Molecular Mechanism of Translocon Assembly into Cell Plasma Membranes

描述（由申请人提供）：蛋白质跨细胞膜易位的复杂过程是细菌发病机制中的常见主题。用于蛋白质递送的特别有效的分子机器是III型分泌（T3S）系统。T3S系统充当注射器，将蛋白质从细菌细胞质直接注射到靶细胞的细胞质中。一旦进入宿主细胞质，易位的毒素破坏真核细胞过程（例如，阻断吞噬作用），调节有利于感染的宿主反应。 在鼠疫耶尔森氏菌和铜绿假单胞菌中收集的证据表明，两种分泌的T3S蛋白插入靶细胞膜并形成一个孔（或易位子），毒素通过该孔易位。尽管最近在这两种蛋白质（易位子）的特征化方面取得了进展，但它们的结构和易位子的组装机制仍然未知。 我已经开发了一套荧光技术，已成功地用于表征各种同源寡聚溶细胞毒素的结构和孔形成机制。我现在建议将这些技术的应用扩展到多蛋白跨膜复合物，如T3S易位子。荧光方法将与其他生物化学和生物物理技术（例如，电生理学测量、单分子技术和冷冻电子显微镜），以明确地解决T3S易位子结构和组装的基本结构方面。通过选择性掺入各种探针（例如，环境敏感荧光团、交联剂、金纳米颗粒、带电基团等）在铜绿假单胞菌的易位蛋白中，我们将通过实验确定这些蛋白质的哪些片段对于确定易位子通道的特征是必需的，哪些片段形成针和易位子之间的接触界面，以及易位蛋白如何排列在哺乳动物细胞膜中形成的易位子复合物中。 除了阐明易位子组装成脂质双层的基本方面外，这些研究可能最终导致阻断蛋白质易位并干扰多种威胁人类病原体的细菌定殖的新治疗策略。

DESCRIPTION (provided by applicant): The complex process of protein translocation across cell membranes is a common theme in bacterial pathogenesis. A particularly efficient molecular machine for pro tein delivery is the type III secretion (T3S) system. The T3S system acts as a syringe that injects proteins from the bacterial cytoplasm directly into the cytoplasm of a target cell. Once into the host cytosol, translocated toxins subvert eukaryotic cellular processes (e.g., blocking phagocytosis), modulating the host response in favor of infection. Collected evidence in Yersinia pestis and Pseudomonas aeruginosa suggests that two secreted T3S proteins insert into the target cell membrane and form a pore (or translocon) through which toxins are translocated. Despite recent advances on the char- acterization of these two proteins (the translocators), their structure and mechanism of assembly of the translocon remain unknown. I have developed a set of fluorescence techniques that have been successfully used to characterize the structure and pore-formation mechanism of various homo- oligomeric cytolytic toxins. I now propose to extent the use of these techniques to multi- protein transmembrane complexes, like the T3S translocon. The fluorescence approach will be combined with other biochemical and biophysical techniques (e.g., electrophysi- ology measurements, single molecule techniques, and cryo-electron microscopy) to un- ambiguously address fundamental structural aspects of the T3S translocon structure and assembly. By selective incorporation of various probes (e.g., environment-sensitive fluorophores, crosslinkers, gold-nanoparticles, charged groups, etc.) in the P. aerugi- nosa translocators, we will experimentally identify, among other things: which segments of these proteins are essential to determine the characteristics of the translocon channel, what segments form the contact interface between the needle and the translocon, and how the translocators are arranged in the translocon complex formed in the mammalian cell membrane. In addition to elucidate fundamental aspects of translocon assembly into lipid bi- layers, these studies may ultimately lead to novel therapeutic strategies that block pro- tein translocation and interfere with bacterial colonization in a broad variety of threaten- ing human pathogens.