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

基本信息

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
项目类别：
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

项目摘要

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 AB5 毒素，由催化 A1 亚基、A2 连接体和细胞结合 B 组成 CTA1 与 CTA2/CTB5 的分离是体内毒素活性所必需的。全毒素通过囊泡载体从质膜传播到目标的内质网 (ER) CTA1/CTA2 二硫键的还原发生在 ER 的常驻氧化还原态，但还原的还原态发生。毒素保持完整。 CTA1 必须主动从还原的全毒素中的非共价组装中转移出来。由蛋白质二硫键异构酶 (PDI) 产生，这是一种内质网定位的蛋白质，具有作为伴侣的相关但不同的功能然后游离的 A1 亚基从 ER 移动到细胞质，在那里启动细胞。导致严重水样腹泻的事件每年导致 1-400 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 介导的全毒素分解提供了基础。为了理解这个过程，我们通过同位素编辑傅里叶变换红外（FTIR）光谱来展示。 PDI 在与 CTA1 接触时展开。实时全毒素分解试验证明了当 PDI 锁定在折叠构象时，不会发生还原的 CTA1 从 CTA2/CTB5 的位移或者当其分子伴侣功能被药物治疗破坏时，PDI 的氧化还原酶活性会降低。 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 的扩大的流体动力学尺寸将推动 CT 全毒素的两个成分，从而充当楔子，将减少的 CTA1 从毒素的其余部分中移出。这种现象也可能发生。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 未表征的神经保护伴侣活性：在淀粉样蛋白形成的情况下展开底物中，PDI 将充当“解聚酶”，从神经毒性聚集体中取代单个蛋白质。 PDI 具有 abb'xa' 组织，由两个硫氧还蛋白样催化结构域 (a 和 a') 组成，中间由两个非催化结构域 (b & b') 和一个 x 连接子根据初步和已发表的数据，我们预测 CTA1。与 PDI 的 b 结构域的结合通过 b'x 结构域传递信号以展开 a' 结构域。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 界面附近，并且 CTA2 的展开将在 CTA1 和 CTA2 之间创建一个楔子，从而导致释放。对该模型的研究将提供对 CTA1 减少的全毒素的详细机制了解。 PDI 独特且以前未被识别的“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广泛的相关性。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

蛋白质二硫键异构酶对大肠杆菌不耐热肠毒素的全毒素分解效率低于霍乱毒素。

DOI：
发表时间：
2022-01-07
期刊：
影响因子：
4.6
作者：
Serrano, Albert;Guyette, Jessica L;Heim, Joel B;Taylor, Michael;Cherubin, Patrick;Krengel, Ute;Teter, Ken;Tatulian, Suren A
通讯作者：
Tatulian, Suren A

Stability and Conformational Resilience of Protein Disulfide Isomerase.

蛋白质二硫键异构酶的稳定性和构象弹性。

DOI：
10.1021/acs.biochem.9b00405
发表时间：
2019-08-08
期刊：
Biochemistry
影响因子：
2.9
作者：
J. Guyette;B. Evangelista;S. Tatulian;K. Teter
通讯作者：
K. Teter

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

霍乱毒素对细胞信号传导和宿主细胞生物学的操纵。

DOI：
发表时间：
2022-12
期刊：
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 Perdeuteration.

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全氘化。

DOI：
发表时间：
2024-03-05
期刊：
影响因子：
2.9
作者：
Mojica, Natalia;Kersten, Flore;Montserrat;Huhn Iii, G Robb;Tislevoll, Abelone M;Cordara, Gabriele;Teter, Ken;Krengel, Ute
通讯作者：
Krengel, Ute

Protein disulfide isomerase does not act as an unfoldase in the disassembly of cholera toxin.

蛋白质二硫键异构酶在霍乱毒素的分解中不充当解折叠酶。

DOI：
发表时间：
2018
期刊：
影响因子：
4
作者：
Cherubin, Patrick;Guyette, Jessica;Taylor, Michael;O'Donnell, Morgan;Herndon, Laura;Burress, Helen;Riad, Aladdin;Tatulian, Suren A;Teter, Ken
通讯作者：
Teter, Ken