BBA - Molecular and Cell Biology of Lipids (v.1485, #2-3)
Physiology and pathophysiology of sphingolipid metabolism and signaling by Andrea Huwiler; Thomas Kolter; Josef Pfeilschifter; Konrad Sandhoff (63-99).
Keywords: Sphingolipid; Glycosphingolipid; Ceramide; Signal transduction; Lysosomal storage disease;
Acyl-CoA dependent acylation of phospholipids in the chloroplast envelope by J.Magnus Kjellberg; Marc Trimborn; Mats Andersson; Anna Stina Sandelius (100-110).
Acyl-CoAs are substrates for acyl lipid synthesis in the endoplasmic reticulum. In addition, they may also be substrates for lipid acylation in other membranes. In order to assess whether lipid acylation may have a role in plastid lipid metabolism, we have studied the incorporation of radiolabelled fatty acids from acyl-CoAs into lipids in isolated, intact pea chloroplasts. The labelled lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol and free fatty acids. With oleoyl-CoA, the fatty acid was incorporated preferably into the sn-2 position of PC and the acylation activity mainly occurred in fractions enriched in inner chloroplast envelope. Added lysoPC stimulated the activity. With palmitoyl-CoA, the fatty acid was incorporated primarily into the sn-1 position of PG and the reaction occurred at the surface of the chloroplasts. As chloroplast-synthesized PG generally contains 16C fatty acids in the sn-2 position, we propose that the acylation of PG studied represents activities present in a domain of the endoplasmic reticulum or an endoplasmic reticulum-derived fraction that is associated with chloroplasts and maintains this association during isolation. This domain or fraction contains a discreet population of lipid metabolizing activities, different from that of bulk endoplasmic reticulum, as shown by that with isolated endoplasmic reticulum, acyl-CoAs strongly labelled phosphatidic acid and phosphatidylethanolamine, lipids that were never labelled in the isolated chloroplasts.
Keywords: Acyl transferase; Chloroplast; Phosphatidylcholine; Phosphatidylglycerol; Lipid synthesis; Pisum sativum;
Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa by Katsuhiro Kita; Nozomu Okino; Makoto Ito (111-120).
Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368–14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.
Keywords: Ceramidase; Reverse hydrolysis reaction; Ceramide; Sphingosine; Sphingolipid;
Long-chain acyl-CoA dehydrogenase is a key enzyme in the mitochondrial β-oxidation of unsaturated fatty acids by Weiping Le; Azfar S. Abbas; Howard Sprecher; Jerry Vockley; Horst Schulz (121-128).
The first reaction of mitochondrial β-oxidation, which is catalyzed by acyl-CoA dehydrogenases, was studied with unsaturated fatty acids that have a double bond either at the 4,5 or 5,6 position. The CoA thioesters of docosahexaenoic acid, arachidonic acid, 4,7,10-cis-hexadecatrienoic acid, 5-cis-tetradecenoic acid, and 4-cis-decenoic acid were effectively dehydrogenated by both rat and human long-chain acyl-CoA dehydrogenases (LCAD), whereas they were poor substrates of very long-chain acyl-CoA dehydrogenases (VLCAD). VLCAD, however, was active with CoA derivatives of long-chain saturated fatty acids or unsaturated fatty acids that have double bonds further removed from the thioester function. Although bovine LCAD effectively dehydrogenated 5-cis-tetradecenoyl-CoA (14:1) and 4,7,10-cis-hexadecatrienoyl-CoA, it was nearly inactive toward the other unsaturated substrates. The catalytic efficiency of rat VLCAD with 14:1 as substrate was only 4% of the efficiency determined with tetradecanoyl-CoA, whereas LCAD acted equally well on both substrates. The conclusion of this study is that LCAD serves an important, if not essential function in the β-oxidation of unsaturated fatty acids.
Keywords: β-Oxidation; Unsaturated fatty acid; Long-chain acyl-CoA dehydrogenase; Very long-chain acyl-CoA dehydrogenase;
Lipoprotein-associated α-tocopheryl-succinate inhibits cell growth and induces apoptosis in human MCF-7 and HBL-100 breast cancer cells by Pirkko J. Pussinen; Helmut Lindner; Otto Glatter; Helga Reicher; Gerhard M. Kostner; Andrea Wintersperger; Ernst Malle; Wolfgang Sattler (129-144).
α-Tocopheryl succinate (α-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent α-TS associates with plasma lipoproteins and if α-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that α-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest α-TS concentrations, lipoproteins carrying 50 000 (VLDL), 5000 (LDL) and 700 (HDL) α-TS molecules per lipoprotein particle were generated. α-TS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects α-TS uptake from apoB/E containing lipoprotein particles human breast cancer cells with low (MCF-7) and normal (HBL-100) LDL-R expression were used. The uptake of free, VLDL- and (apoE-free) HDL3-associated α-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated α-TS by HBL-100 cells (normal LDL-R expression) was about twice as high as compared to MCF-7 cells (low LDL-R expression). VLDL and LDL-associated α-TS inhibited proliferation most effectively at the highest concentration of α-TS used (100% inhibition of MCF-7 growth with 20 μg/ml of lipoprotein-associated α-TS). However, also α-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, α-TS-enriched HDL3 inhibited cell growth by 40–60%. Incubation of both cell lines in the presence of free or lipoprotein-associated α-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) α-TS readily associates with lipoproteins in vitro and in vivo; (2) the lipoprotein-enrichment efficacy was dependent on the particle size and/or the triglyceride content of the lipoprotein; (3) uptake of LDL-associated α-TS was apparently dependent on the level of LDL-R expression; and (4) lipoproteins were efficient α-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug.
Keywords: Vitamin E-succinate; Lipoprotein; Apoptosis; Carcinoma cell;
Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP by E Déziel; F Lépine; S Milot; R Villemur (145-152).
Two rapid and simple methods for the characterisation and quantification of rhamnolipids produced by a growing culture of the Pseudomonas aeruginosa strain 57RP were developed. Two rhamnolipids were purified and their response factors determined. The various rhamnolipids produced were then measured using liquid chromatography/mass spectrometry. The culture supernatants were injected directly, without prior purification, in a HPLC equipped with a C18 reverse-phase column. The complete profile of rhamnolipid congeners produced during a 2 week cultivation period was monitored. In order to shorten the analysis time, another method was developed which did not require chromatographic separation of the rhamnolipids prior to their detection. Quantification of rhamnolipids using the direct infusion method gave results very similar to those obtained with HPLC separation. These two methods were very well correlated with the standard colorimetric orcinol method. The rhamnolipid profiles obtained show that the various rhamnolipid congeners are secreted simultaneously, and that their relative proportion remained unchanged throughout the cultivation period.
Keywords: Rhamnolipid; Mass spectrometry; Electrospray; Liquid chromatography; Pseudomonas aeruginosa;
Activation of astroglial phospholipase D activity by phorbol ester involves ARF and Rho proteins by Katja Kötter; Shenchu Jin; Christoph von Eichel-Streiber; Jong Bae Park; Sung Ho Ryu; Jochen Klein (153-162).
