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作为完整的AB 5蛋白毒素从细胞表面行进到中毒细胞的内质网（ER）。然后，催化A1亚基与毒素的其余部分解离，展开，并通过ER“translocon”孔到达其胞质Gsa靶标。通过ER相关降解质量控制系统（ERAD）促进转运到胞质溶胶中。大多数ERAD底物通过涉及胞质AAA ATP酶p97的机制从ER中提取。然而，p97似乎在CTA 1易位中发挥最小的作用。该项目的总体目标是确定CTA 1易位及其随后在胞质溶胶中激活的分子机制。我们最近报道，CTA 1进入胞质溶胶通道所需的胞质伴侣Hsp 90。这项工作建立了一个新的作用，热休克蛋白90在提取可溶性ERAD基板从ER。Hsp 90与Hop和Hsc 70一起重折叠客户蛋白。基于我们发表的和初步的数据，我们假设热休克蛋白90，啤酒花，和Hsc 70形成一个核心的“易位酶”复合物，直接促进CTA 1易位到胞质溶胶。我们进一步预测，Hsp 90/Hsc 70辅助的无序蛋白质的重折叠与其易位酶功能有关：通过将易位与重折叠耦合，Hsp 90和Hsc 70将阻止（重）折叠的CTA 1蛋白滑回易位子孔。这一过程将为CTA 1易位提供驱动力。我们还预测，热休克蛋白90/Hsc 70辅助的CTA 1的重折叠将放置在一个构象，可以激活宿主ADP-核糖基化因子（ARF）的胞质毒素。最后，我们预测Hsp 90/Hop/Hsc 70复合物也参与了其他毒素和内源性ERAD底物的ER到胞质溶胶的出口，这些毒素和内源性ERAD底物利用p97非依赖性易位途径。在本申请中，我们将（i）定义易位酶复合物的核心组分及其在CTA 1上的结合位点;（ii）证明易位酶复合物的重折叠功能，并检查其对ARF刺激的毒素活性的潜在影响;（iii）确定易位酶复合物的更广泛的毒素和内源性ERAD底物。我们的结构/功能分析的易位酶复合物将采用分子微生物学，细胞生物学和生物物理学的独特组合。该项目将提供分子深入了解CTA 1易位的过程，并将确定一个新的路线ER到胞质溶胶出口ERAD基板。

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.