The Rise and Fall of Anandamide: Processes That Control Synthesis, Degradation, and Storage

Molecular and Cellular Biochemistry https://doi.org/10.1007/s11010-021-04121-5

The rise and fall of anandamide: processes that control synthesis, degradation, and storage

Roger Gregory Biringer1

Received: 18 August 2020 / Accepted: 25 February 2021 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract Anandamide is an endocannabinoid derived from arachidonic acid-containing membrane lipids and has numerous biological functions. Its efects are primarily mediated by the cannabinoid receptors CB1 and CB2, and the vanilloid TRPV1 receptor. Anandamide is known to be involved in sleeping and eating patterns as well as pleasure enhancement and pain relief. This manuscript provides a review of anandamide synthesis, degradation, and storage and hence the homeostasis of the anandamide signaling system.

Keywords Anandamide · Endocannabinoid · Phospholipase · N-acyltransferase · Phosphatase

Introduction notice. This manuscript discusses the key enzymes in AEA homeostasis, in terms of structure, reaction specifcity, enzy- The endocannabinoid anandamide (AEA) is an ethanola- matic activity, regulation, and tissue and cellular expression mide derivative of arachidonic acid (AA) that serves to patterns with a focus on the human isoforms involved. activate primarily cannabinoid and vanilloid receptors. The resulting G-protein signaling initiates a number of biological pathways. The name given to it by its discoverers [1] is Anandamide biosynthesis derived from the Sanskrit word ananda meaning bliss or joy in reference to their fnding that this molecule competes with AEA is synthesized through ligation of a previously mem- exogenous cannabinoids for their specifc receptors in the brane bound arachidonic acid and a membrane bound phos- brain, the frst endogenous molecule found to do so. Subse- phatidylethanolamine forming an N-arachadonoyl phos- quent research reveals that anandamide activates both CB1 phatidylethanolamine (NArPE). Release of AEA from the in the central nervous system, CB2 in the peripheral tissues, composite molecule is accomplished by a series of phos- including immune cells, and TRPV1, a non-selective cation pholipases (Fig. 1). channel ubiquitously expressed in all tissues. The resulting signaling involves a diverse set of biological results includ- Synthesis of N‑acyl phosphatidylethanolamines ing sleep and eating patterns, short-term memory, mood, (NAPEs) as well as modulation of the sensation of heat, acid, and proinfammatory stimulants. There are a number of excellent Overview reviews on these subjects [2–5]. As is typical for signaling molecules, the in vivo con- The synthesis of NAPEs, including NArPE, is accomplished centration of AEA is maintained through the relative rates through the transfer of a fatty acyl group from a glycer- of synthesis and degradation, but also through a less com- ophospholipid, phosphatidylcholine (PC) in particular, to mon storage system, designed to meet high demand on short the primary amine of a phosphatidylethanolamine (PE) by one of a series of N-acyltransferases that are either calcium- * Roger Gregory Biringer independent or calcium-dependent. [email protected] The calcium-independent group of N-acyltransferases are represented by the H-RAS-like suppressor (HRASLS) 1 College of Osteopathic Medicine, Lake Erie College subfamilies of proteins and are members of the H-rev107 of Osteopathic Medicine, Bradenton, FL 34211, USA

Vol.:(0123456789)1 3 Molecular and Cellular Biochemistry

PLAAT1,2,3,4,5

PLA2G4E

NAPE PLC ABHD4

sPLA2

NAPE-PLD Phospho-AEA SHIP1

PTPN22

GP-NAE GDPD1,3 Lyso-NAPE GDPD1,3 GDE1

AEA

Fig. 1 Reactions involved in the formation of Anandamide NAPE-PLD N-Acyl-Phosphatidylethanolamine-hydrolyzing Phos- Metabolites: AEA anandamide, glycero-p-AEA 1-acyl-sn-glyc- pholipase D, PLAAT1,2,3,4,5 phospholipase A-Acyltransferases iso- ero-3-(N-arachidonyl) phosphoethanolamine, and NArPE N-acyl forms 1, 2, 3, 4, and 5, PLC phospholipase C, PTPN22 tyrosine-pro- phosphatidylethanolamine, Enzymes: ABHD4 (lyso)-N-Acylphos- tein phosphatase non-receptor type 22, SHIP1 phosphatidylinositol phatidylethanolamine lipase, GDE1 Glycerophosphodiester phos- 3,4,5-trisphosphate 5-phosphatase 1, and sPLA2 secreted Phospholi- phodiesterase 1, GDPD1,3 Lysophospholipase D isoforms 1 and 3, pase A2 class of proteins. These proteins (PLA/AT subgroup) were Phospholipase A and acyltransferase 1 originally classifed as H-RAS-like tumor suppressors and later found to catalyze phospholipid hydrolysis and There are two known splice isoforms of human phospho- exhibit N-acyltransferase activity. All but PLAAT5 con- lipase A/acyltransferase 1 (hPLAAT1, PLA/AT-1, Uni- tains a putative transmembrane helix but is known to be protKB-Q9HDD0) that are products of alternative splic- membrane associated, nonetheless. Each of the enzymes ing, Isoform 1 (Q9HDD0-1, PLAAT-1S, HRASLS, A-C1) catalyzes the hydrolysis of fatty acids from either the sn-1 and isoform 2 (Q9HDD0-2, PLAAT-1L) that contains an or sn-2 position of glycerophospholipids, the sn-2 posi- additional 105 residues—corresponding to exon 1—at the tion being particularly important for anandamide synthe- N-terminal [7]. Physical properties for all isoforms are given sis, as arachidonic acid is typically found esterifed to the in the supplement Table S1. No coding single nucleotide sn-2 position in glycerophospholipids. Once the fatty acid polymorphism (SNP) variants are reported [8, 9] (https:// is removed it is then a substrate for one of two second- genecards. org ; https://www. ncbi. nlm. nih. gov/ clinv ar ). Pho- ary reactions, O-acyl transfer of the fatty acid to the free bius web server predictions [10] (http://phobius.sbc.su.se/) hydroxyl of a lysophospholipid or an N-acyl transfer to suggest that both isoform 1 and isoform 2 are single-pass the primary amine on a phosphatidylethanolamine. These transmembrane proteins with large cytoplasmic N-terminal enzymes share a similar catalytic mechanism involving a extracellular domains and short 8-residue lumenal C-ter- thioester intermediate formed between an active site Cys minal domains. There is some confusion in the literature and the fatty acid prior to transfer [6]. regarding the expression of the two isoforms. Early work [11] suggests that Isoform 1 is the predominant species

1 3 Molecular and Cellular Biochemistry produced and is expressed in testes, muscle, brain, and for both isoform 1 and 2 posted on the SWISS-MODEL heart. Later work suggests that only the longer Isoform 2 website [14] (https://swissmodel.expasy.org/), template is the endogenous isoform in human tissue and is expressed 4q95.1.A and template 2kyt.1.A. Predicted posttranslational in these same tissues, whereas both isoforms are known to modifcations (PTMs) are given in the supplement Table S1 coexist in murine tissues [7, 12]. The highest expression is [15–17] and no confrmed sites have been reported. found in the testis and skeletal muscle and it is ubiquitously Originally designated as a class II tumor suppressor pro- expressed in most other tissues albeit at very low levels [13]. tein, hPLAAT1 was later found to be involved in calcium- One diference between the two isoforms is the cellular dis- independent phospholipid metabolism with both lipase and tribution, where Isoform 1 is found in the cytosol, whereas acyltransferase activity [13]. The enzymatic activity of both Isoform 2 is found in the both the cytosol and the nucleus. isoforms is essentially the same [7]; however, most of the The latter has been shown to be primarily associated with reported data refer to Isoform 1 (Table 1) [7, 11, 18]. There focal adhesion sites and in the cytosol to a lesser extent [13]. are three enzymatic activities to consider for this enzyme: No X-ray structures have been reported for either isoform. 1) hydrolysis at the sn-1 position, 2) hydrolysis at the sn-2 There are, however, two potential structural model templates position, and 3) the N-acyltransferase activity. Hydrolysis

Table 1 Enzyme Activity for enzymes involved in the formation of N-acylphosphatidylethanolamine (NAPE) Source Reaction Enzyme Specifc activity for given substrate (nmol/min/mg)

PLA1/2 NAT PLA1/PLA2 NAT/PLA1/2 References

Purifed recombinant protein N-acyltransfer hPLA/AT-1L – 14.5 – 2.0–2.5 [7] Purifed recombinant protein N-acyltransfer hPLA/AT-1S – 12.9 – 1.9–2.4 [7] Purifed protein O-acyltransfer hPLA/AT-1S 182 – 6.7–8.9 – [11] COS-7/Human homogenate O and N hPLA/AT-1S 0.15 0.3 – 1.6 [18] COS-7/Human homogenate O and N hPLA/AT-2 0.6 3.6 – 5.6 [18] Purifed recombinant protein O-acyltransfer hPLA/AT-2 – – 0.72 – [20] COS-7/Human homogenatea O-acyltransfer hPLA/AT-2 5.2 – – – [21] COS-7/Human Soluble Fraca O-acyltransfer hPLA/AT-2 3.6 – – – [21] Purifed recombinant protein b O-acyltransfer hPLA/AT-2 440 103 1.9 0.23 [21] COS-7/Human homogenate O and N hPLA/AT-3 1 0.25 0.1 [18] Purifed recombinant protein O-acyltransfer hPLA/AT-3 – – 1.15 – [20] COS-7/Human homogenate O and N hPLA/AT-3 – 0.115 13.8 – [23] COS-7/Human homogenatea O-acyltransfer hPLA/AT-3 12.8 – – – [21] COS-7/Human Soluble Fraca O-acyltransfer hPLA/AT-3 11.3 – – – [21] Purifed recombinant protein b O-acyltransfer hPLA/AT-3 820 4.4 4.4 0.0054 [21] COS-7/Human homogenate O and N hPLA/AT-4 0.5 0.18 – 0.4 [18] Purifed recombinant protein O-acyltransfer hPLA/AT-4 – – 1.5 [20] COS-7/Human homogenatea O-acyltransfer hPLA/AT-4 1.5 – – – [21] COS-7/Human soluble frac a O-acyltransfer hPLA/AT-4 1.6 – – – [21] Purifed recombinant protein b O-acyltransfer hPLA/AT-4 230 4.5 3 0.02 [21] COS-7/Human homogenate O and N hPLA/AT-5 0.1 0.45 – 4 [18] Purifed recombinant protein b Overall hPLA/AT-5 – 50 – – [27] Purifed recombinant protein b Overall hPLA2G4E – 20 – – [28] HEK293/recombinant cell lysate Overall hPLA2G4E – 9 – – [29] Mouse brain lysatec Overall mPLA2G4E – 0.0037 3.3 – [30] HEK293T/mouse recombinantc Overall mPLA2G4E – 6.9 9.6 – [30]

PLA1/2 refers to combined phospholipase A activity without reference to ester positions, NAT refers to N-acyl transferase activity, PLA1/PLA2 ratio of PLA2 activity for position one over that for position 2. NAT/PLA1/2 is the ratio of total PLA2 activity to N-acyltransferase activity. m refers to murine, h refers to human, COS-7 fbroblast-like cell line from monkey kidney, HEK293 human embryonic kidney cell line, PLA/AT phospholipase A/acyltransferase, PLA2G4E cytosolic phospholipase A2 epsilon a Value interpreted from bar graph b Dipalmotoylphosphatidyl choline substrate c Average for Mixed palmitoyl-steroyl substrate: NAT value from sn-1 position, sn-1/sn-1 average of C18 and C16 transfer

