BBA - Molecular and Cell Biology of Lipids (v.1531, #3)

The list of diseases linked to defects in lipid metabolism has recently been augmented by the addition of hyperimmunoglobulinemia D and periodic fever syndrome (HIDS: MIM 260920), which are correlated with depressed levels of mevalonate kinase activity and protein . More specifically, a V377I substitution has been proposed to account for this disease. We observed that V377 appears to be far from invariant in eukaryotic mevalonate kinases. Prokaryotic mevalonate kinases are lower in molecular weight and several terminate prior to residue 377 of the eukaryotic proteins. These observations prompted our direct test of the impact of V377 on activity and protein stability by engineering a V377I mutation in a recombinant human mevalonate kinase. The mutant protein has been isolated and kinetically characterized. In comparison with wild-type enzyme, V377I exhibits only modest differences (notably ≥6-fold inflation of K m(MVA)) that do not account for the diminished mevalonate kinase activity assayed in HIDS cell extracts. Moreover, thermal inactivation (50°C) of isolated wild-type and V377I enzymes demonstrates little difference in stability between these proteins. We conclude that a single V377I substitution is unlikely to explain the observation of depressed mevalonate kinase stability and catalytic activity in HIDS.
Keywords: Mevalonate kinase; EC 2.7.1.36; Hyperimmunoglobulinemia D; Periodic fever syndrome; HIDS; MIM 260920; Cation ligand; Site directed mutagenesis;

Two unusual glycerophospholipids from a filamentous fungus, Absidia corymbifera by Stanislav G Batrakov; Inessa V Konova; Vladimir I Sheichenko; Stanislav E Esipov; Lyudmila A Galanina (169-177).
The chloroform–methanol extractable lipids of the soil filamentous fungus Absidia corymbifera VKMF-965 account for about 20% by weight of dry cells and are composed of low-polarity constituents (about 75% of the total lipids), such as triacylglycerols (mainly), diacylglycerols, sterols and free fatty acids, as well as of glycolipids (about 3%) and phospholipids. The last consist largely of components common to the fungal lipids, namely, phosphatidylethanolamine (38% of the total phospholipids), phosphatidyl-myo-inositol (16%), diphosphatidylglycerol (12%), phosphatidylcholine (7%), phosphatidic acid (6%) and phosphatidylglycerol (3%), and two unusual phospholipids, PL1 (6%) and PL2 (9%). Based on the infrared (IR), 1H-nuclear magnetic resonance (NMR), 13C-NMR and mass spectra along with the results of degradation experiment, these two phospholipids have been established to be 1,2-diacyl-sn-glycero-3-phospho(N-acetylethanolamine), or N-acetyl phosphatidylethanolamine, and 1,2-diacyl-sn-glycero-3-phospho(N-ethoxycarbonyl-ethanolamine), respectively. These structures have been confirmed by preparing similar phospholipids from the phosphatidylethanolamine isolated from the same fungus and correlating their chromatographic behaviour, IR and 1H-NMR spectra with those of PL1 and PL2. So far N-acetyl phosphatidylethanolamine has been detected only in cattle and human brains and a human placenta but its structure was not rigorously proved. PL2 is a novel lipid; to our knowledge no natural phospholipid with an urethane group has yet been found. The main fatty acids of both the phospholipids are n-hexadecanoic, octadecanoic and octadecadienoic ones; PL2 contains in addition a considerable amount of octadecatrienoic acid with its greater portion located at the sn-1 position.
Keywords: Phospholipid; N-Acetyl phosphatidylethanolamine; N-Ethoxycarbonyl phosphatidylethanolamine; Absidia corymbifera;

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture by Naoki Tamasawa; Hiroshi Murakami; Jun Matsui; Kazumi Yamato; Guan JingZhi; Tadaatsu Imaizumi; Koji Fujimoto; Hidemi Yoshida; Kei Satoh; Toshihiro Suda (178-187).
Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7β-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-α-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-α. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-α, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.
Keywords: 7-Ketocholesterol; Endothelial cell; Vascular cell adhesion molecule-1; Tumor necrosis factor-α; Oxidized low density lipoprotein;

