Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments.
Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.
Wadsworth Center, New York State Department of Health, Albany, New York, USA.
David C
DC
Butler
Anne
A
Messer
D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology*; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D005091:Exons; D000071058:Huntingtin Protein; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009955:Ornithine Decarboxylase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis
D018033:Antibodies, Bispecific; D017931:DNA Primers; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase