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Identification of N-acylethanolamines in Dictyostelium discoideum and confirmation of their hydrolysis by fatty acid amide hydrolase[S]

Open AccessPublished:November 27, 2012DOI:https://doi.org/10.1194/jlr.M032219
      N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotypes. This has led to the development of pharmaceutical-based therapies for a variety of conditions targeting FAAH. Recently, our group identified a functional FAAH homolog in Dictyostelium discoideum, leading to our hypothesis that D. discoideum also possesses NAEs. In this study, we provide a further characterization of FAAH and identify NAEs in D. discoideum for the first time. We also demonstrate the ability to modulate their levels in vivo through the use of a semispecific FAAH inhibitor and confirm that these NAEs are FAAH substrates through in vitro studies. We believe the demonstration of the in vivo modulation of NAE levels suggests that D. discoideum could be a good simple model organism in which to study NAE-mediated signaling.
      N-acylethanolamines (NAEs) are lipid-based molecules that play a role in many cell signaling pathways. The most renowned NAE, anandamide (NAE20:4), plays a central role in the endocannabinoid system by being an endogenous agonist for the cannabinoid receptors (
      • Devane W.A.
      • Hanus L.
      • Breuer A.
      • Pertwee R.G.
      • Stevenson L.A.
      • Griffin G.
      • Gibson D.
      • Mandelbaum A.
      • Etinger A.
      • Mechoulam R.
      Isolation and structure of a brain constituent that binds to the cannabinoid receptor.
      ). The enzyme fatty acid amide hydrolase (FAAH) plays an important role in regulating the concentration of anandamide, as well as other NAEs, in vivo by hydrolyzing NAEs to ethanolamine and their corresponding fatty acid (
      • Cravatt B.F.
      • Giang D.K.
      • Mayfield S.P.
      • Boger D.L.
      • Lerner R.A.
      • Gilula N.B.
      Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.
      ). FAAH knockout studies in mice have demonstrated that inhibition of FAAH can produce analgesic (
      • Cravatt B.F.
      • Demarest K.
      • Patricelli M.P.
      • Bracey M.H.
      • Giang D.K.
      • Martin B.R.
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      Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase.
      ), anxiolytic (
      • Moreira F.A.
      • Kaiser N.
      • Monory K.
      • Lutz B.
      Reduced anxiety-like behaviour induced by genetic and pharmacological inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) is mediated by CB1 receptors.
      ), neuroprotective (
      • Aguado T.
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      • Marsicano G.
      • Kokaia Z.
      • Guzmán M.
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      The CB1 cannabinoid receptor mediates excitotoxicity-induced neural progenitor proliferation and neurogenesis.
      ), and anti-inflammatory (
      • Cravatt B.F.
      • Saghatelian A.
      • Hawkins E.G.
      • Clement A.B.
      • Bracey M.H.
      • Lichtman A.H.
      Functional disassociation of the central and peripheral fatty acid amide signaling systems.
      ,
      • Lichtman A.H.
      • Shelton C.C.
      • Advani T.
      • Cravatt B.F.
      Mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptor-mediated phenotypic hypoalgesia.
      ) effects. This has resulted in considerable research into developing highly specific FAAH inhibitors as potential pharmaceutical-based therapies (
      • Boger D.L.
      • Sato H.
      • Lerner A.E.
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      • Austin B.J.
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      Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide.
      ,
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      • Mor M.
      • Tarzia G.
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      Modulation of anxiety through blockade of anandamide hydrolysis.
      ,
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      • Cheng H.
      • Hwang I.
      • Hedrick M.P.
      • Leung D.
      • Acevedo O.
      • et al.
      Discovery of a potent, selective, and efficacious class of reversible α-ketoheterocycle inhibitors of fatty acid amide hydrolase effective as analgesics.
      ,
      • Keith J.M.
      • Apodaca R.
      • Xiao W.
      • Seierstad M.
      • Pattabiraman K.
      • Wu J.
      • Webb M.
      • Kabarz M.J.
      • Brown S.
      • Wilson S.
      • et al.
      Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase.
      ).
      Although much emphasis has been placed on studying NAE-mediated cannabinoid signaling in mammals, NAE signaling is also involved in noncannabinoid signaling pathways in humans and other eukaryotic organisms. For example, N-palmitoylethanolamine (NAE16:0) and N-stearoylethanolamine (NAE18:0) have been shown to be involved in noncannabinoid receptor anti-inflammatory signaling pathways (
      • Berdyshev E.V.
      • Schmid P.C.
      • Kresbach R.J.
      • Hillard C.J.
      • Huang C.
      • Chen N.
      • Dang Z.
      • Schmid H.O.
      Cannabinoid-receptor-independent cell signaling by N-acylethanolamines.
      ,
      • Dalle Carbonare M.
      • Del Giudice E.
      • Stecca A.
      • Colavito D.
      • Fabris M.
      • D'Arrigo A.
      • Bernardini D.
      • Dam M.
      • Leon A.
      A saturated N-acylethanolamine other than N-palmitoyl ethanolamine with anti-inflammatory properties: a neglected story….
      ). Evidence from plant studies have indicated that NAE signaling may be involved in pathogen defense, as evidenced by increased levels of N-myristoylethanolamine (NAE14:0) in the presence of the fungal elicitor xylanase (
      • Tripathy S.
      • Venables B.J.
      • Chapman K.D.
      N-acylethanolamines in signal transduction of elicitor perception. Attenuation of alkalinization response and activation of defense gene expression.
      ) and the down-regulation of defense-related transcripts and increased pathogen susceptibility in FAAH over-expressing plants (
      • Kang L.
      • Wang Y-S.
      • Uppalapati S.
      • Wang K.
      • Tang Y.
      • Vadapalli V.
      • Venables B.J.
      • Chapman K.D.
      • Blancaflor E.B.
      • Mysore K.
      Overexpression of a fatty acid amide hydrolase compromises innate immunity in Arabidopsis.
      ). NAE signaling, specifically N-lauroylethanolamine (NAE12:0), has also been shown to inhibit phospholipase D α activity and abscisic acid-induced stomata closure (
      • Austin-Brown S.L.
      • Chapman K.D.
      Inhibition of phospholipase Dα by N-acylethanolamines.
      ) and to induce microtubule, actin, and endomembrane reorganization (
      • Blancaflor E.B.
      • Hou G.
      • Chapman K.D.
      Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings.
      ,
      • Motes C.M.
      • Pechter P.
      • Yoo C.M.
      • Wang Y.S.
      • Chapman K.D.
      • Blancaflor E.B.
      Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth.
      ).
      NAEs and their precursors have also been shown to be present in several lower eukaryotic organisms. It has long been known that the precursors of NAEs, N-acylphosphatidylethanolamines, are present in Dictyostelium discoideum (
      • Ellingson J.S.
      Identification of (N-acyl)ethanolamine phosphoglycerides and acylphosphatidylglycerol as the phospholipids which disappear as Dictyostelium discoideum cells aggregate.
      ), and NAEs and N-acylphosphatidylethanolamines have been shown to be present in the yeast Saccharomyces cerevisiae (
      • Merkel O.
      • Schmid P.C.
      • Paltauf F.
      • Schmid H.H.O.
      Presence and potential signaling function of N-acylethanolamines and their phospholipids precursors in the yeast Saccharomyces cerevisiae.
      ). Recently, it has been shown that the ciliate Tetrahymena thermophila possesses numerous NAEs (
      • Anagnostopoulos D.
      • Rakiec C.
      • Wood J.
      • Pandarinathan L.
      • Zvonok N.
      • Makriyannis A.
      • Siafaka-Kapadai A.
      Identification of endocannabinoids and related N-acylethanolamines in Tetrahymena. A new class of compounds for Tetrahymena.
      ). Along with evidence of an enzyme similar to FAAH present in Tetrahymena pyriformis (
      • Karava V.
      • Zafiriou P-M.
      • Fasia L.
      • Anagnostopoulos D.
      • Boutou E.
      • Vorgias C.E.
      • Maccarrone M.
      • Siafaka-Kapadai A.
      Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase.
      ), this suggests that NAE signaling is carried out in lower eukaryotes. Furthermore, cannabinoid and cannabinoid-like molecules have been shown to elicit responses in lower eukaryotes, with Δ9-tetrahydrocannabinol affecting cellular growth, movement, and division of T. pyriformis (
      • McClean D.K.
      • Zimmerman A.M.
      Action of Δ9-tetrahydrocannabinol on cell division and macromolecular synthesis in division-synchronized protozoa.
      ,
      • Zimmerman S.
      • Zimmerman A.M.
      • Laurence H.
      Effect of Δ9 tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetrahymena.
      ) and 2-arachidonoyl glycerol inhibiting cellular growth in free-living amoebae (
      • Dey R.
      • Pernin P.
      • Bodennec J.
      Endocannabinoids inhibit the growth of free-living amoebae.
      ). Finally, arachidonic acid, the product of anandamide and 2-arachidonoyl glycerol hydrolysis by FAAH and monoacylglycerol lipase, respectively, is a known chemoattractant for D. discoideum (
      • Schaloske R.H.
      • Blaesius D.
      • Schlatterer C.
      • Lusche D.F.
      Arachidonic acid is a chemoatttractant for Dictyostelium discoideum cells.
      ).
      It is not known whether NAEs are present in D. discoideum. The identification of FAAH in D. discoideum by our group (
      • Neelemagan D.
      • Schoenhofen I.
      • Richards J.C.
      • Cox A.D.
      Identification and recombinant expression of anadamide hydrolyzing enzyme from Dictyostelium discoideum.
      ) led to our hypothesis that these lipids are present in this organism. To test this hypothesis, we further characterized the D. discoideum FAAH enzyme and then, using a targeted lipidomics approach, identified NAEs native to D. discoideum in the presence and absence of a semispecific FAAH inhibitor. These substrates were then confirmed as FAAH substrates through in vitro studies. We believe that this establishes D. discoideum as a suitable model organism in which to study NAE-mediated signaling, which may help elucidate these signaling pathways with broader implications on human health.

