Toxin Thermal Instability and Its Role in Host-Toxin Interactions

描述（由申请人提供）：我的研究项目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的细胞质的分子机制。AB毒素由催化a亚基和细胞结合B亚基组成。AB毒素的一个亚群从细胞表面到达内质网，然后进入细胞质转移链。ab型，ER易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，以便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在与全毒素分离后热不稳定的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，其中毒素的易位、降解和活性都与分离的毒素a链的热不稳定性有关。该模型与普遍认为的erd介导的毒素易位形成鲜明对比，并对宿主-毒素相互作用如何影响中毒过程做出了独特的预测。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和受其与ERAD系统成分和其他已知与毒素相互作用的真核因子的关联的影响。其他er易位毒素的生物物理和生化研究也将用于验证我们的预测，即热不稳定性是利用ERAD进入真核细胞的毒素的共同特性。该项目的工作将对内质网易位毒素的发病机制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。

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.