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亚基和细胞结合B亚基组成。它们被释放到细胞外 但攻击宿主细胞质内的目标。只有在毒素内吞后才能进入细胞质 靶细胞的质膜。部分AB毒素，包括百日咳毒素(PT)和霍乱毒素(CT)， 在毒素分解和A链进入胞浆之前进入内质网(ER)。我们 已经表明，解离的A链在37℃时自发展开，从而触发其输出到胞浆 通过内质网相关降解(ERAD)的机制。大多数ERAD底物被提取到 胞浆通过AAA ATPase p97的作用，但我们已经证明PT(PTS1)的催化亚单位 而CT(CTA1)则需要胞浆伴侣Hsp90进入胞浆。HSP90不是 被认为识别未折叠蛋白质的任何特定特征，但我们确定了RPP结合基序 在PTS1和CTA1的N端有HSP90。最近的研究证明了Grp94的功能作用，即 ER定位的Hsp90家族成员，在PTS1易位和PT中毒中。初步证据进一步 提示Grp94和PTS1之间的紧密联系被内质网定位的亲环素B(CypB)破坏 肽基-脯氨酰顺-反式异构酶(PPI)。我们由此建立的ERAD介导的毒素转位模型表明 PTS1的脯氨酸残基驱动内质网膜两侧的毒素-伴侣相互作用。这部小说 概念将通过细胞培养中的功能丧失研究或毒素结构的体外研究来检验， 内质网微体的毒素-伴侣结合和毒素转位。我们预测CypB及其PPI 从PTS1释放Grp94、PTS1输出到胞浆和PT中毒都需要活性。 PTS1易位和PT中毒都需要Grp94和Hsp90。这两位监护人将 识别PTS1的不同的含有脯氨酸的区域，失去任何一个结合位点都会将PTS1困在内质网中。 Grp94会阻止PTS1的热去折叠，但不会在37℃下重新折叠毒素，而Hsp90会重新折叠 以ATP依赖的方式扰乱PTS1，足以将毒素提取到细胞质中。这部作品 可以通过记录CypB在毒素转运中的作用来确定Pro残基对毒素转运的重要性 PTS1输出，CypB和Grp94之间在处理PTS1过程中的功能链接，以及PTS1如何 易位涉及Hsp90家族成员在内质网两侧的不同功能。 亲环素和Hsp90家族成员可以在复合体中找到并经常作用于相同的底物， 但对于ERAD介导的毒素转位，这些协调功能还没有文献记载。两者都有 蛋白质家族是治疗百日咳的良好药理靶点 PT失活。因此，对PTS1-伴侣相互作用的更深入了解可能会导致更精确的 以及针对PTS1易位所需的特定蛋白的有效治疗。