BBA - Molecular and Cell Biology of Lipids (v.1851, #11)

Inositol-phosphodihydroceramides in the periodontal pathogen Tannerella forsythia: Structural analysis and incorporation of exogenous myo-inositol by Zoë Anne Megson; Ernst Pittenauer; Katarzyna Anna Duda; Regina Engel; Karin Ortmayr; Gunda Koellensperger; Lukas Mach; Günter Allmaier; Otto Holst; Paul Messner; Christina Schäffer (1417-1427).
Unique phosphodihydroceramides containing phosphoethanolamine and glycerol have been previously described in Porphyromonas gingivalis. Importantly, they were shown to possess pro-inflammatory properties. Other common human bacteria were screened for the presence of these lipids, and they were found, amongst others, in the oral pathogen Tannerella forsythia. To date, no detailed study into the lipids of this organism has been performed.Lipids were extracted, separated and purified by HPTLC, and analyzed using GC-MS, ESI–MS and NMR. Of special interest was how T. forsythia acquires the metabolic precursors for the lipids studied here. This was assayed by radioactive and stable isotope incorporation using carbon-14 and deuterium labeled myo-inositol, added to the growth medium. T. forsythia synthesizes two phosphodihydroceramides (Tf GL1, Tf GL2) which are constituted by phospho-myo-inositol linked to either a 17-, 18-, or 19-carbon sphinganine, N-linked to either a branched 17:0(3-OH) or a linear 16:0(3-OH) fatty acid which, in Tf GL2, is, in turn, ester-substituted with a branched 15:0 fatty acid. T. forsythia lacks the enzymatic machinery required for myo-inositol synthesis but was found to internalize inositol from the medium for the synthesis of both Tf GL1 and Tf GL2.The study describes two novel glycolipids in T. forsythia which could be essential in this organism. Their synthesis could be reliant on an external source of myo-inositol.The effects of these unique lipids on the immune system and their role in bacterial virulence could be relevant in the search for new drug targets.
Keywords: Tannerella forsythia; Myo-inositol; Dihydroceramides; Inositol uptake; ESI–MS; NMR;

Molecular mechanism of dietary phospholipid requirement of Atlantic salmon, Salmo salar, fry by G. Carmona-Antoñanzas; J.F. Taylor; L. Martinez-Rubio; D.R. Tocher (1428-1441).
The phospholipid (PL) requirement in fish is revealed by enhanced performance when larvae are provided PL-enriched diets. To elucidate the molecular mechanism underlying PL requirement in Atlantic salmon, Salmo salar, were fed a minimal PL diet and tissue samples from major lipid metabolic sites were dissected from fry and parr. In silico analysis and cloning techniques demonstrated that salmon possess a full set of enzymes for the endogenous production of PL. The gene expression data indicated that major PL biosynthetic genes of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns) display lower expression in intestine during the early developmental stage (fry). This is consistent with the hypothesis that the intestine of salmon is immature at the early developmental stage with limited capacity for endogenous PL biosynthesis. The results also indicate that intact PtdCho, PtdEtn and PtdIns are required in the diet at this stage. PtdCho and sphingomyelin constitute the predominant PL in chylomicrons, involved in the transport of dietary lipids from the intestine to the rest of the body. As sphingomyelin can be produced from PtdCho in intestine of fry, our findings suggest that supplementation of dietary PtdCho alone during early developmental stages of Atlantic salmon would be sufficient to promote chylomicron formation. This would support efficient transport of dietary lipids, including PL precursors, from the intestine to the liver where biosynthesis of PtdEtn, PtdSer, and PtdIns is not compromised as in intestine facilitating efficient utilisation of dietary energy and the endogenous production of membrane PL for the rapidly growing and developing animal.
Keywords: Atlantic salmon; Dietary requirement; Gene expression; Phosphoglycerides; Phospholipids; qPCR;

Electronegative LDL induces priming and inflammasome activation leading to IL-1β release in human monocytes and macrophages by M. Estruch; K. Rajamäki; J.L. Sanchez-Quesada; P.T. Kovanen; K. Öörni; S. Benitez; J. Ordoñez-Llanos (1442-1449).
Background: Electronegative LDL (LDL(−)), a modified LDL fraction found in blood, induces the release of inflammatory mediators in endothelial cells and leukocytes. However, the inflammatory pathways activated by LDL(−) have not been fully defined. We aim to study whether LDL(−) induced release of the first-wave proinflammatory IL-1β in monocytes and monocyte-derived macrophages (MDM) and the mechanisms involved. Methods: LDL(−) was isolated from total LDL by anion exchange chromatography. Monocytes and MDM were isolated from healthy donors and stimulated with LDL(+) and LDL(−) (100 mg apoB/L). Results: In monocytes, LDL(−) promoted IL-1β release in a time-dependent manner, obtaining at 20 h-incubation the double of IL-1β release induced by LDL(−) than by native LDL. LDL(−)-induced IL-1β release involved activation of the CD14-TLR4 receptor complex. LDL(−) induced priming, the first step of IL-1β release, since it increased the transcription of pro-IL-1β (8-fold) and NLRP3 (3-fold) compared to native LDL. Several findings show that LDL(−) induced inflammasome activation, the second step necessary for IL-1β release. Preincubation of monocytes with K+ channel inhibitors decreased LDL(−)-induced IL-1β release. LDL(−) induced formation of the NLRP3-ASC complex. LDL(−) triggered 2-fold caspase-1 activation compared to native LDL and IL-1β release was strongly diminished in the presence of the caspase-1 inhibitor Z-YVAD. In MDM, LDL(−) promoted IL-1β release, which was also associated with caspase-1 activation. Conclusions: LDL(−) promotes release of biologically active IL-1β in monocytes and MDM by induction of the two steps involved: priming and NLRP3 inflammasome activation. Significance: By IL-1β release, LDL(−) could regulate inflammation in atherosclerosis.Display Omitted
Keywords: Electronegative LDL; Interleukin-1β; Inflammasome; Caspase-1; Monocytes; Macrophages;

