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

描述(申请人提供)：霍乱毒素(CT)，由霍乱弧菌产生，可引起危及生命的霍乱腹泻。CT作为完整的AB5蛋白毒素从细胞表面进入中毒细胞的内质网(ER)。然后，催化A1亚单位与毒素的其余部分解离，展开，并通过内质网“转位”孔到达其胞浆GSA靶点。内质网相关降解的质量控制系统(ERAD)促进了转运到细胞质中。大多数ERAD底物是通过胞质AAA ATPase p97的一种机制从内质网中提取的。然而，p97在CTA1易位中的作用似乎很小。该项目的总体目标是确定CTA1易位及其在胞浆中随后激活的分子机制。我们最近报道，CTA1进入胞浆需要胞质伴侣Hsp90。这项工作确立了Hsp90在从内质网中提取可溶性ERAD底物方面的新作用。HSP90与Hop和Hsc70一起作用于客户蛋白的重折叠。根据我们已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”复合体，它直接促进CTA1转位到细胞质。我们进一步预测，Hsp90/Hsc70辅助的无序蛋白质的复性与其转位酶功能有关：通过将易位与复性结合起来，Hsp90和Hsc70将防止(重新)折叠的CTA1蛋白滑回转位子孔。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重折叠将把胞质毒素置于一种可被宿主ADP-核糖化因子(ARF)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底物的胞浆输出，这些底物利用p97独立的易位途径。在本应用中，我们将(I)定义转位酶复合体的核心成分及其在CTA1上的结合位置；(Ii)展示转位酶复合体的重折叠功能并检测其对ARF刺激的毒素活性的潜在影响；以及(Iii)确定转位酶复合体的更广泛的毒素和内源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组合。该项目将提供对CTA1移位鲜为人知的过程的分子洞察，并将为ERAD底物从内质网到胞浆的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.