pH-Induced Changes to Cholera Toxin Interaction with the Eukaryotic Cell

pH 引起的霍乱毒素与真核细胞相互作用的变化

• 批准号: 7142387
• 负责人: KENNETH R TETER
• 金额: $ 7.1万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142387
• 关键词: cholera toxin; cytoplasm; proteasome; proteins; toxin; ubiquitin

DESCRIPTION (provided by applicant): The long-term goal of my research program is to understand the molecular mechanisms that allow certain plant and bacterial toxins to cross the endoplasmic reticulum (ER) membrane and enter the cytosol of an intoxicated eukaryotic cell. One such toxin, cholera toxin (CT), is responsible for the life-threatening watery diarrhea of cholera. CT is internalized by target cells and delivered to the ER by retrograde vesicular transport. The catalytic CTA1 polypeptide then crosses the ER membrane, enters the cytosol, and initiates the major toxic effects of CT. The ER-to-cytosol translocation of CTA1 involves the mechanism of ER- associated degradation (ERAD), a quality control system that recognizes misfolded proteins in the ER and exports them to the cytosol for ubiquitination and degradation by the 26S proteasome. The C-terminal hydrophobic region of 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. Our work has shown that the C-terminal region of CTA1 is not required for toxin entry into the cytosol and that the translocated pool of CTA1 is degraded by a temperature-sensitive, ubiquitin-independent proteasomal mechanism. This degradative mechanism may involve the core 20S proteasome, in contrast to the standard route of ubiquitin-dependent degradation by the 26S proteasome. Both the translocation and degradation of CTA1 may be linked to the heat-labile nature of the isolated CTA1 polypeptide. We believe thermal instability in the CTA1 polypeptide generates a partially unfolded conformational state at 37 degrees C that triggers ERAD activity and renders the cytosolic pool of toxin susceptible to degradation by the 20S proteasome. This model will be tested with experimental conditions involving low pH buffers (pH approximately 6.0) that apparently inhibit the thermal denaturation of CTA1. We predict acidic pH will stabilize the structure of CTA1 and thereby inhibit CTA1 translocation/degradation. Our results will form the basis of a new model for toxin-ERAD interactions with applications to toxin pathogenesis and the development of novel anti-toxin biodefense strategies. Cholera toxin will not function if it cannot enter target cells. Acid-induced changes to the structure of cholera toxin may prevent its entry into target cells and would thus generate resistance to the disease cholera.

描述（由申请人提供）：我的研究计划的长期目标是了解某些植物和细菌毒素穿过内质网（ER）膜并进入中毒真核细胞胞浆的分子机制。其中一种毒素是霍乱毒素 (CT)，它会导致危及生命的霍乱水样腹泻。 CT 被靶细胞内化并通过逆行囊泡转运递送至 ER。然后催化 CTA1 多肽穿过 ER 膜，进入细胞质，并引发 CT 的主要毒性作用。 CTA1 的 ER 至胞质溶胶易位涉及 ER 相关降解 (ERAD) 机制，ERAD 是一种质量控制系统，可识别 ER 中错误折叠的蛋白质并将其输出到胞质溶胶，由 26S 蛋白酶体泛素化和降解。 CTA1 的 C 末端疏水区域被认为可触发 ERAD 活性并刺激 CTA1 易位至胞质溶胶；细胞质中的降解可能是可以避免的，因为 CTA1 缺乏作为泛素附着位点的赖氨酸残基。我们的工作表明，CTA1 的 C 末端区域不是毒素进入细胞质所必需的，并且 CTA1 的易位库会被温度敏感、不依赖于泛素的蛋白酶体机制降解。这种降解机制可能涉及核心 20S 蛋白酶体，这与 26S 蛋白酶体泛素依赖性降解的标准途径相反。 CTA1 的易位和降解可能与分离的 CTA1 多肽的热不稳定性质有关。我们认为，CTA1 多肽的热不稳定性会在 37 摄氏度下产生部分展开的构象状态，从而触发 ERAD 活性，并使胞质毒素库易于被 20S 蛋白酶体降解。该模型将在涉及低 pH 缓冲液（pH 值约为 6.0）的实验条件下进行测试，该缓冲液明显抑制 CTA1 的热变性。我们预测酸性 pH 值将稳定 CTA1 的结构，从而抑制 CTA1 易位/降解。我们的结果将构成毒素-ERAD相互作用新模型的基础，并应用于毒素发病机制和开发新型抗毒素生物防御策略。 如果霍乱毒素无法进入靶细胞，它将无法发挥作用。酸引起的霍乱毒素结构变化可能会阻止其进入靶细胞，从而产生对霍乱疾病的抵抗力。