Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)
Year: 2012
Type: article
Abstract: 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. 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. The human serine hydrolases comprises a large family of enzymes with a predicted number of ∼240 that fall into two subfamilies: the serine proteases (∼125 members) and the metabolic serine hydrolases (∼115 members) (1Simon G.M. Cravatt B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.J. Biol. Chem. 2010; 285: 11051-11055Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 2Long J.Z. Cravatt B.F. The metabolic serine hydrolases and their functions in mammalian physiology and disease.Chem. Rev. 2011; 111: 6022-6063Crossref PubMed Scopus (268) Google Scholar). The metabolic serine hydrolases include lipases and amidases and use a conserved serine nucleophile to hydrolyze amide, ester, and thioester bonds. The majority of serine hydrolases contain an α/β-hydrolase domain (ABHD) fold and use a Ser-His-Asp (SHD) triad for catalysis. Although several members of the metabolic serine hydrolase family are relatively well defined, the majority remain poorly characterized with respect to their physiological substrates and functions. Members of the metabolic serine family are also intimately involved in the generation and degradation of the endocannabinoid 2-arachidonoylyglycerol (C20:4) (2-AG). In brain regions endowed with 2-AG signaling, “on demand” biosynthesis of 2-AG occurs through phospholipase Cβ-catalyzed cleavage of the membrane phospholipid phoshatidylinositol bisphosphate to generate sn-2-arachidonoyl-containing diacylglycerol (DAG) species, which are subsequently hydrolyzed by sn-1-specific lipases (DAGLα and DAGLβ) to generate 2-AG (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. Cell Biol. 2003; 163: 463-468Crossref PubMed Scopus (839) Google Scholar). The endocannabinoids are involved in a broad range of (patho)physiological processes, including neurotransmission, appetite, nociception, addiction, inflammation, peripheral metabolism, and reproduction (4Piomelli D. The molecular logic of endocannabinoid signalling.Nat. Rev. Neurosci. 2003; 4: 873-884Crossref PubMed Scopus (1613) Google Scholar–5Di Marzo V. Bisogno T. De Petrocellis L. Endocannabinoids and related and between and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. of Rev. PubMed Scopus Google Scholar). The of 2-AG are two and that show and is in the The enzymatic for 2-AG is acid and In the three serine monoacylglycerol lipase (MAGL) and the α/β-hydrolase domain proteins ABHD6 and for of 2-AG G.M. Cravatt B.F. of brain enzymes that hydrolyze the endocannabinoid Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, The serine hydrolases ABHD6 and ABHD12 as of 2-arachidonoylglycerol through PubMed Scopus Google Scholar). From these, is relatively well characterized and on a quantitative to be the main 2-AG hydrolase. is a that and and of the catalytic triad of to and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the in and of the catalytic triad of to and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. A. F. lipases and in and Full Text Full Text PDF PubMed Scopus Google Scholar). a and of the catalytic triad of to and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and is thought to functions in D. role for lipase in 2-arachidonoylglycerol PubMed Scopus Google Scholar). In for J.Z. Cravatt B.F. lipase a acid that 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, J.Z. et brain that 2011; PubMed Scopus Google Scholar). is the potent to of brain an of PubMed Scopus Google T. enzymatic activity is for of 2-arachidonoylglycerol in PubMed Scopus Google Scholar, A. T. of of lipase by a 2010; PubMed Scopus Google Scholar). lacks because members of the metabolic serine hydrolase The proteins ABHD6 and ABHD12 remain poorly characterized their physiological substrates and functions. ABHD6 is a membrane with in of F. hydrolase ABHD6 Biol. PubMed Scopus Google Scholar, D. I. of the conserved hydrolase domain containing in PubMed Scopus Google Scholar). on and ABHD6 to be an membrane active site is predicted to the G.M. Cravatt B.F. of brain enzymes that hydrolyze the endocannabinoid Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). an that ABHD6 be well to the of that ABHD6 the and signaling of 2-AG in A. G. et serine hydrolase ABHD6 the and of 2-AG Neurosci. 2010; PubMed Scopus Google Scholar, of domain and acid hydrolase endocannabinoid in Biol. Chem. 2011; Full Text Full Text PDF PubMed Scopus Google is not enzyme substrates as that ABHD12 is a role as a brain 2-AG hydrolase profiling with brain and that the brain ABHD12 for of 2-AG hydrolase activity G.M. Cravatt B.F. of brain enzymes that hydrolyze the endocannabinoid Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). 