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 its 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 leukemia cells with micromolar concentrations of Zn²⁺ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a cell-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn²⁺ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn²⁺ on PARP, a protein with two zinc finger domains, we used recombinant caspase-3 and a chromogenic tetrapeptide substrate containing the caspase-3 cleavage site. In this system, Zn²⁺ inhibited caspase-3 with an IC₅₀ of 0.1 μM. These results identify caspase-3 as a novel target of Zn²⁺ inhibition in apoptosis and suggest a regulatory role for Zn²⁺ in modulating the upstream apoptotic machinery.