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

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.

fl ash 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.

Enzyme purifi cation
The Dd2 gene from D. discoideum AX3 was cloned into Escherichia coli BL21(DE3) as previously described ( 27 ). 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 OD 600 of 0.6, at which time it was induced with 0.1 mM isopropyl-1-thio-␤ -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 ␤ -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 Emulsifl ex 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 fl ow 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 verifi ed using SDS-PAGE, and protein yield was determined by Bradford assay using BSA as protein standard (Bio-Rad Laboratories, Hercules, ( 21 ). Along with evidence of an enzyme similar to FAAH present in Tetrahymena pyriformis ( 22 ), 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 ⌬ 9 -tetrahydrocannabinol affecting cellular growth, movement, and division of T. pyriformis ( 23,24 ) and 2-arachidonoyl glycerol inhibiting cellular growth in freeliving amoebae ( 25 ). 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 ( 26 ).
It is not known whether NAEs are present in D. discoideum . The identifi cation of FAAH in D. discoideum by our group ( 27 ) 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, identifi ed NAEs native to D. discoideum in the presence and absence of a semispecifi c FAAH inhibitor. These substrates were then confi rmed 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.

Preparation of N-acylethanolamines
NAE standards were prepared for NAE12:0 and NAE14:0 as described by Williams et al. ( 28 ). 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide 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 SiO 2 to adsorb the NAE. Dichloromethane was then evaporated, and the SiO 2 was packed into a column, dry-loaded onto a SiO 2 fl ash chromatography column, and purifi ed using a 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 Scientifi c) 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 fl ow 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.

Substrate consumption assays
Measurement of FAAH activity was determined through a modifi ed LC-MS/MS based assay ( 33 ). 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 fi nal 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 fi nal 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 ( ‫ف‬ 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 purifi ed 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.

Phylogenetic characterization of Dictyostelium FAAH
Two putative genes ( Dd1 , Dd2 ) found in the D. discoideum genome containing the amidase signature sequence were compared with confi rmed and potential FAAH protein sequences as identifi ed in silico by McPartland et al. ( 29 ). The analysis resulted in the formation of fi ve distinct phylogenetic groupings ( Fig. 1 ). These groupings can be described as FAAH 1 and its putative homologs, FAAH 2 CA). In the case of further purifi cation 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 (His 6 -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 ␤ -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 fi nal 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 verifi ed using SDS-PAGE, and protein yield was determined by Bradford assay (Bio-Rad Laboratories).

Inhibitor studies
Inhibitor studies were carried out using A p NA 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 A 380 versus mass of p -nitroaniline using the microplate reader. Reactions were carried out in 96-well plates where A p NA (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 fi nal 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 (V max , K M , and K I ) 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 verifi ed on thrombin-digested MBP-FAAH to demonstrate that the maltose binding protein played no signifi cant 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 KH 2 PO 4 , and 0.616 g/L Na 2 HPO 4 ·2H 2 O) at 150 rpm and 21°C to a density of 5 × 10 6 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) ( 32 ). PEA-d 4 and AEA-d 4 (1 ng of each) were added to Dictyostelium cell pellets containing 5 × 10 6 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 Ϫ 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 Scientifi c, Sunnyvale, 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 ).

Inhibition of Dictyostelium FAAH
The effect of known mammalian FAAH inhibitors on Dictyostelium FAAH was determined. The rate of hydrolysis of A p NA 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 K I was determined via a nonlinear regression of reaction velocity versus substrate concentration plots. Dictyostelium FAAH had a V max and K M on A p NA of 127 nmoles/ min/mg and 74 µM, respectively. Results of the inhibitor studies are summarized in Table 1 .
First-generation mammalian FAAH inhibitors ATFMK and MAFP inhibited Dictyostelium FAAH with a K I of 15.4 µM and 658 nM, respectively. ATFMK and MAFP are less effective on Dictyostelium FAAH than mammalian homologs of the enzyme, where IC 50 values of 1.9 µM and 2.5 nM have been reported, respectively ( 33 ). CAY10435, an ␣ -keto oxazolopyridine, was a strong inhibitor of Dictyostelium FAAH with a K I of 835 nM. This compares with a K I 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 ( 8 ). 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) ( 27 ). 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.

Purifi cation 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 ( 27 ). 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 purifi cation of MBP-FAAH was possible via a thrombin digestion to separate the maltose binding protein followed by further purifi cation via a Ni-NTA agarose column. The yield of His 6 -FAAH after the thrombin digestion and purifi cation was 0.8 mg/L liquid media. To ensure the proper protein product was purifi ed,  ( 29 ). After alignment, a minimum evolution phylogeny was constructed. Bootstraps were produced from 500 resamplings. Scale bar represents 0.2 substitutions per amino acid.
PEA-d 4 and AEA-d 4 , 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 confi dent 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).

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 diffi cult 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 signifi cant difference in the amount of the NAE during the 20 min time course.
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 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 K I of 4.2 mM (compared with 900 nM for the mammalian version [ 34 ]), 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 K I value of 34 nM for JNJ1661010 ( 11 ) and with the IC 50 values of 4.6 nM and 12.4 nM for URB597 and LY2183240, respectively ( 9, 35 ), on mammalian FAAH homologs.

D. discoideum AX3 NAEs
NAEs were identifi ed 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 identifi ed 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 confi rm 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, higher reaction velocity on NAE16:0 than NAE14:0 ( 37 ), suggesting the opposite.

DISCUSSION
Previous work by our group had identifi ed 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 signifi cant sequence variation from other FAAH homologs, the conservation of the serine-serine-lysine triad demonstrated to be important for NAE hydrolysis was conserved ( 38,39 ), leading to its identifi cation as Dictyostelium FAAH ( 27 ). 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 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 defi nitive 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 ( 36 ), whereas Arabidopsis FAAH had a  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 K I of several orders of magnitude higher than mammalian FAAH homologs. These results are similar to results observed in inhibitor studies of Arabidopsis FAAH ( 35 ). Of the four highly specifi c 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 identifi ed several NAEs as native to D. discoideum that 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 fi rst-generation substrate-based inhibitors ATFMK and Fig. 4. Multiple reaction monitoring (MRM) chromatograms for each of the seven putative NAEs identifi ed in D. discoideum . The sample was obtained from D. discoideum culture that had been grown to a density of 5 × 10 6 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.
In conjunction with a similar NAE profi le, 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 have been reported to be present in a variety of plant species ( 40 ), human tissues ( 41 ), and the ciliate Tetrahymena thermophila ( 21 ). 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 ( 42 ), NAE16:0, NAE18:0, NAE18:1, and NAE18:2 are widespread in plants, animals, and T. thermophila .  to tightly regulate NAE20:4 due to its binding to the cannabinoid receptors, which have yet to be identifi ed in plants and lower eukaryotes. With a similar NAE profi le 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 ( 43 ) and has been used as a model in other areas, including the study of mitochondrial and neurological diseases ( 44,45 ), pathogen-host interactions ( 46 ), and chemotaxis ( 47 ). 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 ( 48 ). 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 ( 14 ), the anti-infl ammatory effects linked to NAE16:0 ( 49 ), the role of NAE18:0 as an immunomodulator and apoptosis-inducer ( 50 ), and NAE18:1 as a regulator of food intake ( 51 ), 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 profi le 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.
The authors thank Dr. Rishi Kumar for help in producing NAE12:0 and NAE14:0 standards, Dr. Ian Schoenhofen for