Expression, Purification, and Characterization of Histidine-Tagged Mouse Monoglyceride Lipase from Baculovirus-Infected Insect Cells

Karlsson, Marie; Törnqvist, Hans; Holm, Cecilia

doi:10.1006/prep.1999.1194

Cited by 26 publications

(24 citation statements)

References 17 publications

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“…For MAGL, we are not aware of studies that have determined K m for 2-AG as the substrate. Instead, apparent K m values ranging from 0.2 mM to 0.5 mM were reported for purifi ed MAGL preparations using 1 ( 3 )-OG and/or 2-OG as the substrate (23)(24)(25). In one study, however, a K m value of 17 ± 3 µM was reported for hMAGL ( 38 ).…”

Section: Discussionmentioning

confidence: 94%

“…Endocannabinoid hydrolysis is commonly studied using radiolabeled "natural" MAG substrates ( 9,11,14,(23)(24)(25) or colorigenic/fl uorogenic artifi cial substrates ( 9,16 ). In addition, "natural" MAG substrates with mass-spectrometrybased ( 7 ) and HPLC-based ( 15 ) detection have been HEK293 cells were transiently transfected with the cDNAs encoding hABHD6 or hABHD12 as detailed in Materials and Methods.…”

Section: Discussionmentioning

confidence: 99%

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Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

Navia-Paldanius

Savinainen

Laitinen

2012

Journal of Lipid Research

152

199

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amide, ester, and thioester bonds. The majority of serine hydrolases contain an ␣ / ␤ -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 C ␤ -catalyzed cleavage of the membrane phospholipid phoshatidylinositol bisphosphate to generate sn-2-arachidonoyl-containing diacylglycerol (DAG) species, which are subsequently hydrolyzed by sn -1-specifi c lipases (DAGL ␣ and DAGL ␤ ) to generate 2-AG ( 3 ). The endocannabinoids are involved in a broad range of (patho)physiological processes, including neurotransmission, appetite, nociception, addiction, infl ammation, peripheral metabolism, and reproduction ( 4-6 ). The biological actions of 2-AG are mediated via two G protein-coupled receptors (CB1R and CB2R) that show unique and tissue-specifi c distribution. CB1R is highly enriched in the brain.The major enzymatic route for 2-AG inactivation is via hydrolysis, generating arachidonic acid (AA) and glycerol. In the CNS, three serine hydrolases, namely monoacylglycerol lipase (MAGL) and the ␣ / ␤ -hydrolase domain 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 ␣ / ␤ -hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fl uorescent 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 fl uorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verifi ed because none of the mutants showed detectable expression. Inhibitor profi ling revealed striking potency differences between hABHD6 and hABHD12, a fi nding that, when combined with the substrate profi ling 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 compris...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

Navia-Paldanius

Savinainen

Laitinen

2012

Journal of Lipid Research

152

199

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show abstract

“…A series of (5-(2H)-isoxazolonyl) ureas has been developed by Bayer as potent inhibitors of HSL (20). The selectivity of compound 59 (4-isopropyl-3-methyl-2-[1-(3-(S)-methyl-piperidin-1-yl)-meth-anoyl]-2H-isoxazol-5-one), thereafter named BAY, was evaluated on enzymatic activities of purified lipase preparations using 1(3)-monooleoyl-2-0-monooleyl glycerol (MOME) for rat and human HSL, monoolein for mouse monoglyceride lipase, and triolein for bovine lipoprotein lipase and porcine pancreatic lipase as substrates (21,22). MOME is a diacylglycerol analog.…”

