Abstract
Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity.
Highlights
Soluble epoxide hydrolase forms a homodimer that metabolizes a neuroprotective class of lipids termed epoxyeicosatrienoic acids
Generation of Soluble epoxide hydrolase (sEH) Dimerization Mutants—Based on the dimer structure of sEH, Arg-287 is located in the center of the protein (Fig. 1A) and on the dimerization interface (Fig. 1B)
On the basis of the previous observation that the human polymorphism (R287Q) only partially disrupted sEH dimerization [6], we hypothesized that our more severe mutation would completely abolish dimerization. We generated another mutant of sEH, which we hypothesized would abolish dimerization, by changing Glu-254 to arginine (E254R), placing two positively charged amino acids opposite each other
Summary
Soluble epoxide hydrolase (sEH) forms a homodimer that metabolizes a neuroprotective class of lipids termed epoxyeicosatrienoic acids. A common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. We investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme These results indicate that dimerization is required for sEH hydrolase activity. Multiple human missense polymorphisms have been identified throughout the structure of the protein that alter the hydrolase enzymatic activity of sEH [6]. Based on the crystal structure of sEH [7], Arg-287 is localized near the center of the enzyme on the dimerization interface [6] This suggests that the effect of this polymorphism may be functionally linked to its effect on dimerization. This research supports the development of novel therapeutic strategies for inhibiting sEH hydrolase activity
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