COPI interactions mediate toxin entry: a common shared mechanism of translocation

NERCE项目11：COPI相互作用调节毒素进入：一种共同的共同机制 易位，约翰·R·墨菲，博士，波士顿医疗中心。 众所周知，白喉毒素、炭疽毒素和轮状杆菌神经毒素与特定的受体结合。 在它们各自的靶细胞表面上，并通过以下方式内化到内体隔室 受体介导的内吞作用。囊泡(V)-ATPase使内膜腔酸化导致 毒素跨膜结构域的动态构象变化导致其自发插入 进入膜的平面并形成孔或通道。每种毒素的催化域 (例如，片段A、致死因子和/或水肿性因子以及轻链)然后被转移到小泡上 并释放到细胞质中。在白喉催化域和炭疽的情况下 致死因子进入胞浆，COPI辅酶复合体已被证明在早期和 在转位过程中必不可少的一步。在每种情况下，COPI之间的蛋白质相互作用 复杂和毒素是通过一个“进入基序，T1，至少是β-COP成分的 很复杂。炭疽水肿因子和肉毒杆菌神经毒素的生物信息学分析表明 在这些毒素的预测区域中存在T1基序或COPI辅基结合位点 在中毒过程的早期从跨膜区通道中出现。这项拨款申请 建议研究COPI复合体在水肿因子和肉毒杆菌光移位中的作用 在体外从纯化的内容体的管腔中链。我们建议发展新的筛查方法。 结合国家筛查的T1-Motif：：CopI辅酶复合体相互作用的抑制剂 新发传染病和生物防御区域英才中心实验室。 在初步筛选后，小分子多样性集筛选的活性线索将在体外进行 在白喉、炭疽和肉毒杆菌毒素的全细胞细胞毒性试验中得到确认。预计 T1：：CopI相互作用的小分子抑制剂将作为广谱抑制剂发挥作用 白喉、炭疽和肉毒杆菌毒素在体外和体内都有作用。

NERCE Project 11: COPI interactions mediate toxin entry: a common shared mechanism of translocation, John R. Murphy, Ph.D., Boston Medical Center. It is well known that diphtheria toxin, anthrax toxin, and the bptulinum neurotoxins bind to specific receptors on the surface of their respective target cell and are internalized into an endosomal compartment by receptor-mediated endocytosis. Acidification of the endosomal lumen by the vesicular (v)ATPase results in a dynamic conformational changes of the toxins transmembrane domain resulting in its spontaneous insertion into the plane of the membrane and the formation of a pore or channel. The catalytic domain of each toxin (e.g., fragment A, Lethal Factor and/or Edema Factor, and light chain) is then translocated across the vesicle membrane and released into the cytosol. In the case of both the diphtheria catalytic domain and anthrax Lethal Factor delivery into the cytosol, COPI coatomer complex has been shown to play an early and essential step in the translocation process. In each instance, protein protein interaction between COPI complex and the toxin is mediated through an "entry motif, T1, and at least the beta-COP component of the complex. Bioinformatic analysis of anthrax Edema Factor and the botulinum neurotoxins has shown the presence of either the T1-motif or COPI coatomer binding sites in regions of these toxins that are predicted to emerge from the transmembrane domain channel early in the intoxication process. This grant application proposes to investigate the role of COPI complex in the translocation of Edema Factor and botulinum light chain from the lumen of purified endosomes in vitro. We propose the development of screening assays for inhibitors of T1-motif::COPI coatomer complex interactions in conjuction with the National Screening Laboratories for the Regional Centers of Excellence in Emerging Infectious Diseases and Biodefense. Following initial screening, the activity of small molecule diversity set screening leads in in vitro will be confirmed in whole cell cytotoxicity assays for diphtheria, anthrax, and botulinum toxins. It is anticipated that small molecule inhibitors of the T1 ::COPI interactions would function as broad spectrum inhibitors of diphtheria, anthrax, and botulinum toxin action both in vitro and in vivo.