Proline residues are a key determinant for toxin entry into the host cytosol

脯氨酸残基是毒素进入宿主细胞质的关键决定因素

• 批准号: 10740431
• 负责人: KENNETH R TETER
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740431
• 关键词: ADP ribosylation; Acceleration; Affect; Affinity; Amino Acid Sequence; Binding; Binding Sites; Biological Assay; Catalytic Domain; Cell Culture Techniques; Cell membrane; Cells; Cholera Toxin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Couples; Cyclophilin A; Cyclophilins; Cyclosporine; Cytosol; Degradation Pathway; Disease; Dissociation; Endocytosis; Endoplasmic Reticulum; Event; Extracellular Space; Face; Family; Family member; Goals; Heat-Shock Proteins 90; In Vitro; Intoxication; Isomerism; Isotopes; Knowledge; Lead; Length; Link; Mediating; Membrane; Microsomes; Modeling; Molecular Chaperones; N-terminal; Nature; Patients; Peptide Library; Peptidylprolyl Isomerase; Pertussis; Pertussis Toxin; Pilot Projects; Point Mutation; Process; Proline; Protein Family; Proteins; RNA Interference; Reporting; Role; Side; Spectroscopy, Fourier Transform Infrared; Structure; Toxin; Travel; Work; cyclophilin B; inhibitor; loss of function; member; novel; p97 ATPase; pharmacologic; prevent; protein folding; retrograde transport; therapeutically effective

AB toxins consist of a catalytic A subunit and a cell-binding B subunit. They are released into the extracellular space but attack targets within the host cytosol. Entry into the cytosol only occurs after toxin endocytosis from the plasma membrane of the target cell. Some AB toxins, including pertussis toxin (PT) and cholera toxin (CT), travel to the endoplasmic reticulum (ER) before toxin disassembly and A chain passage into the cytosol. We have shown the dissociated A chain spontaneously unfolds at 37°C, which triggers its export to the cytosol through the mechanism of ER-associated degradation (ERAD). Most ERAD substrates are extracted to the cytosol through the action of the AAA ATPase p97, but we have shown that the catalytic subunits of PT (PTS1) and CT (CTA1) instead require the cytosolic chaperone Hsp90 for passage into the cytosol. Hsp90 was not thought to recognize any specific feature of an unfolded protein, but we identified an RPP binding motif for Hsp90 at the N-terminus of PTS1 and CTA1. Recent studies have documented a functional role for Grp94, the ER-localized Hsp90 family member, in PTS1 translocation and PT intoxication. Preliminary evidence further suggests the tight association between Grp94 and PTS1 is disrupted by cyclophilin B (CypB), an ER-localized peptidyl-prolyl cis-trans isomerase (PPI). Our resulting model for ERAD-mediated toxin translocation proposes that PTS1 proline residues drive toxin-chaperone interactions on both sides of the ER membrane. This novel concept will be examined using either loss-of-function studies in cell culture or in vitro studies on toxin structure, toxin-chaperone binding, and toxin translocation from ER-derived microsomes. We predict CypB and its PPI activity will be required for the release of Grp94 from PTS1, PTS1 export to the cytosol, and PT intoxication. Both Grp94 and Hsp90 will be required for PTS1 translocation and PT intoxication. These two chaperones will recognize distinct proline-containing regions of PTS1, and loss of either binding site will trap PTS1 in the ER. Grp94 will prevent the thermal unfolding of PTS1 but will not refold the toxin at 37°C, whereas Hsp90 will refold disordered PTS1 in an ATP-dependent manner that is sufficient for toxin extraction to the cytosol. This work could establish the importance of proline residues for toxin translocation by documenting a role for CypB in PTS1 export, a functional link between CypB and Grp94 in the processing of PTS1, and how PTS1 translocation involves the distinct functions of Hsp90 family members at both sides of the ER membrane. Cyclophilin and Hsp90 family members can be found in a complex and frequently act on the same substrates, but these coordinated functions have not yet been documented for ERAD-mediated toxin translocation. Both protein families represent promising pharmacological targets for treating whooping cough through the inactivation of PT. A deeper understanding of PTS1-chaperone interactions could thus lead to more precise and effective therapeutics against the specific proteins required for PTS1 translocation.

AB毒素由催化A亚基和一个细胞结合B亚基组成。它们被释放到细胞外 空间但攻击目标是宿主细胞质中的目标。进入细胞质仅在毒素内吞作用后才发生 靶细胞的质膜。一些AB毒素，包括百日咳毒素（PT）和霍乱毒素（CT）， 在毒素拆卸之前和进入细胞质的链条通道之前，前往内质网（ER）。我们 已经显示了在37°C下发起的分离的链条，从而触发其出口到细胞质 通过与ER相关降解的机制（ERAD）。大多数ERAD基板被提取到 通过AAA ATPase P97的作用的胞质溶胶，但我们已经证明了PT的催化亚基（PTS1） 而CT（CTA1）则需要胞质伴侣HSP90进入胞质溶胶。 HSP90不是 被认为可以识别展开蛋白质的任何特定特征，但是我们确定了RPP结合基序 PTS1和CTA1的N端的HSP90。最近的研究记录了GRP94的功能作用 pTS1易位和PT中学中的ER定位的HSP90家庭成员。进一步的证据 表明GRP94和PTS1之间的紧密关联受环蛋白B（CYPB）的破坏，这是一种ER定位的 肽基 - 丙酰基传播异构酶（PPI）。我们生成的ERAD介导的毒素易位建议的模型 PTS1脯氨酸恢复了ER膜两侧的毒素 - 链蛋白相互作用。这本小说 将使用细胞培养中的功能丧失研究或毒素结构的体外研究来检查概念， 毒素 - 链蛋白结合和来自ER衍生的微粒体的毒素易位。我们预测CYPB及其PPI 从PTS1，PTS1导出到细胞质和PT中毒将需要活性。 PTS1易位和PT Inxation都需要GRP94和HSP90。这两个伴侣将 公认的PTS1的不同含脯氨酸的区域，两种结合位点的丢失将捕获ER中的PTS1。 GRP94将防止PTS1的热展开，但不会在37°C下重折叠毒素，而HSP90将重新折叠 以ATP依赖性方式的PTS1无序的PTS1足以使毒素提取到细胞质。这项工作 可以通过记录CYPB在 PTS1导出，PTS1处理中CYPB和GRP94之间的功能链接，以及PTS1的方式 易位涉及HSP90家族成员在ER膜两侧的不同功能。 可环蛋白和HSP90家庭成员可以在复杂的同一基材上作用， 但是，这些协调的功能尚未记录在ERAD介导的毒素易位。两个都 代表有前途的药物靶标的蛋白质家族通过 Pt的失活。因此，对PTS1-Chaperone相互作用的更深入了解可能会导致更精确 以及针对PTS1易位所需的特定蛋白质的有效治疗。