Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

Abstract

Calcineurin (CaN) is a Ca 2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe±Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H 2 O 2 was investigated. PAO and MEL inhibited CaN with an IC 50 of 3±8 mm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H 2 O 2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H 2 O 2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H 2 O 2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe 3 ±Zn 2 dinuclear centre. Upon H 2 O 2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe 2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures

“…Cys-299 of protein farnesyltransferase coordinates the zinc finger (38), and zinc finger thiols are well known to be redox-active (39). Cys-228 of calcineurin (protein phosphatase 2B) is recognized as one of two thiols that are most sensitive to oxidation (40). Very recently, Cys-442 of heat-shock protein 60 was reported to be alkylated by a natural electrophilic inhibitor, epolactaene (41).…”

Redox Control of Calcineurin by Targeting the Binuclear Fe2+-Zn2+ Center at the Enzyme Active Site

“…When bovine CaN was isolated as reported (10), its activity in initial experiments, contrary to the homogenate, was found to be insensitive to inhibition by XO/hypoxanthine (data not shown). During the calmodulin affinity chromatography step in the standard procedure of CaN isolation the protein mixture was in contact with high calcium concentrations.…”

“…CaN Isolation-Bovine brain CaN was isolated according to purification procedure of Sharma et al (18) as described in Bogumil et al (10). Porcine brain CaN purification involved the following important modifications; ammonium sulfate precipitation and Affi-Gel blue chromatography steps were omitted, and calmodulin affinity chromatography peak fractions were subjected to gel filtration on a Superdex 200 col-* This work was supported by grants from Deutsche Forschungsgemeinschaft (DFG).…”

“…(6) and hydrogen peroxide (H 2 O 2 ) (7-10) had been suggested as inhibitors of CaN activity, but the complex enzymology of CaN has led to conflicting conclusions about the mechanism of inhibition. With regard to the H 2 O 2 effect, we have postulated a dithiol-disulfide transformation, since inhibition of the isolated enzyme was accompanied by the disappearance of two thiol groups, and reactivation was achieved by thioredoxin and dithiols (10). Furthermore, CaN activity could be blocked by phenylarsine oxide, a selective agent for vicinal dithiols at low concentrations.…”

Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens

“…The center spot was loaded instead with phenylarsine oxide (PAO), a cytotoxic compound that inhibits protein-tyrosine phosphatases and bis-thiol-containing proteins and is cytotoxic to A549 human lung carcinoma cells (41)(42)(43)(44)(45). Subsequently, we seeded A549 cells onto this microarray and incubated the cells on the array for 38 h. The cells grew to confluence throughout the array except around the area containing the PAO-loaded polymer.…”