1 3 Molecular and Cellular Biochemistry at the sn-1 position occurs at 7–9 times the rate for the sn-2 4DOT). PLAAT3 is expressed primarily in the brain, adipose position and the hydrolysis rate of the arachidonic acid tissue, and blood, but is also found ubiquitously expressed ester is slightly slower than observed for the palmitic acid in most other tissues [13]. Its cellular distribution appears to ester (15–35% slower) [11]. The N-acyltransferase rate is be primarily localized to the mitochondria [13] but has also 1.6–2.5 times faster than the overall phospholipase activity. been reported as cytosolic [22] and possibly plasma mem- Sequence comparison to other family members indicate that brane and peroxisome membrane associated by comparison C119 is likely the active site nucleophile. to the rat and mouse counterparts [21]. Predicted PTMs are Regulation of the hPLAAT1 phospholipase or acyltrans- given in the supplement Table S1, none of which have been ferase activity by expression or by posttranslational modi- confrmed experimentally. fcation has not been reported. However, it has been shown Also designated as a tumor suppressor protein, hPLAAT3 that this protein is signifcantly downregulated in tumor cells exhibits a calcium-independent activity. PLA1/2 reaction by unknown processes [19]. rates (Table 1) are faster than the N-acylation reactions by 10–200-fold [18, 20, 21, 23]. There is a preference for the Phospholipase A and acyltransferase 2 sn-1 site that varies from 1.2 to 14 fold (Table 1) and it exhibits tenfold faster reactions with PC substrates as com- Human phospholipase A and acyltransferase 2(hPLAAT2, pared to PE substrates [23]. Mutagenesis studies reveal PLA/AT-2, HRASLS2, UniprotKB-Q9NWW9) presents that C113, H23, and H35 are necessary for activity [24]. as a single isoform with a sequence homology to PLAAT1 Regulation of phospholipase or acyltransferase activity by isoform 1 of 47.6%. Physical properties are given in the sup- expression or by posttranslational modifcation have not plement Table S1. No coding SNP variants are reported [8, been reported. However, although PLAAT3 is active in the 2+ 9]. As expected, structure predictions [10] suggest that it absence of intracellular calcium [Ca ]i, Vmax for lipase is a single-pass membrane protein with a large cytoplas- activity increases up to two fold in the presence of calcium, mic N-terminal domain (residues 1-129) and a small lume- suggesting that this enzyme is a special kind of calcium- nal C-terminal domain (residues 156-162). One partial dependent lipase [25]. Downregulation of the PLAAT3 gene X-ray structure is reported (PDB entry DPZ). hPLAAT2 is through promotor methylation in cancer cells is well docu- expressed primarily in the GI tract and blood but is also mented [6]. Regulation of phospholipase or acyltransferase found ubiquitously expressed in most other tissues [13]. The activity by expression other than in cancer or by posttrans- cellular distribution is localized to mitochondria. Predicted lational modifcation has not been reported. PTMs are given in the supplement Table S1, none of which have been confrmed experimentally. Phospholipase A and acyltransferase 4 Also designated as a tumor suppressor protein, hPLAAT2 exhibits a calcium-independent N-acyltransferase reaction Human phospholipase A and acyltransferase 4 (hPLAAT4, that is about 25% of the combined sn-1 and sn-2 phospholi- PLA/AT-4, HRSL4, RARRES3, HRALSL4, TIG3, Uni- pase (PLA1/2) reaction rate in whole cells, whereas in cell protKB-Q9UL19) presents as two isoforms with a sequence homogenates the opposite is true. Hydrolysis rates for the homology to PLAAT1 isoform 1 of 56.6% for Isoform 1 sn-1 position compared to the sn-2 position vary from 0.7 (Q9UL19-1) and 51.2% for isoform 2 (Q9UL19-2). Physi- to 1.9 (Table 1) [18, 20, 21]. Structural analysis reveals that cal properties for all isoforms are given in the supplement C113 is likely the active site nucleophile [20]. Regulation Table S1. Two coding SNP variants are reported for the of phospholipase or acyltransferase activity by expression canonical isoform, V69L and A162V [8] but no biological or by posttranslational modifcation has not been reported. ramifcations are noted. Phobius web server predictions [10] indicate that Isoform 1 is a single-pass transmembrane pro- Phospholipase A and acyltransferase 3 tein with a large cytoplasmic N-terminal domain (residues 1-136) and a short 8-residue lumenal C-terminal domain, Human phospholipase A and acyltransferase 3 (hPLAAT3, and Isoform 2 shows no transmembrane domains. Two NMR PLA/AT-3, HRASLS3, AdPLA, PLA2G16, H-Rev107, structures are reported for residues 1-125 of Isoform 1 (PDB UniprotKB-P53816) presents as a single isoform with a entry 2LKT and 2MY9). The SWISS-MODEL website [14] sequence homology to PLAAT1 isoform 1 of 46.8%. Physi- selects the same model protein for both isoforms (2LKT), cal properties are given in the supplement Table S1. No cod- suggesting similar structures. PLAAT4 is highly expressed ing SNP variants are reported [8, 9]. Structure predictions in the kidney, thyroid gland, GI tract, and blood, but is also [10] suggest that it is a single-pass membrane protein with found ubiquitously expressed in most other tissues, includ- a larger cytoplasmic N-terminal domain (residues 1-132) ing the brain [13]. Its cellular distribution appears to be and a small lumenal C-terminal domain (residues (155-162). primarily localized to intracellular membranes [13]. Pre- Several partial X-ray structures are reported (e.g., PDB entry dicted PTMs are given in the supplement Table S1, none

1 3 Molecular and Cellular Biochemistry

of which have been confrmed experimentally. PLA1/2 reac- a 70–80% likelihood for a single transmembrane sequence tion rates for purifed protein are 50-fold faster than that for in each of the isoforms. No X-ray structures are available; the N-acylation reactions with a threefold preference for the however, there is one potential structural model template sn-1 site [18, 20, 23] (Table 1) and exhibits twofold faster suggested on the SWISS-MODEL website [14] is template reactions with PC substrates as compared to PE substrates 5iz5.1.A. hPLA2G4E is expressed primarily in the skin and [23]. Regulation of phospholipase or acyltransferase activity although found in other mammal brain tissue it has only by expression or by posttranslational modifcation has not been identifed in the human pituitary tissue [13]. Sequence been reported. However, upregulation of hPLAAT4 mRNA comparison to the murine enzyme indicates that S412 is indirectly in tumor cells by all-trans retinoic acid has been the likely active site nucleophile. Predicted PTMs are given documented [26]. in the supplement Table S1, none of which have been confrmed experimentally. Phospholipase A and acyltransferase 5 Catalytic activity for both purified recombinant and recombinant cell lysates have been reported in terms of Human phospholipase A and acyltransferase 5 (hPLAAT5, overall production of N-acyl fatty acid products [28, 29]. PLA/AT-5, HRSL5, HRALSL5, HRLP5, iNAT, UniprotKB- Catalytic activity is enhanced in the presence of phos- Q96KN8) present as three very similar isoforms with phatidylserine (PS) by 25-fold in the presence of saturat- homologies to Isoform 1 of 96.7% and 96.4% for Isoform ing amounts of calcium ion and the EC 50 for calcium is 2 and Isoform 3, respectively, with only 22.3% homology increased greater than eightfold in the presence of PS [28]. to PLAAT1 Isoform 1. Physical properties for all isoforms Other anionic phospholipids enhance the activity in the fol- are given in the supplement Table S1. Four coding SNP lowing order PS > phosphatidylinositol > phosphatidylglyc- variants are reported for isoform 1, S31G, A93P, Q214R, erol > phosphatidic acid > phosphatidylcholine > phosphati- and A258V [8] but the clinical signifcance of these cod- dylinositol-4,5-bisphosphate [29]. Regulation by expression ing SNPs is unknown. Phobius web server predictions [10] or by posttranslational modifcation has not been reported; indicate that no transmembrane domains exist. No X-ray however, phosphorylation of S800 is predicted by sequence structures are available, however, there are two potential comparison to the murine isoform, suggesting the possibility structural model templates on the SWISS-MODEL website of regulation by phosphorylation. [14], template 2kyt.1.A and template 4q95.1.B. hPLAAT5 The reaction has been characterized in the greatest detail is highly expressed in the testis, granulocytes, pancreas, for the recombinant murine isoform (UniProtK-Q50L42) and expressed at lower amounts throughout the brain, and with a 77.4% sequence homology to the human isoform. appears to be localized to the plasma membrane [13]. Pre- The brain protein expressed in HEK293 cells shows a strong dicted PTMs are given in the supplement Table S1, none of preference (3.3:1 in mouse brain lysate) for transferring the which have been confrmed experimentally. sn-1 O-acyl chain to PE [30] and exhibits little PC-lipase PLA1/2 reaction rates for the purifed protein are about activity in the absence of PE. The enzyme is strongly acti- 25% of that for the N-acylation reactions [22, 27] (Table 1). vated with increasing Ca2+, over 200-fold in cell lysates with Comparison of positional preferences for deacylation have recombinant protein and fourfold in murine brain tissue with not been reported. Regulation of phospholipase of acyltrans- maximal activity at 10 mM Ca2+. Catalytic activity is nearly ferase reactions by expression or by posttranslational modi- fourfold higher in membrane fractions than cytosolic frac- fcation has not been reported. tions obtained from transfected HEK293 cells suggesting that it is membrane associated. The N-terminal region of the Cytosolic phospholipase A2 epsilon murine protein does contain a C2 domain which upon calcium binding is known to target the protein to phospholipid Human cytosolic phospholipase A2 epsilon (hPLA2G4E, membranes [31, 32]. However, unusual for a PLA 2-class Phospholipase A2 group IVE, UniprotKB-Q3MJ16) is enzyme, the murine protein appears to utilize an alterna- a phospholipase with a calcium-requiring N-acyl trans- tive method for membrane targeting where a region of the ferase activity [28, 29]. As reported by Hussain et al. [28] C-terminus containing a number of positively charges amino hPLA2G4E is expressed in two isoforms. Isoform 2 is iden- acid residues bind to the phosphate of several membrane tical to Isoform 1 apart from the frst 376 residues that are phosphoinositides [33]. Characterization of a S420A mutant missing in this isoform, including a membrane associating shows that S420 is the active site nucleophile. Phobius web C2 domain. Physical properties for all isoforms are given in server [10] analysis shows a 70% likelihood for a single the supplement Table S1. There are four coding SNP vari- transmembrane helix near the C-terminal end. The fact ants reported for isoform 1: R81Q, H213Y, K292N, N400S, that the murine enzyme shows a 77.4% sequence homol- M573V and A693T [8, 9]. All coding SNPs are reported ogy with the human form strongly suggests similar structure to be benign. Phobius web server [10] analysis indicates and enzymatic activity.

