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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要本研究的目的是研究延伸的 N 末端磷酸化的作用结核分枝杆菌 SecE 结构域，是 SecYEG 跨膜的重要组成部分蛋白质易位通道，通过一般分泌途径调节蛋白质分泌。证券交易委员会 C 端结构域编码高度保守的跨膜螺旋和两亲性螺旋跨越细菌和古细菌。虽然许多细菌中的 SecE 仅包含约 65 个编码残基这些特征，其他特征，例如大肠杆菌和结核分枝杆菌具有扩展的 N 末端结构域。我们有鉴定出该结构域中的三个 Ser 或 Thr 残基被磷酸化，所有这些残基都显示出高度响应于 PknA 和/或 PknB（两种必需的 PknA 和/或 PknB）耗尽而显着降低磷酸化该病原体的 Ser/Thr 激酶。这些发现为我们的假设提供了前提，即 SecE 延伸的 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。在目标 1 中，我们将执行富含 PknA 和缺乏 PknA 的结核分枝杆菌菌株培养物滤液的定量蛋白质组学。我们还将构建表达天然 SecE 或具有拟磷剂或磷酸消融剂的 SecE 的菌株在确定的磷酸化位点进行取代，并量化这些菌株中的蛋白质分泌。这些实验将确定 SecE N 末端结构域磷酸化对蛋白质分泌的影响。在目标 2 我们将使用两种互补的方法来鉴定与 SecE 相互作用的蛋白质，i) co- 在结核分枝杆菌中使用 SecE 进行免疫沉淀，以及 ii) 检测蛋白质-蛋白质的双杂交测定与天然、拟磷化和磷酸化 SecE 的相互作用。这些实验将表明如何 SecE 及其磷酸化可能在一般分泌途径中发挥作用。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制相关，并可能导致治疗这种疾病的新治疗方法。