Mechanism of Dithiocarbamate Inhibition of Apoptosis: Thiol Oxidation by Dithiocarbamate Disulfides Directly Inhibits Processing of the Caspase-3 Proenzyme

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Abstract
Dithiocarbamates (DCs) have been reported to be potent inhibitors of apoptosis in several different model systems, which suggests a target common to the apoptotic machinery. Without further investigation, this has been assumed to reflect an antioxidant activity of the DCs. However, we have recently shown that DCs exert prooxidant effects on T cells [Nobel et al. (1995) J. Biol. Chem. 270, 26202−26208], which are dependent on their transfer of external copper into the cells and can be inhibited by the inclusion of high-affinity external copper chelators in the medium. Investigating antiapoptotic actions of DCs, we found that inclusion of a membrane-impermeable copper chelator severely compromised the inhibitory activity of reduced DCs. Since copper can promote DC oxidation to the respective DC disulfides, the inhibitory effect on lymphocyte apoptosis might be mediated by the DC disulfides. In agreement with this we observed that DC disulfides were more potent inhibitors of T cell apoptosis than their reduced counterparts. Inhibition of apoptosis by DC disulfides correlated with the inhibition of caspase-3 proenzyme processing and activation. Similar results were obtained in a cell-free model system of caspase-3 activation. Significantly, dithiothreitol reduction of the DC disulfide abolished its inhibition of in vitro proenzyme processing, thereby demonstrating thiol−disulfide exchange between the DC disulfide and a free thiol group on an activator(s) of caspase-3. Since T cell apoptosis involves the generation of mature caspase-3 and requires caspase-3-like activity, we propose that (1) DC disulfides are the active agents behind DC inhibition of apoptosis and (2) their site of action is the proteolytic activation of this enzyme. These findings also reveal the potential for other thiol-oxidizing toxicants to inhibit apoptosis by preventing the proteolytic activation of caspases.