Aldehydic lipid peroxidation products derived from linoleic acid by Peter Spiteller; Werner Kern; Josef Reiner; Gerhard Spiteller (188-208).
Lipid peroxidation (LPO) processes observed in diseases connected with inflammation involve mainly linoleic acid. Its primary LPO products, 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) and 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), decompose in multistep degradation reactions. These reactions were investigated in model studies: decomposition of either 9-HPODE or 13-HPODE by Fe2+ catalyzed air oxidation generates (with the exception of corresponding hydroxy and oxo derivatives) identical products in often nearly equal amounts, pointing to a common intermediate. Pairs of carbonyl compounds were recognized by reacting the oxidation mixtures with pentafluorobenzylhydroxylamine. Even if a pure lipid hydroperoxide is subjected to decomposition a great variety of products is generated, since primary products suffer further transformations. Therefore pure primarily decomposition products of HPODEs were exposed to stirring in air with or without addition of iron ions. Thus we observed that primary products containing the structural element R-CH=CH-CH=CH-CH=O add water and then they are cleaved by retroaldol reactions. 2,4-Decadienal is degraded in the absence of iron ions to 2-butenal, hexanal and 5-oxodecanal. Small amounts of buten-1,4-dial were also detected. Addition of m-chloroperbenzoic acid transforms 2,4-decadienal to 4-hydroxy-2-nonenal. 4,5-Epoxy-2-decenal, synthetically available by treatment of 2,4-decadienal with dimethyldioxirane, is hydrolyzed to 4,5-dihydroxy-2-decenal.
Keywords: Lipid peroxidation; 2,4-Decadienal; 4-Hydroxy-2-nonenal; 2-Butenal; 5-Oxodecanal; 4,5-Epoxy-2-decenal; 4-Oxo-2-nonenal; 4,5-Dihydroxy-2-decenal; Buten-1,4-dial; 9-Hydroxy-12-oxo-10-dodecenoic acid;

The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.
Keywords: Phosphatidylinositol 4-kinase; Plasma membrane; Rat liver; Subcellular localization; Aqueous two-phase partitioning;

Brefeldin A (BFA) causes disassembly of the Golgi apparatus and blocks protein transport to this organelle from the endoplasmic reticulum. However, there still remains considerable ambiguity regarding the involvement of the Golgi apparatus in glycerolipid transport pathways. We examined the effects of BFA upon the intracellular translocation of phosphatidylcholine in alveolar type II cells, that synthesize, transport, store and secrete large amounts of phospholipid for regulated exocytosis. BFA at concentrations as high as 10 μg/ml failed to alter the assembly of phosphatidylcholine into lamellar bodies, the specialized storage organelles for pulmonary surfactant. The same concentration of BFA was also ineffective at altering the secretion of newly synthesized phosphatidylcholine from alveolar type II cells. In contrast, concentrations of the drug of 2.5 μg/ml completely arrested newly synthesized lysozyme secretion from the same cells, indicating that BFA readily blocked protein transport processes in alveolar type II cells. The disassembly of the Golgi apparatus in alveolar type II cells following BFA treatment was also demonstrated by showing the redistribution of the resident Golgi protein MG-160 to the endoplasmic reticulum. These results indicate that intracellular transport of phosphatidylcholine along the secretory pathway in alveolar type II cells proceeds via a BFA insensitive route and does not require a functional Golgi apparatus.
Keywords: Membrane; Organelle; Golgi; Epithelial cells; Lamellar bodies; Secretory pathway;

