BBA - Molecular and Cell Biology of Lipids (v.1821, #2)

Dynamics of arachidonic acid mobilization by inflammatory cells by Alma M. Astudillo; David Balgoma; María A. Balboa; Jesús Balsinde (249-256).
The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.► Cellular utilization of arachidonic acid. ► Methods and strategies for the analysis of arachidonic acid-containing lipids. ► Lipidomic studies of cellular arachidonate metabolism.
Keywords: Arachidonic acid; Phospholipid remodeling; Eicosanoid metabolism; Lipidomics; Mass spectrometry;

Acidity and lipolysis by group V secreted phospholipase A2 strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans by Katariina Lähdesmäki; Katariina Öörni; Mervi Alanne-Kinnunen; Matti Jauhiainen; Eva Hurt-Camejo; Petri T. Kovanen (257-267).
Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A2 (sPLA2-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA2-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased > 60-fold and sPLA2-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA2-V–LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA2-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.► Acidity increases lipolysis of apoB-100-containing lipoproteins by PLA2-V. ► Acidity and PLA2-V increase lipoprotein binding to human aortic proteoglycans (PGs). ► Acidity and PLA2-V increase macrophage uptake of LDL, IDL, sVLDL and LDL-PG complexes.
Keywords: Acidic pH; Atherosclerosis; Macrophage; Triglyceride-rich lipoprotein; sPLA2-V; Aortic proteoglycan;

Distinct cellular pools of perilipin 5 point to roles in lipid trafficking by Sadie R. Bartholomew; Erica Hlavin Bell; Taryn Summerfield; Leslie C. Newman; Erin L. Miller; Brian Patterson; Zach P. Niday; William E. Ackerman; John T. Tansey (268-278).
The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~ 1.15 g/ml) and size (~ 575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~ 15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.► Perilipin 5 is found in two distinct pools in the cell. ► The more dense pool of perilipin 5 also contains perilipin 3 but not perilipin 2. ► The dense pool has a lipid rich core and discrete size and density. ► Expression of perilipin 5 promotes droplet reorganization.
Keywords: Perilipin; Lipolysis; Lipid trafficking; Lipid droplets; Muscle; Adipose;

Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416 by Michael Hörnig; Stavroula Markoutsa; Ann-Kathrin Häfner; Sven George; Joanna M. Wisniewska; Carmen B. Rödl; Bettina Hofmann; Thorsten Maier; Michael Karas; Oliver Werz; Dieter Steinhilber (279-286).
U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.► Covalent modification of Cys416 by U73122 inhibits 5-lipoxygenase. ► New class of irreversible 5-lipoxygenase inhibitors. ► Novel binding site for 5-lipoxygenase inhibitors.
Keywords: 5-Lipoxygenase; Phospholipase C; Leukotriene; U-73122; Cysteine thiol binding;

Green leaf divinyl ether synthase: Gene detection, molecular cloning and identification of a unique CYP74B subfamily member by Yuri V. Gogolev; Svetlana S. Gorina; Natalia E. Gogoleva; Yana Y. Toporkova; Ivan R. Chechetkin; Alexander N. Grechkin (287-294).
Display Omitted► Four CYP74B gene isoforms have been detected in the flax leaf transcriptome. ► The full CYP74B16 (LuDES) sequence has been cloned and expressed in E. coli. ► The preferred substrate of LuDES is α-linolenic acid 13-hydroperoxide, 13(S)-HPOT. ► The divinyl ether (ω5Z)-etherolenic acid is the native product of LuDES. ► LuDES is an unprecedented CYP74B type of divinyl ether synthase.
Keywords: Divinyl ether synthase; Molecular cloning; CYP74B; P450; Oxylipins; Flax (Linum usitatissimum L.);