1 3 Molecular and Cellular Biochemistry

Converting N‑acylphosphatidylethanolamines from malignant endometrial carcinoma (EC) patients in an (NAPEs) to N‑acylethanolamines (NAEs) advanced disease state when compared to peripheral lymphocytes and without signifcant change in the transcription Overview level [46]. This correlates well with the fact that N-acylethanolamine concentrations are higher in post-menopausal Converting NAPEs to N-acylethanolamides (NAEs) such as women with EC compared when with controls. A specifc AEA is accomplished through several pathways (Fig. 1). The mechanism has not been proposed. dominant pathway (75%) [34] is catalyzed by a phospholipase D (NAPE-PLD) where a NAPE is converted directly (lyso)‑N‑acylphosphatidylethanolamine lipase (ABHD4) into the corresponding NAE and a phosphatidic acid. Human (lyso)-N-Acyl-phosphatidylethanolamine lipase N‑Acyl‑phosphatidylethanolamine‑hydrolyzing (hABHD4, alpha/beta- hydrolase 4, UniprotKB-Q8TB40) phospholipase D is expressed as two splice isoforms. Physical properties for all isoforms are given in the supplement Table S1. No Human N-acyl-phosphatidylethanolamine-hydrolyzing coding SNP variants are reported [8, 9]. Only Isoform 1 phospholipase D (hNAPE-PLD, UniprotKB-Q6IQ20) pre- (hABHD4) will be discussed here. hABHD4 is a member sents as a single isoform and is a member of the metallo-β- of the peptidase S33 family, ABHD4/ABHD5 subfamily. No lactamase family. Physical properties are given in the sup- X-ray structures or posttranslational modifcations have been plement Table S1. Two coding SNP variants are reported: reported. There are two potential structural model templates S152A and D389N [8]. Clinical ramifcations for these SNPs suggested on the SWISS-MODEL website [14], template are not reported. hNAPE-PLD is a cytosolic, zinc-requiring 3nwo.1.A and template 5mxp.1.B. hABHD4 is produced protein that is ubiquitously expressed in most tissues with ubiquitously in all tissue types studied, including brain, and particularly high expression in the brain, GI tract, kidney, is localized to the nucleoplasm with cytoplasmic expres- and bladder [13]. One X-ray structure is available (PDB sion in some tissues [13]. Predicted PTMs are given in the entry 4QN9) [22]. Acetylation on the N-terminal methio- supplement Table S1, none of which have been confrmed nine has been reported [35]. Predicted PTMs are given in the experimentally. However, the rat isoform (UniprotKB- supplement Table S1, none of which have been confrmed Q6QA69) with 94.5% sequence homology is known to be experimentally. phosphorylated at S124 which corresponds to S122 on the Enzymatic activity of recombinant proteins for mouse, human isoform [47]. rat and human proteins have been reported for a variety of The enzymatic activity of hABHD4 has not been reported NAPE substrates (Table 2) [36, 37]. The mouse and rat pro- on to date. However, there are several studies available for teins have specifc activities that are an order of magnitude the mouse counterpart which shares a 96.5% sequence higher than the human enzyme against N-palmitoyl-phos- identity to the human protein suggesting a similar value for phatidylethanolamine substrates. The active enzyme acts as the human isoform (Table 2) [34]. ABHD4 catalyzes the a homodimer and requires the presence of a hydrophobic removal of fatty acids from NAPE and the product 1-acyl- cofactor, a bile salt or n-octyl-glucoside [22, 37, 38]. Difer- sn-glycero-3-(N-acyl) phosphoethanolamine (lyso-NAPE) ent bile acids enhance the reaction rate to diferent degrees and does so using an active site serine as the nucleophile that depending on the location of hydroxyl groups on the steroid reacts readily with methoxy arachidonyl fuorophosphonate structure [38], suggesting that the enzyme activity may be (MAFP) inhibitor. Only the latter reaction has been exam- modulated in vivo by the presence of specifc bile acids. The ined directly and only as the combined activity of ABHD4 enzyme also hydrolyzes N-arachidonoyl-phosphatidyletha- and glycerophosphodiester phosphodiesterases (GDEs) [40]. nolamine (NArPE) substrate 7 times faster than N-palmi- Comparison of data from NAPE-PLD−/− mice in the pres- toyl- phosphatidylethanolamine substrate in the presence of ence and absence of inhibitors of either the ABHD4/GDE cholic acid (Table 2). Recently it was reported that a murine arm or the PLC/phosphatase arm of the alternative pathways NAPE-PLD hepatocyte defcient strain develops a high- provides insight regarding the regulation of these two path- fat diet-like phenotype and that is much more sensitive to ways [34]. By comparing results from 1 min incubation to liver infammation, likely due to the associated reduction 1 hr it is clear that the PLC/phosphatase pathway is respon- in almost all endocannabinoid mediators [39]. The authors sible for a rapid production of AEA when the NAPE-PLD hypothesize that NAPE-PLD acts as a “hub” that controls a is blocked and that the ABHD2/GDE pathway is upregu- large array of liver lipids. lated in response to low AEA. Comparison of data for the Regulation via phosphorylation has not been reported; conversion of NAPE and lyso-NAPE to AEA [34, 40] also however, there is one report indicating a 4–14 fold increase indicates that the ABHD2/GDE pathway exhibits a higher in hNAPE-PLD at the protein level in lymphocytes taken specifc activity than the PLC/phosphatase pathway and

1 3 Molecular and Cellular Biochemistry

Table 2 Enzyme activity for enzymes involved in the degradation of N-acylphosphatidylethanolamine (NAPE) Source Substrate product measured Hydrop- Enzyme Specifc activity References hobic cofac- (nmol/min/mg) tor

COS-7/recombinanta N-palmitoy-PE PEA NOG hNAPE-PLD 26.2 [37] COS-7/recombinanta N-palmitoy-PE PEA NOG rNAPE-PLD 297 [37] COS-7/recombinanta N-palmitoy-PE PEA NOG mNAPE-PLD 350 [37] Purifed recombinantb N-palmitoy-PE PEA DCA hNAPE-PLD 156 [38] Purifed recombinantb N-arachadonoyl-PE AEA DCA hNAPE-PLD 1131 [38] COS-7/recombinantc N-palmitoyl-lyso-NAPE GP-AEA – mABHD4 0.1 [40] NAPE-PLD−/− homogenated NAPE AEA – mABHD4 + GDEs 0 [34] NAPE-PLD−/− homogenatee NAPE AEA – mABHD4 + GDEs 0.006 [34] NAPE-PLD−/− homogenatef NAPE AEA – mPLC/phosphatase 0.014 [34] NAPE-PLD−/− homogenateg NAPE AEA – mPLC/phosphatase 0.002 [34] NAPE-PLD−/− homogenatei lyso-NAPE AEA – mABHD4 + GDEs 0.04 [40] NAPE-PLD−/− homogenateh lyso-NAPE AEA – mPLC/phosphatase 0.012 [40] Solubilized Recombinantj lyso-PE Loss of lyso-PE – mGDPD1 0.98 [41] Solubilized Recombinantj lyso-PE Loss of lyso-PE – mGDPD3 14 [41] Solubilized Recombinantj lyso-PC Loss of lyso-PE – mGDPD1 8.2 [41] Solubilized Recombinantj lyso-PC Loss of lyso-PE – mGDPD3 42 [41] HEK293/recombinantk N-Ara-lyso-PE AEA – mGDPD1 2.2 [42] HEK293/recombinantk N-Ara-lyso-PE AEA – hGDPD1 1 [42] HEK293/recombinantk N-Ara-lyso-PE AEA – mGDPD3 7.3 [42] HEK293/recombinantk N-Ara-lyso-PE AEA – hGDPD3 33 [42] HEK293/recombinant N-Pal-lyso-PE PEA – mGDPD1 3.7 [42] HEK293/recombinant N-Pal-lyso-PE PEA – hGDPD1 1.9 [42] HEK293/recombinant N-Pal-lyso-PE PEA – mGDPD3 16 [42] HEK293/recombinant N-Pal-lyso-PE PEA – hGDPD3 46 [42] HEK293/recombinant N-Pal-Lyso-PE PLPA – mGDPD1 0.5 [42] HEK293/recombinant N-Pal-Lyso-PE PLPA – hGDPD1 0.1 [42] HEK293/recombinant N-Pal-Lyso-PE PLPA – mGDPD3 35 [42] HEK293/recombinant N-Pal-Lyso-PE PLPA – hGDPD3 56 [42] HEK293/recombinant GP-NPE PEA – mGDPD1 8.7 [42] HEK293/recombinant GP-NPE PEA – hGDPD1 5.5 [42] HEK293/recombinant GP-NPE PEA – mGDPD3 0 [42] HEK293/recombinant GP-NPE PEA – hGDPD3 2.1 [42] HEK293T/recombinantl lyso-PC, pH 7.5 Choline – rGDE1 170 [43] HEK293T/recombinantl lyso-PC, pH 8.5 Choline – rGDE1 330 [43] HEK293T/recombinantl lyso-PI, pH 7.5 Inositol – rGDE1 74 [43] HEK293T/recombinantl lyso-PI, pH 8.5 Inositol – rGDE1 36 [43] Solubilized recombinant GP-NPE PEA – mGDE1 46,800 [44] Solubilized recombinant GP-NPE PEA – mGDPD1 290 [44] Solubilized recombinant N-Pal-lyso-PE PEA – mGDE1 < 5 [44] Solubilized recombinant N-Pal-lyso-PE PEA – mGDPD1 45 [44] Solubilized recombinant Lyso-PC LPA – mGDE1 0 [44] Solubilized recombinant Lyso-PC LPA – mGDPD1 32 [44] Solubilized recombinant lyso-PE LPA – mGDE1 ≈ 5 [44] Solubilized recombinant lyso-PE LPA – mGDPD1 150 [44] RAW164.7 macrophagem pAEA AEA – mPTPN22 1 [45] RAW164.7 macrophagen pAEA AEA – mSHIP1 0.8 [45] h refers to human, m refers to mouse, r refers to rat, ABHD4 (lyso)-N-Acylphosphatidylethanolamine lipase, AEA anandamide, Ara arachadonoyl, COS-7 fbroblast-like cell line from monkey kidney, DCA deoxycholic acid, GDE Glycerophosphodiester phosphodiesterase, GDPD1 Lysophospholipase D isoform 1, GDPD3 Lysophospholipase D isoform 3, GP-AEA glycerophosphoryl anandamide, GP-NPE glycerophospho- 1 3 Molecular and Cellular Biochemistry

