Monoglyceride lipase mediates tumor-suppressive effects by promoting degradation of X-linked inhibitor of apoptosis protein.

We have previously reported that Monoglyceride Lipase (MGL) expression is absent or reduced in various human malignancies and MGL-deficient mice develop tumors in multiple organs. Evidence also suggests MGL to be a tumor suppressor, however, the mechanisms underlying its tumor-suppressive actions remain to be investigated. Here, we report a novel function of MGL as a negative regulator of XIAP, an important inhibitor of apoptosis. We found that MGL directly interacted with XIAP and enhanced E3-ligase activity and proteasomal degradation of XIAP. MGL overexpression induced cell death that was coupled with caspase activation and reduced XIAP levels. N-terminus of MGL was found to mediate interactions with XIAP and induce cell death. MGL-deficient cells exhibited elevated XIAP levels and exhibited resistance to anticancer drugs. XIAP expression was significantly elevated in tissues of MGL-deficient animals as well as human lung cancers exhibiting reduced MGL expression. Thus, MGL appears to mediate its tumor-suppressive actions by inhibiting XIAP to induce cell death.

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

Therapeutic potential of monoacylglycerol lipase inhibitors.

Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.

Las lipasas: enzimas con potencial para el desarrollo de biocatalizadores inmovilizados por adsorción interfacial: [revisión] / Lipases: enzymes having the potential for developing immobilised biocatalysts by interfacial adsorption: [review]

Las lipasas son enzimas con propiedades funcionales muy interesantes que permiten su utilización práctica en diversos campos de las industrias agroquímica, farmacéutica, de detergentes y alimentaria, así como en química fina. Entre las aplicaciones más importantes de estas moléculas se encuentran: la resolución de mezclas racémicas, la obtención de compuestos ópticamente puros y la bioconversión de principios activos. En este trabajo se presenta una amplia revisión del tema, que abarca desde aspectos estructurales y funcionales de las lipasas, hasta la inmovilización de estas enzimas mediante adsorción interfacial y su empleo en biotecnología.

Lipases are enzymes with very interesting functional properties that allow their practical use in different fields of Agro-Chemical, Pharmaceutical and Food industries, as well as in Fine Chemistry. Among the most relevant applications of these molecules are: racemic mixtures resolution, obtainment of optically pure compounds and bioconversion of active principles. In this work a broad review of this topic is presented. This includes since structural and functional features of lipases until the immobilization of these enzymes by interfacial adsorption and their employment in biotechnology.

High expression of the evolutionarily conserved alpha/beta hydrolase domain containing 6 (ABHD6) in Ewing tumors.

Despite improvements in the treatment of patients with Ewing family tumors (EFT), the prognosis for patients with advanced disease is still unsatisfactory. Recently, we identified lipase I as an EFT-associated gene that might be interesting for the development of new immunological or pharmacological treatment strategies. Lipase I is a member of the large protein superfamilies of alpha/beta hydrolases and serine hydrolases. In the present paper we describe high expression of another member of these superfamilies in EFT. By DNA microarray data base mining we found exceptional high expression of alpha/beta hydrolase domain containing 6 (ABHD6) in EFT but not in other sarcomas. Expression of ABHD6 in EFT correlated with expression of another EFT-associated gene, aristaless. Analysis of ABHD6-associated GGAA microsatellites revealed shorter microsatellites in EFT with lack of ABHD6 expression. ABHD6 homologues were found in varying chordata but not in other animal species. Based on homology modeling we predicted the 3D-structure of ABHD6, which shows high similarity with bacterial homoserine transacetylases. High expression of ABHD6 in EFT in comparison to normal tissues and other tumors suggests that ABHD6 might be an interesting new diagnostic or therapeutic target for EFT. However, knock down of ABHD6 in EFT cells did not inhibit tumor cell growth.

A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

BACKGROUND AND PURPOSE: Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208. EXPERIMENTAL APPROACH: A series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors. KEY RESULTS: The screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC(50)= 88 +/- 12 nM) through a partially reversible mechanism. Initial structure-activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC(50)= 43 +/- 8 nM), while substitution with a methyl group produced the opposite effect (IC(50)= 239 +/- 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC(50); wild-type, 151 +/- 17 nM; C208G, 722 +/- 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242). CONCLUSIONS AND IMPLICATIONS: The results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.

ATP induces a rapid and pronounced increase in 2-arachidonoylglycerol production by astrocytes, a response limited by monoacylglycerol lipase.

