Temporal profiling of the functional phosphoproteome in M. tuberculosis infected

描述（由申请人提供）：估计有20亿人感染结核分枝杆菌，这是一种重要的人类病原体，在世界范围内引起显著的发病率和死亡率。更好地了解结核分枝杆菌发病机理的分子机制对于合理设计下一代药物和疫苗是必要的。我们的目标是阐明结核分枝杆菌在巨噬细胞中生长和持续的宿主信号机制。我们的中心假设是，激酶信号导致感染巨噬细胞中的基因表达程序，促进对感染的反应不足，从而促进结核分枝杆菌的生长和存活。我们已经确定了巨噬细胞中的几种激酶，它们在感染期间支持结核分枝杆菌的复制，表明它们作为毒力策略的一部分被结核分枝杆菌重新激活，或者它们是先天抗微生物途径的负调节因子。与这一建议特别相关的是哺乳动物激酶AKT和EGFR的抑制剂，它们破坏结核分枝杆菌在人原代巨噬细胞中复制的能力。我们的方法是利用全球蛋白质组学和磷酸化蛋白质组学技术的最新进展来表征结核分枝杆菌感染激活的巨噬细胞信号通路。此外，我们将利用这些技术通过鉴定结核分枝杆菌感染巨噬细胞中AKT和EGFR的下游靶点来阐明功能性磷酸化蛋白组。在目标1中，我们描述了使用蛋白质组学和磷酸化蛋白质组学来表征结核分枝杆菌感染人类原代单核细胞源性巨噬细胞引起的信号事件，具有高时间分辨率，使用基于二维凝胶电泳和质谱的蛋白质组学和磷酸化蛋白质组学来监测全球范围内的磷酸化位点。在目标2中，我们使用相同的策略来识别AKT和EGFR抑制背景下蛋白质组和磷蛋白质组的差异。蛋白质组学分析的结果将与现有的转录组学和筛选数据集相结合，以进一步完善结核分枝杆菌感染激活的信号网络结构。此外，这些结果将用于激励未来的假设驱动实验，以寻求描述关键激酶调节因子支持巨噬细胞中结核分枝杆菌毒力的分子机制。

DESCRIPTION (provided by applicant): It is estimated that 2 billion people are infected with M. tuberculosis, an important human pathogen that causes significant morbidity and mortality worldwide. A better understanding of the molecular mechanisms of M. tuberculosis pathogenesis is necessary for the rational design of the next generation of drugs and vaccines. Our goal is to elucidate host signaling mechanisms exploited by M. tuberculosis for growth and persistence in macrophages. Our central hypothesis is that kinase signaling leads to a gene expression program in infected macrophages that promotes an insufficient response to infection thereby facilitating Mtb growth and survival. We have identified several kinases in macrophages that function to support M. tuberculosis replication during infection, indicating either that they re activated by M. tuberculosis as part of a virulence strategy or that they are negative regulators of innate anti-microbial pathways. Of specific relevance to this proposal are inhibitors of the mammalian kinases AKT and EGFR that disrupt the ability of M. tuberculosis to replicate in human primary macrophages. Our approach is to utilize recent advances in global proteomic and phosphoproteomic technologies to characterize macrophage-signaling pathways that are activated by infection with M. tuberculosis. In addition, we will use these technologies to elucidate the functional phosphoproteome by identifying of downstream targets of AKT and EGFR in M. tuberculosis infected macrophages. In aim 1, we describe the use of proteomics and phosphoproteomics to characterize signaling events resulting from M. tuberculosis infection of human primary monocyte-derived macrophages with high temporal resolution using 2D gel electrophoresis and mass-spectrometry based proteomics and phosphoproteomics to monitor phosphorylation sites on a global scale. In aim 2 we utilize the same strategy to identify differences in the proteome and phosphoproteome in the context of inhibition of AKT and, independently, EGFR. Results from the proteomic profiling will be integrated with existing transcriptomic and screening datasets to further refine the architecture of the signaling network activated by M. tuberculosis infection. In addition, the results will be used to motivate future hypothesis driven experiments seeking to describe the molecular mechanisms by which key kinase regulators support M. tuberculosis virulence in macrophages.