Structural basis for recognition of SV2 by type E botulinum neurotoxin

E型肉毒杆菌神经毒素识别SV2的结构基础

• 批准号: 10281936
• 负责人: Rongsheng Jin
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-09 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10281936
• 关键词: Acetylcholine; Achievement; Affect; Affinity; Alpaca; Amino Acid Substitution; Antibodies; Bacteria; Bacterial Toxins; Binding; Binding Sites; Biological; Biological Assay; Bontoxilysin; Botox; Botulism; Cell Surface Receptors; Cell surface; Cells; Chimeric Proteins; China; Cleaved cell; Clinic; Clinical; Clostridium botulinum; Complex; Cosmetics; Crystallization; Custom; Data; Disease; Drug or chemical Tissue Distribution; Esthetics; GTP-Binding Protein alpha Subunits, Gs; Gangliosides; Glycoproteins; Goals; Human; Knowledge; Korea; Link; Mediating; Medicine; Modeling; Molecular; Motor Neurons; Muscle; Mutagenesis; Neuromuscular Junction; Neurons; Paralysed; Pathogenicity; Peptides; Pharmaceutical Preparations; Pharmacology; Polysaccharides; Process; Property; Protein Isoforms; Proteins; Receptor Cell; Resolution; SNAP receptor; Series; Serotyping; Site-Directed Mutagenesis; Specificity; Structure; Synaptic Vesicles; Syndrome; Therapeutic; Toxic effect; Toxin; X-Ray Crystallography; anthrax lethal factor; base; botulinum toxin type B; botulinum toxin type E; clinical application; clinical development; design; ganglioside receptor; improved; in vivo; innovation; nanobodies; novel; novel therapeutics; protein complex; public health relevance; receptor; receptor binding; uptake

Abstract Botulinum neurotoxins (BoNTs) are produced by the bacterium Clostridium botulinum, which are the causative agents of neuroparalytic disease botulism. Nevertheless, type A and type B botulinum neurotoxins (BoNT/A and BoNT/B) have been successfully used in clinic for a variety of aesthetic and therapeutic applications. The high potency of BoNTs relies on the specific and efficient binding and uptake of BoNTs by motor neurons at neuromuscular junctions. Inside the neurons, BoNTs cleave SNARE complex and block the release of acetylcholine, resulting in paralysis of the affected muscles. It is well accepted that most BoNTs synergistically bind specific protein receptors and gangliosides on motor neurons for cell entry. Remarkably, BoNTs develop diverse binding modes for protein receptor recognition, in contrast to a conserved ganglioside-binding mode. In this study, we will focus on the type E toxin (BoNT/E) that is the least studied human pathogenic BoNT in comparison to well-characterized BoNT/A and BoNT/B. Paradoxically, BoNT/E is currently in clinic trial as a new therapeutic and aesthetic product, which displays distinct pharmacological and clinic features when compared to BoNT/A and BoNT/B. At the molecular level, BoNT/E recognizes two of the three isoforms of synaptic vesicle glycoprotein 2 (SV2A and SV2B) as its neuronal receptors for cell entry, but not the closely related SV2C isoform. As SV2A, 2B, and 2C have different tissue distribution in vivo, a better understanding of how BoNT/E recognizes SV2A, 2B, and 2C differentially is crucial to understand the therapeutic profiles of BoNT/E-based drugs as well as to develop new indications. To this end, we propose two Specific Aims using an integrated approach that combines X-ray crystallography, site-directed mutagenesis, and binding assays. In Aim 1, we propose to study the structural basis for recognition of SV2A by BoNT/E. In Aim 2, we aim to understand the affinity and specificity requirements for BoNT/E to recognize three SV2 isoforms, while all the structural findings will be verified by structure-based mutagenesis studies. The achievement of our goals will help to understand the unique pharmacological and clinical profiles of BoNT/E, as well as to facilitate the design of new countermeasures against BoNT/E.

抽象的 肉毒杆菌毒素（BONTS）由肉毒杆菌细菌产生，这是病因 神经分析疾病肉毒杆菌的药物。但是，A型和B型肉毒杆菌神经毒素（BONT/A和 BONT/B）已在诊所成功使用了各种美学和治疗应用。高 BONT的效力取决于运动神经元在 神经肌肉连接。在神经元内，BONTS裂解圈套并阻止释放 乙酰胆碱，导致受影响的肌肉麻痹。大多数BONT在协同上都可以接受 在运动神经元上结合特定的蛋白质受体和神经节的细胞进入。值得注意的是，BONTS发展 与保守的神经节结合模式相比，蛋白质受体识别的各种结合模式。在 这项研究，我们将重点介绍E型毒素（BONT/E），该毒素是研究最少的人类致病性BONT 与特征良好的BONT/A和BONT/B的比较。矛盾的是，BONT/E目前正在诊所试验中作为新的试验 治疗和审美产品，在比较时显示出不同的药理和诊所特征 到bont/a和bont/b。在分子水平上，BONT/E识别突触囊泡的三种同工型中的两个 糖蛋白2（SV2A和SV2B）作为细胞进入的神经元受体，而不是密切相关的SV2C同工型。 由于SV2A，2B和2C在体内具有不同的组织分布，因此可以更好地理解BONT/E的识别方式 SV2A，2B和2C差异对于了解BONT/E基药物的治疗谱至关重要 为了发展新的适应症。为此，我们使用一种集成方法提出了两个具体目标 结合X射线晶体学，定点诱变和结合测定。在AIM 1中，我们建议学习 BONT/E识别SV2A的结构基础。在AIM 2中，我们旨在了解亲和力和特异性 BONT/E识别三个SV2同工型的要求，而所有结构发现将由 基于结构的诱变研究。实现我们的目标将有助于理解独特的 BONT/E的药理和临床轮廓，以及促进新的对策设计 反对BONT/E。