Soluble epoxide hydrolase dimerization is required for hydrolase activity

J Biol Chem. 2013 Mar 15;288(11):7697-7703. doi: 10.1074/jbc.M112.429258. Epub 2013 Jan 28.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8,11,14-Eicosatrienoic Acid / analogs & derivatives
  • 8,11,14-Eicosatrienoic Acid / chemistry
  • DNA Mutational Analysis
  • Dimerization
  • Epoxide Hydrolases / chemistry*
  • Epoxide Hydrolases / physiology
  • Genetic Complementation Test
  • HEK293 Cells
  • Humans
  • Hydrolases / chemistry
  • Kinetics
  • Models, Molecular
  • Mutation
  • Polymorphism, Genetic
  • Solubility
  • Transfection

Substances

  • 14,15-dihydroxyeicosatrienoic acid
  • 14,15-epoxy-5,8,11-eicosatrienoic acid
  • Hydrolases
  • Epoxide Hydrolases
  • EPHX2 protein, human
  • 8,11,14-Eicosatrienoic Acid