The cytoplasm of neural cells contain millimolar amounts of ATP, which flood the extracellular space after injury, activating purinergic receptors expressed by glial cells and increasing gliotransmitter production. These gliotransmitters, which are thought to orchestrate neuroinflammation, remain widely uncharacterized. Recently, we showed that microglial cells produce 2-arachidonoylglycerol (2-AG), an endocannabinoid known to prevent the propagation of harmful neuroinflammation, and that ATP increases this production by threefold at 2.5 min (Witting et al., 2004). Here we show that ATP increases 2-AG production from mouse astrocytes in culture, a response that is more rapid (i.e., significant within 10 sec) and pronounced (i.e., 60-fold increase at 2.5 min) than any stimulus-induced increase in endocannabinoid production reported thus far. Increased 2-AG production from astrocytes requires millimolar amounts of ATP, activation of purinergic P2X7 receptors, sustained rise in intracellular calcium, and diacylglycerol lipase activity. Furthermore, we show that astrocytes express monoacylglycerol lipase (MGL), the main hydrolyzing enzyme of 2-AG, the pharmacological inhibition of which potentiates the ATP-induced 2-AG production (up to 113-fold of basal 2-AG production at 2.5 min). Our results show that ATP greatly increases, and MGL limits, 2-AG production from astrocytes. We propose that 2-AG may function as a gliotransmitter, with MGL inhibitors potentiating this production and possibly restraining the propagation of harmful neuroinflammation.

Phylogenomic and chemotaxonomic analysis of the endocannabinoid system.

The endocannabinoid system consists of two cannabinoid (CB) receptors, seven ligands, and ligand-catabolizing enzymes such as fatty acid amid hydrolase (FAAH) and monoglyceride lipase (MGL). The system's phylogenetic distribution is poorly known. The ligands cannot be molecularly investigated because they are not polypeptides and their specific synthetic enzymes have not been identified, so no sequences are available. Ligand phylogenetics can be inferred, nonetheless, by their presence in a range of extant organisms. Thus a meta-analysis of ligand extraction studies was performed (chemotaxonomy), and compared to a molecular search for homologs of CB receptors, vanilloid receptors (VR1), FAAH, and MGL in the genomes of sequenced organisms (phylogenomics). Putative homologs underwent functional mapping to ascertain the presence of critical amino acid motifs known to impart protein functionality. From an evolutionary perspective it appears that (1) endocannabinoid ligands evolved before CB receptors; (2) the ligands evolved independently multiple times; (3) CB receptors evolved prior to the metazoan-bilaterian divergence (ie, between extant Hydra and leech), but were secondarily lost in the Ecdysozoa; (4) VR1 may predate CB receptors but its affinity for endocannabinoids is a recent acquisition, appearing after the lower vertebrate-mammal divergence; (5) MGL may be as old as the ligands, whereas FAAH evolved recently, after the appearance of vertebrates. FAAH's emergence correlates with VR1's newly-found affinity for anandamide; this overlap in evolutionary time is recapitulated by complementary distribution patterns of FAAH, VR1, and anandamide in the brain. Linking FAAH, VR1, and anandamide implies a coupling among the remaining "older" parts of the endocannabinoid system, MGL, CB receptors, and 2-AG.

A role for monoglyceride lipase in 2-arachidonoylglycerol inactivation.

2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain (where its levels are 170-folds higher than those of anandamide), is produced by neurons in an activity- and calcium-dependent manner, and is rapidly eliminated. The mechanism of 2-AG inactivation is not completely understood, but is thought to involve carrier-mediated transport into cells followed by enzymatic hydrolysis. We examined the possible role of the serine hydrolase, monoglyceride lipase (MGL), in brain 2-AG inactivation. We identified by homology screening a cDNA sequence encoding for a 303-amino acid protein, which conferred MGL activity upon transfection to COS-7 cells. Northern blot and in situ hybridization analyses revealed that MGL mRNA is unevenly present in the rat brain, with highest levels in regions where CB1 cannabinoid receptors are also expressed (hippocampus, cortex, anterior thalamus and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased the degradation of endogenously produced 2-AG in these cells, whereas no such effect was observed on anandamide degradation. These results indicate that hydrolysis via MGL may be a primary route of 2-AG inactivation in intact neuronal cells.

Monoacylglycerol metabolism in rat small intestinal epithelial cells.

1. In various preparations of rat small intestinal epithelial cells the acylation and deacylation of monoacylglycerol were studied. In the in vitro vascularly perfused intestine, of which the lumen was loaded with monoacylglycerol with or without fatty acids, acylation exceeded deacylation. In contrast, deacylation was much faster in isolated microsomes and in isolated whole cells. 2. In vascularly perfused intestine, without long-chain fatty acids present in the lumen, the amount of di- and triacylglycerol formed was found to be half of that formed in perfusions with long-chain fatty acids in the lumen, while the glycerol formation was increased 1.4-fold. 3. The concentration of monoacylglycerol is an important factor in determining the relative rates of monoacylglycerol acylation and deacylation in microsomes: the ratio acylation/deacylation decreases with increasing monoacylglycerol concentrations. 4. The function of the monoacylglycerol lipase in fat resorption is discussed.