Resolving the intoxication mechanism of botulinum neurotoxins using single molecule structural biology

利用单分子结构生物学解析肉毒杆菌神经毒素的中毒机制

• 批准号: 10717466
• 负责人: Mark E Bowen
• 金额: $ 42.74万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10717466
• 关键词: Biophysics; Botulinum Toxins; C-terminal; Cellular Membrane; Cholera Toxin; Classification; Clinical; Clinical Trials; Clostridium botulinum; Cosmetics; Crystallization; Data; Development; Diphtheria Toxin; Drug Kinetics; Electron Microscopy; Electrophysiology (science); Endosomes; Enzymes; Evolution; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Heterogeneity; Imaging technology; Individual; Intoxication; Investigation; Ion Channel; Knowledge; Left; Life; Light; Link; Liposomes; Marketing; Membrane; Membrane Lipids; Methods; Modeling; Molecular; Molecular Conformation; N-terminal; Neurons; Neurotoxins; Peptide Hydrolases; Pharmacologic Substance; Physiological; Process; Protein Dynamics; Protein Engineering; Protein Isoforms; Proteins; Publishing; Reaction; Reagent; Serotyping; Structure; System; Techniques; Tetanus Toxin; Time; Toxin; Visit; biophysical techniques; cell type; clinically relevant; innovation; interest; man; member; molecular imaging; nanobodies; next generation; novel; novel strategies; preservation; prevent; receptor; receptor binding; sensor; simulation; single molecule; single-molecule FRET; stoichiometry; structural biology

Resolving the intoxication mechanism of botulinum neurotoxins using single molecule structural biology. The toxins produced by Clostridium botulinum are some of the deadliest known yet are also revered for their pharmaceutical utility. C. botulinum is classified into seven serotypes (A-G) based on the neurotoxins that they produce. Currently, pharmaceutical development has relied on botulinum neurotoxin type A1 (BoNT/A). However, botulinum neurotoxin type E (BoNT/E) is currently in clinical trials because it provides different pharmacokinetics, faster onset and shorter duration, which enable new treatment regimes. The BoNT proteins are members of the two-component, “AB toxin” family (e.g. tetanus, cholera, and diphtheria toxins), which inject a toxic cargo enzyme (part A) using a proteinaceous transmembrane delivery system (part B). As such, their structure and activity has been well studied. However, several fundamental open questions remain regarding the BoNT delivery mechanism, such as the number of toxins required to deliver the cargo. Additionally, while numerous structures have been solved of the dormant toxins, there is little structural information on the active delivery state(s). AB toxins deliver their cargo across cellular membranes, typically triggered by low pH, which causes structural changes of both parts A and B along with insertion into the membranes. The presence of aggregation at high protein concentrations and membranes provide many experimental challenges for techniques that rely on ensemble averaging. In contrast, single molecule fluorescence can observe individual proteins on single liposomes to revist these classic problems in AB toxin structural biology. These novel approaches will answer long-standing questions in the field and lead to new understanding of the differences between two clinically relevant isoforms.

应用单分子结构技术解析肉毒神经毒素的中毒机制 生物学 肉毒梭菌产生的毒素是已知的最致命的毒素之一，但也因 他们的药物效用。C.肉毒杆菌根据血清型分为七种血清型（A-G）， 它们产生的神经毒素。目前，药物开发一直依赖肉毒杆菌 A1型神经毒素（BoNT/A）。然而，肉毒杆菌神经毒素E型（BoNT/E）目前在临床上 试验，因为它提供了不同的药代动力学，更快的起效时间和更短的持续时间， 新的治疗方案。BoNT蛋白是双组分“AB毒素”家族的成员（例如， 破伤风、霍乱和白喉毒素），其使用注射器注射有毒货物酶（A部分）。 蛋白质跨膜递送系统（部分B）。因此，它们的结构和活动一直是 好好研究。然而，关于BoNT交付，仍存在一些基本的开放问题 机制，如交付货物所需的毒素数量。此外，虽然许多 虽然已经解决了休眠毒素的结构，但活性毒素的结构信息很少。 交付状态。AB毒素通过细胞膜运送它们的货物，通常由低pH触发， 这导致部分A和B沿着插入膜的结构变化。的 在高蛋白浓度和膜下聚集的存在提供了许多实验结果， 对于依赖于系综平均的技术的挑战。相反，单分子荧光可以 观察单个脂质体上的单个蛋白质，以修正AB毒素结构中的这些经典问题 生物学这些新颖的方法将回答该领域长期存在的问题，并带来新的 了解两种临床相关亚型之间的差异。