earticle

영어

The apoptotic caspases have been classified according to their substrate specificities, as the optimal tetrapeptide recognition motifs for various caspases have been determined by the positional scanning substrate combinatorial library technology. Here we chose to focus on two proteolytic recognition motifs, DEVD and IETD, due to their wide use in cell-based apoptosis assay. Although, DEVE and IETD have been considered selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, this work for the non-specificity of the substrates was achieved with caspase-3 via evaluating the difference in proteolytic cleavage between DEVD- and IETD-based substrates. To design a rigorous protocol for this aim, we developed the DEVD- and IETD-based substrate, which are referred to as the GST:DEVD:EGFP and GST:IETD:EGFP, respectively. Using these substrates, the efficiency of caspase-3-induced proteolytic cleavages could be compared via the mass-based (Western blotting and SPR imaging), and fluorescence-based (on-chip visualization and bead assay) methods. Consequently, the GST:IETD:EGFP was partially cleaved in response to caspase-3, whereas GST:DEVD:EGFP completely proteolysed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Therefore, our results revealed that both GST:DEVD:EGFP and GST:IETD:EGFP substrates proved cleavable to caspase-3, although the GST:DEVD:EGFP substrate is more appropriate than the GST:IETD:EGFP substrate in favor of the proteolytic cleavage for caspase-3, which may potentially help to develop more rigorous caspase assay system for the measurement of caspase-3 activation during apoptosis.