Receptor disulfide allosteric regulation of anthrax toxin action

描述(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，并在内体膜上形成一个孔，将有毒的酶转移到宿主细胞的胞浆中。炭疽毒素受体通过为炭疽毒素提供细胞膜上的高亲和力结合锚和进入宿主细胞的途径，在炭疽毒素的作用中起着至关重要的作用。在这项建议中，我们对两个已知的炭疽毒素受体之一ANTXR2的初步研究表明，该受体调节炭疽毒素作用的新机制。我们发现，分子中的二硫键 ANTXR2胞外部分的免疫球蛋白样域(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孔的形成和有毒酶跨膜进入宿主细胞的转移。此外，R2-Ig的二硫键还原引起受体远端PA结合结构域的显著构象变化，即von Willebrand因子A结构域(R2-vWA)。因此，我们的假设是R2-Ig上的二硫键通过对R2-VWA构象的变构作用来调节炭疽毒素的作用，从而干扰PA的前孔到孔的转化和/或膜的插入。在这项建议中，我们将利用脂质体模型膜系统中的荧光技术以及其他方法来研究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，我们还将通过液-质联用进行蛋白质组学分析来确定ANTXR2胞外结构域的二硫键模式，并用单粒子电子显微镜和X射线结晶学来探测二硫键变构。最后，我们将用各种细胞和蛋白质组学方法确定ANTXR2的细胞氧化还原调节因子。这项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，并为开发治疗炭疽病的药物开辟新的途径。从拟议的研究中收集到的结果也将扩展到其他受体-毒素和宿主-病原体系统。 与公共卫生相关：炭疽毒素是炭疽病的主要症状。了解炭疽毒素的作用和毒素与受体的相互作用将为炭疽的治疗提供线索。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，即二硫变构。本项目提出了一系列实验，以阐明二硫变构的机制，并鉴定炭疽毒素受体的氧化还原调节因子。本研究将加深我们对炭疽病发病机制的基本认识，有助于炭疽病新疗法的开发。

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.