Phosphorylation-dependent regulation of protein secretion in Mycobacterium tuberculosis

结核分枝杆菌蛋白质分泌的磷酸化依赖性调节

• 批准号: 10183156
• 负责人: ROBERT N HUSSON
• 金额: $ 22.13万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-08 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183156
• 关键词: Address; Affect; Alleles; Animal Model; Archaea; Arginine; Bacteria; Biological Assay; C-terminal; Cell physiology; Cells; Co-Immunoprecipitations; Complement; Cues; Cytoplasmic Tail; Data; Disease; ELF3 gene; Environment; Escherichia coli; Filtration; Gene Expression; Genes; Genetic; Genetic Structures; Goals; Infection; Integral Membrane Protein; Knowledge; Laboratories; Lead; Mediating; Membrane Proteins; Messenger RNA; Methods; Mycobacterium tuberculosis; N-terminal; Operon; Pathogenesis; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Post-Translational Protein Processing; Process; Protein Biosynthesis; Protein Kinase; Protein Secretion; Protein translocation; Proteins; Proteomics; Publishing; Regulation; Research; Role; Signal Transduction; Structure; System; Testing; Transcriptional Regulation; Tuberculosis; Twin Multiple Birth; Two-Hybrid System Techniques; Work; base; enzyme activity; experimental study; human pathogen; insight; novel; novel therapeutic intervention; pathogen; protein function; protein protein interaction; response; success; transcription factor; yeast two hybrid system

PROJECT SUMMARY The goal of this research is to investigate the role of phosphorylation of the extended N-terminal domain of Mycobacterium tuberculosis SecE, an essential component of the SecYEG transmembrane protein translocation channel, in regulating protein secretion via the general secretory pathway. The SecE C-terminal domain encodes a transmembrane helix and an amphipathic helix that are highly conserved across bacteria and archaea. While SecE in many bacteria comprises only the ~65 residues that encode these features, others, such as E. coli and M. tuberculosis have an extended N-terminal domain. We have identified three Ser or Thr residues in this domain that are phosphorylated, all of which show highly significantly decreased phosphorylation in response to depletion of PknA and/or PknB, the two essential Ser/Thr kinases of this pathogen. These findings provide the premises for our hypothesis that the SecE extended N-terminal domain regulates protein secretion in a phosphorylation-dependent manner. To investigate the regulatory role of this domain, we propose two Aims. In Aim 1 we will perform quantitative proteomics of culture filtrates of PknA replete and PknA depleted M. tuberculosis strains. We will also construct strains expressing native SecE or SecE with phosphomimetic or phosphoablative substitutions at the identified phosphorylation sites, and quantify protein secretion in these strains. These experiments will determine the effects of SecE N-terminal domain phosphorylation on protein secretion. In Aim 2 we will identify proteins that interact with SecE using two complementary methods, i) co- immunoprecipitation with SecE in M. tuberculosis and ii) two-hybrid assays to detect protein-protein interactions with native, phosphomimetic and phosphoablative SecE. These experiments will suggest how SecE and its phosphorylation may function in the general secretion pathway. We anticipate that this research will reveal a novel mechanism for regulating protein secretion that will be relevant for tuberculosis pathogenesis and may lead to new therapeutic approaches to treat this disease.

项目摘要 这项研究的目的是研究扩展N末端的磷酸化的作用 结核分枝杆菌的结构域Sece，secyeg跨膜的重要组成部分 蛋白质易位通道，通过大分泌途径调节蛋白质分泌。 Sece C末端结构域编码高度保守的跨膜螺旋和两亲螺旋 跨细菌和古细菌。而在许多细菌中的sece仅包含约65个编码的残基 这些特征，例如大肠杆菌和结核分枝杆菌具有扩展的N末端结构域。我们有 在该域中鉴定出磷酸化的三个SER或THR残基，所有这些都显示出高度 响应PKNA和/或PKNB的耗竭，显着降低了磷酸化，这两个必不可少 该病原体的Ser/Thr激酶。这些发现为我们的假设提供了前提 扩展的N末端结构域以磷酸化依赖性方式调节蛋白质分泌。 为了研究该领域的监管作用，我们提出了两个目标。在AIM 1中，我们将表演 PKNA灌注和PKNA的培养滤液的定量蛋白质组学消耗了结核分枝杆菌菌株。我们 还将构建用磷酸或磷酸化表达天然sece或sece的菌株 在确定的磷酸化位点取代，并在这些菌株中定量蛋白质分泌。这些 实验将确定二末端结构域磷酸化对蛋白质分泌的影响。在 目的2我们将使用两种互补方法识别与SECE相互作用的蛋白质，i）共同 在结核分枝杆菌中用sece进行免疫沉淀和II）两种杂交测定法以检测蛋白质蛋白质 与天然，磷酸化和磷酸化的SECE相互作用。这些实验将暗示如何 SECE及其磷酸化可能在大分泌途径中起作用。 我们预计这项研究将揭示一种调节蛋白质分泌的新机制 与结核病发病机理有关，并可能导致治疗该疾病的新治疗方法。