Receptor disulfide allosteric regulation of anthrax toxin action

炭疽毒素作用的受体二硫键变构调节

• 批准号: 8255487
• 负责人: Jianjun Sun
• 金额: $ 24.23万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-08 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255487
• 关键词: Affinity; Allosteric Regulation; Anthrax disease; Antigens; Bacterial Toxins; Binding; Biochemistry; Cell Surface Receptors; Cell membrane; Cells; Collaborations; Cytosol; Data; Development; Distal; Disulfides; Electron Microscopy; Endocytosis Pathway; Endosomes; Ensure; Enzymes; Escherichia coli; Fluorescence; Fluorescence Spectroscopy; Glean; Goals; Immune system; Immunoglobulin Domain; Liposomes; Literature; Mediating; Membrane; Metabolism; Molecular; Molecular Conformation; Oxidation-Reduction; Pathogenesis; Pattern; Play; Positioning Attribute; Protein Disulfide Isomerase; Proteins; Proteomics; Protocols documentation; Publishing; Recombinants; Regulation; Research; Role; Series; Structure; Surface; Symptoms; System; Techniques; Testing; Therapeutic; Toxin; Travel; Von Willebrand Factor A Domain; X-Ray Crystallography; anthrax lethal factor; anthrax toxin; anthrax toxin receptors; antigen binding; base; conformational conversion; disulfide bond; disulfide bond reduction; edema factor; experience; extracellular; insight; liquid chromatography mass spectrometry; membrane model; novel; novel strategies; novel therapeutics; particle; pathogen; pathogenic bacteria; public health relevance; receptor; receptor mediated endocytosis; research study; structural biology

DESCRIPTION (provided by applicant): Interaction between bacterial toxins and cellular surface receptors is an important component of host-pathogen interaction. Anthrax toxin protective antigen (PA) binds to cell surface receptor, enters cell through receptor mediated endocytosis, and forms a pore on the endosomal membrane that translocates toxic enzymes into the cytosol of host cell. Anthrax toxin receptors play an essential role in anthrax toxin action by providing the toxin with a high-affinity binding anchor on the cell membrane and a path of entry into the host cell. In this proposal, our preliminary study on one of the two known anthrax toxin receptors, ANTXR2, has suggested a new mechanism by which the receptor regulates anthrax toxin action. We have found that the disulfide bonds in the immunoglobulin-like domain (R2-Ig) within the ANTXR2 extracellular portion are required for anthrax toxin action. Disruption of these disulfide bonds inhibited PA pore formation an d translocation of toxic enzymes across the membrane into the host cell. Moreover, disulfide reduction in R2-Ig induced a significant conformational change in the distal PA-binding domain of the receptor, namely von Willebrand Factor A domain (R2-VWA). Thus, our hypothesis is that the disulfide bonds in R2-Ig regulate anthrax toxin action through allosteric effects on the conformation of R2-VWA, hence interfering with PA prepore-to-pore conversion and/or membrane insertion. In this proposal, we will investigate the mechanism by which disulfide reduction in the R2-Ig domain inhibits anthrax toxin action using fluorescence techniques in a liposomal model membrane system as well as other means. Further, we will determine the disulfide bond pattern of the ANTXR2 ectodomain using proteomic analysis by liquid chromatography-mass spectrometry, and probe disulfide allostery with single-particle electron microscopy and X-ray crystallography. Finally, we will identify the cellular redox regulators of ANTXR2 with a variety of cellular and proteomic approaches. The proposed study will elucidate a novel aspect of receptor-mediated anthrax toxin action and open novel avenues to developing therapeutics against anthrax. The result gleaned from the proposed study will also be extended to other receptor-toxin and host-pathogen systems. PUBLIC HEALTH RELEVANCE: Anthrax toxin is responsible for the major symptoms of anthrax disease. Understanding of anthrax toxin action and toxin-receptor interaction will provide clues to treat anthrax. Our recent studies have discovered a novel mechanism by which anthrax toxin receptor regulates anthrax toxin action, namely disulfide allostery. The current project proposes a series of experiments to elucidate the mechanism of disulfide allostery and to identify the redox regulators of anthrax toxin receptor. This research will enhance our fundamental understanding of anthrax pathogenesis, which will facilitate development of novel therapeutics against anthrax.

描述（由申请人提供）：细菌毒素和细胞表面受体之间的相互作用是宿主-病原体相互作用的重要组成部分。炭疽毒素保护性抗原（PA）与细胞表面受体结合，通过受体介导的内吞作用进入细胞，并在内体膜上形成孔，将有毒酶转移到宿主细胞的胞质溶胶中。炭疽毒素受体通过为毒素提供细胞膜上的高亲和力结合锚以及进入宿主细胞的路径，在炭疽毒素作用中发挥重要作用。在本提案中，我们对两种已知炭疽毒素受体之一 ANTXR2 的初步研究提出了该受体调节炭疽毒素作用的新机制。我们发现二硫键 ANTXR2 胞外部分内的免疫球蛋白样结构域 (R2-Ig) 是炭疽毒素作用所必需的。这些二硫键的破坏抑制了 PA 孔的形成以及有毒酶跨膜易位到宿主细胞中。此外，R2-Ig 中二硫键的还原诱导了受体远端 PA 结合结构域（即冯维勒布兰德因子 A 结构域 (R2-VWA)）的显着构象变化。因此，我们的假设是，R2-Ig 中的二硫键通过对 R2-VWA 构象的变构效应来调节炭疽毒素的作用，从而干扰 PA 前孔到孔的转换和/或膜插入。在本提案中，我们将利用脂质体模型膜系统中的荧光技术以及其他手段，研究 R2-Ig 结构域中的二硫键还原抑制炭疽毒素作用的机制。此外，我们将通过液相色谱-质谱法进行蛋白质组学分析，确定 ANTXR2 胞外域的二硫键模式，并通过单粒子电子显微镜和 X 射线晶体学探测二硫键变构。最后，我们将通过多种细胞和蛋白质组学方法鉴定 ANTXR2 的细胞氧化还原调节因子。拟议的研究将阐明受体介导的炭疽毒素作用的一个新方面，并为开发炭疽疗法开辟新途径。从拟议研究中收集的结果也将扩展到其他受体毒素和宿主病原体系统。 公共卫生相关性：炭疽毒素是造成炭疽病主要症状的原因。了解炭疽毒素作用和毒素-受体相互作用将为治疗炭疽提供线索。我们最近的研究发现了炭疽毒素受体调节炭疽毒素作用的新机制，即二硫键变构。目前的项目提出了一系列实验来阐明二硫键变构机制并鉴定炭疽毒素受体的氧化还原调节剂。这项研究将增强我们对炭疽发病机制的基本了解，从而促进针对炭疽的新型疗法的开发。