A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage

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Abstract

In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione–agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct.

Introduction

Caspases, a family of conserved cysteine proteases that normally cleave after an aspartate residue (D) in their substrates, execute apoptosis, also referred to as programmed cell death (PCD) (Budihardjo et al., 1999, Cohen, 1997, Shi, 2004). The apoptotic caspases have been classified as initiator caspases (including caspases-8 and -9) or executioner caspases (including caspases-3, -6, and -7) (Budihardjo et al., 1999, Grutter, 2000). The caspases involved in apoptosis can be distinguished in accordance with their substrate specificities (Thornberry et al., 1997). Particularly, the primary effector caspases, including caspases-3 and -7, display a preference for substrates harboring the 4-amino acid recognition sequence of the type DEVD, which is detected in many cytosolic and nuclear caspase substrates (Nicholson et al., 1995, Thornberry et al., 1997, Zhivotovsky, 2003). Caspase-3 has been shown to perform a pivotal function in the execution of apoptosis, as this protease is required for the degradation of chromosomes into nucleosomal fragments, and promotes the activation of other effector caspases (Boatright and Salvesen, 2003, Porter and Jänicke, 1999). Activated caspase-3 cleaves a variety of cellular substrates, including poly-(ADP-ribose) polymerase (PARP), the inhibitor of CAD (caspase-activated deoxyribonuclease) (ICAD), and alpha-fodrin, which induces cell death (Counis and Torriglia, 2000, Jänicke et al., 1998, Stroh and Schulze-Osthoff, 1998). Therefore, the modulation of caspase-3 and apoptosis may be a potential therapeutic strategy targeted to the apoptotic pathway.

Thus far, the most frequently utilized method for the measurement of caspase-3 activity is the fluorometric assay (Gurtu et al., 1997). The conventional fluorometric protease assay technique using a fluorogenic substrate, DEVD:AFC (7-amino-4-trifluoromethyl coumarin), was designed in an effort to detect the specific enzymatic activation of caspase-3 (Gurtu et al., 1997). Active caspase-3 cleaves artificial substrates consisting of an appropriate sequence of four amino acids (DEVD) (Nicholson et al., 1995). Caspase-3 cleaves the substrate on the C-terminal side of an aspartate residue which can be bound to a fluorogenic or chromogenic amine, and the cleavage of the Dsingle bondAFC bond subsequently releases the fluorophore AFC. Upon the addition of an active caspase-3 to the well plate, the DEVD:AFC fluorogenic substrate is cleaved, subsequently releasing a fluorescent label which can be detected using a fluorescence plate reader. Likewise, an in vitro assay predicated on changes in the fluorescence properties of the DEVD:MCA (4-methylcoumarinyl-7-amide) substrate has also been developed (Gorman et al., 1999). Although fluorometric assays have been widely utilized to determine the proteolytic activity of proteases due to its high sensitivity and low detection limit, the conventional experimental methods, artificial fluorogenic peptide assays, is not cost-effective, and requires expensive equipment such as a fluorometer or fluorescence microtiter plate reader, thus necessitating more economic and simpler methods.

To this aim, we have developed a newly designed chimeric protein reporter enabling the monitoring of caspase-3 activity. This reporter is based on the caspase-3 specific proteolytic property, which is referred to as GST:DEVD:EGFP, linked together by a linker peptide harboring a consensus DEVD, the sequence specific for caspase-3 cleavage. With this reporter that can be easily and economically produced by in-house purification system, caspase-3 activation could be measured successfully by a variety of bioanalytical methods, including immunoblotting, glutathione–agarose bead assay, and on-chip visualization.

Section snippets

Strains, vectors, and enzymes

Escherichia coli strain D H 5α was utilized as a host for subcloning and E. coli BL21 (DE3) (Novagen, WI) was used for gene expression. The E. coli strain was grown in LB medium at 37 °C and 50 mg/ml of ampicillin was added for the plasmid-harboring strains. pBluescript SK+ (Stratagene, CA) was utilized as a vector for the subcloning and amplification of the full-length gene, and pGEX 4T-1 (Amersham Pharmacia Biotech, CA) for expression. The restriction enzymes and modifying enzymes were obtained

Rationale for GST:DEVD:EGFP reporter-based monitoring of caspase-3 activation

The most commonly utilized experimental method for the monitoring of caspase-3 activation is a fluorometric assay using artificial fluorogenic substrate for caspase-3 (DEVD–AFC) (Los et al., 1995, Enari et al., 1996). Although fluorometric analysis is sensitive, and is suitable for the measurement of small quantities of enzymatic activity, the reagents are substantially expensive, and also necessitate expensive equipment such as a fluorometer or fluorescence microtiter plate reader, thus

Conclusion

The GST:DEVD:EGFP reporter proved useful in the monitoring of caspase-3 activation on the basis of proteolytic properties, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. This study constitutes the first application employing a chimeric protein reporter, with the similar accuracy level compared to the conventional methods such as fluorometric assays. In the current study, we have evaluated only the caspase-3 reporter; however, the

Acknowledgement

This research was supported by grants from the Nano/Bio Science & Technology Program (2005-01333, MEST, Korea), the KRIBB Initiative Research Program (KRIBB, Korea), and the BioGreen21 program (RDA, Korea).

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