Mass spectrometric analysis of nitric oxide-modified caspase-3.
Caspases are a family of cysteine proteases activated during apoptosis. Modification of caspases by nitric oxide and its relevance during apoptosis is currently a controversial subject. In this study we analyzed the S-nitrosated form of caspase-3 at a molecular level. By using electrospray ionization-mass spectrometry, we detected poly-S-nitrosation of caspase-3 with an average of about 2 molecules of NO bound per enzyme. Although NO treatment completely inhibited enzyme activity, S-nitrosation was not restricted to the active site cysteine. Rather, we detected multiple relative mass increases of 30 +/- 1 Da in both the p12 and p17 subunits of caspase-3, corresponding to single to triple S-nitrosation. The stability of these S-nitrosations differed in physiologically relevant concentrations of 5 mM glutathione. Whereas all S-nitroso bonds in the p12 subunit were cleaved with release of NO and partial formation of protein-mixed disulfides with glutathione, a single S-nitrosation in the p17 subunit remained stable. Since this S-nitrosation was not observed in a mutant form of caspase-3 lacking the active site cysteine, we conclude that NO nitrosates the active site cysteine of caspase-3 and that this modification is notably inert to fast trans-nitrosation with glutathione. Furthermore, we provide evidence that treatment of caspase-3 with NO can lead to mixed disulfide formation with glutathione, demonstrating the oxidative character of NO.
Department of Medicine IV, Experimental Division, Faculty of Medicine, University of Erlangen-Nürnberg, Loschgestrasse 8, 91054 Erlangen, Germany.
B
B
Zech
M
M
Wilm
R
R
van Eldik
B
B
Brüne
D053148:Caspase 3; D020169:Caspases / Q000737:chemistry*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D009569:Nitric Oxide / Q000737:chemistry*; D010084:Oxidation-Reduction
D004792:Enzyme Precursors; D009569:Nitric Oxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases