MECHANISM OF BOTULINUM TOXIN ACTION

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 目标的动机是需要了解这种疾病，即 肉毒杆菌中毒和需要找到预防和治疗肉毒杆菌中毒的方法。 因此，工作的重点将是对人类起源细胞的研究， 包括胆碱能神经细胞(这是毒素的靶细胞 临床中毒中的作用)，以及脂肪细胞和肝细胞。 肉毒杆菌毒素作用于胆碱能神经末梢以阻断囊泡融合 和胞吐。这种毒素通过通过一种 涉及与质膜结合的一系列事件， 细胞内毒素的内化和最终表达 行动。已知的最后一步是酶类；肉毒杆菌毒素 是一种锌依赖的金属内蛋白酶，它能裂解多肽，从而控制 胞吐。最近，申请人和他的同事发现， 肉毒杆菌毒素作用于其他类型的细胞(如脂肪、肝脏)以阻断 其他类型的囊泡运动(例如，囊泡穿梭、细胞穿梭)。 因此，将进行实验以确定单个序列是否 可以解释对所有易受攻击的细胞的毒素作用。 肉毒杆菌毒素被认为作用于神经细胞和其他细胞 蛋白水解性裂解四种特定多肽的实验动物起源 (Synaptobrevin、Cellubrevin、SNAP-25和Synaxin)。然而，它还没有 已知这些是否是说明 对人体细胞的毒性作用。因此，将进行实验，以 鉴定和鉴定肉毒杆菌毒素的人体底物。在保持 根据临床中毒的性质，最初的重点将放在 研究人类神经细胞。随后的实验将在人体上进行。 脂肪细胞和人类肝细胞。 一般认为肉毒杆菌毒素对脆弱细胞有作用。 可以完全基于突触缩短蛋白的裂解来解释， Cellubrevin、SNAP-25和Synaxin，但这一点从未得到证实。 因此，申请者和他的同事将使用 选择性地将这些物质中的每一种从靶细胞中移除。其影响 与肉毒杆菌毒素中毒的效果进行比较 选择性地去除特定的底物。这将提供新的见解 转化为毒素作用，以及扩展我们的细胞生物学知识。

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.