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

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，霍乱毒素(CT)，是威胁生命的霍乱水样腹泻的罪魁祸首。CT被靶细胞内化，并通过逆行囊泡运输传递到内质网。催化的CTA1多肽随后穿过ER膜，进入胞浆，启动CT的主要毒性作用。CTA1从内质网到胞浆的易位涉及内质网相关降解(ERAD)的机制，ERAD是一种质量控制系统，它识别内质网中错误折叠的蛋白质，并将它们输出到胞浆中，供26S蛋白酶体泛素化和降解。CTA1的C-末端疏水区被认为可以触发ERAD活性，并刺激CTA1移位到胞浆；胞浆中的降解可能是因为CTA1缺乏作为泛素结合位点的赖氨酸残基。我们的工作表明，CTA1的C-末端区域并不是毒素进入细胞质所必需的，而且CTA1的转位池是通过一种温度敏感的、泛素不依赖的蛋白酶体机制降解的。这种降解机制可能涉及核心20S蛋白酶体，而不是26S蛋白酶体依赖泛素的标准降解途径。CTA1的易位和降解都可能与分离的CTA1多肽的热稳定性有关。我们认为，CTA1多肽的热不稳定会在37℃产生部分展开的构象状态，从而触发ERAD活性，并使胞液中的毒素容易被20S蛋白酶体降解。该模型将在低pH缓冲液(pH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将稳定CTA1的结构，从而抑制CTA1的移位/降解。我们的结果将形成毒素-ERAD相互作用的新模型，并应用于毒素的发病机制和新的抗毒素生物防御策略的开发。 如果霍乱毒素不能进入目标细胞，它就不会发挥作用。酸诱导的霍乱毒素结构变化可能会阻止其进入目标细胞，从而产生对霍乱疾病的抵抗力。

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.