Key result
Engineered viral protease Var26 reduces pathogenic huntingtin aggregation and cytotoxicity in neuroblastoma cells.
p-value: p=<0.05
An engineered viral protease capable of cleaving polyglutamine stretches prevents polyQ-induced neuronal cell death in vitro, offering a potential therapeutic strategy for neurodegenerative diseases like Huntington's.
Hypothesis-generating for polyQ disorders; leaves open whether engineered proteases can be advanced to in vivo or clinical testing.
BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.
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Sellamuthu et al. (2011) studied Polyglutamine diseases (Huntington's disease model). Engineered viral protease (Var26) vs. Wild-type HAV 3C protease or inactive mutant (C172A) was evaluated on Polyglutamine-induced neuronal cell death and protein aggregation (p=<0.05). The engineered viral protease Var26 significantly reduced the aggregation and cytotoxicity of a huntingtin exon1-GFP fusion protein containing a pathogenic polyglutamine stretch in SH-SY5Y neuroblastoma cells.
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