Interaction of Botulinum neurotoxins with presynaptic receptor complexes

描述（由申请人提供）：肉毒杆菌中毒是一种神经麻痹性疾病，可削弱或瘫痪骨骼肌。这种疾病是由7种血清型肉毒杆菌神经毒素（A - G型）中毒引起的。肉毒杆菌神经毒素（BoNTs）是人类毒性最大的蛋白质毒素，被列为A类精选制剂。BoNTs通过多个步骤使神经肌肉连接中毒，包括(a)神经元细胞结合，(b)内化到酸性隔间，(c)酸性隔间的膜移位，以及(d)突触囊泡胞外分泌所需的神经元SNARE蛋白的靶识别和催化裂解。为了了解BoNT如何结合并进入神经元，本应用程序将识别和表征BoNT神经元受体蛋白。利用BoNT的重组受体结合域，一步分离方案表明BoNT神经元受体是突触前受体复合物的一个组成部分。本研究的目的是：目的1，鉴定突触前BoNT受体复合物的蛋白质成分。这将通过蛋白质组学和质谱技术以及使用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，该方法已被用于鉴定BoNT/B与synaptotagmin i的结合。Aim 2将研究BoNT与突触前BoNT受体复合物之间的相互作用。利用生物信息学、分子模型和靶向诱变策略，BoNT和突触前BoNT受体复合物之间的结合域位点将被确定。鉴定不同血清型bont的神经元受体将为开发抑制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，有助于改善利用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.