      MATERIALS AND METHODS

      Chemicals

      N-arachidonoylethanolamine (NAE20:4), N-arachidonoylethanolamine-d4 (AEA-d4), N-palmitoylethanolamine (NAE16:0), N-palmitoylethanolamine-d4 (PEA-d4), N-stearoylethanolamine (NAE18:0), N-oleoylethanolamine (NAE18:1), N-linoleoylethanolamine (NAE18:2), arachidonoyl p-nitroaniline (ApNA), CAY10435 (1-oxazolo [4, 5-b]pyridin-2-yl-1-dodecanone), and URB597 (3′-(aminocarbonyl)[1,1’-biphenyl]3-yl)-cyclohexylcarbamate) were purchased from Cayman Chemicals (Ann Arbor, MI). Arachidonoyltrifluoromethyl ketone (ATFMK), methoxyarachidonoyl fluorophosphonate (MAFP), LY2183240 (5-[(1,1’-Biphenyl]4-yl)methyl]N,N-dimethyl-1H-tetrazole-1-carboxamide), and JNJ1661010 (N-Phenyl-4-(3-phenyl-1,2,4-thiadiazol-5-yl)-1-piperazinecarboxamide) were purchased from Tocris Biosciences (Ellisville, MO). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, dodecanoic acid (FFA12:0), myristic acid (FFA14:0), phenylmethanesulfonyl fluoride (PMSF), and p-nitroaniline were purchased from Sigma-Aldrich (Oakville, ON, Canada).

      Preparation of N-acylethanolamines

      NAE standards were prepared for NAE12:0 and NAE14:0 as described by Williams et al. (
      • Williams J.
      • Wood J.
      • Pandarinathan L.
      • Karanian D.A.
      • Bahr B.A.
      • Vouros P.
      • Makriyannis A.
      Quantitative method for the profiling of the endocannabinoid metabolome by LC-atmospheric pressure chemical ionization-MS.
      ). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.6 mmol) was reacted with a solution containing the corresponding fatty acid (0.5 mmol), ethanolamine (0.75 mmol), and 4-dimethylaminopyridine (0.5 mmol) in 20 ml ice-cold acetonitrile. The reaction was warmed to 21°C and stirred for 6 h. The acetonitrile solvent was then exchanged for dichloromethane to fully dissolve solids before adding SiO2 to adsorb the NAE. Dichloromethane was then evaporated, and the SiO2 was packed into a column, dry-loaded onto a SiO2 flash chromatography column, and purified using a flash chromatography system (Teledyne Isco, Lincoln, NE) using an acetone/hexane gradient from 5-80%. Fractions containing NAEs as determined by UV absorbance were pooled and the solvent evaporated.