2-AG is the substrate for and 2-AG hydrolase activity is the ABHD12 to the endocannabinoid on and data, ABHD12 is an membrane active site is predicted to the G.M. Cravatt B.F. of brain enzymes that hydrolyze the endocannabinoid Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). in the ABHD12 to the and T. D. A. et in ABHD12 the an of endocannabinoid 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). ABHD12 are in brain regions as well as in and related peripheral such as and T. D. A. et in ABHD12 the an of endocannabinoid 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). of ABHD12 is by the of selective inhibitors. on the 2-AG hydrolases a activity profiling of a of substrates by a such as the Although glycerol in use for in as as we are the of not in the of endocannabinoid By applying a sensitive fluorescent to glycerol “on we delineate the substrate of human ABHD6 and ABHD12 in comparison with MAGL. in the three hydrolases a of with substrate and The enzymes detectable acid hydrolase hydrolyze By we the catalytic triad of ABHD12 as and verified as the catalytic nucleophile of profiling revealed striking potency differences between hABHD6 and for the glycerol and the substrates were the 1 (3)- and 2-isomers of and and the 1 (3)- and 2-isomers of and were were the and and and were as in containing in a of containing were to by a with an were in for and activity in Cell were by two with were and for Cell were three in and for were by the in by and for The in by and for were by as a The and of the glycerol is in glycerol a enzymatic to a generation of a The is in Activity-based profiling the fluorophosphonate probe of G.M. 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Full Text Full Text PDF PubMed Scopus Google Scholar). of in were with of the a for 1 serine hydrolases were with of for 1 The by and proteins were in with molecular and with the of serine hydrolases in by fluorescent with The substrate preferences of the endocannabinoid hydrolases were by glycerol in of of of the enzymes in containing with and as in facilitate comparison between the three hydrolases substrate in the three hydrolase in the as well as a glycerol to of the of the and 1 amino acid were mutagenesis to generate hABHD6 mutants and and hABHD12 mutants and by a the for the mutagenesis were of into and for of The of and in by as of enzymes of hABHD6 and of hABHD12 with molecular were by and to a membrane and were with and containing for 1 were with a with a in for in hABHD6 were with a and hABHD12 with a in for 1 by with the of enzyme between and of hABHD6 by with a for in were with a In the case of hABHD12, of by a and with with hABHD6 were by and by a in the of hABHD6 and hABHD12, were for enzyme the of substrates to the and of enzyme between substrate profiling of enzyme and that that the three hydrolases were in in the the of in 1 and and are three The mutants were two to three with and the in are The and and were for comparison between the MAG 1 (3)- and 2-isomers and The and of the endocannabinoid hydrolase is in were with the hABHD6 and were also with the amino acid with the active site probe that the three hydrolases were in as two and hABHD6 as a with a molecular of In hABHD12 as a with a molecular of In with labeling that the a the catalytic serine residues in of the three The also that of ABHD12 because labeling of proteins the of labeling of the with a that of ABHD12 not by that not to of the three hydrolases in and in 2-AG hydrolase activity as with the the enzymatic activity for the three of between and of hABHD6 and hABHD12 activity in membrane with that the two hydrolases are membrane proteins G.M. 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However, because in in and, on the hABHD6 and hABHD12 were detectable also in we of in also the of that a of to of The of the fluorescent to activity in a hydrolase activity to The of 2-AG to that hABHD6 and hABHD12 a broad between and the in the signaling and degradation of 2-AG in T. enzymatic activity is for of 2-arachidonoylglycerol in PubMed Scopus Google Scholar, T. 2-arachidonoylglycerol is a potent in PubMed Scopus Google we also in the that a to the three endocannabinoid hydrolases for further We the substrate preferences of the three hydrolases by glycerol in of the enzymes in containing and with and facilitate comparison between the three hydrolases substrate the three hydrolase in the activity in and and not activity with the in of the glycerol by endocannabinoid hydrolase hABHD6 hydrolyzed a of MAGs with a substrate MAGs with saturated to were with the activity The for 1 (3)- and (C20:4) in hABHD6 preferred the 1 with the activity hABHD6 activity toward and hydrolyze acid hABHD12 a substrate The preferred MAG substrate 1 by 2-AG and which were hydrolyzed to the hABHD12 preferred 1 and a for the 1 of and the the Although hABHD6 hydrolyzed MAGs with saturated to hABHD12 substrates and with a hABHD12 not hydrolyze The substrate of that of hABHD6 with hydrolyzed MAGs with the substrate the best and MAGs with saturated were the activity In to hABHD6 and hABHD12, which preferred the 1 of showed such for the 1 of and (C20:4) However, preferred the 1 of not The activity toward MAGs hABHD6 and hABHD12, not