Molecular Mechanism of Translocon Assembly into Cell Plasma Membranes

描述(由申请人提供)：蛋白质跨细胞膜转移的复杂过程是细菌发病机制中的一个共同主题。蛋白质输送的一种特别有效的分子机器是III型分泌系统(T3S)。T3S系统就像一个注射器，将细菌细胞质中的蛋白质直接注入目标细胞的细胞质中。一旦进入宿主细胞质，转位毒素就会颠覆真核细胞的过程(例如，阻止吞噬作用)，调节宿主的反应，有利于感染。在鼠疫耶尔森氏菌和铜绿假单胞菌中收集的证据表明，两个分泌的T3S蛋白插入到靶细胞膜中，形成一个孔(或转位蛋白)，毒素通过这个孔(或转位蛋白)转运。尽管最近在这两种蛋白(转运子)的特性方面取得了进展，但它们的结构和组装转运子的机制仍不清楚。我已经开发了一套荧光技术，已经成功地用于表征各种同源低聚细胞溶解毒素的结构和成孔机制。我现在建议将这些技术的使用扩展到多蛋白跨膜复合体，如T3S转位蛋白。荧光方法将与其他生化和生物物理技术(例如，电生理测量、单分子技术和冷冻电子显微镜)相结合，以明确地解决T3S转位结构和组装的基本结构方面。通过选择性地掺入各种探针(例如，环境敏感的荧光团、交联剂、金纳米粒子、带电基团等)在铜绿假单胞菌转运子中，我们将通过实验确定这些蛋白质的哪些片段对于确定转运子通道的特性是必不可少的，哪些片段形成针与转位子之间的接触界面，以及转运子在哺乳动物细胞膜中形成的转运子复合体中如何排列。除了阐明转运子组装成脂质双层的基本方面外，这些研究最终可能导致新的治疗策略，阻止蛋白转位并干扰细菌在广泛种类的威胁人类病原体中的定植。

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.