Functional phosphosignaling in Mtb infection

项目总结 磷信号协调宿主巨噬细胞(M‘S)对感染 结核分枝杆菌(Mtb)令人惊讶的是，到目前为止，只有少数几项研究衡量了 Mtb感染后的磷蛋白组质谱分析，而原代人MS中没有。 此外，当今细胞信号领域的一个主要挑战是分离出 功能性的区别于那些不起作用的。缺乏工具，无论是实验的还是计算的，都无法识别功能 非功能性磷酸化事件是目前许多磷信号研究的原因 很大程度上仍然是磷酸盐库存，提供的生物学见解有限。结合磷酸盐和磷酸盐 结构蛋白质组学，我们将检验这样的假设，即蛋白质的结构变化发生在 磷酸化是功能性磷酸化事件的可靠预测因子。我们将通过组合来测试这一想法 详细的亚磷酸盐作图和全球蛋白质结构测量，以比较 未感染结核分枝杆菌感染的原发人MS。我们将关联单个磷酸化位点的变化 蛋白质分解裂解模式的变化是结构变化的读数。磷酸化事件 改变一种蛋白质的结构将测试它们在结核分枝杆菌感染中的功能。我们将击倒(或推倒) 并检测其对细菌载量和M-功能的影响。将挖空效果链接到特定 磷酸化位点，我们将与携带磷酸化和拟态突变的蛋白质互补 并检测对M信号和结核分枝杆菌感染的影响。该项目将为 确定功能磷酸化位点的实验方法，特别是在 感染结核分枝杆菌的M。这些数据将允许对M磷酸化数据集进行更有意义的筛选， 提供感染后细胞重塑的全局、功能视图，突出特定的免疫途径 由结核分枝杆菌感染触发，确定潜在的宿主指导的新治疗靶点，并确定下游 控制或加剧结核分枝杆菌感染的效应器。

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.