Structure of the human acyl-CoA:cholesterol acyltransferase-2 (ACAT-2) gene and its relation to dyslipidemia by Keisuke Katsuren; Toshiya Tamura; Rina Arashiro; Kouki Takata; Toshinobu Matsuura; Norio Niikawa; Takao Ohta (230-240).
Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification in mammalian cells. Two isoforms of ACAT have been reported to date (ACAT-1 and ACAT-2). ACAT-1 is ubiquitously expressed in tissues except the intestine. In contrast, ACAT-2 is expressed mainly in the intestine in humans. To investigate the relationship between ACAT-2 and dyslipidemia, we determined the structure of the human ACAT-2 gene and then studied the relationship between mutations of the ACAT-2 gene and dyslipidemia. To isolate human ACAT-2 genomic DNA, we designed primers based on the human ACAT-2 cDNA sequence: forward primer 5′-ACACCTCGATCTTGGTCCTGCCATA-3′ and reverse primer 5′-GGAATGCAGACAGGGAGTCCT-3′. Using these primers, a human P1-derived artificial chromosome (PAC) library was screened by PCR-based procedures. Isolated PAC clones were completely digested with BamHI and subcloned into plasmid vector. Subclones that contained exons were screened by dot-blot hybridization using partial ACAT-2 cDNA fragments. The coding region of the ACAT-2 gene was encoded in 15 exons from 51 to 265 base pairs on a 21 kilobase span of genomic DNA. The exonic sequences coincided completely with that of ACAT-2 cDNA, and each exon-intron junction conserved splicing consensus sequences. Next, 187 (91 dyslipidemic and 96 normolipidemic) subjects were screened by PCR single-strand conformational polymorphism analysis of the ACAT-2 gene. Three mutations were identified by DNA sequencing: two missense mutations (E14G in exon 1 and T254I in exon 7) and a point mutation in intron 7 (−35G→A). Mutations in exon 1 and intron 7 were not associated with plasma concentrations of lipids and apolipoproteins (apo). However, plasma apoC-III levels in T254I heterozygotes were significantly higher than those in subjects without mutation. Plasma triglyceride (TG) levels in T254I heterozygotes were similar to those in subjects without mutation. Although further studies are needed, our data suggest that ACAT-2 may contribute to apoC-III gene expression and the assembly of apoC-III and TG, possibly in the intestine.
Keywords: Acyl coenzyme A:cholesterol acyltransferase-2; Lipoprotein; Dyslipidemia; Cholesterol esterification; Apolipoprotein C-III; Atherosclerosis;

There is a developmental increase in agonist-induced surfactant secretion in type II cells. The response to the P2Y2 agonist UTP is negligible in early newborn cells but increases with age. The response to terbutaline, N-ethylcarboxyamidoadenosine (NECA), and ATP also increases with age. As glucocorticoids are known to accelerate several aspects of lung maturation we examined the effect of dexamethasone (Dex) on the response of 1-day-old rat type II cells to surfactant secretagogues. Freshly isolated cells were cultured ±10−6 M Dex for 18–20 h after which phosphatidylcholine secretion was measured. Dex slightly decreased the basal secretion rate. However, it significantly increased the response to terbutaline, NECA, ATP and UTP. This effect was dependent on Dex concentration (EC50=2–6×10−9 M) and blocked by the glucocorticoid receptor antagonist RU-486. It is unlikely to be due to increased receptor content as Dex had no effect on adenylate cyclase, phospholipase C or phospholipase D activation and the response to cAMP, forskolin and phorbol ester, secretagogues acting downstream from receptors, was also increased by Dex. These data show that Dex acts directly on the type II cell to enhance the response to surfactant secretagogues, that the effect of the hormone is mediated by the glucocorticoid receptor and suggest induction of a common downstream signaling step(s). Regulation of surfactant secretion may be an important function of glucocorticoids in the developing lung.
Keywords: P2Y2 agonist; Lung development; Mifepristone; Signaling mechanism; Dexamethasone;

To investigate the sequence requirements for apolipoprotein (apo) AI functions, comparisons of human and chicken apoAI were performed. In lipid binding assays, chicken apoAI was capable of transforming phospholipid vesicles into discoidal bilayer structures, similar in both size and apolipoprotein content to those produced with human apoAI under the same conditions. Human and chicken apoAI were indistinguishable in their relative abilities to prevent phospholipase C-induced aggregation of human low density lipoprotein. This activity, which is dependent upon formation of a stable interaction with the modified lipoprotein, represents a sensitive measure of apolipoprotein association with spherical lipoprotein particles. The ability of chicken versus human apoAI to mobilize the regulatory pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase by human fibroblasts was also assessed. Lipid-free chicken and human apoAI were equivalent in their ability to deplete cholesterol from this pool, as were intact chicken high density lipoprotein (HDL) and human HDL3. Based on the overall sequence identity of chicken and human apoAI (48%), and comparison of regions thought to be responsible for key apoAI functions, these data indicate that amphipathic α-helical structure, rather than specific amino acid sequence, is the major determinant of apoAI lipid binding and ability to mobilize the regulatory pool of cellular cholesterol.
Keywords: Apolipoprotein; Sequence; High density lipoprotein; Cholesterol; Efflux;

Author index (260-261).

Contents Vol. 1531 (262-263).