Interaction of Botulinum neurotoxins with presynaptic receptor complexes

描述(申请人提供)：肉毒杆菌中毒是一种神经麻痹疾病，可削弱或瘫痪骨骼肌。这种疾病是由七种血清型肉毒神经毒素(A-G型)中的一种中毒引起的。肉毒杆菌神经毒素(BoNTs)是人类最具毒性的蛋白质毒素，被列为A类精选药物。BoNTs通过一个多步骤的过程毒化神经肌肉接头，包括：(A)神经细胞结合，(B)内化到酸性隔室，(C)从酸性隔室的膜转位，以及(D)突触囊泡吐出所需的神经元SNAR蛋白的靶标识别和催化切割。为了了解BoNT是如何结合和进入神经元的，这项应用将识别和表征BONT神经元受体蛋白。使用重组的BONT受体结合域，一步分离方案表明BONT神经元受体是突触前受体复合体的一个组成部分。本研究的目的是：目的1，鉴定突触前BONT受体复合体的蛋白质组分。这将通过蛋白质组学和质谱学技术实现，并辅之以使用BONT特异性抗体和受体特异性抗体的免疫沉淀方法。作为原理的证明，该方法已被用于鉴定BONT/B与突触聚集蛋白I的结合。目的2将研究BONT与突触前BONT受体复合体之间的相互作用。利用生物信息学、分子建模和定向突变策略，将确定BONT和突触前BONT受体复合体之间的结合结构域的位置(S)。识别不同血清型BoNTs的神经元受体将为开发抑制BoNTs中毒的新疗法提供洞察力和机会。同样，受体识别将通过改进利用BoNTs的临床治疗方案，将这些研究的影响扩大到生物防御之外。

DESCRIPTION (provided by applicant): Botulism is a neuroparalytic disease that can weaken or paralyze skeletal muscle. The disease is caused by intoxication with one of seven serotypes of botulinum neurotoxin (types A - G). Botulinum neurotoxins (BoNTs) are the most toxic protein toxins of humans and are classified as category A select agents. BoNTs intoxicate neuromuscular junctions through a multistep process involving (a) neuronal cell-binding, (b) internalization into acidic compartments, (c) membrane translocation from acidic compartments, and (d) target recognition and catalytic cleavage of neuronal SNARE proteins required for synaptic vesicle exocytosis. To understand how BoNTs bind and enter into neurons, this application will identify and characterize the BoNT neuronal receptor proteins. Using a recombinant receptor binding domain of BoNT, a one-step isolation protocol showed that the BoNT neuronal receptor is a component of a presynaptic receptor complex. The aims of this study will: Aim 1, identify the protein components of the presynaptic BoNT receptor complex. This will be achieved by proteomics and mass spectrometry techniques complemented by immunoprecipitation approaches using BoNT-specific and receptor-specific antibodies. As a proof of principle, this approach has been used to identify the binding of BoNT/B to synaptotagmin I. Aim 2 will study the interaction between BoNTs and the presynaptic BoNT receptor complex. Utilizing bioinformatics, molecular modeling, and targeted mutagenesis strategies the site(s) of binding domain between BoNT and the presynaptic BoNT receptor complex will be defined. Identification of the neuronal receptors for the different serotypes of BoNTs will provide insight and opportunities for the development of novel therapies to inhibit against BoNTs intoxication. Similarly, receptor identification will expand the impact of these studies beyond biodefense, by contributing to improved clinical therapeutic protocols that utilize the BoNTs.