Table 2 (continued) N-palmitoylethanolamine, HEK293 human embryonic kidney cell line, LPA lysophosphatidic acid, NAPE N-acyl phosphatidylethanolamine, NAPE-PLD N-acyl phosphatidylethanolamine phospholipase D, NOG N-octylglucoside, NP-lyso-NAPE 1-olyl-glycerophoso(N-palmitoyl) ethanolamine, Pal palmitoyl, PE phosphatidylethanolamine, PI phosphatidylinositol, pAEA phosphoanandamide, PEA palmitoylethanolamide, PLC phospholipase C, PLPA 2-palmitoylglycerolphosphate, PTPN22 Tyrosine-protein phosphatase non-receptor type 22, RAW164.7 murine leukemia virus transformed murine macrophage, SHIP1 Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1 a Enzyme solubilized in 0.1% N-octylglucoside (NOG) and N-palmitoyl-phosphatidylethanolamine (N-palmitoyl-PC) substrate used b Enzyme tested in the presence of 0.2% deoxycholic acid (DCA) c Specifc activity given as diference between mock and ABHD4 transfected cells d Enzyme activity from NAPE-PLD−/− mouse brain homogenate in the presence of PLC inhibitors after 1 min e Enzyme activity from NAPE-PLD−/− mouse brain homogenate in the presence of PLC inhibitors after 1 h f Enzyme activity from NAPE-PLD−/− mouse brain homogenate in the presence of mABHD4 and GDE inhibitors after 1 min g Enzyme activity from NAPE-PLD−/− mouse brain homogenate in the presence of mABHD4 and GDE inhibitors after 1 h h Specifc activity calculated NAPE-PLD−/− mouse brain homogenate in the presence of mABHD4 inhibitor after 1 h i Specifc activity calculated NAPE-PLD−/− mouse brain homogenate as the diference between AEA production in the absence and presence of mABHD4 inhibitors after 1 h j Mouse recombinant protein produced in transfected Sf9 insect cells solubilized in 2% dodecyl-b-D-maltoside k BHA added to protect arachidonyl moieties from oxidation l Assays performed in 10 mM Mg2+ m Specifc activity calculated as the diference between the SA for wild type and PTPN22−/− RAW264.7 cells n Specifc activity calculated as the diference between controls and SHIP1−/− knockouts is thus the dominant contributor to AEA production once in the highest amounts in the liver followed by all muscle upregulated. tissue, GI tract and female tissues [13]. There are numerous X-ray structures available (e.g., PBL entry 1DB4). Phospholipase C (PLC) and secreted phospholipase A2 Predicted PTMs are given in the supplement Table S1, (sPLA 2) none of which have been confrmed experimentally. Human phospholipase A2 Group V (hPLA2G5) is a Phospholipase C (PLC) represents a class of phospholipases secreted, calcium-dependent lipase. It is translated as that cleave the phospho-head group from phospholipids, 138 residue polypeptide that is processed to 118 residues the most studied of which involve the production of ino- following removal of the signal peptide. Physical prop- sitol 1,4,5-trisphosphate from a 1,2-diacyl-glycero-3-phos- erties are given in the supplement Table S1. There are pho-(1D-myo-inositol-4,5-bisphosphate). The production of 16 coding SNP variants reported [9]. Four of these are phospho-AEA (pAEA) from NAPE has been observed in associated with familial feck retina (G45C, Q128 frame mouse brain homogenates, clearly involving a member of the shift, and two termination mutants W62 and R53), one PLC class [34, 45]. The identity of the particular isoform has is associated with late onset retinal degeneration (V99 yet to be reported. Secreted rat phospholipase A2 group IB frame shift), and the remaining with unknown signifcance (rat sPLA2-IB), secreted human calcium-dependent phos- (P6S, S14T, G45S, G49S, R73L, E76 deletion, C103R, pholipase A2 Group V (hPLA2G5) and human phospholi- Y116C, R123Q, Q130C, Y131C, and N134K). hPLA2G5 pase A2 Group IIA (hPLA2G2A) all are known to produce is expressed in most tissues but is highly expressed in lyso-NAPE from NAPE [48]. Specifc activities for the the retina as well as heart and smooth muscle [13]. X-ray human isoforms are given in Table 1. structures are not available, but a 3D model has been pub- Human phospholipase A2 Group IIA (hPLA2G2A, lished (PDB entry 2GHN). Predicted PTMs are given in phospholipase A2, UniProtKB-P39877) is presented as a the supplement Table S1, none of which have been con- single isoform and is a secreted, calcium-dependent lipase. frmed experimentally. Physical properties are given in the supplement Table S1. Human phospholipase A2 Group IB (hPLA2G1B, PLA2, There are 14 coding SNP variants reported [9]. Five of PLA2A, PPLA2, UniProtKB-P04054) is a secreted, calcium- these are associated with familial feck retina (G45C, dependent lipase. Four SNP variants are reported (R122H, G49S, Q128 frame shift, and two termination mutants D16A, N89T, and N89K) none of which has any known clin- W62 and R53) and the remaining with unknown signif- ical signifcance [8, 9]. hPLA2G1B is expressed exclusively cance (P6S, S14T, R73C, C103R, Y116C, R123Q, Q130C, in the pancreas [13]. Two Xray (PDB entry 3ELO and 6Q42) Y131C and N134K). Its expression is limited but is found and one model structure (PBD entry 1YSK) are available.

1 3 Molecular and Cellular Biochemistry

Predicted PTMs are given in the supplement Table S1, none from cell lysates with similar specifc activities for lyso-PC, of which have been confrmed experimentally. lyso-PE, and N-Pal-lyso-PE [44]. Specifc activities have yet to be reported for purifed Although a specifc catalytic mechanism has not been hPLA2G2A, hPLA2G5 or hPLA2G1B. However, reaction proposed, modeling reveals that D72, E74, and H87 are rates of recombinant forms expressed in HEK293 cells have likely to be required for activity [50]. Further, a proteinase been reported as 0.37 nmol/min/106 cells for hPLA2G2A K protection assay reveals that the catalytic domain faces the and 5.5 nmol/min/106 cells for PLA2G5 [48]. Regulation of lumen/extracellular space. Regulation involving anandamide hPLA2G5 expression is reduced in mast cells when treated metabolism by expression or by posttranslational modifca- with vitamin D (1,25-dihydroxyvitamin D 3) a known mod- tion has not been reported. ulator of the infammatory response, whereas other PLA2 isoforms are upregulated [49]. Regulation involving anan- Lysophospholipase D GDPD3 damide metabolism by expression or by posttranslational modifcation for any of these isoforms has not been reported. Lysophospholipase D GDPD3 is known to convert lyso- NAPEs to AEA or other NAEs in competition with GDPD1 [41, 42]. Human lysophospholipase D GDPD3 (hGDPD3, Lysophospholipase D GDPD1 GDE7, glycerophosphodiester phosphodiesterase 7, Uni- protKB-Q7L5L3) is expressed as two splice isoforms. Iso- Lysophospholipase D GDPD1 is known to catalyze two form 2 is identical to Isoform 1 with the exception of missing reactions leading to the formation of AEA and other NAEs: residues 1-63. Physical properties for all isoforms are given (1) hydrolysis of lyso-NAPE and (2) hydrolysis of glycer- in the supplement Table S1. Only the canonical sequence, ophospho-N-acylethanolamine, each producing the same Isoform 1, will be discussed here. No coding SNP variants amide product [42]. Human lysophospholipase D GDPD1 are reported [8, 9]. hGDPD3 is a metallo-membrane pro- (hGDPD1, GDE4, glycerophosphodiester phosphodiester- tein containing two transmembrane helices with a preference ase 4, UniprotKB-Q8N9F7) is expressed as three splice for Ca2+ and is partially or completely inhibited by other isoforms. Physical properties for all isoforms are given in divalent cations [41, 42]. No X-ray structures are available. the supplement Table S1. Only the canonical sequence, Iso- There is, however, one potential structural model template form 1, will be discussed here. No coding SNP variants are suggested on the SWISS-MODEL website [14], template reported [8, 9]. hGDPD1 is a metallo-membrane protein 5vug.1.B. hGDPD3 is widely expressed with the highest containing two transmembrane helices with a preference expression in tonsils and appendix, tongue, and esophagus, for Mg2+ or Mn 2+ [42, 50] and is inhibited by both Ca 2+ and as well as being expressed in all areas of the brain [13]. EDTA [44]. hGDPD1 is widely expressed in many tissues Intracellular distribution is found on membrane structures with the highest expression in the brain followed by a lower throughout the cytosol and nucleoplasm. Predicted PTMs expression in endocrine tissue, the GI tract, and male and are given in the supplement Table S1, none of which have female tissues [13]. X-ray structures are not available, but been confrmed experimentally. a 3D model has been published [50]. Predicted PTMs are Specifc activities have been reported for both mouse given in the supplement Table S1, none of which have been and human recombinant GDPD3 [42] which share a 79.1% confrmed experimentally. sequence homology. In contrast to GDPD1, both GDPD3 Specifc activities have been reported for both overex- enzymes show a preference for phosphatidylcholine sub- pressed mouse and human recombinant GDPD1 obtained strates over N-acyl-phosphatidylethanolamine substrates. from cell lysates (Table 2) [42]. These two enzymes share The same is true for mGDPD1 hydrolyzing non-N-acylated a 91.9% sequence homology. Both enzymes show a prefer- substates [41]. Specifc activities for both enzymes for the ence for N-acyl-phosphatidylethanolamine substrates over same substrates show the following order: N-Pal-lyso-PE phosphatidylcholine substrates. However, for mGDPD1 the ≈ N-Ole-lyso-PE > > N-Ara-lyso-PE > GP-NPE, an order opposite is true for non-N-acylated substates [41]. Specifc diferent than observed for GDPD1, and the reactions are activities for both enzymes for the same substrates follow the orders of magnitude slower than the corresponding NAPE- following order: glycerophospho-N-palmitoylethanolamine PLD pathway competitor. Regulation of activity involving (GP-NPE) > N-palmitoyl-lysophosphatidylethanolamine anandamide metabolism by expression or by posttransla- (N-Pal-lyso-PE) ≈ N-oleoyl-lysophosphatidylethanolamine tional modifcation has not been reported. (N-Ole-lyso-PE) > N-arachidonoyl-lysophosphatidylethanolamine (N-Ara-lyso-PE), all of which are orders of magnitude Glycerophosphodiester phosphodiesterase 1 slower than the corresponding NAPE-PLD pathway competitor. Purifed recombinant mGDPD1 shows a 5000-fold Glycerophosphodiester phosphodiesterase 1 (GDE1) is increase in specifc activity for GP-NPE over that obtained known to convert GP-NAEs to NAEs as well as exhibiting

1 3 Molecular and Cellular Biochemistry glycerophosphoinositol phosphodiesterase activity [32] indicate that GDE1 activity is both positively and negatively and is a member of the glycerophosphoryldiester phospho- regulated by G-protein signaling. PRAF2 has also been diesterase family (GDE phosphodiesterase family). Human shown to interact with RGS16, which in turn is known to glycerophosphodiester phosphodiesterase 1 (hGDE1, interact with CCR5, a G-protein coupled chemokine recep- MIR16, RGS16-interacting membrane protein, UniprotKB- tor [51]. It has been proposed that a GDE1, RGS16, PRAF2, Q9NZC3) is expressed as a single isoform. Physical prop- CCR5 complex may be involved in cell chemotaxis or per- erties are given in the supplement Table S1. There are two haps desensitization, internalization, and recycling of CCR5 reported coding SNP variants, R208Q and D328K [9]. Nei- [52]. Immunoprecipitation studies and size exclusion chro- ther variant has a known clinical signifcance. hGDE1 is a matography reveals that both Gαq/11 and Gβ proteins associ- metallo-membrane protein containing two transmembrane ate with rat GDE1 [53, 54]. In addition, Gαq/11 knockdown helices with a preference for Mg 2+ and is inhibited by both Neuro2A cells exhibit lysoPLD activity that is reduced by Ca2+ and EDTA [44]. hGDE1 is widely expressed with about 50% compared to wildtype and the endogenous activ- the highest expression in the brain, endocrine tissues, GI ity is partially rescued by overexpression of Gαq in these tract, and muscle tissue. Intracellular distribution is found cells. Taken together all of these results strongly suggest on membrane in the nucleoli, nucleoplasm, and vesicles G-protein modulation of GDE1 activity. Regulation of activ- throughout the cytosol. No X-ray structures are available ity involving anandamide metabolism by expression or by for hGDE1, but 3D model has been proposed [51]. Predicted posttranslational modifcation has not been reported. PTMs are given in the supplement Table S1, none of which have been confrmed experimentally. However, endo H treat- Tyrosine‑protein phosphatase non‑receptor type 22 ment of rat GDE1 (87.9% sequence homology to the human (PTPN22) isoform) confrms that 5–6 kDa of N-glycosylation is present, but specifc sites of attachment are unknown [52]. As the name suggests, tyrosine-protein phosphatase non- Specific activities have not been reported for human receptor type 22 is known primarily for its ability to remove GDE1 but have been reported for both rat and mouse phosphates from tyrosines [55]. However, several reports [43, 44]. These enzymes have an 87.9% and 87.3% clearly show that PTPN22 also catalyzes the dephosphoryla- sequence homology to the human protein, respectively, tion of pAEA to AEA [34, 45]. Human PTPN22 (hPTPN22, and thus these specifc activities can serve as an estimate PEP, LyP, 70Z-PEP, PTPN8, UniprotKB-Q9Y2R2) is for human values. Recombinant rat GDE1 shows a pH- expressed as six splice isoforms. Sequence alignment reveals dependent substrate specifcity where lyso-phosphatidyl- that Isoforms 5 and 6 lack both the known active site nucleo- choline (lyso-PC) is the preferred substrate at pH 7.5 and phile [56] and key sections of the active site structure of the lyso-phosphatidylinositol (lyso-PI) is the preferred sub- canonical sequence and can thus be discounted as biologi- strate at pH 8.5 [43]. Specifc activities for both enzymes cally functional in terms of phosphatase activity. Isoform 3 for the same substrates follow the following order: lacks a key portion of the substrate binding site and thus is GP-NPE > lyso-PE > N-Pal-lyso-PE > lyso-PC. also likely not to be biologically competent for phosphatase Although a specifc catalytic mechanism for hGDE1 has activity. It is unclear from the literature which or if any com- not been proposed, site specifc mutagenesis reveals that bination of the three remaining isoforms is responsible for E97, D99, and H112 are required for activity [43]. Further the phosphatase activity for substrate pAEA. Phobius web detail has been gleaned from a 3D model study [52]. A crude server [10] analysis indicates a 50% likelihood for a single mechanism has been proposed for rat GDE1 where H356 transmembrane sequence in each of the three phosphatase and E341 act as a general acid/base catalyst, generating a competent isoforms noted above. However, hPTPN22 is cyclic phosphate intermediate that is then hydrolyzed to known to be membrane associated rather than an integral form the respective products [43]. The catalytic domain has membrane protein. The proposed transmembrane helix may been shown to face the lumenal/extracellular space [43]. instead represent a helical structure that is partially imbed- Murine GDE1 (mGDE1) was originally identifed as ded in the membrane. Physical properties for all isoforms a RGS16-interacting membrane protein, where RGSs are are given in the supplement Table S1. There are 26 reported known GTPase activating proteins. This fact led to the dis- coding SNP variants of which only one has known clini- covery that the catalytic activity of mGDE1 is regulated by cal signifcance [8, 9]. The variant R620W is implicated in G-protein signaling. When HEK293T cells are transfected susceptibility to insulin dependence, diabetes, rheumatoid with mGDE1 they respond to isoproterenol, a known Gαs arthritis, and other disorders. hPTPN22 is widely expressed coupled β-androgenic receptor agonist, with an ≈ 50% with the highest expression in bone marrow, lymphoid tis- increase in GDE1 activity. When treated with phenylephrine, sues and blood as well as being expressed in all areas of the a known Gαi/q coupled α-androgenic receptor agonist, how- brain. Intracellular distribution is found in the nucleoplasm, ever, GDE1 activity decreases by ≈ 30% [43]. These results vesicles, and on the plasma membrane [13]. Numerous Xray

1 3 Molecular and Cellular Biochemistry structures are available for the frst 300 or so residues which a requirement for its inhibition of T cell receptor signaling contains the N-terminal through the active site regions for all [58]. Phosphorylation of hPTPN22 at S35 by protein kinase three of the isoforms noted above (e.g., PDB entry 2P6X). C has been shown to occur in vivo and in vitro and inhibits One report suggests the presence of second catalytic site the ability to downregulate T cell receptor signaling [48]. could account for the diversity of substrate, lipid vs. peptide Further, phosphorylation impairs hPTPN22’s ability to inac- [57]. Predicted PTMs are given in the supplement Table S1, tivate Src family kinases. Similar studies on AEA production of which only phosphorylation at S751 [58] and S35 [59, 60] have not been reported. on isoform 1 have been confrmed experimentally. Compari- son of the human to murine PTPN22 (UniProtKB-P29352; 70.7% sequence homology) sequences suggest potential Phosphatidylinositol 3,4,5‑trisphosphate 5‑phosphatase 1 phosphorylation at S449, S635, S684, and S692 as well. (SHIP1) Specifc activities have only been reported for mPTPN22 but since there is a 70.7% sequence homology between the As the name suggests, the primary function of Phosphati- human and murine protein, the reported value should serve dylinositol 3,4,5-trisphosphate 5-phosphatase 1 (SHIP1) is as a rough estimate of the human enzyme activity (Table 2). the removal of the 5′-phosphate from phosphatidylinositol Only two studies have reported specifc activities related to 3,4,5-trisphosphate (PtdIns(3,4,5)P3). However, its involve- this enzyme. One study examined mouse brain homogenates ment in AEA metabolism has been observed where SHIP1 from NAPE-PLD−/− mice in the presence of an inhibitor competes with PTPN22 for the hydrolysis of pAEA. Human for ABHD4 and EDTA to knock out any GDE or GDPD SHIP1 (hSHIP1, INPP5D, SIP-45p, 150Ship, hp51CN, Uni- activity [34]. The resulting specifc activity (Table 2) can protKB-Q92835) is expressed as three alternative splicing thus be attributed to a combination of a yet-to-be identi- isoforms. Isoform 2 lacks V117 and is otherwise identical to fed PLC, PTPN22, and SHIP1 (see below) with values in Isoform 1 and thus is very likely to be involved in the AEA the low pmol/mg/min range (discussed below). A second pathway. Isoform 3, however, lacks both the SH2 and SH3 study examined mouse macrophages (RAW264.7) in culture, binding domains essential for its PtdIns(3,4,5)P3 activity comparing the conversion of pAEA to AEA by wildtype and for this reason is not likely to be a biologically com- to PTPN22 −/− knockout strains. The diference in specifc petent isoform for this process. Physical properties for all activity between the two represents the combined activity isoforms are given in the supplement Table S1. Two coding of PTPN22 and SHIP1 (see below) [45]. SNP variants for the canonical isoform have been reported: Regulation of this protein is multifold. One of the frst H1169Y and V685D [8]. None of the variants have any descriptions of the involvement of PTPN22 in anandamide known clinical signifcance. One X-ray structure for resi- metabolism was obtained from a study in which mouse dues 397-857 (PDB entry 6IBD) and one nmr structure for macrophages were treated with the infammation initiator residues 1-112 (PDB entry 2YSX) are available. Predicted lipopolysaccharide (LPS) [45]. Here it was found that LPS PTMs are given in the supplement Table S1, none of which upregulates anandamide synthesis and PTPN22 expression have been confrmed experimentally. while at the same time downregulates NAPE-PLD expres- The only direct reference to the pAEA activity for sion. Further, treatment of these cells with phosphatase SHIP1 comes from a comparison of murine control and inhibitors or the specifc PLC inhibitor neomycin reduces mSHIP1−/− knockout macrophages (Table 2) [45]. The dif- but does not eliminate LPS-induced pAEA dephosphoryla- ference in specifc activities between the knockout and con- tion. These results indicate that the NAPE-PLD pathway is trols is 0.77 nmol/mg/min which is quite comparable to the likely the constitutive pathway to AEA production, whereas 1.0 nmol/mg/min determined for PTPN22. Like mPTPN22, the PLC pathway is invoked during infammation. A sec- mSHIP1 is also upregulated in response to LPS and is tyros- ond regulatory pathway for PTPN22 involves the active site ine phosphorylated [61]. Although specifc phosphorylation C227 and two other Cys residues where C129 protects C227 sites stimulating upregulation were not determined, there are from irreversible oxidation in an oxidizing environment three known mSHIP1 tyrosine phosphorylation sites, Y918, through the formation of a disulfde bond and concomitant Y945, and Y1021 [62], corresponding to Y915, Y944, and reversible loss of activity [56]. On the other hand, formation Y1022 in hSHIP1. There are also fve known mSHIP1 ser- of a disulfde with the local C231 also results in inactiva- ine/threonine phosphorylation sites, S246, S935, T964, tion of the enzyme but suppresses reactivation in a reduc- S967, and S972 [63, 64], corresponding to S243, S934, ing environment, providing negative regulation. A third T963, and S971 in hSHIP1. There is no human counterpart regulatory pathway involves phosphorylation of specifc for S967. The 87.4% sequence homology suggests a similar Ser residues. Phosphorylation is known to prolong the half- likelihood of phosphorylation in human cells. Regulation life of hPTPN22 by inhibiting K48-linked ubiquitination activity involving anandamide metabolism by expression has and also inhibits its recruitment to the plasma membrane, not been reported.

1 3 Molecular and Cellular Biochemistry

Anandamide loss 1 (hFAAH-1, FAAH, anandamide amino hydrolase 1, oleamide hydrolase 1, UniprotKB-O00519) is expressed as Anandamide concentrations are reduced through three gen- a single isoform. Physical properties are given in the sup- eral pathways: (1) degradation to AA and ethanolamine, (2) plement Table S1. There are 6 reported coding SNP vari- conversion to other molecules, and (3) sequestration through ants: G110E, P129T, R295Q, S356V, A476G, and A345D transport to intracellular lipid microvesicles. Figure 2 depicts [8, 9]. The P129T isoform is known to have reduced cel- the known reaction pathways, the enzymes, and other pro- lular stability and has been linked to problem drug use teins known to be involved. [65]. A345D may also have clinical signifcance, as it was obtained from a breast cancer sample. There are no crystal structures for hFAAH-1; however, the rat counterpart Degradation to free AA and ethanolamine (rFAAH-1) with an 82.7% sequence homology has numerous published structures to serve as a model. The crystal Fatty‑acid amide hydrolase 1 (FAAH‑1) structure of a truncated rFAAH-1 (residue 30-579, PDB entry 1MT5) indicates that it is a dimeric enzyme, each The hydrolysis of fatty-acid amides to their correspond- monomer containing two intramembrane hydrophobic heli- ing fatty acid and amine is facilitated by fatty-acid amide ces that are thought to provide a hydrophobic membrane hydrolase 1 (FAAH-1). Human fatty-acid amide hydrolase cap (residues 404-433). Phobius web server predictions

AA + EA

20-HETE-EA PGI2-EA NAAA

FAAH1,2 CYP450s (+/-) 5,6-EET-EA CYP450s

AKR1C3 PTGIS CYP450s PGF -EA PRXL2B 2 COX-2 AEA (+/-) 8,9-EET-EA CYP450s ALOX12 ALOX15 PGH -EA 2 CYP450s PTGDS PTGES1,2,3 TBXAS1 (+/-) 11,12-EET-EA 12S-HPETE-EA

(+/-)14,15-EET-EA

PGD2-EA TXA2-EA PGE2-EA D 15S-HPETE-EA ALOX15 GPX

EXC4-EA EXD4-EA EXE4-EA TXB2-EA EXA4-EA15S-HETE-EA

Fig. 2 Reactions involved in the degradation of anandamide Metabolites: prostaglandin F2a ethanolamide, PGH2-EA prostaglandin H 2 etha- 5,6-EET-EA N-(2-hydroxyethyl)-5,6-epoxyeicosatrienamide, 11,12-EET nolamide, PGI2-EA prostaglandin I2 ethanolamide, TXA2-EA throm- -EA N-(2-hydroxyethyl)-11,12-epoxyeicosatrienamide, 12S-HPETE-EA boxane A2 ethanolamide, TXB2-EA thromboxane B 2 ethanolamide. N-(2-hydroxyethyl)-12S-Hydroperoxyicosatetraenamide, 14,15-EET- Enzymes: AKR1C3 aldo–keto reductase family 1 member C3, ALOX EA N-(2-hydroxyethyl)-14,15-epoxyeicosatrienamide, 15S-HETE-EA 15 arachidonate 15-Lipoxygenase, ALOX12 arachidonate 12-Lipoxy- N-(2-hydroxyethyl)-15S-Hydroxyicosatetraenamide, 15S-HPETE-EA genase, COX-2 cyclooxygenase-2, CYP450s cytochrome P450s (see N-(2-hydroxyethyl)-15S-Hydroperoxyicosatetraenamide, 20-HETE- text for specifc isoforms), FAAH-1,2 fatty-acid amide hydrolase iso- EA N-(2-hydroxyethyl)-20-hydroxyeicosatetraenamide, 8,9-EET-EA forms 1 and 2, GPX glutathione peroxidase, NAAA N-acylethanola- N-(2-hydroxyethyl)-8,9-epoxyeicosatrienamide, AA arachidonic acid, mine-hydrolyzing acid amidase, PRXL2B prostamide/prostaglandin AEA anandamide, EA ethanolamine, EXA4-EA eoxin A4 ethanola- F synthase, PTGDS prostaglandin D2 synthase, PTGES1,2,3 pros- mide, EXC4-EA eoxin C 4 ethanolamide, EXD4-EA eoxin D 4 ethanola- taglandin E2 synthase isoforms 1, 2, and 3, PTGIs prostaglandin I2 mide, EXE4-EA eoxin E4 ethanolamide, PGD2-EA prostaglandin D2 synthase, and TBXAS1 thromboxane A synthase 1 ethanolamide, PGE2-EA prostaglandin E 2 ethanolamide, PGF2a-EA

1 3 Molecular and Cellular Biochemistry

[10] indicate a single transmembrane helix located at the structures for hFAAH-2 and only a 32.1% sequence homol- N-terminus (residue 9-29) and a potential second intramem- ogy to hFAAH-1. However, a threaded model for hFAAH-2 brane helical structure (residue 404-433) for both hFAAH-1 based on rFAAH-1 has been reported and clearly shows the and rFAAH-1 the former is more likely an intramembrane expected Ser-Ser-Lys catalytic triad involving S206, S230, structure. PSORT analysis [66] predicts that the catalytic and K131 [71] in which S230 appears to be the active site domain is located in the cytosolic compartment. Further nucleophile. Interestingly, Phobius web server predictions analysis of rFAAH-1 reveals a unique catalytic triad in the [10] indicate a single transmembrane helix located near the active site consisting of S241-S217-K142 for which S241 is C-terminus (residue 394-414) rather than at the N-terminal the catalytic nucleophile. Sequence comparison shows that as observed for hFAAH-1 but in the same region where a the same residues apply to the human isoform. hFAAH-1 is potential intramembrane helix is predicted for hFAAH-1. In widely expressed with the highest expression in the brain addition, a potential N-terminal intramembrane helix is pre- as well as high expression in male and endocrine tissues dicted for hFAAH-2, albeit with low probability, in the same [13]. Intracellular distribution includes its presence on mem- region the transmembrane helix is predicted for hFAAH- brane structures as well as throughout the cytosol. Treatment 1. PSORT analysis predicts that the catalytic domain is with PNGaseF does not alter gel migration, indicating the located in the lumenal compartment [66]. hFFAH2 is absence of N-linked glycosylation [66]. Predicted PTMs are widely expressed throughout most tissues and is particularly given in the supplement Table S1. abundant in the pancreas and endocrine tissues but is also Specifc activities have been reported for both human and expressed in the brain with highest amounts in the cerebel- rat isoforms with the latter being ≈10 times larger (Table 3) lum [13]. Intracellular distribution appears to be localized [66–68]. FAAH-1 has a catalytic preference for the particu- to lipid droplet membranes [72]. Predicted PTMs are given lar fatty acid present where anandamide > oleamide > N-ole- in the supplement Table S1, none of which have been con- oyl ethanolamine > N-palmitoyl ethanolamine and also has frmed experimentally. the ability to hydrolyze N-acyltaurines (NATs) but at lower Specifc activity has been reported for hFAAH-2 with a rates than ethanolamides [66]. value for AEA hydrolysis that is only 1–2% of that exhib- Regulation of FAAH-1 occurs at both transcriptional and ited by FAAH-1, but comparable activity for hydrolysis of post-transcriptional levels. Physiological levels of proges- oleamide (Table 3). In addition, FAAH-2 has a catalytic pref- terone have been shown to upregulate hFAAH-1 at both the erence for the fatty acid present where oleamide >> N-oleoyl transcriptional and translational levels in T-lymphocytes, up ethanolamine > anandamide > N-palmitoyl ethanolamine to a 270% increase over controls resulting in a 60% decrease and is unable to hydrolyze NATs [66]. These data suggest in anandamide concentrations [69]. It was subsequently that FAAH-1 is primarily involved in anandamide and NAT shown that the increase in hFAAH-1 results from an increase hydrolysis and both FAAH-2 and FAAH-1 contribute to the in the transcription factor Ikaros that subsequently binds to hydrolysis of monosaturated lipid amides [66]. Regulation the FAAH-1 promotor site. These fndings are consistent of FAAH-2 at any level has yet to be reported. with a role for FAAH-1 in modulating immunoendocrine interactions in human pregnancy. Membrane bound non- N‑acylethanolamine‑hydrolyzing acid amidase (NAAA) raft cholesterol in AEA/cholesterol rich membranes such as the ER have been shown to stabilize dimeric FAAH-1 and The hydrolysis of fatty-acid amides to their corresponding directly enhances the enzymatic activity by increasing the fatty acid and amine is also facilitated by an N-acylethanol- accessibility of the membrane port to AEA [70]. Regulation amine-hydrolyzing acid amidase which exhibits a preference of FAAH-1 by phosphorylation has not been reported. for small saturated fatty acids. Human N-acylethanolamine- hydrolyzing acid amidase (hNAAA, ASAHL, PLT, acid Fatty‑acid amide hydrolase 2 (FAAH‑2) ceramidase-like protein, UniprotKB-Q02083) is expressed as three isoforms where isoform 2 is missing the last 35 The hydrolysis of fatty-acid amides to their corresponding residues present in isoform 1 and isoform 3 is missing the fatty acid and amine is also facilitated by fatty-acid amide last 159 residues of isoform 1. Physical properties for all hydrolase 2, but with diferent substrate preferences than isoforms are given in the supplement Table S1. Only Isoform FAAH-1. Human fatty-acid amide hydrolase 2 (hFAAH-2, 1 exhibits demonstrated activity [82] and thus will be the AMDD, anandamide amino hydrolase 2, oleamide hydrolase only isoform discussed further. There are 4 reported coding 2, UniprotKB-Q6GMR7) is expressed as a single isoform. SNP variants (K75R, N107K, V151I, and F334L) none of Physical properties are given in the supplement Table S1. which has a known clinical signifcance [8, 9]. Isoform 1 is There are 7 reported coding SNP variants (R42W, R192Q, processed further to remove the signal peptide, the frst 28 G342R, A458S, A388S, and A418S) of which none have residues and a single cleavage between residues 125 and known clinical signifcance [8, 9]. There are no crystal 126 forming a hetero-dimer consisting of the α-subunit

1 3 Molecular and Cellular Biochemistry

Table 3 Enzyme activity Source Substrate product measured Enzyme Specifc activity References for enzymes involved in the (nmol/min/mg) degradation of anandamide (AEA) and phosphoanandamide COS-7/recombinant AEA AA hFAAH-1 17 [66] (pAEA) COS-7/recombinant AEA AA hFAAH-2 0.46 [66] COS-7/recombinant AEA AA hFAAH-1 30.7 [68] COS-7/recombinant AEA AA rFAAH-1 333 [67] COS-7/recombinant AEA AA hFAAH-2 0.65 [72] COS-7/recombinanta AEA AA hFAAH-1 11.8 [72] HEK293/recombinantb PEA PA hNAAA 8.1 [73] HEK293/recombinantb AEA AA hNAAA 0.24 [73] Solubilized recombinant PEA PA hNAAA 5400 [74] Solubilized recombinant PEA PA hNAAA 330 [75]

Partially purifed recombinant AEA O2 used hCOX-2 12,500 [76]

Partially purifed recombinant AA O2 used hCOX-2 17,300 [76] Recombinant purifed AEA 20-HETE-EA hCYP2D6 0.0037 [77] Recombinant purifed AEA 8,9-EET-EA hCYP2D6 0.0016 [77] Recombinant purifed AEA 11,12-EET-EA hCYP2D6 0.0011 [77] Recombinant purifed AEA 14,15-EET-EA hCYP2D6 0.0013 [77] Recombinant purifed AEA 5,6-EET-EA hCYP2D6 very low [77] Recombinant purifed AEA 20-HETE-EA hCYP2J2 0.00081 [78] Recombinant purifed AEA 8,9-EET-EA hCYP2J2 0.002 [78] Recombinant purifed AEA 11,12-EET-EA hCYP2J2 0.0024 [78] Recombinant purifed AEA 14,15-EET-EA hCYP2J2 0.0042 [78] Recombinant purifed AEA 5,6-EET-EA hCYP2J2 0.0037 [78] Human liver microsomes c AEA 14,15-EET-EA hCYP3A4 0.048 [79] Human liver microsomes c AEA 11,12-EET-EA hCYP3A4 0.044 [79] Human liver microsomes c AEA 8,9-EET-EA hCYP3A4 0.48 [79] Human liver microsomes c AEA 5,6-EET-EA hCYP3A4 0.184 [79] Human liver microsomes c AEA 20-HETE-EA hCYP4F2 0.266 [79] Recombinant purifed AEA 20-HETE-EA hCYP2B6 very low [80] Recombinant purifed AEA 8,9-EET-EA hCYP2B6 0.0031 [80] Recombinant purifed AEA 11,12-EET-EA hCYP2B6 0.0076 [80] Recombinant purifed AEA 14,15-EET-EA hCYP2B6 0.002 [80] Recombinant purifed AEA 5,6-EET-EA hCYP2B6 0.0046 [80] Leukocyte/platelet homogenate AEA 12(S)-HETE-EA hALOX12 1.5 [81]

h refers to human, r refers to rat, 11,12-EET-EA N-(2-hydroxyethyl)-11,12-epoxyeicosatrienamide, 12(S)-HETE-EA 12(S)-hydroxyarachadonoylethanolamide, 14,15-EET-EA N-(2-hydroxyethyl)- 14,15-epoxyeicosatrienamide, 15(S)-HETE-EA 15(S)- hydroxyarachadonoylethanolamide, 20-HETE-EA N-(2-hydroxyethyl)-20-hydroxyeicosatetraenamide, 5,6-EET-EA N-(2-hydroxyethyl)-5,6-epoxyeicosatrienamide, 8,9-EET-EA N-(2-hydroxyethyl)-8,9-epoxyeicosatrienamide, AA arachidonic acid, AEA anandamide, ALOX12 12S-Lipoxygenase, COS-7 fbroblast-like cell line from monkey kidney, COX-2 cyclooxygenase-2, CYP2B6 cytochrome P450 isoform 2B6, CYP2D6 cytochrome P450 isoform 2D6, CYP2J2 cytochrome P450 isoform 2J2, CYP3A4 cytochrome P450 isoform 3A4, CYP4F2 cytochrome P450 isoform 4F2, HEK293 human embryonic kidney cell line, FAAH-1 Fatty-acid amide hydrolase isoform 1, FAAH-2 Fatty-acid amide hydrolase isoform 2, NAAA N-acylethanolamine-hydrolyzing acid amidase, PA palmitic acid, PEA palmitoylylethanolamide a FLAG tagged protein b 0.1% Nonidet P-40 included in the assay c Isoform most likely to catalyze the reaction

