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An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death.

Sellamuthu S, Shin BH, Han HE, Park SM, Oh HJ, Rho SH, Lee YJ, Park WJ - PLoS ONE (2011)

Bottom Line: 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'.Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP.These protective effects of Var26 were proteolytic activity-dependent.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.

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.

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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Related in: MedlinePlus

Engineering scheme for the isolation of polyQ cleaving variants.Screening of the S2 pocket variants for P2-Q cleavage yielded 54 positive candidates (12 from HKKL, 23 from VMHK, and 19 from MHKK libraries). Screening of the S1' pocket variants for P1'-Q cleavage resulted in 23 clones from the LPL library. These variants were combined through shuffling to generate a pool of variants in which each protease possesses S2 as well as S1' pocket mutations. These variants were then screened for Q8 substrate cleavage yielding 11 candidates with low activity. These mutants were further subjected to two rounds of error prone PCR and shuffling coupled with selection through GASP to generate 44 variants with moderate activity.
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pone-0022554-g002: Engineering scheme for the isolation of polyQ cleaving variants.Screening of the S2 pocket variants for P2-Q cleavage yielded 54 positive candidates (12 from HKKL, 23 from VMHK, and 19 from MHKK libraries). Screening of the S1' pocket variants for P1'-Q cleavage resulted in 23 clones from the LPL library. These variants were combined through shuffling to generate a pool of variants in which each protease possesses S2 as well as S1' pocket mutations. These variants were then screened for Q8 substrate cleavage yielding 11 candidates with low activity. These mutants were further subjected to two rounds of error prone PCR and shuffling coupled with selection through GASP to generate 44 variants with moderate activity.

Mentions: The individually selected variants from S2 pocket and S1′ pocket engineering cleaved the P2-Q and P1′-Q substrates respectively, but none of the selected variants could cleave the polyQ substrate (data not shown). Therefore, the variants from the four independent screenings were combined by shuffling to generate a pool of variants that contained various mutations in both the S2 and S1′ pockets (Fig. 2). The resulting library was screened using the Q8 substrate (QQQQQ↓QQQ), which contains glutamine at all eight sites, and eleven positive clones were selected (Table S5). All the selected variants appeared to have relatively low proteolytic activity, as the colonies grew slowly on selective plates and produced a faint blue color on X-gal plates (data not shown). These eleven candidate clones were pooled together and were used as template for further mutagenesis by error prone PCR and DNA shuffling. After two rounds of mutagenesis and selection, 44 variants that appeared to have moderate proteolytic activity against the Q8 substrate were finally obtained (Table S6). The clone that appeared to have the highest proteolytic activity, variant 26 (Var26), was tested for its effects in an in vitro model of HD.


An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death.

Sellamuthu S, Shin BH, Han HE, Park SM, Oh HJ, Rho SH, Lee YJ, Park WJ - PLoS ONE (2011)

Engineering scheme for the isolation of polyQ cleaving variants.Screening of the S2 pocket variants for P2-Q cleavage yielded 54 positive candidates (12 from HKKL, 23 from VMHK, and 19 from MHKK libraries). Screening of the S1' pocket variants for P1'-Q cleavage resulted in 23 clones from the LPL library. These variants were combined through shuffling to generate a pool of variants in which each protease possesses S2 as well as S1' pocket mutations. These variants were then screened for Q8 substrate cleavage yielding 11 candidates with low activity. These mutants were further subjected to two rounds of error prone PCR and shuffling coupled with selection through GASP to generate 44 variants with moderate activity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140514&req=5

pone-0022554-g002: Engineering scheme for the isolation of polyQ cleaving variants.Screening of the S2 pocket variants for P2-Q cleavage yielded 54 positive candidates (12 from HKKL, 23 from VMHK, and 19 from MHKK libraries). Screening of the S1' pocket variants for P1'-Q cleavage resulted in 23 clones from the LPL library. These variants were combined through shuffling to generate a pool of variants in which each protease possesses S2 as well as S1' pocket mutations. These variants were then screened for Q8 substrate cleavage yielding 11 candidates with low activity. These mutants were further subjected to two rounds of error prone PCR and shuffling coupled with selection through GASP to generate 44 variants with moderate activity.
Mentions: The individually selected variants from S2 pocket and S1′ pocket engineering cleaved the P2-Q and P1′-Q substrates respectively, but none of the selected variants could cleave the polyQ substrate (data not shown). Therefore, the variants from the four independent screenings were combined by shuffling to generate a pool of variants that contained various mutations in both the S2 and S1′ pockets (Fig. 2). The resulting library was screened using the Q8 substrate (QQQQQ↓QQQ), which contains glutamine at all eight sites, and eleven positive clones were selected (Table S5). All the selected variants appeared to have relatively low proteolytic activity, as the colonies grew slowly on selective plates and produced a faint blue color on X-gal plates (data not shown). These eleven candidate clones were pooled together and were used as template for further mutagenesis by error prone PCR and DNA shuffling. After two rounds of mutagenesis and selection, 44 variants that appeared to have moderate proteolytic activity against the Q8 substrate were finally obtained (Table S6). The clone that appeared to have the highest proteolytic activity, variant 26 (Var26), was tested for its effects in an in vitro model of HD.

Bottom Line: 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'.Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP.These protective effects of Var26 were proteolytic activity-dependent.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.

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.

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.

Show MeSH
Related in: MedlinePlus