A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage
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 DAFC 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).
References (22)
- et al.
Mechanisms of caspase activation
Curr. Opin. Cell Biol.
(2003) - et al.
Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo
J. Immunol. Methods
(1999) Caspases: key players in programmed cell death
Curr. Opin. Struct. Biol.
(2000)- et al.
Fluorometric and colorimetric detection of caspase activity associated with apoptosis
Anal. Biochem.
(1997) - et al.
Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis
J. Biol. Chem.
(1998) - et al.
Surface plasmon resonance imaging analysis of protein–protein interactions using on-chip-expressed capture protein
Anal. Biochem.
(2006) - et al.
Design of helical proteins for real-time endoprotease assays
Anal. Biochem.
(2000) - et al.
Caspases: preparation and characterization
Methods
(1999) - et al.
A fluorometric assay for HIV-protease activity using high-performance liquid chromatography
Anal. Biochem.
(1990) - et al.
A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis
J. Biol. Chem.
(1997)
Biochemical pathways of caspase activation during apoptosis
Annu. Rev. Cell Dev. Biol.
Cited by (2)
A Tunable, modular approach to fluorescent protease-activated reporters
2013, Biophysical JournalCitation Excerpt :Therefore, although the overall fluorescence intensity is low, cleavage of CA-mNeptune by active caspase-7 results in a >30-fold increase in signal in E. coli (Fig. 5 B), demonstrating that the quenching peptide works on other nonGFP-derived fluorescent proteins and at various locations. All three reporters can clearly be activated and show an improved signal/noise ratio relative to that of comparable reporters (22–30). Nevertheless, the CA-mNeptune, CA-CerFP, and CA-CitFP reporters showed a lower overall fluorescence intensity and a lower relative increase in fluorescence over the background than CA-GFP (Fig. 5 C).
Monitoring of cleavage preference for caspase-3 using recombinant protein substrates
2009, Journal of Microbiology and Biotechnology