Study of Cholera Toxin Entry into the Eukaryotic Cytosol

描述(申请人提供)：这项研究提案的长期目标是剖析允许霍乱毒素(CT)的催化A1多肽进入中毒真核细胞胞浆的分子事件。CTA1 ADP-核糖化并不可逆地激活真核细胞质膜表面异源三聚体G蛋白的刺激性α亚基。由此产生的下游信号事件导致自1995年以来在100多万霍乱病例中出现的危及生命的水样腹泻。霍乱弧菌分泌到细胞外环境后，CT与真核细胞的质膜结合，逆行转运到内质网(ER)。CTA1从内质网中的全毒素中解离出来，然后穿过内质网膜进入胞浆。据推测，内质网相关降解(ERAD)途径促进CTA1从内质网到胞浆的转位。ERAD是一种质量控制机制，它可以识别内质网中错误折叠的蛋白质，并将它们输出到细胞质中进行泛素化和蛋白酶体降解。CTA1中的疏水区被认为能触发ERAD活性，并刺激CTA1移位到胞浆；胞浆中的降解可能是避免的，因为CTA1缺乏作为泛素结合位点的赖氨酸残基。该项目将测试和详细说明ERAD/CTA1易位模型。以前测量CTA1易位的方法是基于CTA1活性的下游胞浆效应，但这项工作将利用最近开发的一种直接监测CTA1易位事件的生化分析方法。该分析将用于(I)确定CTA1转运到胞浆并在胞浆中持续所需的结构特征；(Ii)确定在转运过程中与CTA1相互作用的可能的ERAD因子；(Iii)描述CTA1从内质网转移到胞浆所需的生理参数；以及(Iv)建立一个基于酵母的系统来研究CTA1的转运。这些研究活动将满足建立具有培养本科生、研究生和博士后人才能力的生产性研究实验室的短期目标。拟议的工作将大大有助于理解霍乱的发病机制，将产生与ERAD功能有关的重要观察结果，并将产生与CT贩运和易位路线之后的其他毒素相关的信息。

DESCRIPTION (provided by applicant): The long-term objective of this research proposal is to dissect the molecular events that allow the catalytic A1 polypeptide of cholera toxin (CT) to enter the cytosol of intoxicated eukaryotic cells. CTA1 ADP-ribosylates and irreversibly activates the stimulatory alpha subunit of the heterotrimeric G protein at the cytoplasmic face of the eukaryotic plasma membrane. The resulting downstream signaling events induce the life-threatening watery diarrhea seen in over one million cases of cholera since 1995. After it is secreted into the extracellular milieu by Vibrio cholerae, CT binds to the plasma membrane of eukaryotic cells and is transported in retrograde fashion to the endoplasmic reticulum (ER). CTA1 dissociates from holotoxin in the ER and then crosses the ER membrane to enter the cytosol. It is hypothesized that the ER-associated degradation (ERAD) pathway facilitates CTA1 translocation from the ER to the cytosol. ERAD is a quality control mechanism that recognizes misfolded proteins in the ER and exports them to the cytosol for ubiquitination and proteosomal degradation. A hydrophobic region in CTA1 is thought to trigger ERAD activity and stimulate CTA1 translocation to the cytosol; degradation in the cytosol is presumably avoided because CTA1 has a paucity of the lysine residues that serve as ubiquitin attachment sites. This project will test and elaborate upon the ERAD/CTA1 translocation model. Previous methods to measure CTA1 translocation were based on the downstream cytosolic effects of CTA1 activity, but this work will instead utilize a recently developed biochemical assay that directly monitors the CTA1 translocation event. The assay will be used to (i) identify the structural features of CTA1 that are required for translocation to, and persistence in, the cytosol; (ii) identify the putative ERAD factors that interact with CTA1 during the translocation process; (iii) delineate the physiological parameters required for the ER-to-cytosol transfer of CTA1, and (iv) establish a yeast-based system to study CTA1 translocation. These research activities will meet the short-term goal of establishing a productive research lab with the capacity to train undergraduate, graduate, and post-doctoral personnel. The proposed work will contribute significantly to the understanding of cholera pathogenesis, will generate important observations relating to ERAD function, and will produce information relevant to other toxins that follow the CT trafficking and translocation itinerary.