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: 8444424
• 负责人: Jianjun Sun
• 金额: $ 21.64万
• 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/8444424
• 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.

描述（由申请人提供）：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（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细胞质中。炭疽毒素受体通过在细胞膜上提供高亲和力结合锚固和进入宿主细胞的路径，在炭疽毒素作用中起着至关重要的作用。在该提案中，我们对两个已知的炭疽毒素受体之一AntxR2之一的初步研究提出了一种新的机制，该机制通过该机制调节炭疽毒素作用。我们发现二硫键在 Antxr2毒素作用需要ANTXR2细胞外部分中的免疫球蛋白样结构域（R2-Ig）。这些二硫键的破坏抑制了PA孔的形成，并且跨膜的有毒酶转移到宿主细胞中。此外，R2-Ig的二硫化物还原诱导了受体的远端PA结合域的显着构象变化，即Von Willebrand因子A域（R2-VWA）。因此，我们的假设是，R2-gig中的二硫键通过对R2-VWA构象的变构作用来调节炭疽毒素作用，因此会干扰PA前骨对孔对孔转换率和/或膜插入。在此提案中，我们将研究R2-Ig结构域二硫化物减少的机制，该机制使用脂质体模型膜系统中的荧光技术以及其他方式抑制炭疽毒素作用。此外，我们将使用液相色谱 - 质谱法分析蛋白质组学分析，并确定ANTXR2外生域的二硫键键模式，以及具有单粒子电子显微镜和X射线晶体学的探针二硫化物变构。最后，我们将使用各种细胞和蛋白质组学方法鉴定ANTXR2的细胞氧化还原调节剂。拟议的研究将阐明受体介导的炭疽毒素作用的新颖方面，并开放针对炭疽的治疗疗法的开放途径。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。