(residue 29-125, 14.6 kDa) and the β-subunit (residue 126- PDB entry 6DXW) and one with the 125–126 cleavage 359, 33.3 kDa) [74]. There are two crystal structures for (PDB entry 6DXX). hNAAA is a glycoprotein for which hNAAA, one without the 125–126 cleavage (residue 29-359, N37, N107, N309, N315 and N333 are confrmed sites of

1 3 Molecular and Cellular Biochemistry glycosylation [73–75, 83]. hNAAA is widely expressed in enzyme catalyzed oxidation of the AA moiety itself. The most tissues with highest expression in blood, monocytes reactions discussed below examine the frst steps in such in particular, lymphoid tissues, spleen, and lymph nodes, conversions which involve enzymes that are also associated and to a lesser extent in the gastrointestinal tract and brain with metabolism of AA along numerous eicosanoid meta- [13]. Intracellular distribution favors lysosomes [75, 84]. bolic pathways (Fig. 2). Greater detail on these enzymes and Predicted PTMs are given in the supplement Table S1, but the pathways are found elsewhere [87, 88]. only the N-glycosylations noted above have been confrmed experimentally. Prostaglandin G/H synthase 2 (COX‑2) hNAAA is a cysteine amidase where the active site C126 is responsible for not only substrate hydrolysis, but The primary biological role for prostaglandin G/H Synthase also facilitates auto-cleavage of the 125–126 bond [83] 2 (COX-2) and prostaglandin G/H Synthase 1 (COX1) is along with residues R142, D145, N287 [84], and L325 and the conversion of AA to prostaglandin H2 (PGH2) via pros- T335[82]. Although NAAA is considered to be a cytosolic taglandin G2 (PGG2) through the reaction of substrate with protein, it must also penetrate the lipid bilayer in order to molecular oxygen. PGH 2 is then rapidly converted enzymati- access substrate. It is thought that helix a3 on the α-subunit cally to prostaglandin D2 (PGD2), prostaglandin E 2 (PGE2) and helix a6 from the β-subunit are attracted to the anionic and prostaglandin F 2a (PGF2a), prostaglandin I2 (PGI2) and surface of lysosomal vesicles through their associated cati- thromboxane A2 (TXA2) [87]. PGH2 is quite unstable and onic residues leading to imbedding of the associated hydro- is also rapidly converted to PGD2, PGE2, levuglandin E2 phobic residues [83]. In addition, its activity is stimulated by (LGE2) and levuglandin D2 (LGD2) non enzymatically in both DTT and the detergent Nonidet P-40, the latter shown aqueous solution [88, 89]. COX-2 and not COX1 oxygen- to initiate a conformational change in the structure leading ates anandamide to PGH 2 ethanolamide (PGH2-EA) which to the formation of the active site. is then rapidly converted to PGD2 ethanolamide (PGD2-EA), Specific activity has been reported for hNAAA with PGE 2 ethanolamide (PGE 2-EA), PGF 2a ethanolamide a value for AEA hydrolysis that is only 30–50% of that (PGF 2a-EA), PGI 2 ethanolamide (PGI2-EA), and TXA2 etha- exhibited by FAAH-2 and only 3% of that for PEA hydrol- nolamide (TXA2-EA) enzymatically (Fig. 2) [76, 90–93]. ysis (Table 3). hNAAA has a catalytic preference for the Human cyclooxygenase-2 (COX-2, PTGS2, PGHS-2, smaller fatty-acid amides where PEA > > N-myristyletha- cyclooxygenase-2, UniProtKB-P35354) is a heme-requiring nolamine > > N-steroylethanolamine ≈ N-laurylethanola- enzyme that is expressed as a single isoform. Its physical mine > AEA [73]. Clearly the primary biological function properties and PTMs have been discussed in detail elsewhere of this enzyme is the hydrolysis of PEA but can readily com- [87, 94–96]. During infammation, COX-2 is signifcantly pete with FAAH-2 for AEA. upregulated in the afected tissue with the exception of brain, Regulation of NAAA by lipid and thiol compounds testes, and the macula densa of the kidney where it is consti- has been reported [85]. The presence of phosphatidylcho- tutively expressed [97]. Intracellular distribution favors the line (PC), phosphatidylethanolamine, or sphingomyelin endoplasmic reticulum. increases the hydrolysis rate of PEA by fvefold at 100 µM Specifc activity for partially purifed hCOX-2 towards and increases with phospholipid concentration based on PC AEA has been reported and found to be 70% of that observed results. This compares to the sixfold increase with a similar for the more typical substrate AA (Table 3). Clearly hCOX-2 amount of the nonionic detergent Nonidet P-40. Numerous is a formidable contributor to AEA degradation when endogenous and non-endogenous thiol compounds were expressed in appropriate quantities, for example, during tested where DTT and the endogenous dihydrolipoic acid infammation. Regulation of COX-2 at the transcriptional have the greatest efect. These compounds presumably help level has been reported where a variety of cytokines and keep the catalysis C126 in the reduced state that is required growth factors have been shown to stimulate induction [98]. for activity. Neither additive infuences substrate preference. Tyrosine phosphorylation at Y120 and Y446 are known to Regulation of NAAA expression involving anandamide enhance the activity of hCOX-2 towards AA substrate [95, metabolism has not been reported. N-glycosylation is nec- 99]. Nitrosylation of C526 by an inducible nitric oxide syn- essary for transport of hNAAA from the Golgi, but not thase, a major mediator of infammation, results in a twofold required for catalytic activity [86]. enhancement of activity towards AA [96, 100].

Conversion of anandamide to other compounds Cytochrome P450s

There are numerous pathways involved in the conversion of The primary biological role for cytochrome P450 enzymes anandamide to other compounds that display a wide variety is the oxidation of steroids, lipids, and various xenobiot- of diferent biological efects. The reactions center on the ics. Cytochrome P450 (CYP) represents a large class of

1 3 Molecular and Cellular Biochemistry heme-requiring monooxygenases, too vast to discuss in 12S‑lipoxygenase (ALOX12) detail here. However, it is worthwhile to examine a few that are known to be involved in anandamide catabolism. There 12S-lipoxygenase catalyzes the oxygenation of AEA to are fve major products (Fig. 2) [77]—10 in total—for anan- 12(S)-hydroperoxyarachadonoylethanolamide (12(S)- damide metabolism by CYP enzymes: N-(2-hydroxyethyl)- HPETE-EA) which is rapidly converted to 12(S)- 20-hydroxyeicosatetraenamide (20-HETE-EA), hydroxyarachadonoylethanolamide (12(S)-HETE-EA) N-(2-hydroxyethyl)-5,6-epoxyeicosatrienamide (5,6-EET- as well as numerous other products (Fig. 2) [103, 104]. EA), N-(2-hydroxyethyl)-8,9-epoxyeicosatrienamide Human12S-lipoxygenase (hALOX12, 12S-LOX, 12LO, (8,9-EET-EA), N-(2-hydroxyethyl)-11,12-epoxyeicosatrien- LOG12, UniprotKB-P18054) is translated as a single iso- amide (11,12-EET-EA), N-(2-hydroxyethyl)-14,15-epoxye- form. Physical properties and PTMs are discussed elsewhere icosatrienamide (14,15-EET-EA), each of which is formed [104]. The specifc activity for hALOX12 with AEA was at diferent rates by diferent CYP isoforms (Table 3). Iso- determined for solubilized protein precipitates obtained from forms CYP2D6, CYP3A4, CYP2J2, CYP4F4 are known human leukocyte/platelet homogenates and found to be ≈ anandamide monooxygenases and are found in the human 70% of that found for AA [81] (Table 3). brain as well as other tissues [13, 77–79, 101]. hCYP2D6 As observed for CYP enzymes, the hydroxylated prod- and hCYP3A4 have been shown to be major contributors to uct also binds to the endocannabinoid receptors CB1 and anandamide metabolism in the brain where incubation with CB2. 12(S)-HETE-EA binds to rat brain membranes con- isoform-specifc inhibitory antibodies reduces AEA metabo- taining CB1 with lower efciency (≈ 53%) than AEA and lism by 88% and 42–66%, respectively [77]. Interestingly, human CB2 expressed in CHO cells also binds with lower one of the metabolites, 5,6-EET-EA, is a potent endocan- efciency than AEA (≈ 60%) [103]. A second report indi- nabinoid CB2 receptor agonist with comparable binding cates that 12(S)-HETE-EA binds to both CB1 in rat brain efciency to AEA as well as being more stable than AEA membranes and CB2 in rat spleen membranes with lower [101], the production of which thus increases the potency of efciency than AEA, 60% and 72%, respectively [105]. The the original anandamide signaling. In addition, 5,6-EET-EA binding of 12(S)-HETE-EA to mixed CB1 and CB2 recep- is also an agonist for the endocannabinoid CB1 receptor, tors has been shown to be agonistic but less efective than albeit with > 1000-fold weaker binding. 20-HETE-EA and AEA with an EC50 that is 2.5 times that observed for AEA 14,15-EET-EA also bind to the CB1 receptor with 3.6-fold for the inhibition of electrically initiated twitch response in and 5.7-fold lower efciency than anandamide, respectively, murine deferens [81]. and are more rapidly degraded in the brain than ananda- The catalytic activity of hALOX12 is sensitive to the mide [80]. The biological impact of these and the other P450 redox conditions [106] within the cell and may be regulated metabolites of AEA needs to be clarifed. by phosphorylation at S246 based on the known phosphorylation of the rat isoform [47] for which it shares an 84.5% sequence homology. Upregulation of ALOX12 in porcine Lipoxygenases aortic vascular smooth muscle cells by the proinfammatory cytokines IL-1β, IL-4, and IL-8 has also been reported Lipoxygenases represent a class of non-heme iron-requir- [107]. ing enzymes responsible for oxygenation of a wide array of polyunsaturated fatty acids (PUFAs). The primary bio- 15S‑lipoxygenase (ALOX15) logical role is to oxygenate PUFAs at specifc positions in a stereospecifc manner, producing the corresponding 15S-lipoxygenase catalyzes the oxygenation of AEA to mono-hydroperoxy products that are readily converted to 15(S)-peroxyarachadonoylethanolamide (15(S)-HPETE- mono-hydroxy derivatives by ubiquitous glutathione per- EA) which is rapidly converted to 15(S)-hydroxyaracha- oxidases (GPX). Individual lipoxygenases are responsible donoylethanolamide (15(S)-HETE-EA) by ubiquitous GPXs for hydroperoxidation at specifc positions and associated (Fig. 2). ALOX15 can also convert 15(S)-HPETE-EA to stereochemistry. For example, of the 12 possible peroxy eoxin A 4 ethanolamide (EXA 4-EA) which is then readily positions on arachidonic acid, 10 are found in nature and 5 converted to the ethanolamides of eoxin C4 (EXC4-EA) and found in humans. Both 12S and 15S peroxidations of AEA eoxin D 4 (EXD 4-EA) [108] through the action of leukot- catalyzed by 12S-lipoxygenase and 15S-lipoxygenase, riene C4 synthase (LTC4S) and gamma-glutamyl transami- respectively, have been reported [81, 102, 103]. However, 5S nase (GTT1) in a sequential manner (Fig. 2) [104]. Like and 12R peroxidation, and 15R hydroxylation catalyzed by hALOX12, human 15S-lipoxygenase (hALOX15, 15A- 5S-lipoxygenase, 12R-lipoxygenase, and aspirin-acetylated LOX, 15-LOX, 15-LOX-1, 12/15-lipoxygenase, UniProtKB- COX-2, respectively, have been observed for AA substrate, P16050) is a non-heme, iron-requiring cytosolic protein that but not for AEA. becomes membrane associated in the presence of calcium. It

