title: An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death.
abstract: 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.
  
  
  RESULTS
  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
  These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.
authors.affiliation: College of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Sellamuthu
authors.forename: Bae Hyun
authors.initials: BH
authors.lastname: Shin
authors.forename: Hye-Eun
authors.initials: HE
authors.lastname: Han
authors.forename: Sang Min
authors.initials: SM
authors.lastname: Park
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Oh
authors.forename: Seong-Hwan
authors.initials: SH
authors.lastname: Rho
authors.forename: Yong Jae
authors.initials: YJ
authors.lastname: Lee
authors.forename: Woo Jin
authors.initials: WJ
authors.lastname: Park
mesh_terms: D000086042:3C Viral Proteases; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D030041:Hepatitis A virus / Q000201:enzymology; D006801:Humans; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D010455:Peptides / Q000378:metabolism* / Q000633:toxicity*; D015202:Protein Engineering; D059748:Proteolysis; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D010455:Peptides; D014764:Viral Proteins; C097188:polyglutamine; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases