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

毒素从内质网输出到细胞质的新机制

• 批准号: 8437656
• 负责人: KENNETH R TETER
• 金额: $ 35.2万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8437656
• 关键词: ADP-Ribosylation Factors; Back; Bacterial Toxins; Binding; Binding Sites; Biological Assay; Biophysics; Catalytic Domain; Cell surface; Cells; Cellular biology; Cholera; Cholera Toxin; Client; Complex; Coupling; Cultured Cells; Cytosol; Data; Diarrhea; Disease; Endoplasmic Reticulum; Event; Exhibits; Goals; Humulus; In Vitro; Individual; Intoxication; Isotopes; Life; Link; Mediating; Membrane; Microbiology; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; N-terminal; Pathway interactions; Pertussis Toxin; Pharmaceutical Preparations; Play; Process; Protein C Inhibitor; Proteins; Publishing; Quality Control; RNA Interference; Reporting; Rest; Role; Route; Slide; Small Interfering RNA; Spectroscopy, Fourier Transform Infrared; Structure; Surface Plasmon Resonance; System; Testing; Toxic effect; Toxin; Travel; Vibrio cholerae; Work; base; combinatorial; driving force; in vivo; insight; mutant; novel; p97 ATPase; prevent; translocase

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.

描述（由申请人提供）：由Vibrio Cholerae生产的霍乱毒素（CT）诱导霍乱的生命延伸。 CT作为完整的AB5蛋白毒素从细胞表面传播到醉酒细胞的内质网（ER）。然后，催化A1亚基从其他毒素中分离出来，展开并通过ER“转运”孔以达到其胞质GSA靶标。易位到细胞质的易位，促进了与ER相关降解（ERAD）的质量控制系统。大多数ERAD底物是通过涉及胞质AAA ATPase P97的机制从ER中提取的。但是，p97似乎在CTA1易位中起着最小的作用。该项目的总体目标是定义CTA1易位的分子机制及其在细胞质中的随后激活。我们最近报道说，CTA1传递到胞质溶胶中需要胞质伴侣HSP90。这项工作确立了HSP90在从ER中提取可溶性ERAD底物中的新作用。 HSP90与HOP和HSC70一起工作以重新折叠客户端蛋白。基于我们已发布的初步数据，我们假设HSP90，HOP和HSC70形成了核心“转运酶”复合物，它直接促进CTA1易位到细胞质。我们进一步预测了无序蛋白的HSP90/HSC70辅助重折叠与它们的易位酶功能有关：通过将易位易位与重新折叠，HSP90和HSC70耦合，可以防止（RE）折叠的CTA1蛋白滑回易位孔。此过程将为CTA1易位提供驱动力。我们还预测，CTA1的HSP90/HSC70辅助重折叠将使胞质毒素置于可以通过宿主ADP-ribosylation因子（ARFS）激活的构象中。最后，我们预测HSP90/HOP/HSC70复合物还与其他毒素和使用P97无关的易位途径的内源性ERAD底物的ER到胞质出口有关。在此应用程序中，我们将（i）定义转运酶复合物的核心成分及其在CTA1上的结合位点； （ii）证明了转运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）确定了易位酶配合物的毒素和内源性脱落底物的范围更广泛。我们对转运酶复合物的结构 /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。该项目将为CTA1易位过程不理的过程提供分子洞察力，并将定义ER到胞质溶质出口ERAD底物的新途径。 公共卫生相关性：霍乱毒素和其他细菌毒素必须进入我们身体的细胞，以引起疾病。如果我们了解此事件的分子细节，我们可以开发靶向疗法，以阻止毒素进入宿主细胞，从而阻止毒素介导的疾病。