Toxin Thermal Instability and Its Role in Host-Toxin Interactions

毒素热不稳定性及其在宿主-毒素相互作用中的作用

基本信息

批准号：
7846707
负责人：
KENNETH R TETER
金额：
$ 3.86万
依托单位：
UNIVERSITY OF CENTRAL FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-05 至 2010-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7846707
关键词：
26S proteasome ADP-Ribosylation Factors Affect Affinity Binding Biochemical Biological Assay Catalytic Domain Cell surface Cells Cholera Toxin Circular Dichroism Cytosol Degradation Pathway Dependence Development Dissociation Endoplasmic Reticulum Eukaryotic Cell Event Exhibits Fluorescence Spectroscopy Goals Heating In Vitro Incubated Intoxication Lead Link Mediating Membrane Modeling Molecular Molecular Chaperones Molecular Conformation Nature Pathogenesis Pathway interactions Peptide Hydrolases Pertussis Toxin Phospholipid Interaction Phospholipids Plasmids Process Property Protein Binding Protein Disulfide Isomerase Proteins Public Health Quality Control Research Research Personnel Resistance Rest Ricin Role Shiga Toxin Spectroscopy, Fourier Transform Infrared Surface Plasmon Resonance System Targeted Toxins Techniques Temperature Testing Therapeutic Toxin Travel Ubiquitin Work base direct application holotoxins infrared spectroscopy multicatalytic endopeptidase complex novel physical property polypeptide prevent programs

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of my research program is to understand the molecular mechanisms that allow certain AB-type toxins to cross the endoplasmic reticulum (ER) membrane and enter the cytosol of an intoxicated eukaryotic cell. AB toxins consist of a catalytic A subunit and a cell-binding B subunit. A subset of AB toxins travels from the cell surface to the ER before A chain translocation into the cytosol. AB-type, ER- translocating toxins include cholera toxin (CT), pertussis toxin, Shiga toxin, and ricin. These toxins exploit the quality control mechanism of ER-associated degradation (ERAD) in order to move from the ER to the cytosol. Current models of toxin-ERAD interactions assume the toxin A chain is stable and protease- resistant, but recent work has shown that multiple ER-translocating toxins actually contain A chains that are thermally unstable after dissociation from the holotoxin. Based upon our work with the catalytic subunit of CT (CTA1), we have developed a new model of toxin-ERAD interactions in which toxin translocation, degradation, and activity are all linked to the heat-labile nature of the isolated toxin A chain. This model is in marked contrast to the prevailing view of ERAD-mediated toxin translocation and makes distinct predictions in regards to how host-toxin interactions affect the intoxication process. To test our model, this project will use a variety of biophysical and biochemical techniques to examine how the folding state of CTA1 affects, and is affected by, its association with components of the ERAD system and other eukaryotic factors known to interact with the toxin. Biophysical and biochemical studies of other ER-translocating toxins will also be used to test our prediction that thermal instability is a common property of toxins that exploit ERAD to enter the eukaryotic cell. The work of this project will produce a major conceptual shift in the pathogenesis of ER- translocating toxins, with direct applications to the development of new anti-toxin therapeutic strategies and to a basic understanding of the ERAD mechanism. RELEVANCE TO PUBLIC HEALTH: In order to attack the target cell, certain toxins must first unfold to enter the cell and must then refold inside the cell to become active. Factors associated with the target cell modulate this process, so an understanding of toxin-target interactions could lead to the development of novel anti-toxin therapeutics that prevent the unfolding and/or refolding events required for toxin activity.

描述（由申请人提供）：我的研究项目的长期目标是了解某些 AB 型毒素穿过内质网 (ER) 膜并进入中毒真核细胞胞浆的分子机制。 AB 毒素由催化 A 亚基和细胞结合 B 亚基组成。 AB 毒素的一部分在 A 链易位到细胞质中之前从细胞表面移动到内质网。 AB型、ER转位毒素包括霍乱毒素(CT)、百日咳毒素、志贺毒素和蓖麻毒素。这些毒素利用内质网相关降解 (ERAD) 的质量控制机制，从内质网转移到细胞质。目前的毒素-ERAD 相互作用模型假设毒素 A 链是稳定的且具有蛋白酶抗性，但最近的研究表明，多种 ER 易位毒素实际上含有与全毒素解离后热不稳定的 A 链。基于我们对 CT 催化亚基 (CTA1) 的研究，我们开发了一种新的毒素-ERAD 相互作用模型，其中毒素易位、降解和活性都与分离的毒素 A 链的热不稳定性质有关。该模型与 ERAD 介导的毒素易位的流行观点形成鲜明对比，并对宿主-毒素相互作用如何影响中毒过程做出了不同的预测。为了测试我们的模型，该项目将使用各种生物物理和生化技术来检查 CTA1 的折叠状态如何影响其与 ERAD 系统组件以及已知与毒素相互作用的其他真核因子的关联，以及如何受其影响。其他 ER 易位毒素的生物物理和生化研究也将用于检验我们的预测，即热不稳定性是利用 ERAD 进入真核细胞的毒素的共同特性。该项目的工作将对 ER 易位毒素的发病机制产生重大概念转变，直接应用于开发新的抗毒素治疗策略以及对 ERAD 机制的基本了解。与公众健康的相关性：为了攻击目标细胞，某些毒素必须首先展开进入细胞，然后必须在细胞内重新折叠才能变得活跃。与靶细胞相关的因素调节这一过程，因此对毒素与靶标相互作用的了解可能会导致新型抗毒素疗法的开发，以防止毒素活性所需的解折叠和/或重折叠事件。