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: 7318735
• 负责人: MICHAEL R BALDWIN
• 金额: $ 8.3万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7318735
• 关键词: Address; Affinity; Affinity Chromatography; Antibodies; Binding; Binding Sites; Bioinformatics; Biological Assay; Bontoxilysin; Botulism; C-terminal; CCRL2 gene; Categories; Cells; Clinical; Complement; Complex; Development; Disease; Exhibits; Exocytosis; Family; Generations; Glycosphingolipids; Goals; Human; Immune Sera; Immunoprecipitation; Intervention; Intoxication; Laboratories; Ligand Binding Domain; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Mutagenesis; Neuromuscular Junction; Neurons; Neurotransmitters; One-Step dentin bonding system; Paralysed; Personal Satisfaction; Presynaptic Receptors; Process; Protein Binding; Proteins; Proteomics; Protocols documentation; Recombinants; Research Personnel; Role; SNAP receptor; Serotyping; Site-Directed Mutagenesis; Skeletal Muscle; Specificity; Structure; Synaptic Vesicles; Techniques; Testing; Therapeutic; Toxin; Toxoids; Tropism; Vesicle; base; biodefense; domain mapping; improved; inhibitor/antagonist; insight; member; molecular modeling; novel; presynaptic; programs; receptor; receptor binding; small molecule; synaptotagmin; synaptotagmin I

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.

描述（由申请人提供）：肉毒杆菌中毒是一种神经麻痹性疾病，可以削弱或麻痹骨骼肌。该疾病是由七种肉毒杆菌神经毒素血清型（A - G 型）之一中毒引起的。肉毒杆菌神经毒素 (BoNT) 是对人类毒性最强的蛋白质毒素，被列为 A 类精选药物。 BoNT 通过多步过程使神经肌肉接头中毒，包括 (a) 神经元细胞结合，(b) 内化到酸性区室中，(c) 酸性区室中的膜易位，以及 (d) 突触所需的神经元 SNARE 蛋白的目标识别和催化裂解囊泡胞吐作用。为了了解 BoNT 如何结合并进入神经元，该应用程序将识别和表征 BoNT 神经元受体蛋白。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。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。目标 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域位点。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。