Phosphorylation-dependent regulation of protein secretion in Mycobacterium tuberculosis

项目摘要 本研究的目的是探讨延长的N-末端磷酸化的作用， 结核分枝杆菌SecE的结构域，SecYEG跨膜的重要组成部分 蛋白质转运通道，通过一般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性螺旋 细菌和古细菌之间的关系。虽然许多细菌中的SecE仅包含约65个编码 这些特征，其他，如E. coli和M.结核病具有延伸的N-末端结构域。我们有 鉴定了该结构域中磷酸化的三个Ser或Thr残基，所有这些残基都显示出高度的磷酸化。 显著降低的磷酸化响应于PknA和/或PknB的消耗，这两种必需的磷酸化蛋白质是PknA和/或PknB的磷酸化蛋白质。 该病原体的Ser/Thr激酶。这些发现为我们假设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。 为了研究这个域的调节作用，我们提出了两个目标。在目标1中， PknA充足和PknA缺失的M.结核菌株我们 还将构建表达天然SecE或具有磷酸模拟或磷酸消融的SecE的菌株 在鉴定的磷酸化位点处的取代，并定量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分泌的影响。在 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- 用SecE免疫沉淀法检测M.结核病和ii）检测蛋白质-蛋白质的双杂交测定 与天然、磷酸模拟和磷酸消融SecE的相互作用。这些实验将揭示 SecE及其磷酸化可能在一般分泌途径中起作用。 我们预计这项研究将揭示一种调节蛋白质分泌的新机制， 与结核病发病机制有关，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.