CYP-13A12 of the nematode Caenorhabditis elegans is a PUFA-epoxygenase involved in behavioural response to reoxygenation

Abstract

SHORT TITLE:CYP-13A12 of C. elegans is a PUFA-epoxygenase 4 SYNOPSISA specific behavioural response of Caenorhabditis elegans, the rapid increase of locomotion in response to anoxia/reoxygenation called the O2-ON response, has been used to model key aspects of ischemia/reperfusion injury. A genetic suppressor screen demonstrated a direct causal role of CYP-13A12 in this response and suggested that CYP-eicosanoids, which in mammals influence the contractility of cardiomyocytes and vascular smooth muscle cells, might function in C. elegans as specific regulators of the body muscle cell activity. Here we show that co-expression of CYP-13A12 with the NADPH-CYP-reductase EMB-8 in insect cells resulted in the reconstitution of an active microsomal monooxygenase system that metabolised EPA (eicosapentaenoic acid) and also AA (arachidonic acid) to specific sets of regioisomeric epoxy and hydroxy derivatives. Main products included 17,18-EEQ (epoxyeicosatetraenoic acid) from EPA and 14,15-EET (epoxyeicosatrienoic acid) from AA. Locomotion assays showed that the defective O2-ON response of C20-PUFA-deficient, ∆ -12 and ∆ -6 fatty acid desaturase mutants (fat-2 and fat-3, respectively) can be restored by feeding the nematodes AA or EPA, but not ETYA (eicosatetraynoic acid), a non-metabolisable AAanalogue. Already short-term incubation with 17,18-EEQ was sufficient to rescue the impaired locomotion of the fat-3 strain. The endogenous level of free 17,18-EEQ declined during anoxia and was rapidly restored in response to reoxygenation. Based on these results, we suggest that CYP-dependent eicosanoids such as 17,18-EEQ function as signalling molecules in the regulation of the O2-ON response in C. elegans. Remarkably, the exogenously administered 17,18-EEQ increased the locomotion activity already under normoxic conditions and was effective not only with C20-PUFA mutants but to a lesser extent also with wild-type worms. KEYWORDS:Eicosanoids; Polyunsaturated fatty acids; Caenorhabditis elegans; Cytochrome P450; Reoxygenation; 17,18-EEQ ABBREVIATIONS USED: AA, arachidonic acid; CID, collision-induced dissociation; COD, carbon monoxide difference; CPR, NADPH-CYP reductase; CYP, cytochrome P450; DiHETE, dihydroxyeicosatetraenoic acid; DTT, dithiothreitol; EGL, egg laying defect; EGLN, EGL nine; EEQ, epoxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; EPA, eicosapentaenoic acid; ETYA, eicosatetraynoic acid; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEET, hydroxyepoxyeicosatrienoic acid; HEPE, hydroxyeicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid; HIF, hypoxia inducible factor; LC, liquid chromatography; MC, pharyngeal marginal cell; MS, mass spectrometry; NGM, nematode growth medium; PUFA, polyunsaturated fatty acid; RP, reversed-phase 5 INTRODUCTIONOxygen deprivation upon restriction of blood supply followed by reperfusion and concomitant reoxygenation causes tissue injury and is involved in the initiation of various human pathologies including ischemic stroke, myocardial infarction, and ac...

Glucose or Altered Ceramide Biosynthesis Mediate Oxygen Deprivation Sensitivity Through Novel Pathways Revealed by Transcriptome Analysis inCaenorhabditis elegans

“…Notably, it is not the decreased expression of cyp-25A1 and ugt-63 that contributes to anoxia sensitivity since the knockdown of these genes using RNAi actually enhanced anoxia survival. The ugt-63 and cyp-25A1 genes, part of gene Cluster 4 and 5 ( Figure 4, B and E ), code for proteins that show homology to detoxification system proteins ( Ma et al 2013 ; Keller et al 2014 ; Gems and McElwee 2005 ). The RNA-Seq data indicate that several ugt and cyp gene family members have a differential gene expression profile in the anoxia sensitive animals relative to controls ( Figure 4A and Table S6 ).…”

Epoxides Derived from Dietary Dihomo-Gamma-Linolenic Acid Induce Germ Cell Death in C. elegans

“…In C. elegans , CYP-modified PUFA metabolites appear to regulate pharyngeal pumping, possibly through the activation of ion channels 22 23 . Recently, CYP-modified PUFAs, specifically 17,18- EEQ, was shown to impart protection against ischemic/reperfusion damage 9 35 . Thus, CYP-modified PUFA metabolites perform a range of important physiological functions in both vertebrates and invertebrates that are just beginning to be understood.…”

“…Though strong evidence indicates that epoxide fatty acid metabolites act in signal transduction, no receptors for these molecules have been identified 38 39 . Recently discovered functions for specific eicosanoids in C. elegans , such as the requirement for F-series prostaglandins in sperm guidance 28 29 32 , CYP involvement in response to hypoxia and re-oxygenation 9 35 , and the sensitivity of germ cells to oxygenated metabolites of dietary DGLA, demonstrate the utility of the genetically amenable C. elegans system for the discovery and elucidation of cellular mechanisms of eicosanoid signaling in physiological processes.…”

Using Caenorhabditis elegans to Uncover Conserved Functions of Omega-3 and Omega-6 Fatty Acids

“…The C. elegans study showed that CYP-13A12 responds to the oxygen-dependent enzyme EGL-9 and hypoxia-inducible factor (HIF-1) to facilitate a movement response to reoxygenation after oxygen deprivation [ 86 ]. CYP-13A12 generates epoxy and hydroxyl eicosanoids from AA and EPA, including 14,15-epoxyeicosatrienoic acid (14,15-EET) from AA and 17,18-EEQ from EPA [ 88 ]. This research implicates conserved roles for omega-6 and omega-3 PUFAs and eicosanoid formation in ischemia and reperfusion.…”