The biological activity of ubiquitinated BoNT/B light chain in vitro and in human SHSY-5Y neuronal cells.
BoNT/B light chain is a zinc-dependent endopeptidase. After entering its target, the neuronal cell, BoNT/B is responsible for synaptobrevin-2 (VAMP-2) cleavage. This results in reduced neurotransmitter (acetylcholine) release from synaptic vesicles, yielding muscular paralysis. Since the toxin persists in neuronal cells for an extended period, regeneration of VAMP-2 is prevented. We evaluated therapeutic targets to overcome botulinum persistence because early removal would rescue the neuronal cell. The ubiquitination/proteasome cellular pathway is responsible for removing "old" or undesirable proteins. Therefore, we assessed ubiquitination of BoNT/B light chain in vitro, and characterized the effects of ubiquitination modulating drugs, PMA (phorbol 12-myristate 13-acetate) and expoxomicin, on ubiquitination of BoNT/B light chain in neuronal cells. Both drugs altered BoNT/B light chain ubiquitination. Ubiquitination in vitro and in cells decreased the biological activity of BoNT/B light chain. These results further elucidate BoNT protein degradation pathways in intoxicated neuronal cells and mechanisms to enhance toxin removal.
Shi
Xuerong
X
Department of Regulated Laboratories, Division of Regulated Activities, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910-7500, USA. xuerong.shi@amedd.army.mil
Curran
Jennifer E
JE
Liao
Zhilin
Z
Gordon
Richard K
RK
D015153:Blotting, Western; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D057149:Ubiquitinated Proteins / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
D057149:Ubiquitinated Proteins; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D013755:Tetradecanoylphorbol Acetate