1 3 Molecular and Cellular Biochemistry is transcribed as two isoforms, but only the canonical form, well [113, 114]. Several fatty acid binding proteins, FABP3, Isoform 1, has been observed at the protein level. The physi- FABP5, and FABP7 are known to be involved in intracel- cal properties and PTMs are discussed elsewhere [104, 109]. lular trafcking of AEA [115]. FABP5 was recently shown Specifc activity for hALOX15 with AEA has not been to mediate retrograde endocannabinoid transport, suggest- reported; however, one publication gives a K m for AEA of ing that it may serve as a synaptic carrier for AEA [113]. 108 µM [108]. It should be noted that hALOX15 also pro- heat shock proteins (HSPs) are also involved in intracel- duces 12S-HETE from AA in a 1:9 12S:15S ratio [110]. lular transport of AEA [112]. HSP70, HSP90, HSP60 as The production of 12S-HETE-EA by this enzyme is to be well as several other HSPs (e.g., Grp75, Grp 80) have been expected but has yet to be reported. 15(S)-HETE-EA binds identifed extracellularly [114]. All of these species have to rat brain CB1 with lower efciency than AEA (14%) and been shown to bind lipids and thus may potentially act as does not bind to human CB2 expressed in CHO cells [103]. additional AETs. A second report indicates that 15(S)-HETE-EA binds to Microvesicles and exosomes are also well documented as CB1 in rat brain membranes with a lower efciency than purveyors of both intracellular and extracellular signaling AEA (15%) and does not bind to CB2 in rat spleen mem- through their transport of proteins, peptides, nucleic acids, branes [105]. The binding of 15(S)-HETE-EA to mixed and lipids [116], the latter making them potential candidates CB1 and CB2 receptors has been shown to be agonistic but for both intracellular an extracellular transport of AEA. less efective than AEA with an EC 50 that is 4.9 times that One report supporting vesicular transport of AEA shows for AEA for the inhibition of electrically initiated twitch that microglia produce extracellular vesicles with AEA on response in murine deferens [81]. their surface and in turn stimulate type-1 endocannabinoid Regulation via posttranslational modifcation has not receptors (CB1), facilitating inhibition of presynaptic trans- been reported. However, upregulation of hALOX15 expres- mission [117]. sion by the proinfammatory cytokines IL-4 and IL-13 in human monocytes is well documented [111]. Transport of AEA across plasma membranes

Endocannabinoids are readily transported across plasma Anandamide transport and storage membranes. The mode for transport of AEA has been a hotly debated subject for many years. Model proponents fall into AEA is a well-known paracrine and autocrine signaling three camps, difusion, facilitated transport, or an endocy- molecule. The fact that it is uncharged and hydrophobic totic pathway. It is clear, however, that AEA does not pass necessitates that AEA be transported both extracellularly through membranes by active transport [118]. and intracellularly by carrier molecules. Further, both the Support for difusion control of AEA transport is mul- biosynthetic and degradation machinery are located within tifold. Some cite the lack of direct evidence for a protein- the cell, necessitating the existence of some process or pro- aceous transporter and a simpler model where sequestra- cesses to move AEA out of the cell for extracellular signal- tion of cytosolic AEA by proteins or vesicles necessarily ing and import for degradation. Until recently this process leads to the AEA gradient necessary for difusion across was thought to operate solely on a supply and demand mode. the plasma membrane [119]. Indeed, external addition of However, it has become clear that AEA is also stored in lipid AEA to lipid vesicles containing internalized FAAH results droplets, thus complicating the supply demand model with a in rapid AEA transport and hydrolysis [115]. Further, this “bufer” system that can modulate available AEA. The fol- transport is enhanced by cholesterol and coprostanol but is lowing sections outline each of the processes noted above, not afected by cholesterol sulfate. The equality of choles- including characterizations of the proteins involved. terol and coprostanol transport indicates that the sterol ring enhances transport and coprostanol facilitated transport Extracellular AEA transport proteins (AETs) indicates that lipid rafts are not necessary for this process to occur. The lack of enhancement with cholesterol sulfate Transport of hydrophobic molecules in aqueous solution supports a mechanism whereby sterol ring fip-fopping may in living systems is typically facilitated by specifc binding be involved in the transport process. Earlier work shows proteins. In plasma, albumin, and members of the lipocalin that both AEA insertion into lipid membranes and transport family are involved in transporting lipophilic molecules in across them is cholesterol-dependent [120]. In related work, plasma and may possibly be involved in extracellular AEA AEA present in resealed red blood cell membranes (ghosts) transport. Proteins historically shown to be involved in intra- rapidly exits to external solutions containing bovine serum cellular transport [112] have more recently been shown to albumin (BSA), a known AEA binder, in a BSA-dependent exist extracellularly and one is known to be involved in manner [118]. Similarly, treatment of HeLa cells expressing extracellular AEA transport and others may be involved as mouse FABPs, known AEA binders, with FABP inhibitors

1 3 Molecular and Cellular Biochemistry reduces uptake of [14C] AEA as do FABP knockout cells in Conclusions the absence of inhibitors [121]. Much of the support for AEA membrane transporter Anandamide homeostasis is achieved through the rates of proteins (AMT) comes from observations that a variety of synthesis, degradation, and storage of this molecule. This pharmaceuticals can reduce AEA transport presumably by manuscript discusses the known enzymes and other pro- interacting with an undiscovered AMT. On the other hand, teins that are major contributors to this system. Biosynthe- these inhibitors may instead be interacting with intracellular sis occurs along four parallel pathways, each of which is proteins involved in sequestration or destruction of AEA. A catalyzed by its own set of enzymes and under independent more recent study clearly shows the existence of an AMT control. Degradation can be simple, involving a hydrolysis of that is capable of bi-directional trafcking of AEA [122]. the amide by enzymes, or complex involving the eicosanoid Using specifc inhibitors of both AMT and FAAH they show biosynthesis machinery. Lastly, there is a storage system that inhibition of FAAH results in an increase in intracel- involving lipid microvesicles that modulate available AEA lular AEA and inhibition of the putative AMT results in a through sequestration, regulated release, and degradation. decrease in intracellular AEA, clearly showing the existence One additional item for consideration is the fact that AEA of an AMT. In addition, they also show that the same AMT can be converted to other molecules that are known to acti- inhibitors produce a concentration-dependent decrease in vate the same CB receptors that AEA stimulates, thus con- AEA export, clearly supporting the existence of a yet-to-be tinuing the signaling in the absence of AEA itself. identifed AMT. There are more than a few missing aspects that have yet A third possibility for transmembrane trafcking involves to be uncovered in the laboratory and are required to provide endocytosis. Treatment of rat basophil cells (RBL-2H3) a comprehensive picture of anandamide homeostasis. For with inhibitors of caveola-related (clatherin-independent) example, a relative comparison of the synthesis degrada- endocytosis reduces AEA transport by ≈ 50% compared to tion enzyme rates within particular tissues under defned controls [123]. Further, treatment of these cells with inhibi- metabolic conditions (e.g., healthy vs. infammatory state) tors of clatherin-dependent processes has no efect on AEA would be most enlightening. In addition, exploration of transport, indicating that only the caveola-related transport is potentially new endocannabinoid products formed by oxi- involved. The same study found that AEA is concentrated in dation catalyzed by eicosanoid enzymes other than ALOX12 caveolin-rich membranes following internalization, further and ALOX15 is in order. Lastly, a thorough investigation of supporting the clatherin-independent pathway. how each pathway enzyme is regulated would help to bet- ter defne the condition-dependent roles of each arm of the Storage of AEA multi-armed pathways.

The long-held belief that AEA is synthesized on demand has Supplementary Information The online version contains supplemen- been seriously challenged by more recent reports (see review tary material available at https://doi. org/ 10. 1007/ s11010- 021- 04121-5 . [2]). It has been shown that once AEA is taken up by cells Declarations it is rapidly targeted to adiposomes [112, 124]. Further, it is also observed that larger adiposome compartments have Conflict of interest The author states that he has no confict of interest. an increased capacity to both store and metabolize AEA [124]. Adiposomes not only house intracellular AEA, but also contain degradation enzymes FAAH-1&2 [72, 112] and thus these lipid bodies serve as modulators of intracellular References accumulation and degradation of AEA. Both mobilization and degradation of AEA stored in 1. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, liposomes requires a mechanism by which to move the inter- Grifn G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R nalized AEA from within the liposome to the surface. One (1992) Isolation and structure of a brain constituent that binds mode for this transport has been shown to involve sterols to the cannabinoid receptor. Science 258:1946–1949. https://doi. org/10.1126/science inserted into the liposome membrane [115]. Both lipid-raft 2. Maccarrone M (2017) Metabolism of the endocannabinoid associating cholesterol and lipid-raft avoiding coprostanol anandamide: open questions after 25 years. Front Mol Neurosci enhance transport of AEA across the liposome bilayer indi- 10:166. https://doi.org/10.3389/fnmol.2017.00166 cating that lipid rafts are not necessary for this process to 3. Marsicano G, Chaoulof F (2011) Moving bliss: a new anandamide transporter. Nat Neurosci 15:5–6. https://doi.org/10.1038/ occur. The mode of action is thought to involve a competi- nn.3011 tion for shielding the small headgroups from solvent water 4. Mechoulam R, Parker LA (2013) The endocannabinoid system resulting in increased surface exposure of AEA to FAAH and the brain. Annu Rev Psychol 64:21–47. https://doi.org/10. [115] or to specifc transport proteins. 1146/annurev-psych-113011-143739

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