Zinc Is a Potent Inhibitor of the Apoptotic Protease, Caspase-3

David K. Perry, Miriam J. Smyth, Henning R. Stennicke, Guy S. Salvesen, Patrick Duriez, Guy G. Poirier, Yusuf A. Hannun
1997 Journal of Biological Chemistry  
The prevention of apoptosis by Zn 2؉ 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 Zn 2؉ 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 Zn 2؉ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a
more » ... ll-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn 2؉ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn 2؉ 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, Zn 2؉ inhibited caspase-3 with an IC 50 of 0.1 M. These results identify caspase-3 as a novel target of Zn 2؉ inhibition in apoptosis and suggest a regulatory role for Zn 2؉ in modulating the upstream apoptotic machinery. Apoptosis is a genetically programmed process of cell death characterized by a series of distinct morphological changes (1). Normal development and tissue remodeling of multicellular organisms are dependent on apoptosis, whereas defects in this process have been implicated in a number of pathological conditions. The agents which induce cells to undergo apoptosis are diverse and include extracellular agents such as TNF-␣, 1 the Fas ligand, and chemotherapeutic agents. Intracellular agents which regulate apoptosis include proteases, phosphatases, and kinases, products of lipid metabolism, and the cations Ca 2ϩ and Zn 2ϩ . The influence of Zn 2ϩ on apoptosis is a well known phenomenon (2). In both in vitro and in vivo models, Zn 2ϩ supplementation prevents apoptosis induced by a variety of agents (3, 4). Moreover, cells grown under conditions of Zn 2ϩ deficiency will undergo spontaneous apoptosis (5-7) . The protective effect of Zn 2ϩ has been attributed to its inhibition of a Ca 2ϩ -and Mg 2ϩ -dependent endonuclease (8) , thereby causing inhibition of DNA fragmentation, a terminal step and hallmark of apoptosis. However, a number of observations suggest the existence of other and perhaps more relevant targets for Zn 2ϩ . First, the concentrations of Zn 2ϩ used to demonstrate anti-apoptotic effects in either intact cells or isolated nuclei have ranged from micromolar to millimolar levels. However, the concentrations of Zn 2ϩ used for inhibition of the purified Ca 2ϩ -dependent endonuclease were in the millimolar range (9). Second, it has recently been reported that in L929 cells, Zn 2ϩ inhibited both TNF-␣-and etoposide-induced cytotoxicity, as assessed by methylene blue staining, prior to effects on DNA fragmentation (10). Third, it is becoming more evident that the endonuclease functions in the "execution" rather than "regulation" phase of apoptosis, and therefore its inhibition may not prevent cell death. Fourth, Zn 2ϩ has been demonstrated to inhibit the protease responsible for cleavage of lamins in cell-free extracts (11). Recent studies have pointed to a role for a family of caspase proteases (formerly the ICE/Ced-3 proteases) (12) in apoptosis that act upstream of the endonuclease. Proteases in apoptosis came to the forefront with studies on the proteolysis of poly-(ADP-ribose) polymerase (PARP), initially described in cells induced to undergo apoptosis by various chemotherapeutic agents, including etoposide (13-15). This event was later determined to be catalyzed by the protease resembling ICE (16). The human homolog of this protease has been cloned and is now known as caspase-3 (CPP32/yama/apopain). To determine if Zn 2ϩ inhibits an event upstream of endonuclease activation, we investigated the effect of Zn 2ϩ on etoposide-induced apoptosis using PARP proteolysis as an indicator of this process. We show that Zn 2ϩ is a potent inhibitor of PARP proteolysis in intact cells. We also demonstrate in a cell-free system that Zn 2ϩ potently inhibits PARP proteolysis induced both by a caspase-3-containing apoptotic extract and by purified recombinant caspase-3. These results identify caspase-3 as a novel and proximal site of Zn 2ϩ inhibition in the apoptotic pathway. Moreover, in light of results demonstrating a labile pool of Zn 2ϩ in cells which is in rapid equilibrium with the extracellular medium (17), these findings raise the possibility that the cell may utilize Zn 2ϩ as a regulator of the upstream apoptotic machinery. EXPERIMENTAL PROCEDURES Cell Culture-Molt4 cells from ATCC (Rockville, MD) were maintained under subconfluent conditions in RPMI medium with 10% fetal calf serum. For experiments, cells were diluted to 5 ϫ 10 5 /ml in RPMI
doi:10.1074/jbc.272.30.18530 pmid:9228015 fatcat:xbpd7amdazftxivpx637zhrrju