MECHANISM OF BOTULINUM NEUROTOXIN ACTION

肉毒神经毒素的作用机制

• 批准号: 2431265
• 负责人: BAL RAM SINGH
• 金额: $ 10.17万
• 依托单位: UNIVERSITY OF MASSACHUSETTS DARTMOUTH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2431265
• 关键词: Clostridium; PC12 cells; acetylcholine; atomic absorption spectrometry; binding proteins; botulinum toxins; chemical binding; circular dichroism; dialysis; enzyme linked immunosorbent assay; fluorescence spectrometry; guanine nucleotide binding protein; infrared spectrometry; inhibitor /antagonist; intracellular; laboratory rat; neuromuscular junction; neurotoxins; neurotransmitter transport; nicotinamide adenine dinucleotide; protein structure function; toxicant interaction; zinc

Seven serotypes of botulinum neurotoxin are a group of large proteins (150 kDa) with light (50 kDa) and heavy (100 kDa) chain subunits that act on the presynaptic nerve cells of the neuromuscular junctions. Neurotoxins act intracellulary to block acetylcholine neurotransmitter release leading to the flaccid muscle paralysis in the dreaded botulism disease. In general, the botulism disease results from ingestion of food where anaerobic bacterium, Clostridium botulinum, has grown and produced the neurotoxin. A more frequent form of botulism, infant botulism, occurs from the ingestion of ubiquitous spores of Clostridium botulinum which produces the neurotoxin in the intestinal tract of infants. Biomedical implications of botulinum neurotoxins are not limited to only botulism disease; because of their high potency and specificity at the neuro-muscular junctions, the neurotoxins are being used as therapeutic agents to treat several neuro- muscular diseases such as blepharospasm, strabismus, torticollis, etc., and have potential to be used for many more neuro-muscular disorders. In their mode of action, botulinum neurotoxins bind to the cell surface, and are internalized through endocytosis. At least the light chain (toxic) subunit is translocated into the cytosol, where it blocks the neurotransmitter release through interference with the biochemical and biophysical events of exocytosis. Although it has recently been reported that botulinum neurotoxins act as Zn2+ proteases on the constitutive components of the vesicular exocytosis, the molecular basis and the exact biochemical mechanism of their toxic action is not fully understood. The long-term goals of our study are to investigate (a) the molecular basis of the toxic activity of botulinum neurotoxins, and (b) the intracellular mechanism(s) involved in the neurotoxin-mediated blockage of the neurotransmitter release. Specific aims to be addressed in the next five years are (i) to investigate the role of metal binding on the structure and function of the neurotoxin using spectroscopic methods and neurotransmitter release assay; (ii) to identify peptide segments of neurotoxin light chain that form the toxic site using synthetic peptides as antagonists; and (iii) to investigate the possibility of binding of nicotinamide adenine dinucleotide to the light chain using intrinsic fluorescence quenching and equilibrium dialysis methods.

肉毒杆菌神经毒素的七种血清型是一组大蛋白（150 kDa）与轻链（50 kDa）和重链（100 kDa）亚基，其作用于 神经肌肉接头的突触前神经细胞。神经毒素 作用于细胞内阻断乙酰胆碱神经递质释放， 到可怕的肉毒杆菌中毒引起的肌肉麻痹。在 一般来说，肉毒杆菌中毒是由于摄入食物， 厌氧细菌肉毒梭菌已经生长并产生了 神经毒素一种更常见的肉毒杆菌中毒形式，婴儿肉毒杆菌中毒，发生在 摄入了无处不在的肉毒梭菌孢子， 婴儿肠道中的神经毒素。生物医学意义 肉毒杆菌神经毒素不仅限于肉毒杆菌病;因为 它们在神经肌肉接头处的高效力和特异性， 神经毒素被用作治疗剂来治疗几种神经- 肌肉疾病如眼睑痉挛、斜视、斜颈等， 并且具有用于更多神经肌肉疾病的潜力。 在它们的作用模式中，肉毒杆菌神经毒素结合到细胞表面， 并通过内吞作用内化。至少轻链（有毒） 亚基被转移到胞质溶胶中，在那里它阻断了 神经递质释放通过干扰生物化学和 胞吐作用的生物物理事件。尽管最近有报道称 肉毒杆菌神经毒素作为Zn 2+蛋白酶作用于组成型 泡胞吐的组成部分，分子基础和确切的 其毒性作用的生化机制尚未完全了解。 我们研究的长期目标是研究（a）分子 肉毒杆菌神经毒素毒性活性的基础，和（B） 参与神经毒素介导的阻断的细胞内机制 神经递质的释放下一次会议将讨论的具体目标 五年是（一）调查金属结合的作用， 结构和功能的神经毒素使用光谱方法， 神经递质释放测定;（ii）鉴定神经递质的肽段， 使用合成肽形成毒性位点的神经毒素轻链 作为拮抗剂;和（iii）研究结合的可能性， 烟酰胺腺嘌呤二核苷酸的轻链， 荧光猝灭法和平衡透析法。