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

基本信息

批准号：
10448471
负责人：
Rongsheng Jin
金额：
$ 19.63万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-09 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10448471
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 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 base botulinum toxin type B botulinum toxin type E clinical application clinical development design ganglioside receptor improved in vivo innovation lethal factor nanobodies novel novel therapeutics protein complex public health relevance receptor receptor binding uptake

项目摘要

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.

抽象的肉毒杆菌神经毒素 (BoNT) 由肉毒杆菌产生，是引起肉毒杆菌神经毒素的原因之一。神经麻痹疾病肉毒杆菌中毒剂。然而，A 型和 B 型肉毒杆菌神经毒素（BoNT/A 和 BoNT/B) 已成功应用于临床各种美容和治疗应用。高 BoNT 的效力依赖于运动神经元对 BoNT 的特异性且有效的结合和摄取。神经肌肉接头。在神经元内部，BoNT 裂解 SNARE 复合物并阻止释放乙酰胆碱，导致受影响的肌肉瘫痪。人们普遍认为大多数 BoNT 具有协同作用结合运动神经元上的特定蛋白质受体和神经节苷脂以进入细胞。值得注意的是，BoNT 的发展与保守的神经节苷脂结合模式相比，蛋白质受体识别具有多种结合模式。在在本研究中，我们将重点关注 E 型毒素 (BoNT/E)，它是目前研究最少的人类致病性 BoNT 与充分表征的 BoNT/A 和 BoNT/B 进行比较。矛盾的是，BoNT/E 目前正在作为一种新的药物进行临床试验。治疗和美容产品，相比之下表现出独特的药理和临床特征至 BoNT/A 和 BoNT/B。在分子水平上，BoNT/E 识别突触小泡三种亚型中的两种糖蛋白 2（SV2A 和 SV2B）作为其进入细胞的神经元受体，但不是密切相关的 SV2C 亚型。由于SV2A、2B和2C在体内具有不同的组织分布，更好地了解BoNT/E如何识别 SV2A、2B 和 2C 的差异对于了解基于 BoNT/E 的药物的治疗概况也至关重要以开发新的适应症。为此，我们使用综合方法提出两个具体目标：结合了 X 射线晶体学、定点诱变和结合测定。在目标 1 中，我们建议研究 BoNT/E 识别 SV2A 的结构基础。在目标 2 中，我们旨在了解亲和力和特异性 BoNT/E 识别三种 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。我们目标的实现将有助于了解独特的 BoNT/E 的药理学和临床概况，以及促进新对策的设计反对 BoNT/E。