title: Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase.
abstract: Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr(385), an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress.
authors.affiliation: Department of Pathology, Weill Cornell Medical College, Cornell University, New York, New York;
authors.forename: Ruba S
authors.initials: RS
authors.lastname: Deeb
authors.forename: Tal
authors.initials: T
authors.lastname: Nuriel
authors.forename: Cynthia
authors.initials: C
authors.lastname: Cheung
authors.forename: Barbara
authors.initials: B
authors.lastname: Summers
authors.forename: Brian D
authors.initials: BD
authors.lastname: Lamon
authors.forename: Steven S
authors.initials: SS
authors.lastname: Gross
authors.forename: David P
authors.initials: DP
authors.lastname: Hajjar
mesh_terms: D000222:Adaptation, Physiological / Q000502:physiology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D051545:Cyclooxygenase 1 / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D009566:Nitrates; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D051545:Cyclooxygenase 1; C045258:nitratase
keywords: 3-nitrotyrosine; HPLC with electrochemical detection; cyclooxygenase-1; denitration; liquid chromatograph-tandem mass spectrometry; peroxynitrite; reactive nitrogen species