Seipin is involved in the regulation of phosphatidic acid metabolism at a subdomain of the nuclear envelope in yeast by Heimo Wolinski; Harald F. Hofbauer; Klara Hellauer; Alvaro Cristobal-Sarramian; Dagmar Kolb; Maja Radulovic; Oskar L. Knittelfelder; Gerald N. Rechberger; Sepp.D. Kohlwein (1450-1464).
Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1–Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1–Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site.
Keywords: Phosphatidate phosphatase; Phosphatidic acid; Diacylglycerol; Seipin; Nuclear envelope; Lipid droplet; Yeast; Saccharomyces cerevisiae;

Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP by Ann-Kathrin Häfner; Jana Gerstmeier; Michael Hörnig; Sven George; Ann-Katrin Ball; Mirjam Schröder; Ulrike Garscha; Oliver Werz; Dieter Steinhilber (1465-1472).
Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.
Keywords: Leukotrienes; Inflammation; Lipoxygenase; Subcellular Localization; FLAP;

Anandamide and decidual remodelling: COX-2 oxidative metabolism as a key regulator by M. Almada; F. Piscitelli; B.M. Fonseca; V. Di Marzo; G. Correia-da-Silva; N. Teixeira (1473-1481).
Recently, endocannabinoids have emerged as signalling mediators in reproduction. It is widely accepted that anandamide (AEA) levels must be tightly regulated, and that a disturbance in AEA levels may impact decidual stability and regression. We have previously characterized the endocannabinoid machinery in rat decidual tissue and reported the pro-apoptotic action of AEA on rat decidual cells. Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays a crucial role in early pregnancy, and is also a key modulator in the crosstalk between endocannabinoids and prostaglandins. On the other hand, AEA-oxidative metabolism by COX-2 is not merely a mean to inactivate its action, but it yields the formation of a new class of mediators, named prostaglandin-ethanolamides, or prostamides. In this study we found that AEA-induced apoptosis in decidual cells involves COX-2 metabolic pathway. AEA induced COX-2 expression through p38 MAPK, resulting in the formation of prostamide E2 (PME2). Our findings also suggest that AEA-induced effect is associated with NF-kB activation. Finally, we describe the involvement of PME2 in the induction of the intrinsic apoptotic pathway in rat decidual cells. Altogether, our findings highlight the role of COX-2 as a gatekeeper in the uterine environment and clarify the impact of the deregulation of AEA levels on the decidual remodelling process.
Keywords: Anandamide; Prostamide E2; COX-2; Decidualization; Apoptosis; Signalling pathway;

Regulation of ceramide generation during macrophage apoptosis by ASMase and de novo synthesis by Shih Wei Wang; Payman Hojabrpour; Peng Zhang; Richard N. Kolesnick; Urs P. Steinbrecher; Antonio Gómez-Muñoz; Vincent Duronio (1482-1489).
The survival of macrophages depends on the presence of specific cytokines that activate survival signaling events, as well as suppressing formation of apoptosis-inducing pathways. We have previously shown that macrophages deprived of macrophage colony stimulating factor (M-CSF) produce ceramide that contributes to apoptosis of these cells, a pathway that is suppressed by exposure to oxidized LDL. In this study we have examined macrophages derived from mice lacking acid sphingomyelinase (ASMase) to ask whether these events are altered due to the impaired ability of these cells to break down sphingomyelin and produce ceramide. We found that these cells do survive better than cells from wild type mice, but they still undergo cell death and some ceramide is formed. We show that the ceramide is being produced by a de novo synthetic pathway. Therefore, ceramide production in M-CSF-deprived macrophages arises from a combination of ASMase activity and de novo synthesis.
Keywords: Macrophage; Apoptosis; Ceramide; Sphingomyelinase; Enzyme;