Primary cultures of rat cortical astrocytes express phospholipase D (PLD) isoforms 1 and 2 as determined by RT-PCR and Western blot. Basal PLD activity was strongly (10-fold) increased by 4β-phorbol-12β,13α-dibutyrate (PDB) (EC50: 56 nM), an effect which was inhibited by Ro 31-8220 (0.1–1 μM), an inhibitor of protein kinase C (PKC), and by brefeldin A (10–100 μg/ml), an inhibitor of ADP-ribosylating factor (ARF) activation. Pretreatment of the cultures with Clostridium difficile toxin B-10463 (0.1–1 ng/ml), which inactivates small G proteins of the Rho family, led to a breakdown of the astroglial cytoskeleton; concomitantly, PLD activation by PDB was reduced by up to 50%. In contrast, inactivation of proteins of the Ras family by Clostridium sordellii lethal toxin 1522 did not affect PLD activation. In parallel experiments, serum-induced PLD activation was sensitive to brefeldin A, but not to Ro 31-8220 and not to clostridial toxins. We conclude that, in astrocytes, the PLD isoform which is activated by phorbol ester requires PKC, ARF and Rho proteins for full activity and probably represents PLD1.
Keywords: Astrocyte; Phospholipase D; Phorbol ester; Protein kinase C; ARF protein; Rho protein; Clostridium difficile toxin B; Clostridium sordellii lethal toxin;
Phosphatidylinositol 3-kinase in zymosan- and bacteria-induced signalling to mobilisation of arachidonic acid in macrophages by Gösta Hiller; Malin Sternby; Roger Sundler; Jonny Wijkander (163-172).
Stimulation of mouse peritoneal macrophages with zymosan or bacteria results in activation of 85-kDa cytosolic phospholipase A2 (cPLA2) and release of arachidonate. We have investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the signalling leading to activation of cPLA2 and release of arachidonate in response to zymosan and the bacterium Prevotella intermedia. The specific PtdIns 3-kinase inhibitor wortmannin completely inhibited zymosan- and bacteria-induced release of arachidonate with an IC50 value of 10–20 nM. Wortmannin also completely inhibited the zymosan-induced activation of cPLA2, while the cPLA2 activation by bacteria was partially inhibited by about 50%. Further experiments showed that zymosan-induced activation of extracellular signal-regulated kinase was inhibited, and bacteria-induced activation of the kinase strongly reduced, in the presence of wortmannin. Also zymosan-induced activation of p38 mitogen-activated protein kinase was inhibited by wortmannin, while p38 activation induced by bacteria was not. The zymosan- and bacteria-induced activation of phospholipase C, as determined by the generation of inositol phosphates, was also inhibited by wortmannin. Moreover, zymosan caused activation of PtdIns 3-kinase, which was totally inhibited by wortmannin. In contrast to zymosan and bacteria, arachidonate release induced by calcium ionophore alone, or further amplified by phorbol ester, was not sensitive to wortmannin. These results suggest that PtdIns 3-kinase constitutes a critical component in the zymosan- and bacteria-induced signalling leading to release of arachidonate and that PtdIns 3-kinase is positioned upstream of phospholipase C in this pathway.
Keywords: Cytosolic phospholipase A2; Mitogen-activated protein kinase; Phospholipase C; Lipopolysaccharide; Wortmannin;
Kinetics and plasma concentrations of 26-hydroxycholesterol in baboons by Shengrong Li; Jihai Pang; Evelyn M Jackson; William K Wilson; Glen E Mott; George J Schroepfer (173-184).
26-Hydroxycholesterol (26OHC), a major oxysterol in human blood, is believed to play an important role in reverse cholesterol transport, bile acid formation, and regulation of various cellular processes. Using isotope dilution mass spectrometry, we measured plasma 26OHC concentrations in baboons fed either a high cholesterol/saturated fat (HC-SF) or normal chow diet. Plasma 26OHC levels in baboons were comparable to those reported for humans and were positively correlated with plasma cholesterol concentrations. Animals on the HC-SF diet had significantly higher 26OHC levels (0.274±0.058 μM, mean±S.D.) than those on the chow diet (0.156±0.046 μM). In separate experiments, [3H]26OHC was injected into four tethered baboons, and multiple blood samples drawn over a 1-h period were analyzed for [3H]26OHC and 26OHC. Fitting the specific radioactivity data to a two-pool compartmental model indicated a rapidly turning over plasma compartment (t 1/2 2.9–6.0 min) and a second compartment with slow turnover (t 1/2 76–333 min). The calculated 26OHC production rate was 2.5 μmol/kg body weight/day. Assuming all 26OHC is converted to bile acids, the 26OHC production rate corresponds to about 10% of total bile acid production in adult baboons. These results indicate that rapid turnover of plasma 26OHC at submicromolar concentrations could significantly contribute to bile acid synthesis.
