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 Tyr385, 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.

An overview of mechanisms of redox signaling

“…But, it remains to be seen whether tyrosine nitration is more than a marker of nitrosative stress and plays a significant role in signal transduction. A recent study of reversible tyrosine modification in regulation of cyclooxygenase activity suggests that the latter possibility needs further consideration [26]. …”

Signaling and stress: The redox landscape in NOS2 biology

“…At present, nitration is considered to be a permanent modification, which can impact versatility in signaling. However, the search for denitrases is an active research area (e.g., [159, 160]).…”

Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke

“…Proteinaceous extracts from various tissues contain a putative 'denitrase' activity capable of removing nitrite from the nitrotyrosyl residues of a variety of proteins [42][43][44][45][46][47] including the nitrated Tyr336 on glutamine synthetase [39]. Denitrase activity has been attributed to the E3 component of the a-ketoglutarate dehydrogenase complex [48], glutathione S-transferase, and superoxide dismutase 1 [47]. This activity, however, has not been purified to homogeneity from any of the 'denitrating' extracts and thus the identity of the actual denitrase(s) remains unknown.…”