Transcriptional control by the global regulator Spx

全球监管机构 Spx 的转录控制

• 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
• 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

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）：在病原菌中，复杂的控制系统已经进化出来，可以激活针对免疫细胞用于对抗感染的各种有毒物质的防御。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点。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，其在对毒性氧化剂（包括由吞噬免疫细胞产生的那些）的响应中起作用。Spx是一种转录调节因子，我们在孢子形成细菌枯草芽孢杆菌中发现，但也在金黄色葡萄球菌、肺炎链球菌、单核细胞增生李斯特菌和炭疽芽孢杆菌中发现。Spx已被证明控制S.金黄色葡萄球菌，并且在单核细胞增生李斯特菌和B中产生高水平。炭疽病在宿主细胞入侵。Spx定义了一个家族的监管机构，在结构上类似的酶砷酸还原酶。它具有调节其活性的N-末端CXXC氧化还原二硫化物中心。它的控制机制是独特的，因为游离Spx不表现出DNA结合活性，但当以其氧化二硫化物形式与RNA聚合酶（RNAP）结合时，它指导该酶转录在氧化应激防御中起作用的特定基因。从应激中恢复后，通过蛋白酶ClpXP和Spx结合蛋白YjbH将Spx浓度降低至低水平，这加速了Spx蛋白水解。这项研究的目的是了解B。Subtilis Spx激活转录起始以及YjbH如何发挥功能以介导Spx蛋白水解。在Spx控制的基因的启动子区域中已经鉴定出特定的核苷酸序列元件。这些序列结合Spx和RNAP亚基（CTD）的C-末端结构域的复合物。将确定启动子DNA序列和CTD/Spx对启动子DNA结合的结构要求。此外，亚基定位在RNAP全酶内的Spx激活的转录复合物的动力学将被探讨。高效的蛋白水解需要YjbH，一种与Spx相互作用并加速ClpXP催化的Spx蛋白水解的锌结合蛋白。研究提出了调查氧化剂诱导的稳定的Spx和氧化还原控制YjbH活性。本研究将集中在锌结合位点的衔接子，以测试模型，锌配位半胱氨酸残基的氧化导致失活的YjbH和释放的Spx。将进行差示巯基标记和质谱分析，以研究Spx和YjbH在体内巯基氧化的程度和性质。一个小的蛋白质因子，YirB与YjbH相互作用，将进行研究，以确定它是否是一个不断增长的小蛋白质，防止蛋白酶催化的降解抑制蛋白酶接头蛋白的列表中的一员。 公共卫生关系：复杂的控制系统已经在致病细菌中进化，以动员防御来对抗吞噬免疫细胞产生的有毒氧化剂。调节蛋白Spx控制着细菌对氧化攻击反应的重要组成部分，对其作用机制的研究将揭示中和感染性微生物的靶点。