      Bioinformatics

      FAAH protein sequences were selected based on previous work performed by McPartland et al. (
      • McPartland J.M.
      • Matias I.
      • Di Marzo V.
      • Glass M.
      Evolutionary origins of the endocannabinoid system.
      ). FAAH 1 sequences (or proposed FAAH 1 homologs) were obtained for Homo sapiens (NP_001432), Mus musculus (NP_034303), Takifugu rubripes (FRUP000000146100), Ciona intestinalis (ci0100153926), Caenorhabditis elegans (NP_501368), Saccharomyces cerevisiae S288c (NP_010528), and Tetrahymena thermophila SB210 (159.m00087). FAAH 2 sequences (or proposed FAAH 2 homologs) were obtained for Homo sapiens (NP_777572), Takifugu rubripes (SINFRUP00000168827), and Drosophila melanogaster (NP_725139). Another FAAH sequence was obtained for Arabidopsis thaliana (NP_201249). Other amidase signature sequence proteins, most annotated as glutamyl-tRNAGln amidotransferases, were obtained for Homo sapiens (NP_060762), Mycobacterium tuberculosis (NP_335746), Archaeoglobus fulgidus DSM 4304 (NP_070778), and Plasmodium falciparum 3D7 (NP_702811). Amidase signature sequence-containing protein sequences from D. discoideum AX3, Dd1 (XM_632408), and Dd2 (XM_638290) were obtained from previous sequencing studies by our group (
      • Neelemagan D.
      • Schoenhofen I.
      • Richards J.C.
      • Cox A.D.
      Identification and recombinant expression of anadamide hydrolyzing enzyme from Dictyostelium discoideum.
      ). Takifugu and Ciona sequences were obtained from the Joint Genome Institute (http://genome-jgi-psf.org), and the Tetrahymena sequence originated from the The Institute for Genomic Research (http://www.tigr.org). All other sequences were obtained from GenBank (www.ncbi.nlm.nih.gov). Protein sequences were aligned with ClustalX (
      • Larkin M.A.
      • Blackshields G.
      • Brown N.P.
      • Chenna R.
      • McGettigan P.A.
      • McWilliam H.
      • Valentin F.
      • Wallace I.M.
      • Wilm A.
      • Lopez R.
      • et al.
      Clustal W and Clustal X version 2.0.
      ) using default parameters, and phylogenetic trees were generated using a minimum evolution algorithm using MEGA4 (
      • Tamura K.
      • Dudley J.
      • Nei M.
      • Kumar S.
      MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.
      ). Confidence values for trees were generated by bootstrapping based on 500 resampling replicates.

      Enzyme purification

      The Dd2 gene from D. discoideum AX3 was cloned into Escherichia coli BL21(DE3) as previously described (
      • Neelemagan D.
      • Schoenhofen I.
      • Richards J.C.
      • Cox A.D.
      Identification and recombinant expression of anadamide hydrolyzing enzyme from Dictyostelium discoideum.
      ). Enzyme was produced by diluting a fresh overnight culture of E. coli BL21(DE3) containing pCWMalET-FAAH vector in LB medium containing 0.2% glucose and 100 µg/ml ampicillin. The culture was grown at 25°C and 200 rpm to an OD600 of 0.6, at which time it was induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside to produce a recombinant FAAH linked to a maltose binding protein (MBP-FAAH). Cells were harvested 5 h later by centrifugation at 10,000 g for 10 min. Cell pellets were resuspended in lysis buffer (20 mM Tris-Cl [pH 9.0], 200 mM NaCl, 1 mM EDTA, and 10 mM β-mercaptoethanol) containing Complete® protease inhibitor mixture, EDTA-free (Roche Applied Science, Laval, Quebec, Canada) and 10 µg/ml each of RNaseA and DNaseI (Roche Applied Science). Cell lysis was performed via two passes through an Emulsiflex C5 (Avestin, Ottawa, ON, Canada) at 20,000 psi. Lysates were then centrifuged at 100,000 g for 50 min at 4°C. The supernatant was batch bound to an amylose resin (New England Biolabs, Pickering, Ontario, Canada) for 1 h at 4°C. Protein-bound resin was packed onto a column using gravity flow and washed with 10 column volumes of wash buffer (20 mM Tris-Cl [pH 9.0] and 300 mM NaCl). MBP-FAAH was eluted over 5 column volumes with elution buffer (20 mM Tris-Cl [pH 9.0], 200 mM NaCl, 15 mM maltose). Maltose-eluted fractions containing MBP-FAAH, as determined by absorbance at 280 nm, were pooled and dialyzed overnight in dialysis buffer (20 mM Tris-Cl [pH 9.0], 50 mM NaCl) at 4°C. Protein size was verified using SDS-PAGE, and protein yield was determined by Bradford assay using BSA as protein standard (Bio-Rad Laboratories, Hercules, CA). In the case of further purification of MBP-FAAH, MBP was removed via an overnight digestion at 4°C using a recombinant human thrombin (EMD Chemicals, Gibbstown, NJ). The resulting protein consisted of Dictyostelium FAAH with a hexahistidine tag located at the N-terminus (His6-FAAH), which was constructed in the initial cloning of the gene. Protein was then batch bound to a Ni-NTA resin (Qiagen, Missassauga, Ontario, Canada) for 1 h at 4°C before being packed onto a column. Protein-bound Ni-NTA resin was washed with 10 column volumes of wash buffer (20 mM Tris-Cl [pH 9.0], 400 mM NaCl, 10 mM imidazole, and 10 mM β-mercaptoethanol). To elute recombinant FAAH, a linear gradient was applied from 10 to 100 mM imidazole in wash buffer over 30 volumes followed by a final pulse of 10 volumes of wash buffer containing 200 mM imidazole. Fractions containing recombinant FAAH, as determined by absorbance at 280 nm, were pooled and dialyzed against a dialysis buffer (20 mM Tris-Cl [pH 9.0] and 50 mM NaCl). Protein size was verified using SDS-PAGE, and protein yield was determined by Bradford assay (Bio-Rad Laboratories).

      Inhibitor studies

      Inhibitor studies were carried out using ApNA as the substrate. The rate of hydrolysis by FAAH was determined by monitoring the production of p-nitroaniline at 380 nm with a microplate reader (PowerWave X; Biotech Instruments Inc., Winooski, VT). Substrate conversion was extrapolated from A380 versus mass of p-nitroaniline using the microplate reader. Reactions were carried out in 96-well plates where ApNA (in DMSO) was added to 100 µl reaction buffer (20 mM Tris-Cl [pH 9.0], 50 mM NaCl and 0.5% Triton X-100) to a final concentration of 0–300 µM. Wells were preincubated with inhibitor and preheated to 37°C. Reactions were initiated through the addition of 30 µg of MBP-FAAH (in a 20 mM Tris-Cl [pH 9.0], 50 mM NaCl storage buffer). Final reaction volumes were 200 µL, and reactions were performed in duplicate. Kinetic constants (Vmax, KM, and KI) were determined using three different inhibitor concentrations and calculated using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA). Results of inhibitor studies were verified on thrombin-digested MBP-FAAH to demonstrate that the maltose binding protein played no significant role in enzyme inhibition.

