Diphtheria & Anthrax Toxins: Mechanisms of Cell Entry

白喉毒素、炭疽毒素（保护性抗原[PA]）的催化结构域。致死因子[LF]和 水肿因子[EF]）和肉毒杆菌神经毒素的所有血清型遵循类似的进入胞质溶胶的途径 它们各自的靶向真核细胞。在每种毒素与其特定细胞表面结合后 受体，毒素在网格蛋白包被的小凹中通过受体介导的内吞作用内化到细胞中。 在早期内分泌室酸化后，它们各自的催化酶的移位， 通过至少一个功能性跨膜结构域促进跨膜结构域进入胞质溶胶。使用体外 易位测定，我们最近已经证明，细胞易位因子（CTF）复合物， 白喉毒素C-结构域穿过早期内体细胞膜所需的蛋白质。 囊泡白喉、炭疽毒素和肉毒杆菌毒素A的X射线结构是已知的。BLAST分析 对这些毒素的研究表明，每种毒素中存在12个氨基酸的“进入基序”。本 申请提出继续研究白喉毒素C-结构域，丙氨酸扫描诱变， 假定的“进入基序”，并在炭疽致死因子的体外translocation测定的发展。在 白喉毒素、炭疽毒素和肉毒杆菌毒素的情况， 结构功能域及其作用模式;然而，对分子结构的详细了解 它们各自的催化结构域从酸化的 早期的内体到胞质溶胶仍然是难以捉摸的。只有少数研究直接关注 已经公开了毒素催化结构域进入的易位的分子机制。在 在白喉毒素的情况下，这些研究的结果导致了两种不同的假设机制， 催化结构域转位第一种假设是毒素本身携带足够的 介导其催化结构域跨胞吞囊泡易位的结构信息 膜的相反，第二个假设是基于发现，这两个结构域的 毒素与胞质易位因子（CTF）复合物的组合对于生产性的细胞内毒素是必需的。 易位我们已经使用纯化的早期内体与融合蛋白毒素，DAB 3891 L-2， 仔细研究这两种假设。

The catalytic domains of diphtheria toxin, anthrax toxins (protective antigen [PA]. lethal factor [LF] and edema factor [EF]) and all serotypes of botulinum neurotoxin follow a similar route of entry into the cytosol of their respective targeted eukaryotic cells. Following binding of each toxin to their specific cell surface receptor, the toxin is internalized into the cell by receptor-mediated endocytosis in clathrin coated pits. Following acidification of an early endosmal compartment the translocation of their respective catalytic domains to the cytosol is facilitated by a at least a functional transmembrane domain. Using an in vitro translocation assay, we have recently demonstrated that a cellular translocation factor (CTF) complex is required for the delivery of the diphtheria toxin C-domain across the membrane of early endosomal vesicles. The X-ray structure of diphtheria, anthrax toxin, and botulinum toxin A is known. BLAST analysis of these toxins has suggested the presence of 12 amino acid "entry motif" in each toxin. The present application proposes the continuing study of diphtheria toxin C-domain, alanine scanning mutagenesis of the putative "entry motif", and the development of an in vitro transloaction assay for anthrax lethal factor. In the case of diphtheria toxin, anthrax toxins, and botulinum toxins a great deal is known of their respective structure function domains and their mode of action; however, a detailed understanding of the molecular mechanism(s) required for translocation of their respective catalytic domains from the lumen of acidified early endosomes to the cytosol have remained elusive. Only a few studies have focused directly on the molecular mechanism(s) of translocation f the toxin catalytic domain entry have been published. In the case of diphtheria toxin, the results of these studies have led to two divergent hypothetical mechanisms of catalytic domain translocation. The first is based on the hypothesis that the toxin itself carries sufficient structural information to mediate translocation of its catalytic domain across the endocytic vesicle membrane. In contrast, the second hypothesis is based on findings that both structural domains of the toxin in combination with a cytosolic translocation factor (CTF) complex are necessary for productive translocation. We have used purified early endosomes charged with the fusion protein toxin, DAB3891L-2, to critically examine both hypotheses.