YscE/YscG chaperone-dependent secretion of Yersinia pestis YscF

描述（由申请人提供）：鼠疫耶尔森氏菌（鼠疫病原体）使用III型分泌系统（T3 SS）将效应蛋白注入真核细胞。耶尔森氏菌T3 S装置的组装和功能依赖于21种必需的耶尔森氏菌分泌（Ysc）蛋白。在功能性基础结构组装之后，T3 S装置仅分泌早期底物（YscF、YscI和YscP），其功能是组装杆（YscI）和针（YscF）结构;然而，对YscF和其他早期底物的独特特征知之甚少，这些特征使得它们能够选择性识别和分泌。在这个提议中，我们将表征识别YscF作为早期T3 S底物的独特分子特征;以及识别这些特征的T3 S装置的组件。重要的是，细胞质YscF仅存在于与其异源二聚体YscE/YscG分子伴侣的1：1：1复合物中，这种相互作用对于防止YscF的蛋白水解降解至关重要。我们还提供了实验证据，YscE/YscG伴侣是必不可少的YscF分泌独立的作用，稳定YscF。首先，我们将研究YscF N-末端分泌信号在YscF分级分泌中的作用。此外，将鉴定结合并稳定YscF但不分泌YscF的YscE和YscG点突变体。总之，这些研究将定义识别YscEFG复合物和选择性分泌YscF所需的YscF和YscE/YscG分子伴侣的独特分子特征。最后，我们将使用表征YscKYscQYscL分选平台和YscN ATP酶在天然和突变体YscEFG复合物的识别中的作用。这些研究将定义识别事件，允许T3 S装置区分早期和晚期T3 S底物，这是建立分泌层次结构所必需的过程。 公共卫生相关性：该项目的总体目标是了解病原菌如何组装专门的蛋白质分泌系统，使它们能够将毒素直接注入人体细胞。干扰这些组装事件的药物有望解除这些病原体的武装，并允许自然防御机制消除细菌。

DESCRIPTION (provided by applicant): Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. The assembly and function of the Yersinia T3S apparatus is dependent upon 21 essential Yersinia secretion (Ysc) proteins. Following assembly of a functional base structure, the T3S apparatus secretes only early substrates (YscF, YscI and YscP) that function to assemble the rod (YscI) and needle (YscF) structures; however, little is known about the unique features of YscF and other early substrates that enable their selective recognition and secretion. In this proposal, we will characterize the unique molecular features that identify YscF as an early T3S substrate; as well as the components of the T3S apparatus that recognize these features. Importantly, cytoplasmic YscF is found exclusively in a 1:1:1 complex with its heterodimeric YscE/YscG chaperone, an interaction that is essential to prevent proteolytic degradation of YscF. We also provide experimental evidence that the YscE/YscG chaperone is essential for YscF secretion independent of its role in stabilizing YscF. Initially, we will investigate the role of the YscF N-terminal secretion signal in the hierarchical secretion of YscF. In addition, YscE and YscG point mutants that bind and stabilize YscF, but do not secrete YscF will be identified. Together, these studies will define the unique molecular features of YscF and the YscE/YscG chaperone that are required for recognition of the YscEFG complex and selective secretion of YscF. Finally, we will use characterize the role of a YscKYscQYscL sorting platform and YscN ATPase in the recognition of native and mutant YscEFG complexes. These studies will define the recognition events that allow the T3S apparatus to differentiate early and late T3S substrates, a process that is essential for establishing a secretion hierarchy. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to understand how pathogenic bacteria assemble specialized protein secretion systems that allow them to inject toxins directly into human cells. Drugs that interfere with these assembly events would be expected to disarm these pathogens and allow natural defense mechanisms to eliminate the bacteria.