Preferential interactions between ApoE-containing lipoproteins and Aβ revealed by a detection method that combines size exclusion chromatography with non-reducing gel-shift by Mary Jo LaDu; Gregory W. Munson; Lisa Jungbauer; Godfrey S. Getz; Catherine A. Reardon; Leon M. Tai; Chunjiang Yu (295-302).
The association between apolipoprotein E (apoE) and amyloid-β peptide (Aβ) may significantly impact the function of both proteins, thus affecting the etiology of Alzheimer's disease (AD). However, apoE/Aβ interactions remain fundamentally defined by the stringency of the detection method. Here we use size exclusion chromatography (SEC) as a non-stringent approach to the detection of apoE/Aβ interactions in solution, specifically apoE and both endogenous and exogenous Aβ from plasma, CSF and astrocyte conditioned media. By SEC analysis, Aβ association with plasma and CNS lipoproteins is apoE-dependent. While endogenous Aβ elutes to specific human plasma lipoproteins distinct from those containing apoE, it is the apoE-containing lipoproteins that absorb excess amounts of exogenous Aβ40. In human CSF, apoE, endogenous Aβ and phospholipid elute in an almost identical profile, as do apoE, exogenous Aβ and phospholipid from astrocyte conditioned media. Combining SEC fractionation with subsequent analysis for SDS-stable apoE/Aβ complex reveals that apoE-containing astrocyte lipoproteins exhibit the most robust interactions with Aβ. Thus, standardization of the methods for detecting apoE/Aβ complex is necessary to determine its functional significance in the neuropathology characteristic of AD. Importantly, a systematic understanding of the role of apoE-containing plasma and CNS lipoproteins in Aβ homeostasis could potentially contribute to identifying a plasma biomarker currently over-looked because it has multiple components.► SEC is a non-stringent approach to the detection of apoE/Aβ interactions in solution. ► By SEC analysis, Aβ association with CNS and plasma lipoproteins is apoE-dependent. ► ApoE-containing human plasma lipoproteins absorb excess amounts of exogenous Aβ40. ► Aβ and apoE-containing astrocyte lipoproteins exhibit the most robust interaction. ► Aβ/lipoprotein interactions are important for clearance and as a potential biomarker.
Keywords: Alzheimer's disease; Amyloid-beta peptide (Aβ); Apolipoprotein E (apoE); apoE/Aβ; Interaction; Lipoprotein;

Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages by Natalie Fournier; Nesrine Attia; Delphine Rousseau-Ralliard; Benoît Vedie; Frédéric Destaillats; Alain Grynberg; Jean-Louis Paul (303-312).
Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34 h, 70 μM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~ 23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (− 36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA + polyunsaturated FA):(saturated FA + TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.► We studied the impact of membrane trans fatty acids incorporation on cholesterol efflux from mouse and human macrophages. ► The industrially produced trans elaidic acid specifically reduced ABCA1 cholesterol efflux pathway. ► The naturally produced trans vaccenic acid had no impact on cholesterol efflux. ► In both cellular models, these deleterious effects were observed in cholesterol-normal or cholesterol-loaded macrophages. ► Elaidic acid may contribute to atherogenesis by impairing the ABCA1 cholesterol efflux pathway in macrophages.
Keywords: trans fatty acids; Cholesterol efflux; J774 mouse macrophages; Human monocyte-derived macrophages; Elaidic acid; ATP binding cassette transporter A1;

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells by Ruchi Chaube; Vasantha Madhuri Kallakunta; Michael Graham Espey; Ryan McLarty; Adam Faccenda; Sirinart Ananvoranich; Bulent Mutus (313-323).
Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~ 3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~ 1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~ 74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.► NSMase2 inhibition by ER stress-results in elevated cellular cholesterol. ► NO-levels in ER stressed cells were severely attenuated (~ 70%) within 24 h. ► NSMase2 overexpression increased eNOS phosphorylation but did not affect NO levels. ► NSMase2 knock down decreases eNOS phosphorylation (~ 50%) and NO formation (~ 40%).
Keywords: Neutral sphingomyelinase 2; Nitric oxide synthase; Nitric oxide; ER stress; Endothelial dysfunction;

