Temporal profiling of the functional phosphoproteome in M. tuberculosis infected

感染结核分枝杆菌的功能性磷酸蛋白质组的时间分析

• 批准号: 8791881
• 负责人: Sarah A Stanley
• 金额: $ 19.6万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8791881
• 关键词: AKT inhibition; Antibiotics; Apoptosis; Architecture; Attenuated; Cell physiology; Cessation of life; Complement; Complex; Data Set; Development; Disease; Drug resistance; Environment; Epidermal Growth Factor Receptor; Event; Future; Gene Expression; Goals; Growth; Health; Human; Immune; Immune response; Infection; Knowledge; Lead; Mammalian Cell; Mass Spectrum Analysis; Mediator of activation protein; Molecular; Monitor; Morbidity - disease rate; Mycobacterium tuberculosis; Nutrient; Outcome; Pathogenesis; Pathway interactions; Phagosomes; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Phosphotransferases; Proteins; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Public Health; Publishing; RNA Interference; Regulation; Regulatory Pathway; Research; Resources; Role; Signal Pathway; Signal Transduction; Technology; Time; Tuberculosis; Two-Dimensional Gel Electrophoresis; Vaccine Adjuvant; Vaccines; Virulence; Work; antimicrobial; base; combat; design; inhibitor/antagonist; interest; kinase inhibitor; macrophage; monocyte; mortality; next generation; novel strategies; novel therapeutics; pathogen; prevent; programs; research study; response; screening; small molecule; stem; targeted treatment; temporal measurement; therapeutic development; therapeutic target; therapeutic vaccine; transcriptomics

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.

描述（由申请人提供）：据估计，有20亿人感染M。结核病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。对M.结核病发病机制的研究对于合理设计下一代药物和疫苗是必要的。我们的目标是阐明宿主信号机制利用M。结核病在巨噬细胞中的生长和持久性。我们的中心假设是激酶信号传导导致感染的巨噬细胞中的基因表达程序，其促进对感染的反应不足，从而促进Mtb生长和存活。我们已经确定了巨噬细胞中的几种激酶，其功能是支持M。结核复制过程中，这表明它们被M。结核病作为毒力策略的一部分，或者它们是先天性抗微生物途径的负调节剂。与该提议特别相关的是破坏M.结核病在人类原代巨噬细胞中复制。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感染M.结核此外，我们将利用这些技术来阐明功能磷酸化蛋白质组通过确定下游靶点的AKT和EGFR在M。结核感染的巨噬细胞。在目的1中，我们描述了使用蛋白质组学和磷酸蛋白质组学来表征由M.结核病感染的人原发性单核细胞衍生的巨噬细胞与高时间分辨率使用二维凝胶电泳和质谱为基础的蛋白质组学和磷酸化蛋白质组学监测磷酸化位点在全球范围内。在目标2中，我们利用相同的策略来鉴定在抑制AKT和独立地抑制EGFR的情况下蛋白质组和磷酸化蛋白质组的差异。蛋白质组分析的结果将与现有的转录组和筛选数据集相结合，以进一步完善由M激活的信号网络的结构。肺结核感染。此外，这些结果将被用来激励未来的假设驱动实验，试图描述关键激酶调节剂支持M的分子机制。巨噬细胞中的结核毒力。