April 2013

Document Type


Degree Name



Dept. of Molecular and Medical Genetics


Oregon Health & Science University


Soluble epoxide hydrolase (sEH) is a promising therapeutic target for multiple cardiovascular diseases, including stroke. Available inhibitors of sEH target the enzyme’s hydrolase catalytic site, which metabolizes lipid molecules shown to be protective against ischemic brain damage called epoxyeicosatrienoic acids. Recently, a human missense polymorphism (R287Q), in the gene that encodes for sEH, designated ephx2, was shown to afford a reduction in risk from stroke, despite the fact that the residue is not localized near the hydrolase catalytic site. This suggests the existence of an alternative strategy to reduce sEH enzymatic activity. Based on the localization of the R287 residue on the homodimerization interface of sEH, and previous work showing sEH protein harboring the R287Q polymorphism forms a greater amount of monomers than the wildtype enzyme, I hypothesized that the R287Q polymorphism was conferring protection from ischemia by disrupting dimerization. Specifically, I hypothesized that dimerization was a key regulator for two important aspects of sEH function, hydrolase activity and subcellular localization. To test these hypotheses, I developed a series of mutations predicted to either stabilize or destabilize sEH dimerization by manipulating an inter-monomeric salt-bridge localized at the sEH dimerization interface. I used a splitfirefly luciferase complementation system to evaluate the dimerization status of each mutation before quantifying their hydrolase activity. I found that monomeric sEH has decreased activity compared to dimeric sEH, leading to the conclusion that dimerization regulates sEH hydrolase enzymatic activity. Next, I used the previously characterized dimerization constructs, fused to green fluorescent protein (GFP), to test the effect of dimerization on the subcellular localization of sEH. I found that disrupting sEH dimerization increases peroxisomal localization in primary cortical mouse neurons. Finally, I tested the hypothesis that peroxisomal localization of sEH is protective against ischemic injury. I injected TAT-fused sEH proteins with either an intact or defective peroxisome targeting sequence (PTS) into sEH knockout mice before subjecting them to experimental stroke. I found that mice treated with sEH, which was capable of translocating to peroxisomes, had less brain damage compared to mice treated with sEH protein that is restricted to the cytosol. Taken together, this data suggests that disrupting dimerization may be a novel therapeutic approach for targeting sEH.




School of Medicine



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