease ClpXP and the Spx-binding protein YjbH, which accelerates Spx proteolysis. The goal of the proposed research is to learn how B. subtilis Spx activates transcription initiation and how YjbH functions to mediate Spx proteolysis. Specific nucleotide sequence elements have been identified in the promoter regions of genes controlled by Spx. These sequences bind a complex of Spx and the C-terminal domain of RNAP subunit (CTD). The promoter DNA sequence and the structural requirements of CTD/Spx for promoter DNA binding will be determined. Furthermore, the dynamics of subunit positioning in RNAP holoenzyme within the Spx-activated transcription complex will be explored. Efficient proteolysis requires YjbH, a zinc-binding protein that interacts with Spx and accelerates ClpXP-catalyzed Spx proteolysis. Studies are proposed to investigate the oxidant-induced stabilization of Spx and redox control of YjbH activity. This study will focus on the Zn-binding site of the adaptor to test the model that oxidation of Zn-coordinating cysteine residues causes inactivation of YjbH and release of Spx. Differential thiol labeling and mass spectrometry will be performed to investigate the extent and nature of thiol oxidation of Spx and YjbH in vivo. A small protein factor, YirB that interacts with YjbH, will be studied to determine if it is a member of a growing list of small proteins that prevent protease-catalyzed degradation by inhibiting protease adaptor proteins. PUBLIC HEALTH RELEVANCE: Complex systems of control have evolved in disease-causing bacteria to mobilize defenses that counter the toxic oxidizing agents produced by phagocytic immune cells. The regulatory protein Spx controls important components of the bacterial response to oxidative attack, and the study of its mechanism of action will uncover targets for neutralizing infectious microorganisms.

描述（由申请人提供）：在致病细菌中，复杂的控制系统已经发展起来，激活了针对免疫细胞用来打击感染的各种有毒剂的防御措施。这些系统调节重要的毒力决定因素，这些决定因素是旨在减轻传染病的药物的靶标。低GC革兰氏阳性细菌具有由蛋白SPX介导的独特的转录控制系统，该系统在对毒性氧化剂的反应中起作用，包括吞噬免疫细胞产生的氧化剂。 SPX是一种转录调节剂，我们在形成孢子的细菌（枯草芽孢杆菌）中发现，但也可以在金黄色葡萄球菌，肺炎链球菌，单核细胞增生李斯特菌和炭疽菌中发现。 SPX已被证明可以控制金黄色葡萄球菌中与毒力相关活性的表达，并在宿主细胞浸润过程中产生的单核细胞增生李斯特菌和炭疽芽孢杆菌在高水平中产生。 SPX定义了一个在结构上类似于砷酸酶还原酶的调节剂家族。它具有N端CXXC氧化还原二硫化物中心，可调节其活性。它的控制机制是独一无二的，因为自由SPX不会表现出DNA结合活性，但是当与RNA聚合酶（RNAP）绑定在其氧化二硫化物形式时，它将酶指导该酶转录在氧化应激防御中起作用的特定基因。通过蛋白酶CLPXP和SPX结合蛋白YJBH从应激中恢复后，SPX浓度降低到低水平，这加速了SPX蛋白水解。拟议的研究的目的是了解枯草芽孢杆菌SPX如何激活转录启动以及YJBH如何介导SPX蛋白水解的功能。在由SPX控制的基因的启动子区域中鉴定了特定的核苷酸序列元件。这些序列结合了SPX的复合物和RNAP亚基（CTD）的C末端结构域。将确定CTD/SPX对启动子DNA结合的启动子DNA序列和结构要求。此外，将探索SPX激活的转录复合物中RNAP全酶中亚基定位的动力学。有效的蛋白水解需要YJBH，这是一种锌结合蛋白，与SPX相互作用并加速ClpxP催化的SPX蛋白水解。提出了研究来研究氧化剂诱导的SPX稳定和YJBH活性的氧化还原控制。这项研究将集中于适配器的Zn结合位点，以测试模型，即氧化锌协调性半胱氨酸残基会导致YJBH失活和SPX的释放。将进行差异硫醇标记和质谱法，以研究体内SPX和YJBH的硫醇氧化的程度和性质。将研究一个与YJBH相互作用的小蛋白质因子YIRB，以确定它是否是越来越多的小蛋白列表的成员，该蛋白质通过抑制蛋白酶适配器蛋白来预防蛋白酶诱导的降解。公共卫生相关性：复杂的控制系统已经在引起疾病的细菌中进化，以动员吞噬免疫细胞产生的有毒氧化剂的防御能力。调节蛋白SPX控制细菌对氧化攻击的重要组成部分，其作用机理的研究将发现中和传染性微生物的靶标。