Interaction of Botulinum neurotoxins with presynaptic receptor complexes

肉毒杆菌神经毒素与突触前受体复合物的相互作用

• 批准号: 7940858
• 负责人: MICHAEL R BALDWIN
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940858
• 关键词: Address; Affinity; Affinity Chromatography; Antibodies; Axilla; Binding; Binding Sites; Blepharospasm; Bontoxilysin; Botulism; CCRL2 gene; Categories; Cells; Cervical Dystonia; Clinical; Complement; Complex; Development; Disease; Exhibits; Exocytosis; Family; Gangliosides; Glycosphingolipids; Goals; Human; Hyperhidrosis disorder; Immune Sera; Immunoprecipitation; Integral Membrane Protein; Intervention; Intoxication; Laboratories; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Molecular Models; Motor Neurons; Mutagenesis; Neuromuscular Diseases; Neuromuscular Junction; Neurons; Neurotransmitters; One-Step dentin bonding system; Paralysed; Physiological; Presynaptic Receptors; Process; Protein Binding; Protein Isoforms; Proteins; Proteomics; Protocols documentation; Recombinants; Role; SNAP receptor; Serotyping; Site; Site-Directed Mutagenesis; Skeletal Muscle; Structure; Surface; Synaptic Vesicles; Techniques; Testing; Therapeutic; Toxin; Toxoids; Tropism; Vesicle; base; biodefense; improved; insight; member; molecular modeling; novel; novel therapeutics; presynaptic; protein complex; receptor; receptor binding; synaptotagmin; tetanospasmin; tool

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.

肉毒杆菌中毒是一种神经麻痹性疾病，可以削弱或麻痹骨骼肌。该病是由 七种肉毒神经毒素血清型（A - G 型）之一中毒。肉毒杆菌神经毒素 (BoNT) 是人类毒性最强的蛋白质毒素，被归类为 A 类精选药物。肉毒毒素 通过涉及 (a) 神经元细胞结合、(b) 的多步骤过程使神经肌肉接头中毒 内化到酸性区室中，（c）从酸性区室中膜易位，以及（d）突触小泡胞吐作用所需的神经元 SNARE 蛋白的目标识别和催化裂解。 BoNT 被认为通过双受体机制与神经元表面结合，其中生理受体是由神经节苷脂和蛋白质双受体组成的复合物。使用 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组成部分。本研究的目的是： 目标 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并辅以使用 BoNT 特异性和受体特异性抗体的免疫沉淀方法来实现。 Mm 2 将研究 BoNT 与突触前 BoNT 受体复合物之间的相互作用。利用 BoNT 受体结合域 (HCR) 的晶体结构，HCR 域的靶向诱变将用于鉴定毒素-神经元受体相互作用位点。 鉴定不同血清型 BoNT 的神经元受体将提供洞察力和 开发抑制 BoNT 中毒的新疗法的机会。同样，受体鉴定将有助于改进利用 BoNT 的临床治疗方案，从而将这些研究的影响扩展到生物防御之外。