Publications

• Inactivation of lipid glyceryl ester metabolism in human THP1 monocytes/macrophages by activated organophosphorus insecticides: role of carboxylesterases 1 and 2.

  Xie S., Borazjani A., Hatfield M.J., Edwards C.C., Potter P.M., Ross M.K.

  Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad su ... >> More

  Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 μM; and kcat/Km, 1.2 and 0.93 μM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 μM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels. << Less

  Chem. Res. Toxicol. 23:1890-1904(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol.

  Nithipatikom K., Endsley M.P., Pfeiffer A.W., Falck J.R., Campbell W.B.

  Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is ... >> More

  Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway. << Less

  J. Lipid Res. 55:2093-2102(2014) [PubMed] [EuropePMC]

• Biochemical and pharmacological characterization of the human lymphocyte antigen B-associated transcript 5 (BAT5/ABHD16A).

  Savinainen J.R., Patel J.Z., Parkkari T., Navia-Paldanius D., Marjamaa J.J., Laitinen T., Nevalainen T., Laitinen J.T.

  <h4>Background</h4>Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown. ... >> More

  <h4>Background</h4>Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown.<h4>Methodology/principal findings</h4>Amino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (≥95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14:0), long-chain unsaturated (C18:1, C18:2, C20:4) monoacylglycerols (MAGs) and 15-deoxy-Δ12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)-vs. 2-isomers of MAGs C18:1, C18:2 and C20:4. Inhibitor profiling revealed that β-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions.<h4>Conclusions/significance</h4>This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to lipase inhibitors suggest that BAT5 is a genuine lipase with preference for long-chain unsaturated MAGs and could in this capacity regulate glycerolipid metabolism in vivo as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors. << Less

  PLoS ONE 9:E109869-E109869(2014) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase.

  Vila A., Rosengarth A., Piomelli D., Cravatt B., Marnett L.J.

  Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variet ... >> More

  Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH. << Less

  Biochemistry 46:9578-9585(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.

  Blankman J.L., Simon G.M., Cravatt B.F.

  Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anan ... >> More

  Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system. << Less

  Chem. Biol. 14:1347-1356(2007) [PubMed] [EuropePMC]

• Human neuropathy target esterase catalyzes hydrolysis of membrane lipids.

  van Tienhoven M., Atkins J., Li Y., Glynn P.

  A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that ... >> More

  A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A(2) (iPLA(2)) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA(2) inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V(max) approximately 0.01 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of (14)C-labeled products from two differentially labeled (14)C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V(max) approximately 20 micromol/min/mg and K(m) approximately 0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V(max) approximately 1 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking. << Less

  J. Biol. Chem. 277:20942-20948(2002) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Mutation of Cys242 of human monoacylglycerol lipase disrupts balanced hydrolysis of 1- and 2-monoacylglycerols and selectively impairs inhibitor potency.

  Laitinen T., Navia-Paldanius D., Rytilahti R., Marjamaa J.J., Karizkova J., Parkkari T., Pantsar T., Poso A., Laitinen J.T., Savinainen J.R.

  Considerable progress has been made in recent years in developing selective, potent monoacylglycerol lipase (MAGL) inhibitors. In the investigations of measures to inhibit this enzyme, less attention has been paid to improving our understanding of its catalytic mechanisms or substrate preferences. ... >> More

  Considerable progress has been made in recent years in developing selective, potent monoacylglycerol lipase (MAGL) inhibitors. In the investigations of measures to inhibit this enzyme, less attention has been paid to improving our understanding of its catalytic mechanisms or substrate preferences. In our study, we used site-directed mutagenesis, and we show via versatile activity assays combined with molecular modeling that Cys242 and Tyr194, the two opposing amino acid residues in the catalytic cavity of MAGL, play important roles in determining the rate and the isomer preferences of monoacylglycerol hydrolysis. In contrast to wild-type enzymes that hydrolyzed 1- and 2-monoacylglycerols at similar rates, mutation of Cys242 to alanine caused a significant reduction in overall activity (maximal velocity, Vmax), particularly skewing the balanced hydrolysis of isomers to favor the 2-isomer. Molecular modeling studies indicate that this was caused by structural features unfavorable toward 1-isomers as well as impaired recognition of OH-groups in the glycerol moiety. Direct functional involvement of Cys242 in the catalysis was found unlikely due to the remote distance from the catalytic serine. Unlike C242A, mutation of Tyr194 did not bias the hydrolysis of 1- and 2-monoacylglycerols but significantly compromised overall activity. Finally, mutation of Cys242 was also found to impair inhibition of MAGL, especially that by fluorophosphonate derivatives (13-to 63-fold reduction in potency). Taken together, this study provides new experimental and modeling insights into the molecular mechanisms of MAGL-catalyzed hydrolysis of the primary endocannabinoid 2-arachidonoylglycerol and related monoacylglycerols. << Less

  Mol. Pharmacol. 85:510-519(2014) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.

• Biochemical and pharmacological characterization of human alpha/beta-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12).

  Navia-Paldanius D., Savinainen J.R., Laitinen J.T.

  In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG ... >> More

  In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors. << Less

  J. Lipid Res. 53:2413-2424(2012) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis.

  Nomura D.K., Long J.Z., Niessen S., Hoover H.S., Ng S.W., Cravatt B.F.

  Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) i ... >> More

  Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK: << Less

  Cell 140:49-61(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.