Functional exploration of a deep Mycobacterium tuberculosis phosphoproteome

结核分枝杆菌深层磷酸蛋白质组的功能探索

• 批准号: 10656957
• 负责人: Christoph Grundner
• 金额: $ 61.73万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-03 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656957
• 关键词: Affect; Arginine; Bacillus subtilis; Bacteria; Biology; Chromosomes; DNA Binding; Data; Environment; Eukaryota; Event; Family; Gene Expression; Genes; Genetic Transcription; Gram-Positive Bacteria; Individual; Maps; Mass Spectrum Analysis; Measures; Mediating; Modification; Mutation; Mycobacterium tuberculosis; Organism; Output; Pathogenesis; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Post-Translational Protein Processing; Proteins; Proteolysis; Regulation; Role; Signal Pathway; Signal Transduction; Substrate Interaction; System; Testing; Transcriptional Regulation; Tuberculosis; Virulence; Virulence Factors; endopeptidase Clp; gain of function; loss of function; mimetics; mutant; novel; phosphoproteomics; preservation; protein degradation; transcription factor; transcriptomics

ABSTRACT Phosphosignaling provides the major conduit for bacterial adaptation. The two component systems (TCSs) have long been viewed as the canonical phosphosignaling systems in bacteria, but increasingly, O- phosphorylation mediated by Ser/Thr kinases is recognized as a relevant bacterial phosphosignaling mechanism as well. We now show that in fact, Mycobacterium tuberculosis (Mtb) has an expansive, distributed, and cooperative O-phosphorylation system of a size and complexity that is typically only associated with eukaryotes. By using a comprehensive STPK loss- and gain-of-function mutant panel and quantitative mass spectrometry, we show that >70% of Mtb proteins are phosphorylated on Ser/Thr/Tyr, identify thousands of individual Ser/Thr kinase substrates, and show that the Ser/Thr kinases collectively control the expression of ~30% of Mtb genes. Here, we will test a new and extensive regulatory connection between the Ser/Thr kinases and the His kinases of the TCSs and test the role of O-phosphorylation on the regulation of transcription factors. As a result of our exhaustive analysis of Mtb phosphorylation, we also obtained the first evidence of Arg phosphorylation in Mtb- the first bacterial Arg phosphoproteome outside of gram-positive bacteria. We will identify the relevant phosphoenzymes and test the idea that pArg functions as a degradation tag for ClpP- mediated proteolysis.

抽象的 磷酸信号传导提供了细菌适应的主要途径。两部分系统 (TCS) 长期以来一直被视为细菌中典型的磷酸信号系统，但越来越多地，O- Ser/Thr 激酶介导的磷酸化被认为是相关的细菌磷酸信号传导 机制也是如此。我们现在证明，事实上，结核分枝杆菌 (Mtb) 具有广泛的、 分布式、协作式 O-磷酸化系统的大小和复杂性通常仅相关 与真核生物。通过使用全面的 STPK 功能丧失和功能获得突变体面板和定量 通过质谱分析，我们发现 >70% 的 Mtb 蛋白在 Ser/Thr/Tyr 上被磷酸化，识别出数千种 单个 Ser/Thr 激酶底物的分析，并表明 Ser/Thr 激酶共同控制 约 30% 的 Mtb 基因。在这里，我们将测试 Ser/Thr 激酶之间新的广泛的监管联系 和 TCS 的 His 激酶并测试 O-磷酸化在转录调节中的作用 因素。通过对 Mtb 磷酸化的详尽分析，我们还获得了第一个证据 Mtb 中的精氨酸磷酸化——革兰氏阳性菌之外的第一个细菌精氨酸磷酸化蛋白质组。我们将 识别相关的磷酸酶并测试 pArg 作为 ClpP- 降解标签的想法 介导的蛋白水解作用。