Diphtheria & Anthrax Toxins: Mechanisms of Cell Entry

描述（由申请人提供）：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])和所有血清型肉毒杆菌神经毒素都遵循类似的途径进入其各自靶向真核细胞的细胞质。在每种毒素与它们特定的细胞表面受体结合后，毒素通过受体介导的胞吞作用在网格蛋白包覆的窝中被内化到细胞中。在早期内体腔室酸化后，它们各自的催化结构域向细胞质溶胶的易位至少由一个功能性跨膜结构域促进。利用体外易位试验，我们最近证明了细胞易位因子（CTF）复合体是白喉毒素c结构域跨越早期内体囊泡膜传递所必需的。白喉、炭疽毒素和肉毒毒素A的x射线结构是已知的。对这些毒素的BLAST分析表明，每种毒素中存在12个氨基酸“进入基序”。本申请建议继续研究白喉毒素c结构域，丙氨酸扫描诱变假定的“进入基序”，并开发炭疽致死因子的体外易位试验。在白喉毒素、炭疽毒素和肉毒杆菌毒素的情况下，对它们各自的结构、功能域和作用方式已经有了大量的了解；然而，对它们各自的催化结构域从酸化的早期内体的管腔转移到细胞质溶胶所需的分子机制的详细了解仍然是难以捉摸的。只有少数研究直接关注毒素催化结构域进入易位的分子机制已经发表。在白喉毒素的情况下，这些研究的结果导致了催化结构域易位的两种不同的假设机制。第一种是基于毒素本身携带足够的结构信息来介导其催化结构域跨内吞囊泡膜的易位的假设。相比之下，第二种假设是基于毒素的两个结构域与细胞质易位因子（CTF）复合物的结合对于生产易位是必要的。我们使用纯化的早期内体携带融合蛋白毒素DAB3891L-2，对这两种假设进行了严格的检验。

DESCRIPTION (provided by applicant): 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 endosomal compartment, the translocation of their respective catalytic domains to the cytosol is facilitated by 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 translocation 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 of 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.