Study of Cholera Toxin Entry into the Eukaryotic Cytosol

霍乱毒素进入真核细胞质的研究

• 批准号: 6600322
• 负责人: KENNETH R TETER
• 金额: $ 15.66万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6600322
• 关键词: CHO cells; Saccharomyces cerevisiae; Vibrio cholerae; bacteria infection mechanism; cell membrane; cell migration; cholera toxin; cytoplasm; endoplasmic reticulum; enzyme activity; eukaryote; host organism interaction; intracellular transport; protein disulfide isomerase; tissue /cell culture

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.

描述（由申请人提供）：本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质溶胶的分子事件。CTA 1 ADP-核糖基化并不可逆地激活真核细胞质膜细胞质面处异源三聚体G蛋白的刺激性α亚基。自1995年以来，在超过100万例霍乱病例中，由此产生的下游信号事件诱发了危及生命的水样腹泻。在被霍乱弧菌分泌到细胞外环境中后，CT结合到真核细胞的质膜上，并以逆行方式转运到内质网（ER）。CTA 1在ER中与全毒素解离，然后穿过ER膜进入细胞质。据推测，ER相关降解（ERAD）途径促进CTA 1从ER易位到胞质溶胶。ERAD是一种质量控制机制，其识别ER中错误折叠的蛋白质并将其输出到胞质溶胶以进行泛素化和蛋白质体降解。人们认为CTA 1中的疏水性区域会触发ERAD活性并刺激CTA 1转移到胞质溶胶中;细胞质溶胶中的降解大概是可以避免的，因为CTA 1缺乏作为遍在蛋白附着位点的赖氨酸残基。该项目将测试和阐述ERAD/CTA 1易位模型。以前的方法来衡量CTA 1易位是基于下游的CTA 1活性的胞质效应，但这项工作将利用最近开发的直接监测CTA 1易位事件的生化测定。该试验将用于（i）鉴定CTA 1易位至胞质溶胶并在胞质溶胶中持续存在所需的结构特征;（ii）鉴定在易位过程中与CTA 1相互作用的推定ERAD因子;（iii）描述CTA 1 ER至胞质溶胶转移所需的生理参数，以及（iv）建立基于酵母的系统以研究CTA 1易位。这些研究活动将满足建立一个有能力培养本科生，研究生和博士后人员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了解霍乱的发病机制，将产生重要的意见有关ERAD功能，并将产生相关的其他毒素，遵循CT贩运和易位行程的信息。