Title

Mechanism of peroxisomal targeting of human soluble epoxide hydrolase (hsEH) and its effect on hsEH stability and activity

Date of Completion

January 2008

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Soluble epoxide hydrolase (sEH) is a homodimeric enzyme with a C-terminal epoxide hydrolase domain and an N-terminal phosphatase domain. sEH has been suggested to play a role in a variety of biological processes, like inflammation, hypertension, cell signaling and cholesterol synthesis, by modulating levels of its endogenous substrates. However, its biological significance in human remains unclear. Part of this unknown is due to controversy regarding human soluble epoxide hydrolase (hsEH) subcellular localization. Despite the putative peroxisomal targeting sequences on the protein sequence, hsEH is dual localized in both peroxisomes and cytosol in human tissues. The unusual dual localization of hsEH, together with the differential localization of the substrates of hsEH C-terminal domain and N-terminal domain, suggests that gaining insight into the hsEH dual localization would pave way to understanding the clinical relevance of this enzyme.^ In this study, we utilized a GFP-fusion protein model to track movement of hsEH inside cells. We demonstrated that the peroxisomal localization of hsEH is determined not only by peroxisomal targeting sequences, but also modulated to some extent by hsEH quaternary structure and protein expression levels. In addition we also demonstrated that the peroxisomal localization protects hsEH from cytosolic degradation, thus stabilizing and increasing half-life and activities of enzyme localized to peroxisomes compared to the enzyme localized to cytosol. Surprisingly, we found that the C-terminal domain and the N-terminal domain of hsEH regulate cholesterol conversely. The C-terminal domain of hsEH decreases cholesterol, while N-terminal domain increases cholesterol. In addition, we also demonstrated that the role of hsEH in cholesterol regulation is influenced by subcellular localizations. Cholesterol regulating function of the C-terminal domain of hsEH is lost when it is located in the peroxisomes, whereas the N-terminal domain localization in the peroxisomes, increases cholesterol Levels. The RT-PCR results suggests that cholesterol regulation by hsEH is through regulation of key genes in cholesterol metabolism, such as 3-hydroxy-3-methylglutaryl coenzyme A, apolipoprotein Al and the ATP-binding cassette transporter Al. Our results, for the first time, provide a possible explanation of how hsEH is involved in human diseases through differential subcellular localization. ^