Interaction of Botulinum neurotoxins with presynaptic receptor complexes

描述（由申请人提供）：肉毒中毒是一种神经麻痹性疾病，可以削弱或麻痹骨骼肌。这种疾病是由肉毒杆菌神经毒素的七种血清型（A - G型）之一中毒引起的。肉毒神经毒素（BoNT）是人类毒性最大的蛋白质毒素，被归类为A类选择剂。BoNT通过涉及（a）神经元细胞结合，（B）内化到酸性区室，（c）从酸性区室的膜移位，和（d）突触囊泡胞吐所需的神经元SNARE蛋白的靶标识别和催化裂解的多步骤过程来毒害神经肌肉接头。为了了解BoNT如何结合并进入神经元，本申请将鉴定和表征BoNT神经元受体蛋白。使用重组受体结合结构域的BoNT，一步分离协议表明，BoNT神经元受体是一个组件的突触前受体复合物。本研究的主要目的是：目的1、鉴定突触前BoNT受体复合物的蛋白组分。这将通过蛋白质组学和质谱技术，辅以使用BoNT特异性和受体特异性抗体的免疫沉淀方法来实现。作为原理证明，该方法已用于鉴定BoNT/B与突触结合蛋白I的结合。目的2研究突触前BoNT受体复合物与BoNT的相互作用。利用生物信息学、分子建模和靶向诱变策略，将定义BoNT和突触前BoNT受体复合物之间的结合结构域的位点。不同血清型BoNT的神经元受体的鉴定将为开发抑制BoNT中毒的新疗法提供见解和机会。同样，受体鉴定将扩大这些研究的影响，超越生物防御，通过促进改善利用BoNT的临床治疗方案。

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.