Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.
Calcineurin (CaN) is a Ca2+-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 H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM 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 H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 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 H2O2 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 Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.
Bogumil
R
R
Fakultät für Biologie, Universität Konstanz, Germany. bogumil@voodoo.gk-biochem.uni-konstanz.de
Namgaladze
D
D
Schaarschmidt
D
D
Schmachtel
T
T
Hellstern
S
S
Mutzel
R
R
Ullrich
V
V
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D065095:Calcineurin Inhibitors; D002417:Cattle; D004220:Disulfides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D010084:Oxidation-Reduction; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism
D001152:Arsenicals; D065095:Calcineurin Inhibitors; D004220:Disulfides; D004791:Enzyme Inhibitors; C029341:oxophenylarsine; D014050:Toluene; D006861:Hydrogen Peroxide; C004848:dithiol