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

描述（由申请人提供）：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质溶胶的分子机制。其中一种毒素，霍乱毒素（CT），是造成危及生命的霍乱水样腹泻的原因。CT被靶细胞内化并通过逆行囊泡运输递送到ER。然后催化性CTA 1多肽穿过ER膜，进入胞质溶胶，并启动CT的主要毒性作用。CTA 1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，其识别ER中的错误折叠蛋白并将其输出至胞质溶胶以用于26 S蛋白酶体的泛素化和降解。CTA 1的C-末端疏水区被认为触发ERAD活性并刺激CTA 1易位至胞质溶胶;由于CTA 1缺乏作为泛素附着位点的赖氨酸残基，因此可能避免了胞质溶胶中的降解。我们的工作表明，CTA 1的C-末端区域是不需要的毒素进入胞质溶胶和CTA 1的易位池是由温度敏感的，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20 S蛋白酶体，与26 S蛋白酶体的泛素依赖性降解的标准途径相反。CTA 1的易位和降解可能与分离的CTA 1多肽的热不稳定性质有关。我们认为CTA 1多肽的热不稳定性在37 ℃时产生部分未折叠的构象状态，其触发ERAD活性并使毒素的胞质池易于被20 S蛋白酶体降解。该模型将在涉及明显抑制CTA 1热变性的低pH缓冲液（pH约6.0）的实验条件下进行测试。我们预测酸性pH将稳定CTA 1的结构，从而抑制CTA 1的易位/降解。我们的研究结果将形成一个新的模型的基础上毒素ERAD相互作用的应用毒素发病机制和新的抗毒素生物防御策略的发展。 霍乱毒素如果不能进入靶细胞就不能发挥作用。酸诱导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。

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.