Keywords: Oxysterol; Gas chromatography-mass spectrometry; Selected-ion monitoring; 27-Hydroxycholesterol; Plasma cholesterol; Turnover;
Differential effects of acyl-CoA binding protein on enzymatic and non-enzymatic thioacylation of protein and peptide substrates by Julianne T. Dunphy; Hans Schroeder; Rania Leventis; Wendy K. Greentree; Jens K. Knudsen; John R. Silvius; Maurine E. Linder (185-198).
Both enzymatic and autocatalytic mechanisms have been proposed to account for protein thioacylation (commonly known as palmitoylation). Acyl-CoA binding proteins (ACBP) strongly suppress non-enzymatic thioacylation of cysteinyl-containing peptides by long-chain acyl-CoAs. At physiological concentrations of ACBP, acyl-CoAs, and membrane lipids, the rate of spontaneous acylation is expected to be too slow to contribute significantly to thioacylation of signaling proteins in mammalian cells (Leventis et al., Biochemistry 36 (1997) 5546–5553). Here we characterized the effects of ACBP on enzymatic thioacylation. A protein S-acyltransferase activity previously characterized using G-protein α-subunits as a substrate (Dunphy et al., J. Biol. Chem., 271 (1996) 7154–7159), was capable of thioacylating short lipid-modified cysteinyl-containing peptides. The minimum requirements for substrate recognition were a free cysteine thiol adjacent to a hydrophobic lipid anchor, either myristate or farnesyl isoprenoid. PAT activity displayed specificity for the acyl donor, efficiently utilizing long-chain acyl-CoAs, but not free fatty acid or S-palmitoyl-N-acetylcysteamine. ACBP only modestly inhibited enzymatic thioacylation of a myristoylated peptide or G-protein α-subunits under conditions where non-enzymatic thioacylation was reduced to background. Thus, protein S-acyltransferase remains active in the presence of physiological concentrations of ACBP and acyl-CoA in vitro and is likely to represent the predominant mechanism of thioacylation in vivo.
Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice by Maki Tsujita; Shigehiro Tomimoto; Kuniko Okumura-Noji; Mitsuyo Okazaki; Shinji Yokoyama (199-213).
Helical apolipoprotein(apo)s generate pre-β-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell–apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo–cell interaction is a major source of plasma HDL in mice.
Keywords: High density lipoprotein; apoA-I; Probucol; Cholesterol efflux;
Modifications of glycosphingolipid profile and synthesis in normal rat fibroblasts and in syngeneic neoplastic cells at different subculture stages by Irma Colombo; Elena Sottocornola; Simona Moretti; Maria Antonia Meloni; Proto Pippia; Bruno Berra (214-224).
Glycosphingolipids are plasma membrane macromolecules involved in diversified recognition functions on the cell surface resulting in modulation of cell adhesion and differentiation. As the in vitro cellular system of the neoplastic cell line SGS/4A and syngeneic normal fibroblasts (FG) represents a useful tool for studies on molecular mechanisms regulating cell adhesion, neoplastic transformation and cellular ageing, we studied the changes of glycosphingolipid and of the enzymes involved in their metabolism in both cultured cells at different subculture stages. The FG subculture progression induces a drastic decrease of total glycosphingolipid content with consistent alterations in the molecular composition. In particular, a significant decrease of GM3, a slight increase of GD1a, the disappearance of ‘b’-series gangliosides and the drastic reduction of triosylceramides were observed. On the contrary, the increasing number of SGS/4A subcultures, characterized by a specific and different glycosphingolipid composition as compared with FG cells, does not cause modifications. Although glycosyltransferase activity levels quite well parallel the glycosphingolipid patterns and can account for the noted variations, the mRNA expression analysis of two glycosyltransferases suggests that the in vitro cell ageing of normal rat fibroblasts causes drastic changes in the glycosphingolipid profile through the regulation, at either the transcriptional or post-translational level, of some biosynthetic enzymes.