Section: Methodsmentioning

confidence: 99%

Adipocyte Lipases and Defect of Lipolysis in Human Obesity

et al. 2005

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The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine-and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine-and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity. Diabetes 54:3190 -3197, 2005 O besity, which is characterized by an excess of fat stores, is the most important risk factor for type 2 diabetes. Adipose tissue lipolysis leads to the hydrolysis of triglycerides and release of free fatty acids (FFAs). Because of the link between elevated circulating FFA levels and the development of insulin resistance and the metabolic syndrome (1,2), adipose tissue lipolysis constitutes a target for the drug industry. Nicotinic acid, which acts by inhibiting adipose tissue lipolysis, was the first extensively used lipid-lowering agent (3). Catecholamines and natriuretic peptides are the major hormones stimulating this catabolic pathway in humans (4). Resistance to catecholamine-induced lipolysis in subcutaneous adipose tissue has been demonstrated in obese adults and children (5,6) and is attributed to decreased expression of lipolytic ␤ 2 -adrenoceptors (7), increased antilipolytic properties of ␣ 2 -adrenoceptors (8), and decreased expression of hormone-sensitive lipase (HSL) (9). It is possible that the HSL defect is the most important factor because it is also observed in nonobese first-degree relatives to obese subjects (10) and because there is a positive relationship between lipolytic capacity and HSL expression in human subcutaneous fat cells (11).The rate-limiting role of HSL in adipose tissue lipolysis has been challenged by the data from HSL knockout mice (12-15). Catecholamine-induced lipolysis is abrogated, but residual basal lipolysis is observed in adipocytes from HSL-null mice. These data suggest the existence of non-HSL lipases in adipos...

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“…The enzyme specifically hydrolyzes MG and has no activity against DG or TG. Cloning of the mouse cDNA encoding MGL revealed that the enzyme is composed of 302 amino acids with a molecular mass of 33 kDa (200)(201)(202). Although not directly related to other lipases in the human genome, MGL belongs to the large family of a/b hydrolase fold proteins with a GXSXG motif.…”

Section: Mgl the Final Step In Tg Hydrolysismentioning

confidence: 99%

Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores

Zechner

Kienesberger

Haemmerle

et al. 2009

Journal of Lipid Research

484

377

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Fatty acids (FAs) are essential components of all lipid classes and pivotal substrates for energy production in all vertebrates. Additionally, they act directly or indirectly as signaling molecules and, when bonded to amino acid side chains of peptides, anchor proteins in biological membranes. In vertebrates, FAs are predominantly stored in the form of triacylglycerol (TG) within lipid droplets of white adipose tissue. Lipid droplet-associated TGs are also found in most nonadipose tissues, including liver, cardiac muscle, and skeletal muscle. The mobilization of FAs from all fat depots depends on the activity of TG hydrolases. Currently, three enzymes are known to hydrolyze TG, the well-studied hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL), discovered more than 40 years ago, as well as the relatively recently identified adipose triglyceride lipase (ATGL). The phenotype of HSL-and ATGL-deficient mice, as well as the disease pattern of patients with defective ATGL activity (due to mutation in ATGL or in the enzymeʼs activator, CGI-58), suggest that the consecutive action of ATGL, HSL, and MGL is responsible for the complete hydrolysis of a TG molecule. The complex regulation of these enzymes by numerous, partially uncharacterized effectors creates the "lipolysome," a complex metabolic network that contributes to the control of lipid and energy homeostasis. This review focuses on the structure, function, and regulation of lipolytic enzymes with a special emphasis on ATGL.-Zechner, R., P. C. Kienesberger, G. Haemmerle, R. Zimmermann, and A. Lass. Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. J. Lipid Res. 2009. 50: 3-21.Supplementary key words lipolysis • hydrolase • neutral lipid storage disease Lipid homeostasis reflects a balance of processes, designed to generate fatty acids (FAs) and lipids, deliver them from their site of origin to target tissues, and catabolize them for metabolic purposes. Innumerable genes and signal components are responsible for an integrated communication network between many tissues and organs, including adipose tissue, liver, muscles, the digestive tract, pancreas, and the nervous system. This network ultimately accounts for the accurate regulation of lipid and energy homeostasis. Despite the central physiological importance of these processes for human health, many basic mechanisms regulating the synthesis, uptake, storage, and utilization of lipids remain insufficiently characterized.FAs are vital components of essentially all known organisms. They are important substrates for oxidation and the production of cellular energy. FAs are essential precursors for all lipid classes, including those forming biological membranes. Finally, they are important for protein function in acylated proteins and as ligands for nuclear receptor transcription factors. In contrast to these "beneficial" characteristics, unesterified FAs can become deleterious for cells when present even at relatively low concentrations. The chronic exposure of nona...

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Expression, Purification, and Characterization of Histidine-Tagged Mouse Monoglyceride Lipase from Baculovirus-Infected Insect Cells

Cited by 26 publications

References 17 publications

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

Adipocyte Lipases and Defect of Lipolysis in Human Obesity

Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores

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