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Calcineurin activity in T-lymphocytes has been shown to be sensitive to H(2)O(2). In this report, we investigate the effects of H(2)O(2)and other physiological oxidants on calcineurin activity both in vivo and in vitro. Intracellular calcineurin activity, as determined by NF-AT phosphorylation state and activity, is inhibited by H(2)O(2) but is insensitive to superoxide and NaOCl. Similarly, treatment of T-lymphocytes with NaOCl and paraquat does not drastically alter the activity of calcineurin in crude cell lysate, while H(2)O(2) causes significant inhibition. Sensitivity to H(2)O(2) and NaOCl in vivo correlates with the half-life of each species in cell medium. The intracellular redox potential is unaffected by H(2)O(2) (100 micro M) or NaOCl (600 micro M), indicating that H(2)O(2) inhibits calcineurin via a direct mechanism that does not involve a change in the cytosolic redox potential. In contrast, calcineurin activity in cell lysate is inhibited by all three oxidants. H(2)O(2) inactivation of calcineurin is rapid, with inactivation occurring in

For three decades, oxidative stress and the role of reactive oxygen species in biology have been extensively studied. Recently, a new interest in these areas has emerged with the discovery of superoxide reductases, a family of familiar bacterial metalloenzymes whose heretofore unknown function has now been apparently revealed. In a series of experiments utilizing genetic, molecular biological, and biochemical methods, these enzymes have been shown to be physiologically competent at removing superoxide. The role of these enzymes and their biological relationship to the well-known superoxide dismutases is discussed.

The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure. These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized. Both recombinant fragments behaved as independent metal-binding domains. The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif. The C-terminal fragment, which accommodates a Fe(Nepsilon-His)3(Ndelta-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide. The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c. The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

To explore whether the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin is subject to redox regulation in vivo, we used a luciferase reporter gene construct whose expression was controlled by the transcription factor NF-AT (the nuclear factor of activated T-cells) to monitor intracellular calcineurin activity following redox state perturbations. The NF-AT reporter construct was transfected into Jurkat cells, and luciferase activity was assessed following treatment with phorbol ester and ionomycin in the presence of either hydrogen peroxide or dithiothreitol (DTT). While DTT had no effect, H(2)O(2) completely abrogated NF-AT transactivation in response to stimulation. The inhibitory effect was specific for NF-AT as comparable levels of H(2)O(2) had only minor effects on constitutive transcription factors while an analogous construct under AP-1 control showed a 5-fold stimulation in transactivation in the presence of H(2)O(2). The inhibitory effect of H(2)O(2) was observed up to approximately 3 h following mitogen stimulation, a time point where NF-AT activity begins to increase under normal conditions. Protein serine/threonine phosphatase activities from Jurkat lysate indicated that calcineurin activity was inhibited not only by H(2)O(2) but also by high concentrations of DTT. These results indicate that calcineurin activity is subject to redox regulation in vivo and are discussed in the context of redox reactions involving active site metal ions.

Dephosphorylation of Ca2+ channels by the Ca(2+)-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca(2+)-dependent inactivation of Ca2+ current in rat pituitary tumor (GH3) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca2+ to the channel protein, the alternative "calcineurin hypothesis" has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH3 cells. To determine if calcineurin plays a part in the voltage- and/or Ca(2+)-dependent components of dihydropyridine-sensitive Ca2+ current decay, we rapidly altered the intracellular Ca2+ buffering capacity of GH3 cells by flash photolysis of DM-nitrophen, a high affinity Ca2+ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca2+ current inactivation in a two-pulse voltage protocol with Ca2+ as the charge carrier, but had no effect when Ba2+ was substituted for Ca2+. Despite confirmation of the abundance of calcineurin in the GH3 cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca(2+)-dependent inactivation of Ca2+ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca(2+)-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca(2+)-dependent inactivation after photolysis. The results provide strong evidence against Ca(2+)-dependent dephosphorylation as the mechanism of Ca2+ current inactivation in GH3 cells, but support the alternative idea that Ca(2+)-dependent inactivation reflects a direct effect of intracellular Ca2+ on channel gating.