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（由申请方提供）：细菌毒素与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（Anthrax toxin protective antigen，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，并在胞内体膜上形成一个孔，将有毒酶转运到宿主细胞的胞质溶胶中。炭疽毒素受体在炭疽毒素的作用中起着重要作用，它为毒素提供了细胞膜上的高亲和力结合锚和进入宿主细胞的途径。在这个提议中，我们对两种已知的炭疽毒素受体之一ANTXR 2的初步研究提出了一种新的机制，该受体通过这种机制调节炭疽毒素的作用。我们已经发现， ANTXR 2胞外部分内的免疫球蛋白样结构域（R2-IG）是炭疽毒素作用所需的。这些二硫键的破坏抑制PA孔的形成和有毒酶的跨膜转运到宿主细胞中。此外，二硫键还原R2-IG诱导受体的远端PA结合结构域，即von Willebrand因子A结构域（R2-VWA）的显着构象变化。因此，我们假设R2-IG中的二硫键通过对R2-VWA构象的变构效应调节炭疽毒素作用，从而干扰PA prepore-to-pore转换和/或膜插入。在这个建议中，我们将调查的机制，在R2-IG域的二硫键还原抑制炭疽毒素的作用，使用荧光技术在脂质体模型膜系统以及其他手段。此外，我们将确定的ANTXR 2胞外域的二硫键模式，使用蛋白质组学分析液相色谱-质谱，探针二硫键变构与单粒子电子显微镜和X-射线晶体学。最后，我们将通过多种细胞和蛋白质组学方法确定ANTXR 2的细胞氧化还原调节剂。这项研究将阐明受体介导的炭疽毒素作用的一个新方面，并开辟新的途径，开发治疗炭疽。从拟议的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：炭疽毒素是炭疽病的主要症状。了解炭疽毒素的作用及毒素与受体的相互作用，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一种新机制，即二硫键变构。本研究拟通过一系列实验来阐明炭疽毒素受体的二硫键变构机制，并鉴定炭疽毒素受体的氧化还原调节剂。这项研究将提高我们对炭疽发病机制的基本认识，这将有助于开发新的治疗炭疽病的药物。