hydrolyze hABHD6 hABHD12 acid hydrolase substrate were not by hydrolases The catalytic triad on mutagenesis and of the catalytic triad of to and Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). is the predicted catalytic of ABHD6 and acid of the and that the are well conserved between the for ABHD6 between human and between human and and between and the were and α/β-hydrolase including the lipase and the predicted catalytic are We the amino of the predicted catalytic of hABHD6 and hABHD12 and, in the of the mutants to 2-AG the enzymes hydrolyzed whereas mutants were of enzymatic activity were two three and time the the to that the proteins were and in to those of their to be the case for hABHD12 with the that the as three to of toward enzymatic to the of the enzyme and two with the whereas none of the mutants showed detectable is that to degradation in the that we the of the hydrolases to of which the catalytic with the that none of the mutants by probe We the three hydrolases further by the and for 2-AG as the substrate The were for for hABHD12, and for hydrolase the were and We the potency of a of toward hABHD6 and hABHD12 2-AG as the The the (C20:4) and the the lipase and the and the were for and the were of We that the potent of by and were potent toward hABHD6 hABHD12, whereas for and the potency hABHD12, the potency of hABHD6 and were with the hABHD6 hABHD12 as in and were and were for hydrolase activity as in were for with with of the and glycerol containing 2-AG and glycerol for Inhibitor were and the were as for are three in a were with the hABHD6 hABHD12 as in and were and were for hydrolase activity as in were for with with of the and glycerol containing 2-AG and glycerol for Inhibitor were and the were as for are three is MAG substrates and of the catalytic triad of to and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. role for lipase in 2-arachidonoylglycerol PubMed Scopus Google Scholar, of brain an of PubMed Scopus Google Scholar, T. A. and of monoacylglycerol lipase in PubMed Scopus Google A. A. and of a monoacylglycerol lipase in human PubMed Scopus Google Scholar, and of lipase PubMed Scopus Google substrates and of the catalytic triad of to and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. of of lipase by a 2010; PubMed Scopus Google Scholar). In MAG substrates with G.M. Cravatt B.F. of brain enzymes that hydrolyze the endocannabinoid Biol. Full Text Full Text PDF PubMed Scopus Google and T. enzymatic activity is for of 2-arachidonoylglycerol in PubMed Scopus Google Although such as of to and of the substrates toward an of the to toward the substrates could be in a Although glycerol to in for and of a monoacylglycerol lipase the PubMed Scopus Google Scholar, G. D. et lipase in and Biol. Chem. 2011; Full Text Full Text PDF PubMed Scopus Google we for the first time and the sensitive glycerol for of endocannabinoid hydrolase The activity to delineate the substrate of the endocannabinoid hydrolases by a of for the first time the substrate preferences of hABHD6 and We are not of that to a in the case of MAGL. that the three hydrolases are genuine MAG lipases activity toward acid of the hydrolases the MAG glycerol acid that, in to brain membrane that to generate and glycerol by the of and MAG lipases G. acid in brain PubMed Scopus Google the three endocannabinoid hydrolases are of activity that is for the first of We that hABHD6 and hABHD12 the 1 (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. 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I. of the conserved hydrolase domain containing in PubMed Scopus Google Scholar). with ABHD6 should on From the hABHD12 best hydrolyzed the of the endocannabinoid 2-AG for 1 (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. Cell Biol. 2003; 163: 463-468Crossref PubMed Scopus (839) Google hydrolyzed also signaling in 2-AG is on and on the (4Piomelli D. The molecular logic of endocannabinoid signalling.Nat. Rev. Neurosci. 2003; 4: 873-884Crossref PubMed Scopus (1613) Google Scholar–5Di Marzo V. Bisogno T. De Petrocellis L. Endocannabinoids and related and between and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. of Rev. PubMed Scopus Google Scholar). is well that in the 2-AG to 1 (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. Cell Biol. 2003; 163: 463-468Crossref PubMed Scopus (839) Google T. enzymatic activity is for of 2-arachidonoylglycerol in PubMed Scopus Google Scholar). could an for 1 (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. Cell Biol. 2003; 163: 463-468Crossref PubMed Scopus (839) Google is not of is potent 2-AG in the T. 2-arachidonoylglycerol is a potent in PubMed Scopus Google Scholar). ABHD12 further of the endocannabinoid by 1 (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. 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Source: Journal of Lipid Research
DOI: 10.1194/jlr.m030411
Institution: University of Eastern Finland
Language: English
Subfield: Pharmacology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2163086339