      Dictyostelium strain growth and lipid extraction

      D. discoideum AX3 cells were grown axenically in liquid culture (18 g/L maltose, 14.3 g/L peptone, 7.15 g/L yeast extract, 0.486 g/L KH2PO4, and 0.616 g/L Na2HPO4·2H2O) at 150 rpm and 21°C to a density of 5 × 106 cells/ml. To study the effect of FAAH inhibition, cells were grown for a further 24 h in the presence of 10 µM MAFP. Cells were then harvested via centrifugation at 500 g for 10 min. To remove phospholipids, which have been demonstrated to be potentially problematic in downstream analysis of NAEs, neutral lipids were extracted from Dictyostelium cell pellets using a mixture of ethyl acetate and hexane (9:1, v/v) (
      • Zoerner A.A.
      • Batkai S.
      • Suchy M-T.
      • Gutzki F.M.
      • Engeli S.
      • Jordan J.
      • Tsikas D.
      Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.
      ). PEA-d4 and AEA-d4 (1 ng of each) were added to Dictyostelium cell pellets containing 5 × 106 cells and resuspended in 250 µL of PBS. A 9:1 (v/v) mixture of ethyl acetate and hexane (750 µL) was then added, and the solution was vortexed at maximum speed for 30 s before separating the phases via centrifugation at 4,500 g for 5 min. The organic layer (top) was then transferred to a clean glass vial and dried under nitrogen and stored at −20°C until analysis by LC-MS/MS, at which time dried samples were reconstituted in a 50% ethanol/water mixture.

      LC-MS/MS analysis

      Analyses were performed on an LC-MS/MS system consisting of an Ultimate3000 HPLC system (Thermo Scientific, Sunnyvale, CA) linked to a 4000 Qtrap triple quadrupole linear ion trap mass spectrometer equipped with a Micro Ion Spray interface (AB/Sciex, Concord, Ontario, Canada). Samples were loaded from an autosampler that was kept at ambient temperature. Chromatographic separation was performed on a Kappa BioBasic C18 column (Thermo Scientific) with dimensions of 150 × 0.32 mm (5 µm particle size with 300 Å pore size). Mobile phase A consisted of 10 mM ammonium acetate in water. Mobile phase B consisted of 10 mM ammonium acetate in methanol. Gradient elution was performed at a flow rate of 5 µL/min. The initial composition of the gradient was 50% B for 3 min before a ballistic gradient raised the composition to 96% B over 1 min. The gradient was held at 96% B for 26 min. At 30 min, the gradient was returned to 50% B to reequilibrate the column for 10 min. The total run time was 40 min. The mass spectrometer was operated in positive ionization mode. Nitrogen was used as the curtain and nebulizer gas with the following source conditions: capillary voltage: 5.1 kV, temperature: 100°C, GS1: 12. The declustering potential was set at 70 eV for all masses. The collision energy and dwell times were set at 30 eV and 100 ms, respectively, for all multiple reaction monitoring transitions. Q1 and Q3 were operated at unit resolution. The following MS parameters were used to measure NAEs (precursor ion, product ion): NAE12:0 (244.5, 62.1), NAE14:0 (272.5, 62.1), NAE16:0 (300.5, 62.1), NAE18:0 (328.5, 62.1), NAE18:1 (326.5, 62.1), NAE18:2 (324.5, 62.1), NAE20:4 (348.5, 62.1), PEA-d4 (304.5, 66.1), and AEA-d4 (352.5, 66.1). Detection limits for all NAEs were determined during standard curve construction and were 5 fmol on column for NAE12:0 and 1 fmol on column for NAE14:0, NAE16:0, NAE18:0, NAE18:1, NAE18:2, and NAE20:4.

      Substrate consumption assays

      Measurement of FAAH activity was determined through a modified LC-MS/MS based assay (
      • King A.R.
      • Duranti A.
      • Tontini A.
      • Rivara S.
      • Rosengarth A.
      • Clapper J.R.
      • Astarita G.
      • Geaga J.A.
      • Luecke H.
      • Mor M.
      • et al.
      URB602 inhibits monoacylglycerol lipase and selectivity blocks 2-arachidonoylglycerol degradation in intact brain slices.
      ). The rate of hydrolysis of putative FAAH substrates was determined by measuring the depletion of reactant via LC-MS/MS. NAEs tested include NAE12:0, NAE14:0, NAE16:0, NAE18:0, NAE18:1, NAE18:2, and NAE20:4. Reactions were carried out in 1.5 ml microcentrifuge tubes where the addition of the corresponding NAE (in DMSO) to a final concentration of 300 µM was added to reaction buffer (20 mM Tris-Cl [pH 9.0], 50 mM NaCl, 10% DMSO, and 0.5% Triton X-100) to a final volume of 1.0 ml and preincubated at 21°C. Enzymatic reactions were initiated through the addition of 10 µg of MBP-FAAH (in a 20 mM Tris-Cl [pH 9.0], 50 mM NaCl storage buffer) and incubated at 21°C. Aliquots (100 µL) were taken at 0, 1, 2, 5, 10, and 20 min. Another 100 µL aliquot was taken for an overnight reaction (∼16 h) to determine reaction completion. Enzymatic reactions were stopped by placing aliquots in 300 µL of a 9:1 (v/v) mixture of ethyl acetate and hexane and purified and stored as described previously. For analysis by LC-MS/MS, dried samples were reconstituted in a 50% ethanol/water mixture. Kinetic assays were performed in triplicate.

