Interaction of Botulinum neurotoxins with presynaptic receptor complexes

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 肉毒神经毒素(A-G型)七种血清型中的一种中毒。肉毒杆菌神经毒素 (BoNTs)是对人类毒性最大的蛋白质毒素，被归类为A类精选制剂。BoNTs 通过多步骤过程使神经肌肉接头中毒，包括(A)神经细胞结合，(B) 内化进入酸性隔室，(C)膜从酸性隔间移位，以及(D)突触囊泡吐出所需的神经元SNARE蛋白的靶标识别和催化切割。BoNTs被认为是通过双受体机制与神经元表面结合的，其中生理受体是由神经节苷脂和蛋白质(S)双受体组成的复合体。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。M2将研究BoNTs与突触前BONT受体复合体之间的相互作用.利用BONT受体结合域(HCR)的晶体结构，对HCR结构域进行定向突变，以确定毒素与神经元受体的相互作用部位。 识别不同血清型BoNTs的神经元受体将提供洞察力和 开发抑制BoNTs中毒的新疗法的机会。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。

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. BoNTs are thought to bind to the surface of neurons via a dual receptor mechanism in which the physiologic receptor is a complex composed of gangliosides and protein(s) dual receptors. 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. Mm 2 will study the interaction tietween BoNTs and the presynaptic BoNT receptor complex. Utilizing the crystal structures of BoNT receptor binding domains (HCRs), targeted mutagenesis of the HCR domain will be employed to identify the toxin- neuronal receptor interaction sites. 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.