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

• 批准号: 7585327
• 负责人: KENNETH R TETER
• 金额: $ 34.83万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-15 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7585327
• 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

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(申请人提供)：我的研究计划的长期目标是了解允许某些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。在A链移位到胞浆之前，AB毒素的一个子集从细胞表面转移到内质网。AB型内质网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，对蛋白酶具有抗性，但最近的工作表明，多种内质网转位毒素实际上含有A链，这些A链在与全息毒素解离后是热不稳定的。基于我们对CT催化亚单位(CTA1)的工作，我们开发了一个新的毒素-ERAD相互作用模型，在该模型中，毒素的移位、降解和活性都与分离的毒素A链的热不稳定性有关。该模型与ERAD介导的毒素转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明确的预测。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研究CTA1的折叠状态如何影响它与ERAD系统组件的关联，以及它与其他已知与毒素相互作用的真核因子的关系。对其他ER转运毒素的生物物理和生化研究也将被用来检验我们的预测，即热不稳定性是利用ERAD进入真核细胞的毒素的共同属性。该项目的工作将在内质网转运毒素的发病机制方面产生重大的概念转变，直接应用于开发新的抗毒素治疗策略和对ERAD机制的基本理解。 与公众健康相关：为了攻击目标细胞，某些毒素必须首先解开才能进入细胞，然后必须在细胞内重新折叠才能激活。与靶细胞相关的因素调节这一过程，因此了解毒素与靶细胞的相互作用可能导致开发新的抗毒素疗法，以防止毒素活性所需的展开和/或折叠事件。