The "Disaggregase" Mechanism of Holotoxin Disassembly by Protein Disulfide Isomerase

霍乱毒素(CT)是一种AB5毒素，由催化A1亚基、A2连接子和细胞结合B组成 五聚体。体内毒素活性需要将CTA1与CTA2/CTB5分离。这发生在 全毒素通过囊泡载体从质膜进入靶的内质网。 手机。CTA1/CTA2二硫键的还原发生在内质网的驻留氧化还原状态，但被还原的 毒素仍然完好无损。CTA1必须从还原的全毒素中的非共价组装中被积极地取代 通过蛋白质二硫键异构酶(PDI)，一种内质网定位的蛋白质具有连锁但不同的伴侣功能 和氧化还原酶。然后，游离的A1亚基从内质网移动到胞浆，在那里它启动了细胞 导致大量水样腹泻的事件，每年导致100-400万人患病和10万人死亡。 这个项目的目标是确定霍乱中一个基本但鲜为人知的事件的分子细节。 中毒：PDI介导的全毒素分解。我们最近的生物物理分析提供了基础 来理解这一过程。我们已经通过同位素编辑的傅里叶变换红外(FTIR)光谱显示 当与CTA1接触时，PDI就会展开。实时全毒素分解试验证明 当PDI被锁定在折叠构象中时，还原的CTA1不会从CTA2/CTB5移位 或者当它的伴侣功能被药物治疗扰乱时。相比之下，PDI的氧化还原酶活性是 CT拆卸不需要。PDI的部分去折叠为CT解离提供了分子基础： 展开的PDI的扩展流体动力学尺寸将推动CT全毒素的两个组分， 从而起到了楔子的作用，将还原的CTA1从其余的毒素中分离出来。这种现象也可能 适用于PDI与其他AB毒素的相互作用，它为已建立的但结构上的 PDI的未知神经保护性伴侣活性：在淀粉样蛋白形成的情况下展开 底物，PDI将扮演一个“解聚酶”的角色，从神经毒性聚合体中取代单个蛋白质。 PDI有一个ABB‘xa’组织，它由两个硫氧还蛋白样催化结构域(a&a‘)组成，由 两个非催化结构域(b&b‘)和一个x连接子。根据初步和公布的数据，我们预测CTA1 与PDI的B结构域的结合通过B‘x结构域发送信号以展开A’结构域。我们 进一步预测PDI与CTA1的一个区域结合，该区域将其a‘结构域定位在CTA1和 CTA2。A‘结构域的展开将在CTA1和CTA2之间形成一个楔形，从而导致释放 从其还原的全毒素中分离出CTA1。对此模型的审问将提供详细的机械洞察 负责CT分解的PDI的独特和以前未被识别的“解聚体”活性，具有 与毒素生物学、神经生物学和分子伴侣的细胞生物学具有潜在的广泛相关性。

Cholera toxin (CT) is an AB5 toxin that consists of a catalytic A1 subunit, an A2 linker, and a cell-binding B pentamer. The separation of CTA1 from CTA2/CTB5 is required for in vivo toxin activity. This occurs after the holotoxin travels by vesicle carriers from the plasma membrane to the endoplasmic reticulum (ER) of a target cell. Reduction of the CTA1/CTA2 disulfide bond occurs at the resident redox state of the ER, but the reduced toxin remains intact. CTA1 must be actively displaced from its non-covalent assembly in the reduced holotoxin by protein disulfide isomerase (PDI), an ER-localized protein with linked but distinct functions as a chaperone and oxidoreductase. The free A1 subunit then moves from the ER to the cytosol where it initiates the cellular events leading to a profuse watery diarrhea that causes 1-4 million illnesses and 100,000 deaths per year. The goal of this project is to define the molecular details of an essential but poorly understood event in cholera intoxication: PDI-mediated holotoxin disassembly. Our recent biophysical analysis has provided the foundation to understand this process. We have shown by isotope-edited Fourier transform infrared (FTIR) spectroscopy that PDI unfolds upon contact with CTA1. A real-time holotoxin disassembly assay demonstrated the displacement of reduced CTA1 from CTA2/CTB5 does not occur when PDI is locked in a folded conformation or when its chaperone function is disrupted by drug treatment. In contrast, the oxidoreductase activity of PDI is not required for CT disassembly. The partial unfolding of PDI provides a molecular basis for CT disassembly: the expanded hydrodynamic size of unfolded PDI would push against two components of the CT holotoxin, thus acting as a wedge to dislodge reduced CTA1 from the rest of the toxin. This phenomenon could also apply to PDI interactions with other AB toxins, and it provides a basis for the established but structurally uncharacterized neuroprotective chaperone activity of PDI: by unfolding in the presence of an amyloid-forming substrate, PDI would act as a “disaggregase” to displace individual proteins from the neurotoxic aggregate. PDI has an abb'xa' organization that consists of two thioredoxin-like catalytic domains (a & a') separated by two non-catalytic domains (b & b') and an x linker. Based on preliminary and published data, we predict CTA1 binding to the b domain of PDI transmits a signal through the b'x domains for unfolding of the a' domain. We further predict that PDI binds to a region of CTA1 that positions its a' domain near the interface of CTA1 and CTA2. Unfolding of the a' domain would then create a wedge between CTA1 and CTA2, leading to the release of CTA1 from its reduced holotoxin. Interrogation of this model will provide detailed mechanistic insight into the unique and previously unrecognized “disaggregase” activity of PDI that is responsible for CT disassembly, with potentially broad relevance to toxin biology, neurobiology, and the cell biology of molecular chaperones.