MECHANISM OF BOTULINUM NEUROTOXIN ACTION

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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.