期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 102, 期 1, 页码 117-122出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0405989102
关键词
milochondria; proteins; nitric oxide
资金
- NHLBI NIH HHS [R01 HL061795, R01 HL058976, R01 HL 61795, N01HV28178, P50 HL055993, R01 HL 58976, N01 HV 28178, P01 HL 55993] Funding Source: Medline
Protein S-nitrosation represents a recently described form of posttranslational 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 5-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 approximate to1 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, beta-galactosiclase, peroxiredoxin 1, beta-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.
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