      RESULTS

      Phylogenetic characterization of Dictyostelium FAAH

      Two putative genes (Dd1, Dd2) found in the D. discoideum genome containing the amidase signature sequence were compared with confirmed and potential FAAH protein sequences as identified in silico by McPartland et al. (
      • McPartland J.M.
      • Matias I.
      • Di Marzo V.
      • Glass M.
      Evolutionary origins of the endocannabinoid system.
      ). The analysis resulted in the formation of five distinct phylogenetic groupings (Fig. 1). These groupings can be described as FAAH 1 and its putative homologs, FAAH 2 and its putative homologs, other FAAH homologs, eukaryotic amidotransferases, and prokaryotic amidotransferases. Neither of the two D. discoideum proteins containing the amidase signature domain clustered among the FAAH 1 or FAAH 2 clades. The Dd2 protein clustered with the well characterized FAAH protein from Arabidopsis thaliana in a separate grouping to both the FAAH 1 and FAAH 2 clades. The identity between Dd2 and Arabidopsis FAAH is only 32%, but this increases to 50% across the amidase signature sequence. By comparison, Dd2 has only 20% identity with human FAAH 1 (40% over amidase signature sequence) (
      • Neelemagan D.
      • Schoenhofen I.
      • Richards J.C.
      • Cox A.D.
      Identification and recombinant expression of anadamide hydrolyzing enzyme from Dictyostelium discoideum.
      ). This suggests that Dd2 is a FAAH homolog but is more closely related to Arabidopsis FAAH than to human FAAH. The other putative D. discoideum protein analyzed, Dd1, clustered among known eukaryotic amidotransferases, suggesting that Dd1 is not a homolog of FAAH. From this point on, this paper will refer to the protein Dd2 as Dictyostelium FAAH.
      Figure thumbnail gr1
      Fig. 1Phylogenetic tree of FAAH and putative FAAH homologs. Two D. discoideum proteins containing the amidase signature sequence were analyzed along with FAAH and putative FAAH protein sequences identified by McPartland et al. (
      • McPartland J.M.
      • Matias I.
      • Di Marzo V.
      • Glass M.
      Evolutionary origins of the endocannabinoid system.
      ). After alignment, a minimum evolution phylogeny was constructed. Bootstraps were produced from 500 resamplings. Scale bar represents 0.2 substitutions per amino acid.

      Purification of a recombinant FAAH from D. discoideum

      The Dictyostelium FAAH gene from D. discoideum was cloned into E. coli BL21(DE3) and expressed as a recombinant fusion protein linked to a maltose binding protein (
      • Neelemagan D.
      • Schoenhofen I.
      • Richards J.C.
      • Cox A.D.
      Identification and recombinant expression of anadamide hydrolyzing enzyme from Dictyostelium discoideum.
      ). Typical yields of the MBP-FAAH fusion protein from E. coli BL21(DE3) cells were in the order of 3 mg/L of liquid media. Because the fusion protein was designed with an internal thrombin cleavage site and a hexahistidine tag, further purification of MBP-FAAH was possible via a thrombin digestion to separate the maltose binding protein followed by further purification via a Ni-NTA agarose column. The yield of His6-FAAH after the thrombin digestion and purification was 0.8 mg/L liquid media. To ensure the proper protein product was purified, the proteins were analyzed using SDS-PAGE to verify protein size. The calculated molecular weight of Dictyostelium FAAH was approximately 70 kDa. Given that the maltose binding protein has an approximate molecular weight of 42.5 kDa, the approximate molecular weight of the MBP-FAAH fusion protein is 112.5 kDa. All proteins migrated near their theoretical weights on the SDS-PAGE gel (Fig. 2).
      Figure thumbnail gr2
      Fig. 2SDS-PAGE of purification of FAAH from D. discoideum. A recombinant N-terminal hexahistidine tagged FAAH from D. discoideum linked to a maltose-binding protein (MBP-FAAH) was expressed in E. coli BL21(DE3). After lysis, MBP-FAAH was purified using an amylose column. MBP-FAAH could be further purified via an overnight digestion at 4°C with a recombinant thrombin, which separated the maltose binding protein from His6-FAAH. His6-FAAH could then be further purified using a Ni-NTA column. Lane 1, protein standard; Lane 2, MBP-FAAH post amylose column; Lane 3, thrombin-digested MBP-FAAH; Lane 4, His6-FAAH post Ni-NTA column.

      Inhibition of Dictyostelium FAAH

      The effect of known mammalian FAAH inhibitors on Dictyostelium FAAH was determined. The rate of hydrolysis of ApNA was measured by monitoring the release of p-nitroaniline at 380 nm with each inhibitor being tested at three different concentrations. The mode of inhibition was determined through Lineweaver-Burk analysis (Fig. 3), whereas KI was determined via a nonlinear regression of reaction velocity versus substrate concentration plots. Dictyostelium FAAH had a Vmax and KM on ApNA of 127 nmoles/min/mg and 74 µM, respectively. Results of the inhibitor studies are summarized in Table 1.
      Figure thumbnail gr3
      Fig. 3Inhibitor studies of Dictyostelium FAAH. Saturation curves of the reaction of Dictyostelium FAAH hydrolyzing arachidonoyl p-nitroaniline to arachidonic acid and p-nitroaniline with four different inhibitors at three different inhibitor concentrations. Lines of best fit determined by nonlinear regression of either a competitive or noncompetitive inhibition model and errors bars represent ± 1 SD. Inset: Lineweaver-Burk plot of inhibition of Dictyostelium FAAH. A: MAFP (squares, 0 µM; triangles, 100 nM inverted triangles, 1 µM; diamonds 2.5 µM). B: CAY10435 (squares, 0 µM; triangles, 100 nM; inverted triangles, 1 µM; diamonds, 2.5 µM). C: ATFMK (squares, 0 µM; triangles, 14 µM; –inverted triangles, 70 µM; diamonds, 140 µM). D: PMSF (squares, 0 mM; triangles, 1 mM; inverted triangles, 5 mM; diamonds, 10 mM).
      TABLE 1Summary of inhibitor studies detailing mode of inhibition on Dictyostelium FAAH, KI on Dictyostelium FAAH, and either KI or IC50 on mammalian versions of FAAH
      InhibitorMode of Inhibition for Dictyostelium FAAHKI DictyosteliumKI or IC50 Mammalian
      nM
      ATFMKCompetitive15,400IC50= 1900 (
      • Deutsch D.G.
      • Omeir R.
      • Arreaza G.
      • Salehani D.
      • Prestwich G.D.
      • Huang Z.
      • Howlett A.
      Methyl arachidonoyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase.
      )
      MAFPIrreversible648IC50= 2.5 (
      • Deutsch D.G.
      • Omeir R.
      • Arreaza G.
      • Salehani D.
      • Prestwich G.D.
      • Huang Z.
      • Howlett A.
      Methyl arachidonoyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase.
      )
      CAY10435Noncompetitive835KI= 0.57 (8)
      PMSFIrreversible4,160,000IC50= 900 (
      • Deutsch D.G.
      • Omeir R.
      • Arreaza G.
      • Salehani D.
      • Prestwich G.D.
      • Huang Z.
      • Howlett A.
      Methyl arachidonoyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase.
      )
      First-generation mammalian FAAH inhibitors ATFMK and MAFP inhibited Dictyostelium FAAH with a KI of 15.4 µM and 658 nM, respectively. ATFMK and MAFP are less effective on Dictyostelium FAAH than mammalian homologs of the enzyme, where IC50 values of 1.9 µM and 2.5 nM have been reported, respectively (
      • King A.R.
      • Duranti A.
      • Tontini A.
      • Rivara S.
      • Rosengarth A.
      • Clapper J.R.
      • Astarita G.
      • Geaga J.A.
      • Luecke H.
      • Mor M.
      • et al.
      URB602 inhibits monoacylglycerol lipase and selectivity blocks 2-arachidonoylglycerol degradation in intact brain slices.
      ). CAY10435, an α-keto oxazolopyridine, was a strong inhibitor of Dictyostelium FAAH with a KI of 835 nM. This compares with a KI of 0.57 nM for the mammalian enzyme. Furthermore, CAY10435 was shown to be a noncompetitive inhibitor of Dictyostelium FAAH, whereas for the mammalian enzyme the inhibition was competitive (
      • Boger D.L.
      • Sato H.
      • Lerner A.E.
      • Hedrick M.P.
      • Fecik R.A.
      • Miyauchi H.
      • Wilkie G.D.
      • Austin B.J.
      • Patricelli M.P.
      • Cravatt B.F.
      Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide.
      ).
      Other mammalian FAAH inhibitors tested in this study were far less effective inhibitors of Dictyostelium FAAH. The irreversible serine protease inhibitor PMSF inhibited Dictyostelium FAAH with a KI of 4.2 mM (compared with 900 nM for the mammalian version [
      • Deutsch D.G.
      • Omeir R.
      • Arreaza G.
      • Salehani D.
      • Prestwich G.D.
      • Huang Z.
      • Howlett A.
      Methyl arachidonoyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase.
      ]), whereas the highly potent mammalian FAAH inhibitors JNJ1661010 (a thiadiazolopiperazinyl urea-based inhibitor), URB597 (a carbamate-based inhibitor), and LY2183240 (a heterocyclic urea-based inhibitor) were all poor inhibitors of Dictyostelium FAAH, with negligible inhibition at inhibitor concentrations of 500 µM, 150 µM, and 250 µM, respectively. These values compare with a KI value of 34 nM for JNJ1661010 (
      • Keith J.M.
      • Apodaca R.
      • Xiao W.
      • Seierstad M.
      • Pattabiraman K.
      • Wu J.
      • Webb M.
      • Kabarz M.J.
      • Brown S.
      • Wilson S.
      • et al.
      Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase.
      ) and with the IC50 values of 4.6 nM and 12.4 nM for URB597 and LY2183240, respectively (
      • Kathuria S.
      • Gaetani S.
      • Fegley D.
      • Valiño F.
      • Duranti A.
      • Tontini A.
      • Mor M.
      • Tarzia G.
      • La Rana G.
      • Calignano A.
      • et al.
      Modulation of anxiety through blockade of anandamide hydrolysis.
      ,
      • Alexander J.P.
      • Cravatt B.F.
      The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases.
      ), on mammalian FAAH homologs.

