Roles of the epoxygenase CYP2J2 in the endothelium.
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ABSTRACT:
SUBMITTER: Askari A
PROVIDER: S-EPMC3711961 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
Publications
Roles of the epoxygenase CYP2J2 in the endothelium.
Askari Ara A Thomson Scott J SJ Edin Matthew L ML Zeldin Darryl C DC Bishop-Bailey David D
Prostaglandins & other lipid mediators 20130306
Cytochrome p450 (CYP)2J2 is an epoxygenase enzyme that metabolises arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are inactivated by soluble epoxide hydrolase (sEH), which converts them in to their corresponding dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is highly expressed in cardiovascular tissue including the heart and vascular endothelial cells. CYP2J2 and the EETs it produces have been shown to have a diverse range of effects on the vasculature, including the regulation of in ...[more]
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Project description:The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.
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Project description:CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N-terminus was used to express full length CYP2J2 in E. coli while maintaining wild-type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N-terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native-like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.
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Project description:Ample evidences demonstrate that cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) exert diverse biological activities, which include potent vasodilatory, anti-inflammatory, and cardiovascular protective effects. In this study, we investigated the effects of endothelium-specific CYP2J2 overexpression on age-related insulin resistance and metabolic dysfunction. Endothelium-specific targeting of the human CYP epoxygenase, CYP2J2, transgenic mice (Tie2-CYP2J2-Tr mice) was utilized. The effects of endothelium-specific CYP2J2 overexpression on aging-associated obesity, inflammation, and peripheral insulin resistance were evaluated by assessing metabolic parameters in young (3 months old) and aged (16 months old) adult male Tie2-CYP2J2-Tr mice. Decreased insulin sensitivity and attenuated insulin signaling in aged skeletal muscle, adipose tissue, and liver were observed in aged adult male mice, and moreover, these effects were partly inhibited in 16-month-old CYP2J2-Tr mice. In addition, CYP2J2 overexpression-mediated insulin sensitization in aged mice was associated with the amelioration of inflammatory state. Notably, the aging-associated increases in fat mass and adipocyte size were only observed in 16-month-old wild-type mice, and CYP2J2 overexpression markedly prevented the increase in fat mass and adipocyte size in aged Tie2-CYP2J2-Tr mice, which was associated with increased energy expenditure and decreased lipogenic genes expression. Furthermore, these antiaging phenotypes of Tie2-CYP2J2-Tr mice were also associated with increased muscle blood flow, enhanced active-phase locomotor activity, and improved mitochondrial dysfunction in skeletal muscle. Collectively, our findings indicated that endothelium-specific CYP2J2 overexpression alleviated age-related insulin resistance and metabolic dysfunction, which highlighted CYP epoxygenase-EET system as a potential target for combating aging-related metabolic disorders.
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Project description:BackgroundThe cytochrome P-450 2J2 (CYP2J2) is known to be one of the major enzymes of epoxygenase pathway of arachidonic acid in extrahepatic tissues, which produces series of regioisomeric cis-epoxyeicosatrienoic acids (EETs) such as 5,6-, 8,9-, 11,12-, and 14,15-EETs. In the present study, we analyzed the impact of a genetic variant in CYP2J2 on coronary artery disease (CAD) in the Telangana region of Indian population.Material and methodsThe case-control study consisted of 100 CAD cases and 110 healthy controls. The deoxyribonucleic acid was extracted using the salting out method. Genotyping and gene expression was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and real-time-PCR methods.ResultsIn the present study, the percentage of smokers, alcoholics, hypertensive patients, and diabetics was high. Increase in fasting glucose, urea, creatinine, fasting triglycerides, total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), total cholesterol/high-density lipoprotein (TC/HDL), LDL/HDL, homocysteine, and C-reactive protein levels were significantly higher in patients with CAD than in controls (p < 0.001). CYP2J2 G-50T was associated with CAD (p = 0.04). The mRNA expression of CYP2J2 showed altered gene expression in this study among CAD patients in comparison with control (p = 0.01).ConclusionsA functionally relevant polymorphism of the CYP2J2 gene was independently associated with an increased risk of CAD.
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Project description:Cytochrome P450 epoxygenases (CYP) metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which exhibit vasodilatory, anti-inflammatory and neuroprotective actions in experimental cerebral ischemia. We evaluated the effect of endothelial-specific CYP overexpression on cerebral blood flow, inflammatory cytokine expression and tissue infarction after focal cerebral ischemia in transgenic mice.Male and female wild-type and transgenic mice overexpressing either human CYP2J2 or CYP2C8 epoxygenases in vascular endothelium under control of the Tie2 promoter (Tie2-CYP2J2 and Tie2-CYP2C8) were subjected to 60-min middle cerebral artery occlusion (MCAO). Microvascular cortical perfusion was monitored during vascular occlusion and reperfusion using laser-Doppler flowmetry and optical imaging. Infarct size and inflammatory cytokines were measured at 24h of reperfusion by TTC and real-time quantitative PCR, respectively. Infarct size was significantly reduced in both Tie2-CYP2J2 and Tie2-CYP2C8 transgenic male mice compared to corresponding WT male mice (n=10 per group, p<0.05). Tie2-CYP2J2, but not Tie2-CYP2C8 male mice maintained higher blood flow during MCAO; however, both Tie2-CYP2J2 and Tie2-CYP2C8 had lower inflammatory cytokine expression after ischemia compared to corresponding WT males (n=10 per group for CBF and n=3 for cytokines, p<0.05). In females, a reduction in infarct was observed in the caudate-putamen, but not in the cortex or hemisphere as a whole and no differences were observed in blood flow between female transgenic and WT mice (n=10 per group).Overexpression of CYP epoxygenases in vascular endothelial cells protects against experimental cerebral ischemia in male mice. The mechanism of protection is in part linked to enhanced blood flow and suppression of inflammation, and is both sex- and CYP isoform-specific.
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Project description:CYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein-lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2.
| S-EPMC4559526 | biostudies-literature