ZINC IS A POTENT INHIBITOR OF THE APOPTOTIC PROTEASE, CASPASE-3 - A NOVEL TARGET FOR ZINC IN THE INHIBITION OF APOPTOSIS

The prevention of apoptosis by Zn2+ has generally been attributed to i ts inhibition of an endonuclease acting in the late phase of apoptosis . In this study we investigated the effect of Zn2+ on an earlier event in the apoptotic process, the proteolysis of the ''death substrate'' poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4 leuke mia cells with micromolar concentrations of Zn2+ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide, U sing a cell-free system consisting of purified bovine PARP as a substr ate and an apoptotic extract or recombinant caspase-3 as the PARP prot ease, Zn2+ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn2+ on PARP, a protein with two zinc finger dom ains, we used recombinant caspase-3 and a chromogenic tetrapeptide sub strate containing the caspase-3 cleavage site. In this system, Zn2+ in hibited caspase-3 with an IC50 of 0.1 mu M. These results identify cas pase-3 as a novel target of Zn2+ inhibition in apoptosis and suggest a regulatory role for Zn2+ in modulating the upstream apoptotic machine ry.