Diphtheria & Anthrax Toxins: Mechanisms of Cell Entry

描述(申请人提供)：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])和所有血清型肉毒杆菌神经毒素都遵循类似的途径进入各自目标真核细胞的胞浆。在每种毒素与其特定的细胞表面受体结合后，毒素通过受体介导的内吞作用内化到细胞内，这些内吞作用是在笼状蛋白包裹的凹坑中进行的。在早期的内体隔室酸化后，它们各自的催化结构域至少通过一个功能跨膜结构域促进了它们向胞浆的移位。利用体外易位试验，我们最近证明了白喉毒素C结构域需要一个细胞易位因子(CTF)复合体才能穿过早期内体囊泡的膜。白喉、炭疽毒素和A型肉毒毒素的X射线结构是已知的。对这些毒素的BLAST分析表明，每种毒素中都存在12个氨基酸的“进入基序”。本申请建议继续研究白喉毒素C区，对假定的“进入基序”进行丙氨酸扫描诱变，并建立炭疽致死因子的体外易位试验。就白喉毒素、炭疽毒素和肉毒杆菌毒素而言，人们对它们各自的结构、功能结构域和作用模式知之甚少；然而，对于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中所需的分子机制(S)仍然缺乏详细的了解。只有少数研究直接集中在毒素催化结构域进入的分子机制(S)上。在白喉毒素的情况下，这些研究的结果导致了两种不同的催化域移位的假设机制。第一种是基于这样的假设，即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泡膜的移位。相反，第二种假设是基于这样的发现，即毒素的两个结构域与胞浆转位因子(CTF)复合体相结合是产生转位所必需的。我们使用纯化的早期内吞体内充满融合蛋白毒素DAB3891L-2，以批判性地检验这两种假说。

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.