Diphtheria & Anthrax Toxins: Mechanisms of Cell Entry

白喉毒素、炭疽毒素(保护性抗原[PA])的催化域。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都遵循类似的进入胞浆的途径 它们各自的靶向真核细胞。在每种毒素与其特定的细胞表面结合后 在受体的作用下，毒素通过受体介导的内吞作用在笼罩在胞核中的细胞内进入细胞。 在早期内膜室酸化后，它们各自的催化转位 至少一个功能跨膜结构域促进了结构域到胞浆的结合。使用体外培养技术 易位分析，我们最近证明了细胞易位因子(CTF)复合体是 白喉毒素C结构域跨早期内质体膜转运所必需的 水泡。白喉、炭疽毒素和A型肉毒毒素的X射线结构是已知的。BLAST分析 这些毒素中的一种表明每种毒素中存在12个氨基酸的“进入基序”。现在 应用建议继续研究白喉毒素C区、丙氨酸扫描诱变 推测的“进入基序”，以及炭疽致死因子体外反式作用试验的发展。在……里面 白喉毒素、炭疽毒素和肉毒杆菌毒素的大量案例都知道它们各自的情况 结构功能区及其作用方式；然而，对分子的详细理解 它们各自的催化结构域从酸化的管腔移位所需的机制(S) 细胞质的早期内含体仍然难以捉摸。只有几项研究直接集中在 毒素催化结构域进入易位的分子机制(S)已经发表。在 白喉毒素的案例，这些研究的结果导致了两种不同的假设机制 催化结构域易位。第一种是基于毒素本身携带足够的 结构信息介导其催化结构域跨内吞小泡的移位 薄膜。相比之下，第二种假设是基于两个结构域的发现 毒素与胞浆转位因子(CTF)复合体的结合是生产所必需的 易位。我们已经使用纯化的早期内吞体充填融合蛋白毒素DAB3891L-2，以 批判性地检查这两种假说。

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.