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AB5毒素，由催化A1亚基，A2接头和细胞结合B组成 五角星。体内毒素活性需要从CTA2/CTB5中分离CTA1。这发生在 从囊泡载体从质膜到靶的内质网（ER）的囊泡载体传播 细胞。 CTA1/CTA2二硫键的降低发生在ER的氧化还原状态，但减少了 毒素保持完整。 CTA1必须在减少的Holotoxin中积极从其非共价组件中移位 通过蛋白质二硫化物异构酶（PDI），一种具有链接但独特功能作为链酮的ER定位蛋白 和氧化还原酶。然后，自由A1亚基从ER移动到胞质溶胶，在该胞浆中启动细胞 导致大量水状腹泻的事件每年造成1-4百万疾病和100,000人死亡。 该项目的目的是定义霍乱中必不可少但不理解的事件的分子细节 中毒：PDI介导的Holotoxin拆卸。我们最近的生物物理分析为基础提供了 了解这个过程。我们已经通过同位素编辑的傅立叶变换感染（FTIR）光谱显示 PDI在与CTA1接触后展开。实时的全毒素拆卸测定法证明了 当PDI锁定在折叠构象中时，不会发生减少CTA1/CTB5的CTA1的位移 或当其伴侣功能被药物治疗破坏时。相反，PDI的氧化还原酶活性为 CT拆卸不需要。 PDI的部分展开为CT拆卸提供了分子基础： 展开的PDI的扩大流体动力大小将推向CT Holotoxin的两个成分，即 这种现象也可以充当楔形物，使从其他毒素中将CTA1降低。 适用于与其他AB毒素的PDI相互作用，它为已建立但结构上的基础提供了基础 PDI的未表征神经保护伴侣活性：通过在淀粉样蛋白形成的情况下展开 底物PDI将充当“分解酶”，以使单个蛋白质从神经毒性骨料中取代。 PDI具有一个ABB'XA'组织，该组织由两个硫氧还蛋白样催化域（A＆A'）组成 两个非催化域（B＆B'）和一个X连接器。根据初步和已发布的数据，我们预测CTA1 与PDI的B域结合，通过B'X域传输A信号，以展开A'域。我们 进一步预测PDI与CTA1区域结合，该区域将其A'域定位在CTA1和CTA1界面附近 CTA2。然后，A'域的展开将在CTA1和CTA2之间产生一个楔子，从而导致释放 CTA1的降低全毒素。对该模型的审问将提供详细的机械洞察力 PDI的独特且以前未被认可的“分类酶”活性，负责CT拆卸， 与毒素生物学，神经生物学和分子链烷的细胞生物学有可能广泛的相关性。

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