Poster No. 8
Title:
Therapeutics Which Inhibit Botulinum Toxin Function and Accelerate Its Degradation Within Neurons
Authors:
Chueh-Ling Kuo, George Oyler, Jorge Sepulvedal, Saul Tzipori, Charles Shoemaker
Presented by:
Chueh-Ling Kuo
Departments:
Department of Biomedical Sciences, Cummings School of Veterinary Medicine; Synaptic Research
Abstract:
Botulinum neurotoxin (BoNT) is the most potent bacterial toxin, causing disease and creating a serious Category
A bioterror threat. Seven BoNT serotypes A-G inhibit neurotransmission by delivery of a protease to neurons
that cleave proteins necessary for exocytosis. BoNT/A is the most persistent and dangerous serotype, with
symptoms lasting up to a year in humans. In this project, we seek to accelerate intraneuronal BoNT protease
degradation by exploiting the host proteasome ubiquitin system. A series of constructions were prepared and
tested that are composed of a BoNT protease targeting domain and an E3 ligase/E3 ligase targeting domain.
These ubiquitin E3-ligases, once delivered to intoxicated neurons, should bind the BoNT protease and target it
for accelerated ubiquitination and turnover. The BoNT protease targeting domains are camelid heavy-chain
antibody fragments (VHH). Some of the VHHs are also potent inhibitors of the protease activity. Among all of
the E3 ligase targeting domains tested, we found that the F box protein, TrCP, was most effective. To date, we
developed E3 ligases consisting of an anti-BoNT/A protease domain fused to the TrCP F-box domain. These
ligases substantially accelerate BoNT/A protease degradation in cell based assay and protect neuronal cells from
the consequences of BoNT/A intoxication. Replacement of the BoNT protease targeting domain a VHH specific
to BoNT serotype B protease resulted in an E3 ligase promoting BoNT/B protease degradation. We are currently
developing a means to deliver the designer ligases to the cytosol of intoxicated neurons by fusing them to atoxic
mutants of Clostridial toxins. If successful, it should be possible to develop therapeutic E3 ligases against all
serotypes by selecting appropriate BoNT protease targeting fragments.
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