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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

Var26 prevents polyQ aggregation.A. SH-SY5Y cells were cotransfected with pcDNA-HttEx1(25Q) or (97Q)-GFP together with an empty vector (Vec) or, pcDNA3-WT 3CP (WT), pcDNA3-Var26 (Var26), or pcDNA3-C172A (C172A). After 48hrs of incubation, the cells were fixed and observed under a fluorescence microscope. The percentage of GFP-aggregation positive cells was plotted (n = 3). Error bars represent the SD. Significance was determined by student's t test. *p<0.05, **p<0.01. B. SH-SY5Y cells were treated as in panel A. After 48hrs of incubation, the cells were fixed and immunostained with the HP12 antibody. The percentage of cells containing amino-terminal aggregation was plotted (n = 4). Error bars represent the SD. Significance was determined by student's t test. **p<0.01.
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pone-0022554-g004: Var26 prevents polyQ aggregation.A. SH-SY5Y cells were cotransfected with pcDNA-HttEx1(25Q) or (97Q)-GFP together with an empty vector (Vec) or, pcDNA3-WT 3CP (WT), pcDNA3-Var26 (Var26), or pcDNA3-C172A (C172A). After 48hrs of incubation, the cells were fixed and observed under a fluorescence microscope. The percentage of GFP-aggregation positive cells was plotted (n = 3). Error bars represent the SD. Significance was determined by student's t test. *p<0.05, **p<0.01. B. SH-SY5Y cells were treated as in panel A. After 48hrs of incubation, the cells were fixed and immunostained with the HP12 antibody. The percentage of cells containing amino-terminal aggregation was plotted (n = 4). Error bars represent the SD. Significance was determined by student's t test. **p<0.01.

Mentions: Aggregation of the HttEx1-GFP fusion proteins was observed under a fluorescent microscope. Consistent with previous reports, no aggregation was observed with HttEx1(25Q)-GFP but significant aggregation was observed with HttEx1(97Q)-GFP. This protein aggregation was significantly blocked by Var26 but not by WT 3CP or C172A (Fig. 4A). This result indicates that cleavage of the polyQ stretch by Var26 leads to the dispersion of the resulting carboxy-terminus of the fusion protein. To test whether the amino-terminal fragment of the fusion protein that results from Var26-mediated cleavage is also dispersed, immunostaining was performed with the antibody HP12, which is specific for the amino-terminal 17 amino acids of Htt. The aggregation of the amino-terminal fragments was also significantly blocked. Moreover, HP12 immunofluorescence was indistinguishable from GFP fluorescence in all of the cells observed (Fig. 4B). This result indicates that the cleavage of HttEx1(97Q)-GFP by Var26 reduced aggregation of the fusion protein.


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)

Var26 prevents polyQ aggregation.A. SH-SY5Y cells were cotransfected with pcDNA-HttEx1(25Q) or (97Q)-GFP together with an empty vector (Vec) or, pcDNA3-WT 3CP (WT), pcDNA3-Var26 (Var26), or pcDNA3-C172A (C172A). After 48hrs of incubation, the cells were fixed and observed under a fluorescence microscope. The percentage of GFP-aggregation positive cells was plotted (n = 3). Error bars represent the SD. Significance was determined by student's t test. *p<0.05, **p<0.01. B. SH-SY5Y cells were treated as in panel A. After 48hrs of incubation, the cells were fixed and immunostained with the HP12 antibody. The percentage of cells containing amino-terminal aggregation was plotted (n = 4). Error bars represent the SD. Significance was determined by student's t test. **p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140514&req=5

pone-0022554-g004: Var26 prevents polyQ aggregation.A. SH-SY5Y cells were cotransfected with pcDNA-HttEx1(25Q) or (97Q)-GFP together with an empty vector (Vec) or, pcDNA3-WT 3CP (WT), pcDNA3-Var26 (Var26), or pcDNA3-C172A (C172A). After 48hrs of incubation, the cells were fixed and observed under a fluorescence microscope. The percentage of GFP-aggregation positive cells was plotted (n = 3). Error bars represent the SD. Significance was determined by student's t test. *p<0.05, **p<0.01. B. SH-SY5Y cells were treated as in panel A. After 48hrs of incubation, the cells were fixed and immunostained with the HP12 antibody. The percentage of cells containing amino-terminal aggregation was plotted (n = 4). Error bars represent the SD. Significance was determined by student's t test. **p<0.01.
Mentions: Aggregation of the HttEx1-GFP fusion proteins was observed under a fluorescent microscope. Consistent with previous reports, no aggregation was observed with HttEx1(25Q)-GFP but significant aggregation was observed with HttEx1(97Q)-GFP. This protein aggregation was significantly blocked by Var26 but not by WT 3CP or C172A (Fig. 4A). This result indicates that cleavage of the polyQ stretch by Var26 leads to the dispersion of the resulting carboxy-terminus of the fusion protein. To test whether the amino-terminal fragment of the fusion protein that results from Var26-mediated cleavage is also dispersed, immunostaining was performed with the antibody HP12, which is specific for the amino-terminal 17 amino acids of Htt. The aggregation of the amino-terminal fragments was also significantly blocked. Moreover, HP12 immunofluorescence was indistinguishable from GFP fluorescence in all of the cells observed (Fig. 4B). This result indicates that the cleavage of HttEx1(97Q)-GFP by Var26 reduced aggregation of the fusion protein.

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