Abstract
An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.
Highlights
Under oxidative stress, a number of mechanisms have been proposed to trigger reactive oxygen species generation, with the enzymes xanthine oxidase, cyclooxygenase, leukocyte NADPH oxidase, mitochondria and uncoupled endothelial NOS as putative sources [9, 10]
We have demonstrated that endothelial NOS (eNOS) can be S-glutathionylated under oxidative stress in vivo and in vitro, leading to an uncoupling of the enzyme [13]
Several reports have shown that protein S-glutathionylation plays an important role in redox signaling and can be protective against irreversible oxidation of the protein thiols in cardiovascular diseases [17, 49]
Summary
A number of mechanisms have been proposed to trigger reactive oxygen species generation, with the enzymes xanthine oxidase, cyclooxygenase, leukocyte NADPH oxidase, mitochondria and uncoupled endothelial NOS (eNOS) as putative sources [9, 10]. This eNOS protein radical formation leads to modification of the function and coupling of the enzyme through subsequent S-glutathionylation or disulfide formation and is shown to occur in endothelial cells under oxidant stress. Immunoprecipitation with anti-eNOS and antiDMPO followed by immunoblotting with anti-DMPO or antieNOS antibodies confirmed that eNOS protein radical formation occurred in these cells (Fig. 6c).
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