A Novel Mechanism for Toxin Export from the Endoplasmic Reticulum to the Cytosol

描述（由申请人提供）：霍乱毒素（CT），由霍乱弧菌产生，引起危及生命的霍乱腹泻。CT作为一个完整的AB5蛋白毒素从细胞表面传播到中毒细胞的内质网（ER）。催化A1亚基随后与毒素的其余部分分离，展开，并通过内质网“转座子”孔到达其胞质Gsa目标。er相关降解（ERAD）的质量控制系统促进了转运到细胞质中。大多数ERAD底物是通过细胞质AAA atp酶p97的机制从内质网中提取的。然而，p97似乎在CTA1易位中起很小的作用。该项目的总体目标是确定CTA1易位及其随后在细胞质溶胶中的激活的分子机制。我们最近报道了细胞质伴侣Hsp90是CTA1进入细胞质所必需的。这项工作确定了Hsp90在从内质网中提取可溶性ERAD底物中的新作用。Hsp90与Hop和Hsc70共同作用，重新折叠客户蛋白。根据我们发表的和初步的数据，我们假设Hsp90、Hop和Hsc70形成一个核心的“转位酶”复合物，直接促进CTA1易位到细胞质。我们进一步预测，Hsp90/Hsc70辅助的紊乱蛋白的重折叠与其易位酶功能有关：通过易位与重折叠的耦合，Hsp90和Hsc70将阻止（重新）折叠的CTA1蛋白滑入易位孔。这一过程将为CTA1易位提供驱动力。我们还预测，Hsp90/ hsc70辅助的CTA1重折叠将使胞质毒素处于可被宿主adp -核糖基化因子（ARFs）激活的构象中。最后，我们预测Hsp90/Hop/Hsc70复合体还参与其他毒素和内源性ERAD底物的ER-to-cytosol输出，这些底物利用不依赖p97的易位途径。在本应用中，我们将(i)定义转位酶复合物的核心组分及其在CTA1上的结合位点；（ii）证明转位酶复合体的重折叠功能，并检查其对arf刺激的毒素活性的潜在影响；（iii）为转位酶复合体识别更广泛的毒素和内源性ERAD底物。我们对转位酶复合体的结构/功能分析将采用分子微生物学、细胞生物学和生物物理学的独特结合。该项目将为目前尚不清楚的CTA1易位过程提供分子视角，并将为ERAD底物从er到细胞质出口确定一条新的途径。

DESCRIPTION (provided by applicant): Cholera toxin (CT), produced by Vibrio cholerae, induces the life-threatening diarrhea of cholera. CT travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic A1 subunit then dissociates from the rest of the toxin, unfolds, and passes through an ER "translocon" pore to reach its cytosolic Gsa target. Translocation into the cytosol is facilitated y the quality control system of ER-associated degradation (ERAD). Most ERAD substrates are extracted from the ER through a mechanism involving the cytosolic AAA ATPase p97. However, p97 appears to play a minimal role in CTA1 translocation. The overall goal of this project is to define the molecular mechanism for CTA1 translocation and its subsequent activation in the cytosol. We recently reported that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. This work established a new role for Hsp90 in the extraction of a soluble ERAD substrate from the ER. Hsp90 works with Hop and Hsc70 to refold client proteins. Based upon our published and preliminary data, we hypothesize Hsp90, Hop, and Hsc70 form a core "translocase" complex that directly facilitates CTA1 translocation to the cytosol. We further predict the Hsp90/Hsc70-assisted refolding of disordered proteins is linked to their translocase function: by coupling translocation with refolding, Hsp90 and Hsc70 would prevent the (re)folded CTA1 protein from sliding back into the translocon pore. This process would provide the driving force for CTA1 translocation. We also predict the Hsp90/Hsc70-assisted refolding of CTA1 will place the cytosolic toxin in a conformation that can be activated by host ADP-ribosylation factors (ARFs). Finally, we predict the Hsp90/Hop/Hsc70 complex is also involved with the ER-to-cytosol export of other toxins and endogenous ERAD substrates that utilize a p97-independent translocation pathway. In this application, we will (i) define the core components of the translocase complex and their binding sites on CTA1; (ii) demonstrate the refolding function of the translocase complex and examine its potential effect on ARF-stimulated toxin activity; and (iii) identify a broader range of toxins and endogenous ERAD substrates for the translocase complex. Our structure / function analysis of the translocase complex will employ a unique combination of molecular microbiology, cell biology, and biophysics. This project will provide molecular insight into the poorly understood process of CTA1 translocation and will define a new route for the ER-to-cytosol export of ERAD substrates. PUBLIC HEALTH RELEVANCE: Cholera toxin and other bacterial toxins must enter the cells of our body in order to cause disease. If we understand the molecular details of this event, we can develop targeted therapies to block toxin entry into the host cell and, thus, toxin-mediated disease.