Functional phosphosignaling in Mtb infection

结核分枝杆菌感染中的功能性磷酸信号传导

基本信息

批准号：
10040388
负责人：
Christoph Grundner
金额：
$ 27.95万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-02 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10040388
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

项目摘要

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.

项目概要磷酸信号传导协调宿主巨噬细胞 (M’s) 对感染的大部分反应令人惊讶的是，迄今为止只有少数研究测量了结核分枝杆菌（Mtb）的全球变化。通过质谱分析发现 Mtb 感染后的磷酸化蛋白质组，而在原代人类 Ms 中没有发现磷酸化蛋白质组。此外，当今细胞信号传导领域的一个主要挑战是分离磷酸化事件缺乏区分功能性和非功能性的实验或计算工具。非功能性磷酸化事件是当前许多磷酸信号传导研究的原因大部分仍然是磷酸位点的库存，提供有限的生物学见解。结构蛋白质组学，我们将测试蛋白质结构变化发生的假设磷酸化是功能性磷酸化事件的可靠预测因子，我们将通过结合来测试这个想法。详细的磷酸位点图谱和整体蛋白质结构测量，以比较蛋白质组我们将把单个磷酸化位点的变化关联起来。将蛋白水解裂解模式的变化作为结构变化的读数。改变蛋白质的结构将测试其在结核分枝杆菌感染中的功能。磷蛋白并测试对细菌负荷和 M 功能的影响将敲除的影响与特定的效果联系起来。磷酸化位点，我们将用带有磷酸化和模拟突变的蛋白质进行补充并测试对 M 信号传导和 Mtb 感染的影响。该项目将为一个方法提供原理验证。一般情况下，特别是在以下情况下识别功能性磷酸化位点的实验方法感染 Mtb 的 M。这些数据将有助于对 M 磷酸化数据集进行更有意义的管理，提供细胞感染后重塑的全局功能视图，突出特定的免疫途径由 Mtb 感染触发，确定潜在的新宿主导向治疗靶点，并确定下游控制或恶化 Mtb 感染的作用。