An Engineered Viral Protease Exhibiting Substrate Specificity for a Polyglutamine Stretch Prevents Polyglutamine-Induced Neuronal Cell DeathReport as inadecuate




An Engineered Viral Protease Exhibiting Substrate Specificity for a Polyglutamine Stretch Prevents Polyglutamine-Induced Neuronal Cell Death - Download this document for free, or read online. Document in PDF available to download.

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 HttEx197Q-GFP in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx197Q-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.



Author: Saravanan Sellamuthu , Bae Hyun Shin , Hye-Eun Han, Sang Min Park, Hye Jin Oh, Seong-Hwan Rho, Yong Jae Lee, Woo Jin Park

Source: http://plos.srce.hr/



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