Cytochrome P450 2J2 mediated metabolism of omega-6 and omega-3 endocannabinoids (2016)

Cytochrome P450 2J2 (CYP2J2) is membrane bound enzyme that is highly expressed in the cardiovascular system and brain where it mediates the metabolism of polyunsaturated fatty acids into oxygenated bioactive metabolites. This enzyme is most well-known for the conversion of arachidonic acid into epoxyeicosatrienoic acids (EETs) regioisomers, 14,15-, 11,12-, 8,9-, and 5,6-EET that mediate vasodilatory, pro-angiogenic and anti-inflammatory effects. More recently, CYP2J2 has been identified as a key enzyme for the metabolism of omega-3 fatty acids, such as eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), producing epoxygenated metabolites that provide cardioprotective benefits. \n\nThe biochemical study of the CYP2J2 mediated metabolism of xenobiotics and endogenous fatty acids have been difficult due to challenges with protein expression and solubilization. To circumvent these issues, a series of N-terminal modifications were introduced in the CYP2J2 N-terminus that enabled heterologous expression in E. coli. Importantly, to address the propensity of CYP2J2 to aggregate in solution, each construct was incorporated into the native membrane bilayers of nanodiscs for rigorous spectroscopic and functional studies. Notably, all N-terminal constructs were stably bound within the nanodisc bilayers, which prompted efforts to elucidate additional motifs that impart membrane binding. These observations were further explored in a study using molecular dynamics simulations which revealed CYP2J2 membrane insertion 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 prepared from the truncated CYP2J2 construct (Δ34), which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Interestingly, the expression of the CYP2J2 F-G loop mutants in E. coli were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34 while functional characterization revealed that the double mutant, Δ34-I236D/F239H, exhibited the greatest solubility while maintaining native-like enzymatic activity. Further, the membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, all three F-G loop mutants exhibited lower Trp quenching indicating reduced membrane insertion. \n\nThe original CYP2J2 expression and characterization studies enabled metabolic screening of endogenous polyunsaturated fatty acid (PUFA) derived substrates that included derivatives of AA, EPA and DHA. Notably, these efforts resulted in the discovery of the CYP2J2-nanodisc mediated oxygenation of omega-6 derived endocannabinoids, thereby revealing a previously unknown metabolic pathway. The endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), are endogenous lipid mediators that are agonists of cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) with effects similar to the active ingredient of marijuana. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Additionally, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. \n\nThe demonstrated ability of CYP2J2 to oxygenate AEA and 2-AG raised questions whether CYP2J2 could also accommodate the omega-3 derived endocannabinoids, eicosapentaenoic ethanolamide (EPEA) and docosahexaenoic ethanolamide (DHEA), as substrates. Indeed, subsequent metabolism studies of CYP2J2 with EPEA/DHEA revealed the production of a new class of ω-3 derived metabolites that we termed the omega-3 endocannabinoid epoxides – epoxyeicosatetraenoic-ethanolamide (EEQ-EA) and epoxydocosapentaenoic-ethanolamide (EDP-EA). These newly discovered endogenous metabolites share structural similarity to both their endocannabinoid and epoxide parent compounds, with potential to exert physiological effects mediated through both signaling pathways. Both EEQ-EAs and EDP-EAs were endogenously present in rat brain and peripheral organs as determined via LC-MS/MS with 19,20-EDP-EA also detectable in pooled human plasma. Using LC-MS/MS we demonstrated the direct production of these lipids by recombinant human CYP2J2 epoxygenase, rat brain microsomes and activated BV-2 microglia cells when supplemented with the parent EPEA or DHEA lipids. The discovery of these metabolites prompted a panel of in vitro assays to provide a first ever examination of their biological effects. Most notably, upon LPS stimulation of BV-2 microglia cells, both 17,18-EEQ-EA and 19,20-EDP-EA decreased pro-inflammatory IL-6 and nitric oxide whilst increasing anti-inflammatory IL-10. Additional studies revealed that these metabolites exerted anti-angiogenic effects in HMVEC cells, vasodilatory properties in bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the following thesis details new methods for the study of CYP2J2 within the membrane bilayers of nanodiscs. These techniques were used to reveal a new role for CYP2J2 in the metabolism of endocannabinoids into novel endocannabinoid metabolites with important physiological functions in the heart and brain.