      D. discoideum AX3 NAEs

      NAEs were identified in D. discoideum AX3 through a targeted lipidomics approach consisting of the extraction of neutral lipids with a mixture of ethyl acetate and hexane (9:1, v/v) followed by LC-MS/MS analysis. Initially, potential NAE candidates were identified via a precursor scan (m/z = 62.1), with electrospray ionization carried out in positive mode of uninhibited and MAFP-inhibited (10 µM) AX3 neutral lipid extracts. These scans revealed peaks consistent with the expected peaks of NAE12:0, NAE14:0, NAE16:0, NAE18:0, NAE18:1, NAE18:2, and NAE20:4. To confirm the identity of these peaks as NAEs, LC-MS/MS was performed on lipid extracts from an MAFP-inhibited culture. One sample was spiked with a 1 nmol mixture of the seven NAE standards, and the other was not. In terms of lipophilicity, all seven peaks in the unspiked sample had the same retention time as their corresponding NAE standard (Fig. 4). Also, the two deuterated internal standards, PEA-d4 and AEA-d4, coeluted with their corresponding nondeuterated NAE (data not shown). Furthermore, MS/MS was carried out on each putative NAE to produce a collision-induced dissociation spectrum (Fig. 5; supplementary Fig. I). The peaks in the unspiked sample produce a similar spectrum to that of the synthetic NAE standards for six out of the seven NAEs. The lone exception was NAE20:4, where the MS/MS spectral quality of unspiked sample did not allow for a confident spectral match to the standard. The presence of NAEs in D. discoideum has been demonstrated by lipophilicity (i.e., retention time in HPLC) and gas-phase ion chemistry (i.e., MS/MS fragmentation patterns).
      Figure thumbnail gr4
      Fig. 4Multiple reaction monitoring (MRM) chromatograms for each of the seven putative NAEs identified in D. discoideum. The sample was obtained from D. discoideum culture that had been grown to a density of 5 × 106 cells and then exposed to MAFP at a concentration of 10 µM for 24 h. A: Chromatograms from a sample that was not spiked with a 1 nmol mixture of each NAE. B: Chromatograms from a sample from the same culture spiked with a 1 nmol mixture. Elution times are denoted in minutes, and MRM transitions are indicated for each run.
      Figure thumbnail gr5
      Fig. 5Collision-induced dissociation (CID) mass spectra for the three C18 NAEs identified in D. discoideum. The putative NAEs in D. discoideum underwent CID, and their spectra were compared with the CID spectra of their respective synthetic NAE standard. The spectrum obtained from the unspiked D. discoideum lipid extract (left) is compared with that of the synthetic standard (right). The x axis denotes fragment size in amu.
      After the confirmation of the identity of NAEs in D. discoideum, they were quantified in uninhibited and MAFP-inhibited lipid extracts using multiple reaction monitoring (Table 2). The uninhibited AX3 strain produced quantifiable levels of NAE12:0, NAE14:0, NAE16:0, and NAE18:0, with quantities ranging from 0.4 pmol per 108 cells for NAE16:0 to 9.0 pmol per 108 cells for NAE12:0. There were no detectable levels of NAE18:1, NAE18:2, or NAE20:4 in the uninhibited AX3 extracts. As for the MAFP-inhibited extracts, addition of 10 µM MAFP for 24 h resulted in detectable levels of NAE18:1 and NAE18:2 (3.6 and 9.3 pmol per 108 cells, respectively). The addition of MAFP also resulted in a significant increase in the level of NAE16:0 from 0.41 to 1.2 pmol per 108 cells (P < 0.0001).
      TABLE 2.Quantitation of NAEs in D. discoideum in the presence and absence of 10 µM MAFP
      NAE Concentration
      NAE12:0NAE14:0NAE16:0NAE18:0NAE18:1NAE18:2
      pmol per 108 cells
      AX3 (n = 8)9.0 (4.5)
      Numbers in parentheses represent SD.
      2.6 (2.2)0.41 (0.21)1.1 (0.5)
      MAFP (n = 6)4.5 (3.1)1.3 (1.2)1.2* (0.3)0.88 (0.14)3.6* (1.9)9.3* (6.7)
      * P < 0.0001.
      a Numbers in parentheses represent SD.