Phosphatidylcholine (PC) homeostasis is important for maintaining cellular growth and survival. Cellular growth and apoptosis may also be influenced by the PC to phosphatidylethanolamine (PE) ratio as a reduction in this ratio can result in a loss of membrane integrity. To investigate whether a reduced PC:PE ratio influences cellular growth and apoptosis, we utilized the MT58 cell line, which contains a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase-α, the rate-limiting enzyme for PC biosynthesis. Incubation of MT58 cells at the restrictive temperature of 41 °C results in a reduction of cellular PC and induces apoptosis. Furthermore, MT58 cells have a 50% reduction in the PC:PE ratio when incubated at 41 °C. In an attempt to normalize the PC:PE ratio, which may stabilize cellular membranes and rescue MT58 cells from apoptosis, the cells were treated with either silencing RNA to impair PE biosynthesis or lysophosphatidylcholine to increase PC mass. Impairing PE biosynthesis in MT58 cells reduced cellular PE and PC concentrations by 30% and 20%, but did not normalize the PC:PE ratio. Loss of both phospholipids enhanced the onset of apoptosis in MT58 cells. Lysophosphatidylcholine normalized cellular PC, increased PE mass by 10%, restored cellular growth and prevented apoptosis of MT58 cells without normalizing the PC:PE ratio. Furthermore, total amount of cellular PC and PE, but not the PC:PE ratio, correlated with cellular growth (R 2  = 0.76), and inversely with cellular apoptosis (R 2  = 0.97). These data suggest the total cellular amount of PC and PE, not the PC:PE ratio, influences growth and membrane integrity of MT58 cells.► MT58 cells have impaired PC biosynthesis and growth at 41°C ► Incubation of MT58 cells at 41°C causes a 50% decrease in the PC/PE ratio ► SiRNA decrease of PE biosynthesis did not normalize the PC/PE ratio ► Lysophosphatidylcholine normalized PC and restored growth, but not PC/PE ratio ► Concentration of PC and PE, but not PC/PE ratio, influences growth of MT-58 cells
Keywords: Apoptosis; ChoK1; Phospholipids; Lysophosphatidylcholine; CTP:phosphocholine cytidylyltransferase;

Apo J/clusterin expression and secretion: Evidence for 15-deoxy-Δ12,14-PGJ2-dependent mechanism by Damien Gates; Kiera Dollin; Roisin Connolly; Ian Young; Lesley Powell; Jane McEneny; Martin Gleave; Ann McGinty (335-342).
Cyclooxygenase-2 (Cox-2) and Apo J/clusterin are involved in inflammatory resolution and have each been reported to inhibit NF-κB signalling. Using a well-validated rat pheochromocytoma (PC12) cell culture model of Cox-2 over-expression the current study investigated inter-dependence between Cox-2 and clusterin with respect to induction of expression and impact on NF-κB signalling. Both gene expression and immunoblot analysis confirmed that intracellular and secreted levels of clusterin were elevated in Cox-2 over-expressing cells (PCXII). Clusterin expression was increased in control (PCMT) cells in a time- and dose-dependent manner by 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), but not PGE2, and inhibited in PCXII cells by pharmacological Cox inhibition. In PCXII cells, inhibition of two transcription factors known to be activated by 15d-PGJ2, heat shock factor 1 (HSF-1) and peroxisome proliferator activated receptor (PPAR)γ, by transcription factor oligonucleotide decoy and antagonist (GW9662) treatment, respectively, reduced clusterin expression. While PCXII cells exhibited reduced TNF-α-induced cell surface ICAM-1 expression, IkB phosphorylation and degradation were similar to control cells. With respect to the impact of Cox-2-dependent clusterin upregulation on NF-κB signalling, basal levels of IκB were similar in control and PCXII cells, and no evidence for a physical association between clusterin and phospho-IκB was obtained. Moreover, while PCXII cells exhibited reduced NF-κB transcriptional activity, this was not restored by clusterin knock-down. These results indicate that Cox-2 induces clusterin in a 15d-PGJ2-dependent manner, and via activation of HSF-1 and PPARγ. However, the results do not support a model whereby Cox-2/15d-PGJ2-dependent inhibition of NF-κB signalling involves clusterin.► Direct modulation of clusterin expression by Cox-2. ► Pre-secretory/secretory and pre-nuclear clusterin isoforms upregulated. ► Cox-2-dependent clusterin was normally processed and secreted. ► Cox-2 induced clusterin via activation of HSF1 and PPARγ. ► Cox-2/15d-PGJ2, but not clusterin, inhibited NF-κB signalling at the nuclear level.
Keywords: Peroxisome proliferator activated receptor; GW9662; Inflammation; Heat shock factor 1; NF-κB;