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

AB毒素由催化A亚单位和细胞结合B亚单位组成。它们被释放到细胞外 空间，但攻击宿主胞质溶胶内的目标。进入胞质溶胶只发生在毒素内吞后， 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）， 在毒素分解和A链进入胞质溶胶之前行进到内质网（ER）。我们 已经显示解离的A链在37°C自发地展开，这触发其输出到胞质溶胶 通过ER相关降解（ERAD）机制。大多数ERAD底物被提取到 通过AAA ATP酶p97的作用，细胞质中的PT（PTS 1）的催化亚基， 而CT（CTA 1）则需要胞质分子伴侣Hsp 90进入胞质。HSP 90不是 我们认为它可以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， Hsp 90位于PTS 1和CTA 1的N末端。最近的研究已经证明了Grp 94的功能作用， ER定位的Hsp 90家族成员，在PTS 1易位和PT中毒中。初步证据进一步 提示Grp 94和PTS 1之间的紧密联系被亲环素B（CypB）破坏，CypB是一种ER定位的蛋白， 肽基脯氨酰顺反异构酶（PPI）。我们得到的ERAD介导的毒素易位模型建议 PTS 1脯氨酸残基驱动内质网膜两侧的毒素-伴侣相互作用。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-伴侣结合，以及来自ER衍生微粒体的毒素易位。我们预测CypB及其PPI 从PTS 1释放Grp 94、PTS 1输出到胞质溶胶和PT中毒将需要活性。 PTS 1易位和PT中毒都需要Grp 94和Hsp 90。这两位监护人 识别PTS 1的不同含脯氨酸区域，并且任一结合位点的缺失将PTS 1捕获在ER中。 Grp 94将阻止PTS 1的热解折叠，但在37°C下不会使毒素再折叠，而Hsp 90将使毒素再折叠。 以ATP依赖的方式将PTS 1无序化，这足以将毒素提取到胞质溶胶中。这项工作 可以通过记录CypB在以下过程中的作用来确定脯氨酸残基对毒素易位的重要性： PTS 1导出，CypB和Grp 94在PTS 1处理中的功能链接，以及PTS 1如何 易位涉及ER膜两侧Hsp 90家族成员的不同功能。 亲环蛋白和Hsp 90家族成员可以在复合物中发现，并且经常作用于相同的底物， 但是这些协调功能对于ERAD介导的毒素易位还没有记载。两 蛋白质家族代表了有希望的药理学靶点，用于通过 PT失活。因此，对PTS 1-分子伴侣相互作用的更深入理解可以导致更精确的 以及针对PTS 1易位所需的特异性蛋白质的有效治疗剂。

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.