Regulation of hepatic cardiolipin metabolism by TNFα: Implication in cancer cachexia by Laure Peyta; Kathleen Jarnouen; Michelle Pinault; Cedric Coulouarn; Cyrille Guimaraes; Caroline Goupille; Jean-Paul Pais de Barros; Stephan Chevalier; Jean-François Dumas; François Maillot; Grant M. Hatch; Pascal Loyer; Stephane Servais (1490-1500).
Cardiolipin (CL) content accumulation leads to an increase in energy wasting in liver mitochondria in a rat model of cancer cachexia in which tumor necrosis factor alpha (TNFα) is highly expressed. In this study we investigated the mechanisms involved in liver mitochondria CL accumulation in cancer cachexia and examined if TNFα was involved in this process leading to mitochondrial bioenergetics alterations. We studied gene, protein expression and activity of the main enzymes involved in CL metabolism in liver mitochondria from a rat model of cancer cachexia and in HepaRG hepatocyte-like cells exposed to 20 ng/ml of TNFα for 12 h. Phosphatidylglycerolphosphate synthase (PGPS) gene expression was increased 2.3-fold (p < 0.02) and cardiolipin synthase (CLS) activity decreased 44% (p < 0.03) in cachectic rat livers compared to controls. CL remodeling enzymes monolysocardiolipin acyltransferase (MLCL AT-1) activity and tafazzin (TAZ) gene expression were increased 30% (p < 0.01) and 50% (p < 0.02), respectively, in cachectic rat livers compared to controls. Incubation of hepatocytes with TNFα increased CL content 15% (p < 0.05), mitochondrial oxygen consumption 33% (p < 0.05), PGPS gene expression 44% (p < 0.05) and MLCL AT-1 activity 20% (p < 0.05) compared to controls. These above findings strongly suggest that in cancer cachexia, TNFα induces a higher energy wasting in liver mitochondria by increasing CL content via upregulation of PGPS expression.
Keywords: Cardiolipin biosynthesis; Cytokines; Mitochondria; Energy wasting; Liver; Cardiolipin remodeling; Inflammation;

Alterations of plasma glycerophospholipid and sphingolipid species in male alcohol-dependent patients by Martin Reichel; Stefanie Hönig; Gerhard Liebisch; Anja Lüth; Burkhard Kleuser; Erich Gulbins; Gerd Schmitz; Johannes Kornhuber (1501-1510).
Alcohol abuse is a major risk factor for somatic and neuropsychiatric diseases. Despite their potential clinical importance, little is known about the alterations of plasma glycerophospholipid (GPL) and sphingolipid (SPL) species associated with alcohol abuse.Plasma GPL and SPL species were quantified using electrospray ionization tandem mass spectrometry in samples from 23 male alcohol-dependent patients before and after detoxification, as well as from 20 healthy male controls.A comparison of alcohol-dependent patients with controls revealed higher phosphatidylcholine (PC; P-value = 0.008) and phosphatidylinositol (PI; P-value = 0.001) concentrations in patients before detoxification, and higher PI (P-value = 0.001) and phosphatidylethanolamine (PE)-based plasmalogen (PE P; P-value = 0.003) concentrations after detoxification. Lysophosphatidylcholines (LPC) were increased by acute intoxication (P-value = 0.002). Sphingomyelin (SM) concentration increased during detoxification (P-value = 0.011). The concentration of SM 23:0 was lower in patients (P-value = 2.79 × 10− 5), and the concentrations of ceramide Cer d18:1/16:0 and Cer d18:1/18:0 were higher in patients (P-value = 2.45 × 10− 5 and 3.73 × 10− 5). Activity of lysosomal acid sphingomyelinase (ASM) in patients correlated positively with the concentrations of eight LPC species, while activity of secreted ASM was inversely correlated with several PE, PI and PC species, and positively correlated with the molar ratio of PC to SM (Pearson's r = − 0.432; P-value = 0.039).Plasma concentrations of numerous GPL and SPL species were altered in alcohol-dependent patients. These molecules might serve as potential biomarkers to improve the diagnosis of patients and to indicate health risks associated with alcohol abuse. Our study further indicates that there are strong interactions between plasma GPL concentrations and SPL metabolism.Display Omitted
Keywords: Acid sphingomyelinase; Alcohol dependence; Anxiety; Cardiovascular; Case–control study; Ceramide; Clinical; Depression; Diagnostic; Disease; Glycerophospholipids; Lysophosphatidylcholines; Mass spectrometry; Phosphatidylcholines; Phosphatidylinositols; Plasma; Plasmalogens; Sphingolipids; Sphingomyelin; Tandem mass spectrometry;

G protein–membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein–lipid interactions and its implications in membrane microdomain localization by Rafael Álvarez; David J. López; Jesús Casas; Victoria Lladó; Mónica Higuera; Tünde Nagy; Miquel Barceló; Xavier Busquets; Pablo V. Escribá (1511-1520).
G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.Display Omitted
Keywords: G protein–lipid interactions; Membrane microdomain; Lipid structure; Myristoylation; Palmitoylation; Cell signaling;