      Substrate consumption rate of Dictyostelium FAAH

      An attempt to determine the in vitro kinetics of Dictyostelium FAAH on a variety of NAE substrates was made by measuring the rate of substrate depletion in 300 µM reactions via LC-MS/MS. The determination of true initial reaction rates was difficult due to the variance of LC-MS/MS measurements at low substrate consumption levels. To reduce this variance, we report our results as a substrate consumption rate from time 0 to 20 min. At this time, approximately 30% of the substrate was consumed. All seven NAE substrates tested were completely degraded by FAAH in overnight reactions, demonstrating that Dictyostelium FAAH is capable of hydrolyzing NAE substrates with a variety of acyl chain lengths and levels of unsaturation. Substrate consumption rates were determined for six of the seven substrates (Table 3). A consumption rate was not reported for NAE12:0 because there was no significant difference in the amount of the NAE during the 20 min time course.
      TABLE 3.Substrate consumption rate of Dictyostelium FAAH on NAE substrates at 300 µM substrate concentration and comparison of acyl chain length on FAAH reaction rates for three different species (n = 3)
      Relative Rate to NAE20:4
      SubstrateSubstrate Consumption RateDictyosteliumArabidopsis (
      • Alexander J.P.
      • Cravatt B.F.
      The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases.
      )
      Rat
      In this study, corresponding amide substrates were used (i.e., arachidonamide).
      (
      • Boger D.L.
      • Fecik R.A.
      • Patterson J.E.
      • Miyauchi H.
      • Patricelli M.P.
      • Cravatt B.F.
      Fatty acid amide hydrolase susbstrate specificity.
      )
      nmol/min/mg
      N-arachidonoylethanolamine (NAE20:4)111.4 (58.9)
      Numbers in parentheses represent standard deviations.
      1.001.001.00
      N-linoleoylethanolamine (NAE18:2)83.4 (58.5)0.750.790.33
      N-oleoylethanolamine (NAE18:1)112.2 (56.3)1.01n.d.0.32
      N-stearoylethanolamine (NAE18:0)39.9 (18.7)0.36n.d.0.22
      N-palmitoylethanolamine (NAE16:0)10.1 (0.5)0.090.680.23
      N-myristoylethanolamine (NAE14:0)20.7 (6.6)0.190.510.27
      n.d., not determined.
      a In this study, corresponding amide substrates were used (i.e., arachidonamide).
      b Numbers in parentheses represent standard deviations.
      Dictyostelium FAAH displayed a preference for unsaturated NAE substrates, with consumption rates of 111.4, 83.4, and 112.2 nmol/min/mg for NAE20:4, NAE18:2, and NAE18:1, respectively. By comparison, saturated NAE substrates were hydrolyzed at less than half those rates at 39.9, 10.1, and 20.7 nmol/min/mg for NAE18:0, NAE16:0, and NAE14:0, respectively. The preference of Dictyostelium FAAH for unsaturated NAEs is consistent with results reported for the Arabidopsis and rat FAAH homologs. However, although anandamide is still a preferred NAE substrate for all three homologs, neither Dictyostelium FAAH nor Arabidopsis FAAH have as strong a preference for the arachidonoyl side chain as exhibited by the rat FAAH homolog. Although the presence of an unsaturated site in the acyl chain had a definitive impact on reaction velocity, there was no clear effect of acyl chain length on reaction velocity. The hydrolysis of NAE18:0 was higher than NAE16:0 and NAE14:0. However, NAE14:0 had a higher reaction velocity than NAE16:0. Previous reports have shown that rat FAAH slightly prefers shorter acyl chain substrates (
      • Boger D.L.
      • Fecik R.A.
      • Patterson J.E.
      • Miyauchi H.
      • Patricelli M.P.
      • Cravatt B.F.
      Fatty acid amide hydrolase susbstrate specificity.
      ), whereas Arabidopsis FAAH had a higher reaction velocity on NAE16:0 than NAE14:0 (
      • Shrestha R.
      • Dixon R.A.
      • Chapman K.D.
      Molecular identification of a functional homologue of the mammalian fatty acid amide hydrolase in Arabidopsis thaliana.
      ), suggesting the opposite.

