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Abstract
Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.
Highlights
85727 and by NIEHS, National Institutes of Health Grants R37 ES02710, P42 ES004699, R01 ES013933
We assessed the consequences of incubating soluble epoxide hydrolase (sEH)-expressing cell lysates with the ONOOϪ donor, SIN-1
We observed that while the c-Myctagged sEH recovered from human embryonic kidney (HEK) cell lysates treated with inactivated ONOOϪ (i.e. ONOOϪ incubated in bicarbonatecontaining buffer for 24 h; 37 °C) demonstrated only a weak signal, tyrosine nitration of the protein was clearly evident in samples treated with fresh ONOOϪ (Fig. 4A)
Summary
85727 and by NIEHS, National Institutes of Health Grants R37 ES02710, P42 ES004699, R01 ES013933. The latter reaction was unaffected by the microsomal epoxide hydrolase inhibitor, elaidamide, but was significantly attenuated (by 61 Ϯ 4% and 70 Ϯ 3% in human and murine endothelial cells, respectively) by the selective sEH inhibitor, ACU (10 mol/liter, Fig. 1B). The sEH inhibitor reduced activity in cell lysates to levels comparable with those measured in lung endothelial cells derived from sEHϪ/Ϫ mice.
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