Limits...
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.

Show MeSH

Related in: MedlinePlus

Principle of GASP.A. A fusion protein containing the Ste2 transmembrane domain followed by a polyQ substrate linker and a LexA-b42 transcription factor was expressed in yeast EGY48 cells using a constitutive ADH promoter. Co-expression of galactose-inducible WT HAV 3CP does not result in cleavage of the polyQ substrate sequence (upper panel), while a conceptual 3CP variant cleaves the polyQ linker, causing the release of the transcription factor from the membrane, which in turn activates the reporter genes, Leu2 and LacZ (lower panel). B. EGY48 cells were transformed with pGAL-HAV3CP and pADH-Ste2-Substrate-Lex in which glutamine is substituted in the P5-P3′ positions of the 3CP substrate sequence (see Table S1). Cleavage of the indicated substrate sequence was evident by the growth of yeast cells in leucine-deficient medium and the formation of a blue color on X-gal medium.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3140514&req=5

pone-0022554-g001: Principle of GASP.A. A fusion protein containing the Ste2 transmembrane domain followed by a polyQ substrate linker and a LexA-b42 transcription factor was expressed in yeast EGY48 cells using a constitutive ADH promoter. Co-expression of galactose-inducible WT HAV 3CP does not result in cleavage of the polyQ substrate sequence (upper panel), while a conceptual 3CP variant cleaves the polyQ linker, causing the release of the transcription factor from the membrane, which in turn activates the reporter genes, Leu2 and LacZ (lower panel). B. EGY48 cells were transformed with pGAL-HAV3CP and pADH-Ste2-Substrate-Lex in which glutamine is substituted in the P5-P3′ positions of the 3CP substrate sequence (see Table S1). Cleavage of the indicated substrate sequence was evident by the growth of yeast cells in leucine-deficient medium and the formation of a blue color on X-gal medium.

Mentions: The principle underlying GASP was previously published and is illustrated in Fig. 1A. Cleavage in the substrate site of a membrane-anchored fusion protein (STE2-substrate-LexA-b42) induces the release of the LexA-b42 moiety from the plasma membrane and activates the expression of the reporter genes, Leu2 and LacZ. Protease expression is under the control of the Gal1 promoter and is, thus induced by the addition of galactoseto the media. Growth on selective plates and formation of a blue color on X-gal plates indicates site-specific proteolysis. Using this method, the ability of the HAV 3C protease (3CP) to cleave substrates containing glutamine substituted at different sites was analyzed. Substrate linker oligonucleotides encoding the wild-type (WT) substrate sequence (ELRTQ↓SFS) and mutated substrate sequences (P5-Q ∼ P3′-Q) in which glutamine residues replaced the native residues from P5 through P3' (Table S1) were prepared and cloned into the pADH-Ste2-Lex vector. Each of these substrate vectors was transformed, along with the protease vector, pGAL-3CP, into the yeast strain EGY48. The results from the selective plates and the X-gal plates suggested that glutamine is tolerated at the P5, P4, P3, P1, P2′, and P3′, but not at the P2 and P1′ positions (Fig. 1B). An oligonucleotide encoding a substrate (P5-3′Q(TS)) containing glutamine residues at all sites except the P2 and P1′ positions (QQQTQ↓SQQ) was then prepared. This substrate was cleaved by 3CP (Fig. 1B). Therefore, the S2 and S1′ pockets were subjected to further engineering to generate PQP.


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)

Principle of GASP.A. A fusion protein containing the Ste2 transmembrane domain followed by a polyQ substrate linker and a LexA-b42 transcription factor was expressed in yeast EGY48 cells using a constitutive ADH promoter. Co-expression of galactose-inducible WT HAV 3CP does not result in cleavage of the polyQ substrate sequence (upper panel), while a conceptual 3CP variant cleaves the polyQ linker, causing the release of the transcription factor from the membrane, which in turn activates the reporter genes, Leu2 and LacZ (lower panel). B. EGY48 cells were transformed with pGAL-HAV3CP and pADH-Ste2-Substrate-Lex in which glutamine is substituted in the P5-P3′ positions of the 3CP substrate sequence (see Table S1). Cleavage of the indicated substrate sequence was evident by the growth of yeast cells in leucine-deficient medium and the formation of a blue color on X-gal medium.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022554-g001: Principle of GASP.A. A fusion protein containing the Ste2 transmembrane domain followed by a polyQ substrate linker and a LexA-b42 transcription factor was expressed in yeast EGY48 cells using a constitutive ADH promoter. Co-expression of galactose-inducible WT HAV 3CP does not result in cleavage of the polyQ substrate sequence (upper panel), while a conceptual 3CP variant cleaves the polyQ linker, causing the release of the transcription factor from the membrane, which in turn activates the reporter genes, Leu2 and LacZ (lower panel). B. EGY48 cells were transformed with pGAL-HAV3CP and pADH-Ste2-Substrate-Lex in which glutamine is substituted in the P5-P3′ positions of the 3CP substrate sequence (see Table S1). Cleavage of the indicated substrate sequence was evident by the growth of yeast cells in leucine-deficient medium and the formation of a blue color on X-gal medium.
Mentions: The principle underlying GASP was previously published and is illustrated in Fig. 1A. Cleavage in the substrate site of a membrane-anchored fusion protein (STE2-substrate-LexA-b42) induces the release of the LexA-b42 moiety from the plasma membrane and activates the expression of the reporter genes, Leu2 and LacZ. Protease expression is under the control of the Gal1 promoter and is, thus induced by the addition of galactoseto the media. Growth on selective plates and formation of a blue color on X-gal plates indicates site-specific proteolysis. Using this method, the ability of the HAV 3C protease (3CP) to cleave substrates containing glutamine substituted at different sites was analyzed. Substrate linker oligonucleotides encoding the wild-type (WT) substrate sequence (ELRTQ↓SFS) and mutated substrate sequences (P5-Q ∼ P3′-Q) in which glutamine residues replaced the native residues from P5 through P3' (Table S1) were prepared and cloned into the pADH-Ste2-Lex vector. Each of these substrate vectors was transformed, along with the protease vector, pGAL-3CP, into the yeast strain EGY48. The results from the selective plates and the X-gal plates suggested that glutamine is tolerated at the P5, P4, P3, P1, P2′, and P3′, but not at the P2 and P1′ positions (Fig. 1B). An oligonucleotide encoding a substrate (P5-3′Q(TS)) containing glutamine residues at all sites except the P2 and P1′ positions (QQQTQ↓SQQ) was then prepared. This substrate was cleaved by 3CP (Fig. 1B). Therefore, the S2 and S1′ pockets were subjected to further engineering to generate PQP.

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