Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

摘要

Botulinum neurotoxin has no cure once it has entered neurons. One approach to finding a treatment is to determine the molecules that regulate the half-life of the toxin in the cell. The serotype BoNT/A has a very long half- life (months) whereas the serotype BoNT/E has a short half-life (days). We have made a chimeric protein consisting of the light chains (LCs) of both toxins and found the half-life to be short, similar to light chain E (LcE). This finding suggests that the molecules controlling intracellular degradation of LcE are dominant over those that control LcA. We have also completed an assessment of neuronal proteins that bind each LC by Yeast 2 Hybrid (Y2H) analysis. We have assessed the contributions of these molecules to LC half-life by knock-down experiments. Furthermore, we have produced a designer ligase which is comprised of a VHH antibody binding region specific for LcA that is coupled to a ubiquitin ligase molecule. When delivered intracellularly as a plasmid by transfection, this molecule has been shown to shorten the half-life of LcA by ubiquitination and targeting to the proteasome. We have inserted the designer ligase into a protein delivery system and have found the protein to be unstable. We are working on producing a more stable chimeric protein and producing adequate amounts for in vitro testing.