Soluble epoxide hydrolase dimerization is required for hydrolase activity.
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.
Departments of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, Portland, Oregon 97239-3098; Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon 97239-3098.
Jonathan W
JW
Nelson
Departments of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, Portland, Oregon 97239-3098.
Rishi M
RM
Subrahmanyan
Departments of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, Portland, Oregon 97239-3098.
Sol A
SA
Summers
Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239-3098.
Xiangshu
X
Xiao
Departments of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, Portland, Oregon 97239-3098. Electronic address: alkayedn@ohsu.edu.
Nabil J
NJ
Alkayed
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004252:DNA Mutational Analysis; D019281:Dimerization; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000502:physiology; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D006801:Humans; D006867:Hydrolases / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011110:Polymorphism, Genetic; D012995:Solubility; D014162:Transfection
C107016:14,15-dihydroxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D006867:Hydrolases; D004851:Epoxide Hydrolases; C506428:EPHX2 protein, human; D015126:8,11,14-Eicosatrienoic Acid