Defining the role of ClpX in mycobacterial cell cycle progression

定义 ClpX 在分枝杆菌细胞周期进程中的作用

• 批准号: 8983021
• 负责人: Jemila Caplan Kester
• 金额: $ 3.67万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8983021
• 关键词: ATP phosphohydrolase; Antibiotics; Bacteria; Biochemical; Biochemistry; Biological Assay; Caulobacter; Caulobacter crescentus; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Shape; Cell division; Cell physiology; Cells; Cessation of life; Complex; DNA biosynthesis; Data; Disease; Drug Targeting; Failure; Family; Fluorescence; Gammaproteobacteria; Genus Mycobacterium; Growth; Housekeeping; Immunoprecipitation; In Vitro; Inherited; Knowledge; Lead; Life; Literature; Manuscripts; Mediating; Methodology; Methods; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Mycobacterium smegmatis; Mycobacterium tuberculosis; Parents; Participant; Pathogenesis; Pattern; Peptide Hydrolases; Physiological; Preparation; Process; Proteins; Proteolysis; Reproduction; Resistance; Resolution; Role; Shapes; Techniques; Testing; Tuberculosis; Work; base; cell growth; cellular imaging; daughter cell; experience; global health; in vivo; method development; mycobacterial; novel; public health relevance; response; retinal rods; single cell analysis; stressor; time use; unfoldase

 DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a global health burden, causing over 1.5 million deaths annually. Pathogenesis requires, among many other factors, the bacteria to grow and divide. Yet regulators of the cell cycle for Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, remain undiscovered. In my thesis work, I seek to identify the molecular mechanisms governing the cell cycle in mycobacteria. Mycobacteria grow and divide asymmetrically, in part reflecting a unique cell growth cycle characterized by windows of differential growth rates at the old and new poles of the rod shaped cell (manuscript in preparation). In mycobacteria, asymmetric growth and division is important because it rapidly generates phenotypically different cells able to withstand different stressors like antibiotics. Th model bacterium, Caulobacter crescentus, also generates phenotypically distinct daughter cells in part through asymmetric division and partitioning of key cell cycle regulators. In Caulobacter, the essential housekeeping protease complex ClpXP degrades a key cell cycle regulator to initiate this process. I postulated that similar mechanisms might underlie asymmetric growth and division in mycobacteria. ClpX, an AAA+ ATPase of the HSP100 family of molecular chaperones, can either function alone to unfold proteins or deliver unfolded substrates to ClpP for degradation. I have demonstrated that depletion of ClpX in mycobacteria results in cell cycle arrest during division using dynamic live cell imaging, supporting a role for ClpX in cell cycle regulation. Based on my phenotypic studies and the known cellular functions of ClpX, I hypothesized there is a substrate of ClpX that is a key cell cycle component. To identify ClpX substrates required for cell cycle regulation, I used a pseudo-trap in Mycobacterium smegmatis (Msm) and uncovered 200 high-confidence putative ClpX substrates. I have selected 10 putative substrates with a role in cell cycle progression to validate as substrates of ClpX. I propose ClpX regulates the mycobacterial cell cycle by acting on one or more of these identified putative substrates. I will confirm a role in cell cycle progression for the interaction of ClpX an one or more of these substrates through these aims. First, I will determine which substrates ClpXP degrades and which ClpX regulates as a chaperone. Then I will show physiologic relevance of ClpX and its substrates in cell cycle progression. These studies will provide a methodology for biochemical identification of high-confidence ATPase/chaperone substrates, and lead to a better understanding of the mycobacterial cell cycle. In completing these steps, I will gain expertise in biochemistry, molecular biology, live cell microscopy, analysis, and scientific method development.

 描述（由申请人提供）：结核病（TB）仍然是全球健康负担，每年造成150多万人死亡。致病过程需要细菌生长和分裂，以及许多其他因素。然而，结核病病原体结核分枝杆菌（Mtb）细胞周期的调节因子仍未被发现。在我的论文工作中，我试图确定分枝杆菌细胞周期的分子机制。分枝杆菌的生长和分裂是不对称的，部分反映了独特的细胞生长周期，其特征是在杆状细胞的新旧两极有不同的生长速率窗口（手稿在准备中）。在分枝杆菌中，不对称生长和分裂很重要，因为它能快速产生表型不同的细胞，能够承受不同的压力，如抗生素。Th模型细菌新月柄杆菌（Caulobacter crescentus）也部分地通过关键细胞周期调节剂的不对称分裂和分配产生表型不同的子细胞。在柄杆菌属中，必需的管家蛋白酶复合物ClpXP降解关键的细胞周期调节剂以启动该过程。我推测类似的机制可能是分枝杆菌不对称生长和分裂的基础。 ClpX是HSP 100分子伴侣家族的AAA+ ATP酶，可以单独发挥功能以解折叠蛋白质或将解折叠的底物递送至ClpP进行降解。我已经证明，消耗ClpX在分枝杆菌的分裂过程中使用动态活细胞成像的细胞周期停滞的结果，支持ClpX在细胞周期调控中的作用。根据我的表型研究和ClpX已知的细胞功能，我假设ClpX的底物是细胞周期的关键成分。为了确定细胞周期调控所需的ClpX底物，我在耻垢分枝杆菌（Msm）中使用了假陷阱，并发现了200个高置信度的推定ClpX底物。我已经选择了10个推定的基板与细胞周期进程中的作用，以验证作为底物的ClpX。 我建议ClpX调节分枝杆菌细胞周期的一个或多个这些公认的底物。我将通过这些目的确认ClpX与一种或多种这些底物相互作用在细胞周期进程中的作用。首先，我将确定哪些底物ClpXP降解，哪些ClpX作为分子伴侣进行调节。然后，我将展示ClpX及其底物在细胞周期进程中的生理相关性。这些研究将提供一种高置信度的ATP酶/伴侣底物的生化鉴定方法，并导致更好地了解分枝杆菌细胞周期。在完成这些步骤，我将获得生物化学，分子生物学，活细胞显微镜，分析和科学方法开发的专业知识。