Keywords: Glycosphingolipid; Glycosyltransferase; Sialyltransferase; Cell ageing; Sarcoma Galliera; (Rat fibroblast);
Isolevuglandin–protein adducts in humans: products of free radical-induced lipid oxidation through the isoprostane pathway by Robert G. Salomon; Eugenia Batyreva; Kamaljit Kaur; Dennis L. Sprecher; Martin J. Schreiber; John W. Crabb; Marc S. Penn; Angela M. DiCorleto; Stanley L. Hazen; Eugene A. Podrez (225-235).
A family of extremely reactive electrophiles, isolevuglandins (isoLGs), is generated in vivo by free radical-induced lipid oxidation and rearrangement of endoperoxide intermediates of the isoprostane pathway. Protein adducts of two different oxidized lipids, isoLGE2 and isoLGE2, and the corresponding autoantibodies are present in human blood. Western blot analysis of a polyacrylamide gel electrophoresis gel detects several immunoreactive plasma proteins. Only a minor fraction of the isoLG–protein modifications is associated with low density lipoprotein since mean levels were decreased only 20–22% by immunoprecipitation of apolipoprotein B (apoB). Mean levels of both isoLGE2 and isoLGE2–protein adducts in plasma from patients with atherosclerosis (AS) (n=16) or end-stage renal disease (RD) (n=8) are about twice those in healthy individuals (n=25). These elevated levels are not related to variations in age, total cholesterol or apoB. A linear correlation (r=0.79) between plasma isoLGE2 and isoLGE2–protein adduct levels in all 49 individuals is consistent with a common free radical-induced mechanism for the production of both oxidized lipids in vivo. The correlation is even stronger (r=0.86) for patients with AS or RD. That isoLG–protein adduct levels are more strongly correlated with disease than are total cholesterol or apoB suggests an independent defect that results in an abnormally high level of oxidative injury associated with AS and RD.
Keywords: Levuglandin; Isolevuglandin; Isoprostane; Atherosclerosis; Renal disease;
Cloning and expression of rat neutral sphingomyelinase: enzymological characterization and identification of essential histidine residues by Yukiko Mizutani; Keiko Tamiya-Koizumi; Fumitoshi Irie; Yoshio Hirabayashi; Masao Miwa; Shonen Yoshida (236-246).
Using cross-species sequence homology, we cloned a cDNA for rat neutral sphingomyelinase (nSMase) composed of 422 amino acids that shares 87.6 and 79.0% identity with the mouse and human forms respectively. The rat nSMase expressed in Escherichia coli catalyzed sphingomyelin hydrolysis at neutral pH in a Mg2+-dependent manner, and required Triton X-100, dithiothreitol, and KCl for its full activity. The cloned rat enzyme shares conserved sequences with nSMases from both eukaryotes and prokaryotes. Introduction of single mutations into either of the histidine residues at positions 136 and 272, putative active sites, entirely abolished the activity, supporting a common mechanism for the nSMase family independent of the species. However, mutation in histidine 151, conserved only in eukaryotes, also abolished the activity, suggesting eukaryote-specific control of nSMase linked to this histidine 151. This enzyme also catalyzed the hydrolysis of lyso-platelet activating factor to yield 1-alkylglycerol at a rate that is slightly lower than that with sphingomyelin.
Keywords: Neutral sphingomyelinase; cDNA cloning; Lyso-platelet activating factor; Site-directed mutagenesis; Active site; Rat;