BBA - Molecular and Cell Biology of Lipids (v.1821, #11)
Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP) by Sawsan Amara; Vincent Delorme; Michel Record; Frédéric Carrière (1379-1385).
Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351 Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides > PC > MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level.Display Omitted► MAFP inhibits the lipase, phospholipase and galactolipase activities of HPLRP2. ► MAFP therefore inhibits both phospholipase A1 (HPLRP2) and A2 (cPLA2) activities. ► These findings provide additional evidence that MAFP specificity is weak. ► These findings challenge the common use of MAFP as an inhibitor of PLA2s.
Keywords: Enzyme inhibition; Galactolipase; Lipase; Phospholipase A1; Phosphonate; PLRP2;
Phospholipid sources for adrenic acid mobilization in RAW 264.7 macrophages. Comparison with arachidonic acid by Carlos Guijas; Alma M. Astudillo; Luis Gil-de-Gómez; Julio M. Rubio; María A. Balboa; Jesús Balsinde (1386-1393).
Cells metabolize arachidonic acid (AA) to adrenic acid (AdA) via 2-carbon elongation reactions. Like AA, AdA can be converted into multiple oxygenated metabolites, with important roles in various physiological and pathophysiological processes. However, in contrast to AA, there is virtually no information on how the cells regulate the availability of free AdA for conversion into bioactive products. We have used a comparative lipidomic approach with both gas chromatography and liquid chromatography coupled to mass spectrometry to characterize changes in the levels of AA- and AdA-containing phospholipid species in RAW 264.7 macrophage-like cells. Incubation of the cells with AA results in an extensive conversion to AdA but both fatty acids do not compete with each other for esterification into phospholipids. AdA but not AA, shows preference for incorporation into phospholipids containing stearic acid at the sn-1 position. After stimulation of the cells with zymosan, both AA and AdA are released in large quantities, albeit AA is released to a greater extent. Finally, a variety of phosphatidylcholine and phosphatidylinositol molecular species contribute to AA; however, AdA is liberated exclusively from phosphatidylcholine species. Collectively, these results identify significant differences in the cellular utilization of AA and AdA by the macrophages, suggesting non-redundant biological actions for these two fatty acids.► Cells convert arachidonic acid into adrenic acid via 2-carbon elongation reactions. ► Changes in arachidonate- and adrenoate-containing phospholipids are characterized. ► Adrenic acid shows preference for phospholipids containing sn-1 stearic acid. ► Adrenic acid is liberated exclusively from phosphatidylcholine species.
Keywords: Arachidonic acid; Adrenic acid; Lipid mediator; Inflammation; Monocyte/macrophage;
Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK1/2 MAP kinases by Pierre-Christian Violet; Emmanuelle Billon-Denis; Philippe Robin (1394-1405).
The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK1/2 MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK1/2 occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK1/2 was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK1/2 activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK1/2 activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK1/2 activation.► LPA receptor antagonist DGPP 8:0 stimulates ERK1/2 by unknown mechanism. ► DGPP effect is not mediated by LPA receptors. ► Lipid phosphate phosphatase dephoshporylates DGPP. ► LPA receptor antagonist VPC32183 inhibits DGPP dephosphorylation. ► LPP1 siRNA or VPC32183 suppresses DGPP-induced ERK1/2 activation.
Keywords: Diacylglycerol pyrophosphate; Lipid phosphate phosphatase; ERK; Phosphatidic acid; Lysophosphatidic acid; ELT3 cell;
Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice by Sascha Obrowsky; Prakash G. Chandak; Jay V. Patankar; Thomas Pfeifer; Silvia Povoden; Renate Schreiber; Guenter Haemmerle; Sanja Levak-Frank; Dagmar Kratky (1406-1414).
Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4 h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.► HSLiKO mice accumulate cholesteryl esters in the small intestine. ► HSLiKO mice exhibit accelerated cholesterol absorption. ► HSLiKO mice show decreased intestinal cholesterol biosynthesis. ► Intestinal HSL deficiency does not affect triglyceride metabolism.
Keywords: Hormone sensitive lipase; Cholesterol absorption; Triglyceride absorption; Intestine-specific HSL-deficient mice; Small intestine;
Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS by Martin Giera; Andreea Ioan-Facsinay; Rene Toes; Fei Gao; Jesmond Dalli; André M. Deelder; Charles N. Serhan; Oleg A. Mayboroda (1415-1424).
Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A4 and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.► LC-MS/MS profiling of lipids in synovial fluid of rheumatoid arthritis patients. ► LC-MS/MS platform incorporating a ion trap MS and a core-shell particle column. ► Targeted investigation of pro-resolving lipid mediators in patient samples. ► Quantification of MaR1, 5S,12S-diHETE, RvD5 and LXA4 in synovial fluid. ► First description of pro-resolving lipid mediators in rheumatoid arthritis patients.
Keywords: Lipid mediator; LC–MS/MS; Lipidomics; Rheumatoid arthritis; Special pro-resolving mediator; Synovial fluid;
Rat and human fatty acid amide hydrolases: Overt similarities and hidden differences by Almerinda Di Venere; Enrico Dainese; Filomena Fezza; Beatrice Clotilde Angelucci; Nicola Rosato; Benjamin F. Cravatt; Alessandro Finazzi-Agrò; Giampiero Mei; Mauro Maccarrone (1425-1433).
Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a relevant role in the metabolism of fatty acid amides and esters. It degrades important neurotransmitters such as oleamide and anandamide, and it has been involved in a number of human pathological conditions, representing therefore a valuable target for biochemical and pharmacological research. In this study, we have investigated in vitro the structure–function relationship of rat and human FAAHs. In particular circular dichroism, fluorescence spectroscopy and light scattering measurements have been performed, in order to characterize the structural features of the two proteins, both in the presence and absence of the irreversible inhibitor methoxyarachidonyl-fluorophosphonate. The results demonstrate that the structural dynamics of the two FAAHs are different, despite their high sequence homology and overall similarity in temperature-dependence. Additionally, membrane binding and kinetic assays of both FAAHs indicate that also the functional properties of the two enzymes are different in their interaction with lipid bilayers and with exogenous inhibitors. These findings suggest that pre-clinical studies of FAAH-dependent human diseases based only on animal models should be interpreted with caution, and that the efficacy of new drugs targeted to FAAH should be tested in vitro, on both rat and human enzymes.► Structural and functional characterization of rat and human fatty acid amide hydrolase (FAAH) ► Rat and human FAAH differ in their structural and functional properties. ► Their conformational differences might explain dissimilarities in membrane binding capability and inhibitor efficiency. ► The animal model alone might not be sufficient to test new drugs against FAAH-dependent human diseases.
Keywords: Anandamide hydrolase; Endocannabinoid; Fluorescence; Membrane binding; Protein aggregation and stability;
The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition by Ángel Pérez-Lara; Antonio L. Egea-Jiménez; Alessio Ausili; Senena Corbalán-García; Juan C. Gómez-Fernández (1434-1442).
Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP2 in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP2. The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP2, the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP2. We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP2 although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.Display Omitted
Keywords: PKCα; Rapid kinetics; PIP2; stopped-flow;
Anti-inflammatory and metabolic actions of FXR: Insights into molecular mechanisms by Danielle A.A. Hollman; Alexandra Milona; Karel J. van Erpecum; Saskia W.C. van Mil (1443-1452).
The farnesoid X receptor (FXR) is a ligand-activated transcription factor belonging to the nuclear receptor (NR) superfamily. FXR plays an important role in positively regulating genes (transactivation) involved in bile acid homeostasis, fat and glucose metabolism. Recently, it has become clear that an additional important role for FXR consists of downregulating genes involved in inflammation. Because of this broad spectrum of regulated genes, therapeutically targeting FXR with full agonists will likely result in adverse side effects, in line with what is described for other NRs. It may therefore be necessary to develop selective FXR modulators. However, the molecular mechanisms that distinguish between FXR-mediated transactivation and transrepression are currently unknown. For other NRs, post-translational modifications such as SUMOylation and phosphorylation have been reported to be unique to either transactivation or transrepression. Here, we review current knowledge on post-translational regulation of FXR with respect to transactivation and transrepression. Ultimately, increased understanding of the different mechanisms of transactivation and transrepression of nuclear receptors will aid in the development of NR drugs with fewer side effects.► FXR transactivates genes involved in bile acid, glucose and fat homeostasis. ► FXR negatively regulates genes (via transrepression) involved in inflammation. ► Therefore, side effects will probably occur when full FXR agonists are used in clinic. ► Post-translational modifications differentiate between transactivation/-repression. ► Unraveling the mechanisms of FXR signaling enables selective FXR ligand design.
Keywords: FXR; Post-translational modification; Transactivation; Transrepression; Selective ligand; Nuclear receptor;