Functional phosphosignaling in Mtb infection

结核分枝杆菌感染中的功能性磷酸信号传导

• 批准号: 10177868
• 负责人: Christoph Grundner
• 金额: $ 21.15万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-02 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10177868
• 关键词: Affect; Biological; Cells; Complement; Data; Data Set; Docking; Endopeptidase K; Enzyme-Linked Immunosorbent Assay; Equipment and supply inventories; Event; Foundations; Global Change; Human; Immune; Immune response; Immunity; Individual; Infection; Knock-out; Lead; Link; Lip structure; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Modeling; Molecular Conformation; Mutation; Mycobacterium tuberculosis; Natural Immunity; Outcome; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Phenotype; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Predisposition; Primary Infection; Proteins; Proteolysis; Proteome; Proteomics; RNA Interference; Role; Sampling; Shotguns; Signal Transduction; Site; Structure; Surface; System; Testing; Trypsin; Tuberculosis; base; cytokine; insight; knock-down; macrophage; mimetics; mutant; phosphoproteomics; protein protein interaction; protein structure; protein structure function; response; targeted treatment; three dimensional structure; tool; Affect; Biological; Cells; Complement; Data; Data Set; Docking; Endopeptidase K; Enzyme-Linked Immunosorbent Assay; Equipment and supply inventories; Event; Foundations; Global Change; Human; Immune; Immune response; Immunity; Individual; Infection; Knock-out; Lead; Link; Lip structure; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Modeling; Molecular Conformation; Mutation; Mycobacterium tuberculosis; Natural Immunity; Outcome; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Phenotype; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Predisposition; Primary Infection; Proteins; Proteolysis; Proteome; Proteomics; RNA Interference; Role; Sampling; Shotguns; Signal Transduction; Site; Structure; Surface; System; Testing; Trypsin; Tuberculosis; base; cytokine; insight; knock-down; macrophage; mimetics; mutant; phosphoproteomics; protein protein interaction; protein structure; protein structure function; response; targeted treatment; three dimensional structure; tool

PROJECT SUMMARY Phosphosignaling coordinates much of the host macrophage’s (M’s) response to infection with Mycobacterium tuberculosis (Mtb). Surprisingly, only few studies to date have measured global changes in the phosphoproteome upon infection with Mtb by mass spectrometry, and none in primary human Ms. In addition, a major challenge in the cell signaling field today is to separate phosphorylation events that are functional from those that are not. The lack of tools, experimental or computational, to d istinguish functional from non-functional phosphorylation events is the reason why many current studies of phosphosignaling largely remain inventories of phosphosites that provide limited biological insight. Combining phospho- and structural proteomics, we will test the hypothesis that the occurrence of a structural change of a protein upon phosphorylation is a reliable predictor of functional phosphorylation events. We will test this idea by combining detailed phosphosite mapping and global protein structure measurements to compare the proteomes of uninfected to Mtb-infected primary human Ms. We will correlate changes in individual phosphorylation sites with changes in the proteolytic cleavage pattern as a readout of structural change. Phosphorylation events that change a protein’s structure will be tested for their function in Mtb infection. We will knock out (or down) the phosphoprotein and test the effect on bacterial loads and M function. To link effects of knockout to specific phosphorylation sites, we will complement with the protein bearing phosphoablative and -mimetic mutations and test for effects on M signaling and Mtb infection. This project will provide a proof-of-principle for an experimental method to identify functional phosphorylation sites in general, and in particular in the context of the Mtb-infected M. These data will allow for more meaningful curation of M phosphorylation datasets, provide a global, functional view of the cell’s remodeling upon infection, highlight the specific immune pathways triggered by Mtb infection, identify potential new host-directed therapy targets, and identify downstream effectors that control or exacerbate Mtb infection.

项目摘要 磷酸化仪坐标会辅助宿主巨噬细胞对感染的反应 结核分枝杆菌（MTB）。令人惊讶的是，迄今为止只有很少的研究衡量了全球变化 通过质谱法感染MTB后，磷酸蛋白酶在原代人MS中没有。在 此外，当今细胞信号传导场的主要挑战是分开磷酸化事件 来自那些没有的功能。缺乏实验性或计算的工具，以distinguish功能 来自非功能磷酸化事件是许多当前磷酸化的研究的原因 在很大程度上仍然是提供有限的生物学见解的磷酸材料的库存。结合磷酸和 结构蛋白质组学，我们将检验以下假设：蛋白质上发生结构变化 磷酸盐是功能磷酸盐事件的可靠预测指标。我们将通过组合来测试这个想法 详细的磷材料图和全球蛋白质结构测量值，以比较 未感染对MTB感染的原代人M。我们将关联单个磷酸化位点的变化 随着结构变化的读数，蛋白水解裂解模式的变化。磷酸化事件 更改蛋白质的结构将在MTB感染中的功能进行测试。我们将淘汰（或向下） 磷蛋白并测试对细菌负荷和M功能的影响。将淘汰的效果与特定的效果联系起来 磷酸化位点，我们将使用具有磷酸化和模拟突变的蛋白质 并测试对M信号传导和MTB感染的影响。该项目将为一个项目提供原则证明 实验方法通常鉴定功能磷酸化位点，尤其是在 MTB感染的M。这些数据将允许M磷酸化数据集的更有意义的策划， 在感染后提供细胞重塑的全球功能视图，突出显示特定的免疫途径 由MTB感染触发，识别潜在的新宿主定向治疗靶标，并识别下游 控制或加剧MTB感染的效应器。