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

蛋白质二硫键异构酶分解全毒素的“解聚酶”机制

• 批准号: 10326796
• 负责人: KENNETH R TETER
• 金额: $ 36.51万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-03 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326796
• 关键词: Amyloid; Amyloid Fibrils; Binding; Binding Proteins; Binding Sites; Biological Assay; Biology; C-terminal; Catalytic Domain; Cell Line; Cell membrane; Cell surface; Cells; Cellular biology; Cessation of life; Cholera; Cholera Toxin; Communicable Diseases; Cytosol; Data; Diarrhea; Disease; Endoplasmic Reticulum; Event; Exhibits; Foundations; GTP-Binding Protein alpha Subunits, Gs; Goals; Health; Human; Hybrids; Hydrogen; Individual; Intoxication; Isotopes; Length; Link; Mass Spectrum Analysis; Mediating; Membrane; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; NMR Spectroscopy; Nerve Degeneration; Neurobiology; Neurodegenerative Disorders; Oxidation-Reduction; Oxidoreductase; Pharmacotherapy; Play; Positioning Attribute; Process; Property; Protein Disulfide Isomerase; Proteins; Publishing; Rest; Role; Signal Transduction; Site; Spectroscopy, Fourier Transform Infrared; TXN gene; Tertiary Protein Structure; Testing; Therapeutic Intervention; Time; Toxin; Travel; Vesicle; Work; X-Ray Crystallography; base; biophysical analysis; disulfide bond; holotoxins; in vivo; insight; mutant; neurotoxic; novel; novel therapeutic intervention; protein function; response; transmission process

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.

霍乱毒素（CT）是一种AB 5毒素，由催化A1亚基、A2接头和细胞结合B组成 五聚体。CTA 1与CTA 2/CTB 5的分离是体内毒素活性所必需的。这发生在 全毒素通过囊泡载体从质膜行进到靶标的内质网（ER cell. CTA 1/CTA 2二硫键的还原发生在ER的驻留氧化还原态，但还原的CTA 1/CTA 2二硫键在ER的驻留氧化还原态发生。 毒素仍然完好无损CTA 1必须从其在还原全毒素中的非共价组装中被主动置换 蛋白质二硫键异构酶（PDI），一种ER定位的蛋白质，具有连接但不同的伴侣功能 和氧化还原酶。然后游离的A1亚基从ER移动到胞质溶胶，在那里它启动细胞内的 导致大量水样腹泻的事件，每年导致1-4百万人患病和10万人死亡。 该项目的目标是确定霍乱中一个重要但知之甚少的事件的分子细节 中毒：PDI介导的全毒素分解。我们最近的生物物理学分析为 来理解这个过程。我们已经通过同位素编辑的傅里叶变换红外（FTIR）光谱表明， PDI在与CTA 1接触时会展开。实时全毒素分解试验表明， 当PDI锁定在折叠构象时，不发生还原的CTA 1从CTA 2/CTB 5的置换 或者当其伴侣蛋白功能被药物治疗破坏时。相比之下，PDI的氧化还原酶活性是 CT拆卸不需要。PDI的部分解折叠为CT分解提供了分子基础： 未折叠的PDI的扩大的流体动力学尺寸将推动CT全毒素的两种组分， 从而作为楔子将还原的CTA 1从毒素的其余部分中除去。这种现象也可能 适用于PDI与其他AB毒素的相互作用，它为建立但结构上 PDI的未表征的神经保护性伴侣活性：通过在淀粉样蛋白形成蛋白存在下解折叠 在神经毒性底物中，PDI将充当“解聚酶”以从神经毒性聚集体中置换单个蛋白质。 PDI具有abb'xa'结构，其由两个硫氧还蛋白样催化结构域（a & a '）组成，所述催化结构域被 两个非催化结构域（B & b '）和一个X接头。根据初步和已发表的数据，我们预测CTA 1 与PDI的B结构域的结合通过b ′ x结构域传递信号，用于a ′结构域的解折叠。我们 进一步预测PDI结合CTA 1的一个区域，该区域将其α ′结构域定位在CTA 1的界面附近， CTA 2。然后，a'结构域的解折叠将在CTA 1和CTA 2之间产生一个楔形，导致释放。 从其还原的全毒素中提取这个模型的询问将提供详细的机械洞察力， PDI的独特且先前未被识别的“解聚酶”活性负责CT分解， 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。

项目成果

Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin.

• DOI: 10.1038/s41598-021-03939-9
• 发表时间: 2022-01-07
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Serrano A;Guyette JL;Heim JB;Taylor M;Cherubin P;Krengel U;Teter K;Tatulian SA
• 通讯作者: Tatulian SA

The manipulation of cell signaling and host cell biology by cholera toxin.

• DOI: 10.1016/j.cellsig.2022.110489
• 发表时间: 2022-12
• 期刊: CELLULAR SIGNALLING
• 影响因子: 4.8
• 作者: White, Christopher;Bader, Carly;Teter, Ken
• 通讯作者: Teter, Ken

Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration.

使用纳特里根弧菌高产生产具有挑战性的表达靶标并进行蛋白质氘化。

• DOI: 10.1101/2023.11.03.565449
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Mojica,Natalia;Kersten,Flore;Montserrat-Canals,Mateu;Huhn3rd,GRobb;Tislevoll,AbeloneM;Cordara,Gabriele;Teter,Ken;Krengel,Ute
• 通讯作者: Krengel,Ute