Functions and Mechanisms of M. Tuberculosis S/T Kinases

描述（由申请人提供）：本研究项目的长期目标是了解丝氨酸/苏氨酸蛋白激酶（STPKs）在致病性结核分枝杆菌（Mtb）中的功能。结核分枝杆菌引起结核病，它感染了世界三分之一的人口。结核病每年造成约200万人死亡，耐药菌株正在迅速出现。为了针对Mtb STPKs提供新的治疗方法，需要进行基础研究以确定这些酶的特定生物学功能和调控机制。在第一个资助期，我们率先开展了Mtb STPKs的结构研究，并建立了目前的范式，即由两个结构界面介导的二聚化和磷酸化激活细菌STPKs。我们还开发了新的方法来鉴定Mtb中的STPK底物。这些研究提供了集中生化、生物物理、结构和遗传方法来建立细菌STPK信号传导新原理的机会。方法的广度和新颖性是该计划的优势。本研究有四个具体目的：1。定义通过结核分枝杆菌跨膜受体激酶信号传导的基础。2. 发现激酶交叉磷酸化和调控的网络。3. 发现Mtb STPKs候选蛋白底物。4. 确定STPKs如何调节肽聚糖前体的Mtb翻转酶。我们的初步研究建立了概念框架和这些目标的可行性。我们提出了几项开创性的研究，包括激酶定位、二聚化和体内激活的空间和时间定位。我们将验证以下假设：二聚化激活细菌STPKs，完整的结核分枝杆菌激酶形成一个分层网络，STPKs调节结核分枝杆菌的大部分生理机能，PknB感知细胞壁片段并调节肽聚糖生物合成。由于激酶信号在细胞生理学中的核心作用，STPKs作为药物靶点的关注日益增加，以及结核病对全球健康的影响，我们提出的研究将在分子生物学和医学上具有很高的意义。公共卫生相关性：通过发现结核病（TB）和其他细菌感染的环境感知机制，该项目将直接与正在进行的开发针对重大威胁生命疾病的新抗生素的努力联系起来。人类细胞也通过类似的分子传感器发出信号，称为蛋白激酶，这些激酶是治疗糖尿病、炎症、疼痛和癌症等疾病的药物的重要靶点。因此，这一规划对人类健康具有广泛和直接的影响。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to understand the functions of Ser/Thr protein kinases (STPKs) in the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb causes tuberculosis (TB), and it infects one third of the world's population. TB kills ~2 million people annually, and drug resistant strains are emerging rapidly. To target the Mtb STPKs with new therapeutics, basic research is needed to define the specific biological functions and regulatory mechanisms of these enzymes. In the first grant period, we pioneered structural studies of the Mtb STPKs and established the current paradigm that dimerization and phosphorylation mediated by two structural interfaces activate bacterial STPKs. We also developed novel approaches to identify STPK substrates in Mtb. These studies afford the opportunity to focus biochemical, biophysical, structural and genetic methods to establish new principles of bacterial STPK signaling. The breadth and novelty of approaches are strengths of this program. This research has four specific aims: 1. Define the basis for signaling through Mtb transmembrane receptor kinases. 2. Discover networks of kinase cross-phosphorylation and regulation. 3. Discover candidate protein substrates of Mtb STPKs. 4. Determine how the STPKs regulate the Mtb flippase for peptidoglycan precursors. Our preliminary studies established the conceptual framework and the feasibility of these aims. We propose several groundbreaking studies, including spatial and temporal mapping of kinase localization, dimerization and activation in vivo. We will test the hypotheses that dimerization activates bacterial STPKs, that the complete Mtb kinome forms a hierarchical network, that the STPKs regulate much of Mtb physiology, and that PknB senses cell wall fragments and regulates peptidoglycan biosynthesis. Because of the central roles of kinase signaling in cellular physiology, the increasing focus on STPKs as pharmaceutical targets, and the worldwide health impact of TB, our proposed studies will have high significance for molecular biology and medicine. PUBLIC HEALTH RELEVANCE: By discovering the mechanisms of environmental sensing underlying tuberculosis (TB) and other bacterial infections, this program will connect directly to ongoing efforts to develop new antibiotics against major life-threatening diseases. Human cells also signal through similar molecular sensors, called protein kinases, and these kinases are important targets for drugs to treat diseases including diabetes, inflammation, pain and cancer. Consequently, this program has broad and direct implications for human health.