Proc Natl Acad Sci U S A 102(1): 117-122

<em>S</em>-nitrosoprotein formation and localization in endothelial cells

Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA 02118

^{To whom correspondence should be addressed. E-mail:}ude.ub@zlacsolj.

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA

Received 2004 Aug 13; Accepted 2004 Nov 5.

Abstract

Protein S-nitrosation represents a recently described form of post-translational modification that is rapid and reversible. However, the analysis of protein S-nitrosation in situ has been difficult because of the absence of specific probes and the instability of cellular protein S-nitrosothiols. We developed a rapid and specific method for detecting endothelial S-nitrosoproteins patterned after the biotin switch method that involves thiol alkylation followed by reductive generation of thiols from S-nitrosothiols, which are then labeled with either a biotin- or Texas red-derivative of methanethiosulfonate. When we used this methodology, we found that S-nitrosated proteins can form within endothelial cells from an exogenous S-nitrosothiol donor or from endogenous production of NO by endothelial NO synthase. When we used confocal microscopy, we found that these S-nitrosoproteins exist mainly in the mitochondria and peri-mitochondrial compartment, and that their half-life is ≈1 h. Cellular S-nitrosated protein abundance changed as expected, with changes in activity of NO synthase, and with impairment of mitochondrial function and scavenging of peroxynitrite. We used a proteomic approach involving two-dimensional gel electrophoresis and mass spectrometry, and found that a limited number of S-nitrosoproteins exist in endothelial cells (S-nitrosoproteome) and identified GAPDH, vimentin, β-galactosidase, peroxiredoxin 1, β-actin, and ubiquitin-conjugating enzyme E2 among them. The most abundant S-nitrosated protein in the resting endothelial cell is GAPDH, suggesting a regulatory function for NO in glycolysis. These data offer methods and insights into identifying the protein targets of S-nitrosation reactions and their potential role in cell function and phenotype.

Keywords: mitochondria, proteins, nitric oxide

Abstract

Posttranslational modification of proteins, including glycation, phosphorylation, and disulfide bond formation, modulates protein folding, trafficking, and function. Protein S-nitrosation represents a recently described form of posttranslational modification (1) that is rapid and reversible. S-nitrosoprotein formation can occur through N₂O₃- or peroxynitrite-based oxidation of the thiol functionality of cysteinyl side chains (2, 3), trans-S-nitrosation reactions from low-molecular-weight donor S-nitrosothiols (4, 5), direct reaction of NO to form RSNOH, followed by electron abstraction (6), or catalysis at transition metal centers (7).

The analysis of protein S-nitrosation in situ, derived from either endogenous NO production or exogenous NO donors, has been difficult because of technical limitations. A polyclonal antibody raised against an S-nitrosated glutaraldehyde conjugate of BSA and cysteine has been used in immunoblotting and immunochemistry (8); however, the lack of specificity of the antibody limits its application to this problem. Although many different, specific proteins have been shown to undergo S-nitrosation in vitro, there is little direct evidence for S-nitrosation of cellular proteins that can be ascribed specifically to NO-derived from endogenous NO synthase activity.

S-nitrosothiols are stable in the absence of reduced metal ions or thiols. Reduced metal ions catalyze the decomposition of S-nitrosothiols, with contaminant transition metal ions likely accounting for the highly variable published values for the half-lives of low-molecular-weight S-nitrosothiols. Within cells, these substituted thiols may react with the high intracellular concentration of glutathione to generate a mixed disulfide and facilitate release of NO. Trans-S-nitrosation reactions within cells may also facilitate redistribution of NO from one thiol pool to another, possibly in a compartment-specific manner.

Here, we use a recently developed methodology, patterned after the biotin switch approach of Jaffrey and colleagues (9, 10), to show that S-nitrosated proteins can form within endothelial cells from an exogenous S-nitrosothiol donor or from endogenous production of NO by endothelial NO synthase. We find a limited number of S-nitrosated proteins, and identify them by mass spectrometry. We also show that S-nitrosated proteins exist mainly in the mitochondria and peri-mitochondrial compartment of endothelial cells, and that their half-life is ≈1 h. These data offer methods and insights into identifying the protein targets of S-nitrosation reactions and their potential role in cell function and phenotype.

NCBI, National Center for Biotechnology Information.

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Acknowledgments

We thank Dr. Catherine Costello for assistance with the mass spectrometry and Ms. Stephanie Tribuna for expert secretarial assistance. This work was supported in part by National Institutes of Health Grants P01HL55993, R01HL58976, R01HL61795, and N01HV28178 (Boston University Cardiovascular Proteomics Center grant).

Acknowledgments

Notes

Author contributions: J.L. designed research; Y.Y. and J.L. performed research; J.L. contributed new reagents/analytic tools; Y.Y. and J.L. analyzed data; and Y.Y. and J.L. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: MMTS, methyl methanethiosulfonate; GSNO, S-nitrosoglutathione; DEANONOate, 2-(N,N-dimethylamino)-diazenolate-2-oxide·Na; MTSEA, 2-((6-((biotinoyl)amino)-hexanoyl)amino) ethylmethanethiosulfonate; WGA, wheat germ agglutinin; DPBS, dextrose-PBS; BAEC, bovine aortic endothelial cell; HAEC, human aortic endothelial cell.

Notes

Author contributions: J.L. designed research; Y.Y. and J.L. performed research; J.L. contributed new reagents/analytic tools; Y.Y. and J.L. analyzed data; and Y.Y. and J.L. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

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