      DISCUSSION

      Previous work by our group had identified two amidase signature sequence genes in the D. discoideum genome, which were named Dd1 (GenBank: XM_632408) and Dd2 (GenBank: XM_638290). Initial cloning and expression of these genes as recombinant proteins in E. coli had revealed that of the two proteins only Dd2 was capable of hydrolyzing anandamide (NAE20:4). Despite significant sequence variation from other FAAH homologs, the conservation of the serine-serine-lysine triad demonstrated to be important for NAE hydrolysis was conserved (
      • Bracey M.H.
      • Hanson M.A.
      • Masuda K.R.
      • Stevens R.C.
      • Cravatt B.F.
      Structural adaptations in a membrane enzyme that terminates endocannabiond signaling.
      ,
      • McKinney M.K.
      • Cravatt B.F.
      Evidence for distinct roles in catalysis for residues of the serine-serine-lysine catalytic triad of fatty acid amide hydrolase.
      ), leading to its identification as Dictyostelium FAAH (
      • Neelemagan D.
      • Schoenhofen I.
      • Richards J.C.
      • Cox A.D.
      Identification and recombinant expression of anadamide hydrolyzing enzyme from Dictyostelium discoideum.
      ). Further phylogenetic characterization of Dd1 and Dd2 with known FAAH sequences revealed that, although Dd2 was indeed a FAAH homolog being most closely related to Arabidopsis FAAH, Dd1 clustered among other eukaryotic amidotransferases. The presence of a functional FAAH in D. discoideum led to the hypothesis that the natural substrates of FAAH, NAEs, are also present in D. discoideum. To verify this, we further characterized FAAH to identify inhibitors of FAAH, which should elevate NAE levels in D. discoideum and aid in identifying NAEs before attempting targeted lipidomic analyses of D. discoideum lipid extracts.
      Inhibitor studies were carried out in vitro to verify if known FAAH inhibitors were effective on Dictyostelium FAAH and to identify inhibitors that could potentially allow for the modulation of NAE levels in vivo. Although the first-generation substrate-based inhibitors ATFMK and MAFP inhibited Dictyostelium FAAH, they were slightly less effective than on mammalian FAAH homologs. The serine protease inhibitor PMSF was also capable of inhibiting Dictyostelium FAAH but with a KI of several orders of magnitude higher than mammalian FAAH homologs. These results are similar to results observed in inhibitor studies of Arabidopsis FAAH (
      • Alexander J.P.
      • Cravatt B.F.
      The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases.
      ). Of the four highly specific mammalian FAAH inhibitors tested, only CAY10435 inhibited Dictyostelium FAAH, although through a noncompetitive mechanism as opposed to the competitive mechanism in which it inhibits mammalian FAAH homologs.
      Targeted lipidomic analyses of D. discoideum lipid extracts identified several NAEs as native to D. discoideum that have been reported to be present in a variety of plant species (
      • Chapman K.D.
      • Venables B.
      • Blair Jr, R.
      • Bettinger C.
      N-acylethanolamines in seeds. Quantification of molecular species and their degradation upon imbibition.
      ), human tissues (
      • Long J.Z.
      • LaCava M.
      • Jin X.
      • Cravatt B.F.
      An anatomical and temporal portrait of physiological substrates for fatty acid amide hydrolase.
      ), and the ciliate Tetrahymena thermophila (
      • Anagnostopoulos D.
      • Rakiec C.
      • Wood J.
      • Pandarinathan L.
      • Zvonok N.
      • Makriyannis A.
      • Siafaka-Kapadai A.
      Identification of endocannabinoids and related N-acylethanolamines in Tetrahymena. A new class of compounds for Tetrahymena.
      ). Although the short-chain saturated NAEs NAE12:0 and NAE14:0 are typically associated with plants where, functionally, NAE12:0 has been shown to competitively inhibit lipoxygenase activity and manipulate oxylipin metabolism (
      • Keereetaweep J.
      • Kilaruna A.
      • Feussner I.
      • Venables B.J.
      • Chapman K.D.
      Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity.
      ), NAE16:0, NAE18:0, NAE18:1, and NAE18:2 are widespread in plants, animals, and T. thermophila. In conjunction with a similar NAE profile, substrate kinetic studies revealed that Dictyostelium FAAH, like other FAAH homologs, hydrolyzes a broad spectrum of NAE substrates. The principal difference in substrate kinetics is that Dictyostelium FAAH hydrolyzes NAE20:4 at a similar rate to other NAEs, whereas mammalian homologs hydrolyze NAE20:4 at a 3-fold elevated rate compared with other NAE substrates. This could be due to the need for mammals to tightly regulate NAE20:4 due to its binding to the cannabinoid receptors, which have yet to be identified in plants and lower eukaryotes. With a similar NAE profile and FAAH activity, this raises the possibility that many of the NAE-signaling pathways present in D. discoideum may be conserved in higher eukaryotic organisms.
      The characterization of Dictyostelium FAAH, the identification of native NAEs, and the ability to modulate their levels in vivo provides an opportunity to use D. discoideum as a model system in which to study NAE-mediated signaling. The use of D. discoideum as a model system has many advantages. D. discoideum is already an extensively studied protozoan in developmental biology due to its unusual life cycle that sees it switch from a unicellular grazer to a multicellular slug upon starvation (
      • Devreotes P.
      Dictyostelium discoideum: a model system for cell-cell interactions in development.
      ) and has been used as a model in other areas, including the study of mitochondrial and neurological diseases (
      • Francione L.M.
      • Anneslev S.J.
      • Carilla-Latorre S.
      • Escalante R.
      • Fisher P.R.
      The Dictyostelium model for mitochondrial disease.
      ,
      • Meyer I.
      • Kuhnert O.
      • Gräf R.
      Functional analyses of lissencephaly-related proteins in Dictyostelium.
      ), pathogen-host interactions (
      • Steinert M.
      • Heuner K.
      Dictyostelium as host model for pathogenesis.
      ), and chemotaxis (
      • Devreotes P.N.
      Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium.
      ). Due to its well-characterized nature, genetic and biochemical techniques in D. discoideum are already well developed, including gene knockout by homologous recombination, RNAi-mediated knockdowns, and genetic overexpression (
      • Kuhlmann M.
      • Popova B.
      • Nellen W.
      RNA interference and antisense-mediated gene silencing in Dictyostelium.
      ). Finally, many of the roles linked to NAEs in higher eukaryotes, such as the elevated levels of NAE14:0 in the presence of fungal xylanase in plants (
      • Tripathy S.
      • Venables B.J.
      • Chapman K.D.
      N-acylethanolamines in signal transduction of elicitor perception. Attenuation of alkalinization response and activation of defense gene expression.
      ), the anti-inflammatory effects linked to NAE16:0 (
      • Lambert D.M.
      • Vandevoorde S.
      • Diependaele G.
      • Govaerts S.J.
      • Robert A.R.
      Anticonvulsant activity of N-palmitoylethanolamide, a putative endocannabinoid, in mice.
      ), the role of NAE18:0 as an immunomodulator and apoptosis-inducer (
      • Maccarrone M.
      • Pauselli R.
      • Di Rienzo M.
      • Finazzi-Agrò A.
      Binding, degradation and apoptotic activity of stearoylethanolamide in rat C6 glioma cells.
      ), and NAE18:1 as a regulator of food intake (
      • Fu J.
      • Kim J.
      • Oveisi F.
      • Astarita G.
      • Piomelli D.
      Targeted enhancement of oleoylethanolamide production in proximal small intestine induces across-meal satiety in rats.
      ), are subsets of larger research areas already being modeled in D. discoideum.
      We have demonstrated that the slime mold D. discoideum possesses a similar NAE profile and a FAAH that acts on a similarly broad set of NAE substrates as those found in higher eukaryotic organisms. Furthermore, we have demonstrated the ability to modulate the levels of NAEs in vivo. Given its genetic tractability and broad use as a model organism to study many cellular processes, we propose that D. discoideum would be a suitable model in which to study NAE-mediated signaling. Given the large number of human physiological conditions associated with NAE-mediated signaling, the elucidation of these pathways in D. discoideum could have much broader implications on human health.

      Acknowledgments

      The authors thank Dr. Rishi Kumar for help in producing NAE12:0 and NAE14:0 standards, Dr. Ian Schoenhofen for help in protein purification, and Dr. Susan Logan for the use of laboratory space.

      Supplementary Material

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