Abstract
Nitric oxide (NO) is produced by different isoforms of nitric oxide synthases (NOSs) and operates as a mediator of important cell signaling pathways, such as the cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated through S-nitrosation of target proteins. To explore thiol-based protein modifications in a situation of defined nitrosative stress, we used a transgenic mouse model with cardiac specific overexpression of inducible nitric oxide synthase (iNOS) and concomitant myoglobin deficiency (iNOS(+)/myo(-/-)). In comparison with the wild type hearts, protein glutathiolation detected by immunoblotting was significantly enhanced in iNOS(+)/myo(-/-) hearts, whereas protein S-nitrosation as measured by the biotin switch assay and two-dimensional PAGE revealed that nearly all of the detected proteins ( approximately 60) remained unchanged with the exception of three proteins. Tandem mass spectrometry revealed these proteins to be peroxiredoxins (Prxs), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite, and a wide range of organic hydroperoxides are reduced and detoxified. Immunoblotting with specific antibodies revealed up-regulation of Prx VI in the iNOS(+)/myo(-/-) hearts, whereas expression of Prx II and Prx III remained unchanged. Furthermore, the analysis of the cardiac S-nitrososubproteome identified several new proteins possibly being involved in NO-signaling pathways. Our data indicate that S-nitrosation and glutathiolation of cardiac proteins may contribute to the phenotype of NO-induced heart failure. The up-regulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, S-nitrosation of Prxs may serve a new function in the signaling cascade of nitrosative stress.
Highlights
17440 JOURNAL OF BIOLOGICAL CHEMISTRY of vascular tone, cardiac contraction, relaxation, and energetics [1, 2]
In order to evaluate which of these modifications dominates in a mixture of proteins derived from a mouse heart homogenate, samples were incubated with GSNO and examined by application of the biotin switch method (Fig. 2) and use of an antibody directed against S-glutathiolated proteins (PSSG) (Fig. 3)
By application of an improved biotin switch method, we demonstrate that a massive increase of nitric oxide synthases (NOSs) activity in the heart (Ͼ200-fold above WT control [7]) does not lead to a global rise of S-nitrosated proteins
Summary
17440 JOURNAL OF BIOLOGICAL CHEMISTRY of vascular tone, cardiac contraction, relaxation, and energetics [1, 2]. For detection and identification of endogenously S-nitrosated proteins in WT and iNOSϩ/ myoϪ/Ϫ mouse hearts, four samples per group were analyzed.
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