Abstract 484: Nitrosative Stress Enhances S-nitrosation Of Peroxiredoxins In The Heart
Nitric oxide (NO) is produced by different isoforms of NO-synthases and operates as a mediator of important cell signaling pathways. To explore thiol-based protein modifications in a situation of nitrosative stress, two transgenic mouse models recently generated in our laboratory were used:
Cardiac specific overexpression of inducible nitric oxide synthase (iNOS) (tg-iNOS+) and
tg-iNOS+ with concomitant myoglobin-deficiency (tg-iNOS+/myo−/−).
Protein S-nitrosation, an important redox-based posttranslational modification, revealed no differences between WT and tg-iNOS+ hearts as measured by the biotin-switch assay and 2-D PAGE. Even in the absence of myoglobin - an efficient endogenous NO-oxidase - the protein S-nitrosation pattern for nearly all the detected proteins (>40) remained unchanged in the tg-iNOS+/myo−/−hearts, with the exception of three proteins. Tandem mass spectrometry uncovered these proteins as peroxiredoxins (Prx II, III, VI), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides are reduced and detoxified. To prove whether the higher abundance of the Prxs was due to enhanced S-nitrosation or due to changes in their basal expression levels, immunoblotting with specific antibodies was applied and revealed upregulation of Prx VI in tg-iNOS+/myo−/− hearts. The other proteins found to be S-nitrosated were identified as well. Data mining indicated a significant overlap of these proteins with proteins becoming glutathiolated. Protein glutathiolation detected by immunoblotting was enhanced in the tg-iNOS+ hearts and even more so in the tg-iNOS+/myo−/− hearts. We conclude that protein glutathiolation in our transgenic model of nitrosative stress is important to protect protein thiols from irreversible oxidation. The upregulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Enhanced S-nitrosation of the Prxs may serve a new function in the signalling cascade coping with nitrosative stress.