Figure 5
Substitution of Met-44 by Leu attenuates the proteolytic maturation of caspase-3 in E. coli and MCF-7 cells. (a and b) Purified recombinant procaspase-3-D3A and its Met-to-Leu mutants (Met-27, Met-39 and Met-44) were treated with PDT using 28 μg/ml of Photofrin, followed by caspase-3 activity assays using Ac-DEVD-pNA (a) or endogenous PARP from A431 cell lysates (b). In (b), A431 cell lysates (100 μg) were digested with procaspase-3-D3A and its mutants at 37°C for 1 h, resolved by 10% SDS-PAGE, and subjected to immunoblotting with an anti-PARP antibody. ‘Act Casp3’ denotes the recombinant active form of caspase-3 purified from E. coli. (c) Wild-type procaspase-3 and its Met-to-Leu mutants were cloned into the pET23a vector, expressed in E. coli BL-21(DE3) cells via IPTG induction for different time periods, and analyzed by immunoblotting with an anti-caspase-3 antibody. FL, full-length form of procaspase-3; Ac, activated form of caspase-3. (d) Schematic representation of the structures of EGFP-tagged full-length procaspase-3, prodomain-deleted (ΔN) procaspase-3 and the Met-to-Leu mutants (Met-27, Met-39 and Met-44). The lower panel denotes the predicted MWs of the unprocessed and possible proteolytic forms of caspase-3 found in MCF-7 cells after the application of UV irradiation to trigger apoptosis. (e and f) Vectors encoding EGFP-tagged full-length procaspase-3, ΔN-procaspase-3 or the Met-to-Leu mutants were individually transfected to MCF-7 cells, and the transfected cells were selected with G418 (800 μg/ml) for 2 weeks. The stably selected cells were left untreated or irradiated with UV light (200 J/m2). Cell lysates were collected at 4, 8 and 24 h and subjected to 15% SDS-PAGE, followed by immunoblot analysis using antibodies against GFP, cleaved caspase-3 (D175) or actin (e), or by caspase-3 activity assay using Ac-DEVD-pNA (f). The experiments were repeated three times with reproducible results
