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）：在致病菌中，复杂的控制系统已经进化，可以激活免疫细胞对各种有毒物质的防御，以对抗感染。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目标。低GC革兰氏阳性细菌具有独特的转录控制系统，由蛋白Spx介导，在对有毒氧化剂（包括吞噬免疫细胞产生的氧化剂）的反应中起作用。Spx是一种转录调节因子，我们在芽孢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、单核增生李斯特菌和炭疽芽孢杆菌中发现。Spx已被证明可以控制金黄色葡萄球菌毒力相关活性的表达，并且在单核增生李斯特菌和炭疽杆菌侵袭宿主细胞时产生高水平的Spx。Spx定义了一个在结构上类似于砷酸盐还原酶的调节因子家族。它具有一个n端CXXC氧化还原二硫中心，调节其活性。它的控制机制是独特的，因为游离Spx不表现出dna结合活性，但当它以氧化二硫形式结合到RNA聚合酶（RNAP）时，它指导酶转录在氧化应激防御中起作用的特定基因。蛋白酶ClpXP和Spx结合蛋白YjbH在胁迫恢复后使Spx浓度降低到较低水平，从而加速Spx蛋白的水解。本研究的目的是了解枯草芽孢杆菌Spx如何激活转录起始，以及YjbH如何介导Spx蛋白水解。在Spx控制的基因的启动子区域已经鉴定出特定的核苷酸序列元件。这些序列结合了Spx复合物和RNAP亚基（CTD）的c端结构域。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，在spx激活的转录复合体中，RNAP全酶的亚基定位动力学将被探索。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。研究建议探讨氧化诱导的Spx稳定和YjbH活性的氧化还原控制。本研究将重点研究接头的zn结合位点，以验证zn配位半胱氨酸残基氧化导致YjbH失活和Spx释放的模型。差分硫醇标记和质谱将被用于研究体内Spx和YjbH的硫醇氧化的程度和性质。将研究与YjbH相互作用的小蛋白因子YirB，以确定它是否是通过抑制蛋白酶适应蛋白来阻止蛋白酶催化降解的小蛋白列表中的一员。