Phosphorylation-dependent regulation of protein secretion in Mycobacterium tuberculosis

项目总结 本研究的目的是研究延伸的N-末端的磷酸化作用 结核分枝杆菌SecE结构域，SecYEG跨膜的重要组成部分 蛋白质转运通道，通过总的分泌途径调节蛋白质的分泌。《密室》 C末端结构域编码高度保守的跨膜螺旋和两亲性螺旋 跨越细菌和古生菌。而许多细菌中的SecE只包含编码的~65个残基 这些特征，其他如大肠杆菌和结核分枝杆菌，都有一个扩展的N-末端结构域。我们有 鉴定了该结构域中三个被磷酸化的丝氨酸或苏氨酸残基，所有这些残基都高度显示出 在耗尽PounA和/或PounB后，磷酸化水平显著降低，这两种必需的 这种病原体的丝氨酸/苏氨酸蛋白激酶。这些发现为我们的假设提供了前提，即 延伸的N-末端结构域以一种磷酸化依赖的方式调节蛋白质的分泌。 为了研究这个领域的调节作用，我们提出了两个目标。在目标1中，我们将表演 结核分枝杆菌全株和缺失株培养滤液的定量蛋白质组学研究。我们 还将构建表达天然SecE或SecE的菌株，这些菌株具有拟磷化或磷化 确定的磷酸化位点的替换，并量化这些菌株的蛋白质分泌。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……里面 目的2我们将使用两种互补的方法鉴定与SecE相互作用的蛋白质：i)共刺激 SECE免疫沉淀法检测结核分枝杆菌及II)双杂交法检测蛋白质 与天然的、拟磷的和磷化的SecE的相互作用。这些实验将说明如何 SecE及其磷酸化可能在一般的分泌途径中发挥作用。 我们预计，这项研究将揭示一种新的调节蛋白质分泌的机制， 与结核病的发病机制相关，并可能导致治疗这种疾病的新的治疗方法。

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.