MECHANISM OF BOTULINUM TOXIN ACTION

肉毒杆菌毒素的作用机制

• 批准号: 2702962
• 负责人: LANCE L SIMPSON
• 金额: $ 40.35万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2702962
• 关键词: adipocytes; botulinum toxins; chemical binding; cytotoxicity; electrophysiology; exocytosis; fluorescence microscopy; hepatotoxin; human tissue; laboratory mouse; laboratory rabbit; laboratory rat; liver cells; mixed tissue /cell culture; molecular cloning; nerve endings; neurons; neurotoxicology; neurotoxins; nucleic acid hybridization; syntaxin; toxicant interaction; transport proteins; western blottings

The long-term objective of the proposed research is to determine the cellular, subcellular and molecular actions of botulinum toxin. This objective is motivated by the need to understand the disease known as botulism and by the need to find ways to prevent and to treat botulism. Therefore, the focus of the work will be studies on cells of human origin, including cholinergic nerve cells (which are the target cells for toxin action in clinical poisoning), as well as fat cells and liver cells. Botulinum toxin acts on cholinergic nerve endings to block vesicle fusion and exocytosis. The toxin exerts this effect by proceeding through a sequence of events that involves binding to the plasma membrane, internalization, and eventual expression of an intracellular poisoning action. The final step is known to be enzymatic in nature; botulinum toxin is a zinc-dependent metalloendoprotease that cleaves peptide, that govern exocytosis. Recently, the applicant and his colleagues have found that botulinum toxin acts on other types of cells (e.g., fat, liver) to block other types of vesicle movement (e.g., vesicle shuttling, transcytosis). Therefore experiments will be done to determine whether a single sequence of events can account for toxin action on all vulnerable cells. Botulinum toxin is believed to act on nerve cells and other cells of laboratory animal origin by proteolytically cleaving four specific peptides (synaptobrevin, cellubrevin, SNAP-25 and syntaxin). However, it is not yet known whether these are the substrates that are the substrates that account for toxin action on human cells. Therefore, experiments will be done to identify and characterize human substrates for botulinum toxin. In keeping with the nature of clinical poisoning, initial emphasis will be on the study of human nerve cells. Subsequent experiments will be done on human fat cells and human liver cells. It is generally assumed that botulinum toxin action on vulnerable cells can be fully explained on the basis of cleavage of synaptobrevin, cellubrevin, SNAP-25 and syntaxin, but this has never been proved. Therefore, the applicant and his colleagues will use techniques that selectively remove each of these substances from target cells. The effects of poisoning with botulinum toxin will then be compared with the effects of selectively removing specific substrates. This will provide novel insights into toxin action, as well as expanding our knowledge of cell biology.

拟议研究的长期目标是确定 肉毒杆菌毒素的细胞、亚细胞和分子作用。 这 目的是出于需要了解这种疾病， 肉毒杆菌中毒以及寻找预防和治疗肉毒杆菌中毒的方法的需要。 因此，工作的重点将是对人类起源的细胞的研究， 包括胆碱能神经细胞（其是毒素的靶细胞 在临床中毒中起作用），以及脂肪细胞和肝细胞。 肉毒毒素作用于胆碱能神经末梢阻断囊泡融合 和胞吐作用。 毒素通过一个 涉及与质膜结合的事件序列， 内化，并最终表达细胞内中毒 行动上 最后一步是已知的酶的性质;肉毒杆菌毒素 是一种锌依赖性金属内切蛋白酶，它切割肽， 胞吐作用 最近，申请人和他的同事发现， 肉毒杆菌毒素作用于其它类型的细胞（例如，脂肪、肝脏）阻断 其它类型的囊泡运动（例如，囊泡穿梭、转胞吞作用）。 因此，将进行实验以确定单个序列是否 可以解释毒素对所有脆弱细胞的作用。 肉毒杆菌毒素被认为作用于神经细胞和其他细胞， 通过蛋白水解切割四种特异性肽的实验室动物来源 （小突触泡蛋白、小纤维泡蛋白、SNAP-25和突触融合蛋白）。 然而，现在还不是 知道这些是否是基质， 对人体细胞的毒性作用。 因此，将进行实验， 鉴定和表征肉毒杆菌毒素人底物。 一致 由于临床中毒的性质，最初的重点将放在 人类神经细胞的研究。 随后的实验将在人类身上进行。 脂肪细胞和人类肝细胞。 一般认为肉毒杆菌毒素作用于脆弱细胞 可以根据小突触泡蛋白的裂解得到充分解释， cellubrevin，SNAP-25和syntaxin，但这从未得到证实。 因此，申请人及其同事将使用技术， 选择性地从靶细胞中去除这些物质。 的影响 肉毒杆菌毒素中毒的效果将与 选择性地去除特定的衬底。 这将提供新的见解 毒素的作用，以及扩大我们的细胞生物学知识。