BBA - Molecular and Cell Biology of Lipids (v.1530, #1)

Nuclear phospholipase C and signaling by Lucio Cocco; Alberto M. Martelli; R.Stewart Gilmour; Sue Goo Rhee; Francesco A. Manzoli (1-14).
Keywords: Nucleus; Inositol lipid; Phospholipase; Signaling;

Abundant expression of uncoupling protein-2 in the small intestine: up-regulation by dietary fish oil and fibrates by Takatoshi Murase; Hidehiko Kondo; Tadashi Hase; Ichiro Tokimitsu; Masayuki Saito (15-22).
Mitochondrial uncoupling protein-2 (UCP-2) is widely expressed in various mammalian tissues, although its physiological functions are not well understood. We examined the effects of dietary fish oil on UCP-2 expression in the rat small intestine, in which UCP-2 mRNA levels are higher than in other organs. Feeding with fish oil (20%) up-regulated UCP-2 mRNA within 6 days in the small intestine as well as the liver, compared to feeding with soybean oil. This was mimicked by feeding with agonists for peroxisome proliferator-activated receptor α (PPARα) such as fenofibrate and bezafibrate, but not the PPARγ agonist troglitazone. The bezafibrate-induced increase in UCP-2 expression was found within 2 days in the small intestine, but only after 6 days in the liver. The up-regulation of UCP-2 was also found in cultured intestinal epithelial cells (IEC-6) treated for 24 h with various long-chain fatty acids and PPARα agonists. These results indicated that intestinal UCP-2 is up-regulated through direct activation of PPARα by dietary fatty acids.
Keywords: Uncoupling protein; Fish oil; Fatty acid; Peroxisome proliferator-activated receptor; Small intestine; IEC-6;

Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity by Chiaki Ogino; Yukinari Negi; Hidenori Daido; Masayuki Kanemasu; Akihiko Kondo; Shun’ichi Kuroda; Katsuyuki Tanizawa; Nobuaki Shimizu; Hideki Fukuda (23-31).
A membrane-bound phospholipase D (PLD) has been identified and isolated in a soluble form from an actinomycete, Streptoverticillium cinnamoneum. The enzyme has a monomeric structure with a molecular size of about 37 kDa, being the smallest among the enzymes so far reported. The enzyme catalyzes the hydrolysis of phosphatidylethanolamine and phosphatidylserine as preferred substrates, but not the transphosphatidylation reaction of their phospholipid groups to ethanol. Together with the absence of immunochemical cross-reactivity, these enzymatic properties demonstrate that the membrane-bound enzyme is distinct from the extracellular enzyme recently characterized and cloned from the same bacterial strain [C. Ogino et al., J. Biochem. 125 (1999) 263–269] and is therefore regarded as a novel prokaryotic PLD.
Keywords: Phospholipase D; Actinomycete; Membrane-bound enzyme; Hydrolysis; Phosphatidylethanolamine;

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif by Cristiana Griffoni; Pavel P. Laktionov; Elena Y. Rykova; Enzo Spisni; Massimo Riccio; Spartaco Santi; Anton Bryksin; Natalia Volodko; Regine Kraft; Valentin Vlassov; Vittorio Tomasi (32-46).
The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A2 (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD+ co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.
Keywords: Antisense oligonucleotide; Cytosolic phosholipase A2; Nucleus; Glyceraldehyde-3-phosphate dehydrogenase;

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells by Massimo Di Vito; Luisa Lenti; Arno Knijn; Egidio Iorio; Federica D’Agostino; Agnese Molinari; Annarica Calcabrini; Annarita Stringaro; Stefania Meschini; Giuseppe Arancia; Argante Bozzi; Roberto Strom; Franca Podo (47-66).
The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r=0.993, P<0.001). Similar ML levels were measured in drug-induced apoptotic cells (A≈30–40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter≤1.0 μm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, ‘biochemically active’ phase of programmed cell death.
Keywords: Apoptosis; Lymphoblast activation; Mobile lipid; 1H nuclear magnetic resonance; Anthracycline; Dexamethasone;

Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages by Akihiko Saiga; Yasuhide Morioka; Takashi Ono; Kazumi Nakano; Yoshikazu Ishimoto; Hitoshi Arita; Kohji Hanasaki (67-76).
We have previously shown the expression of group X secretory phospholipase A2 (sPLA2-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA2-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA2-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA2s. In resting macrophages, sPLA2-X elicited a modest production of prostaglandin E2 and thromboxane A2. After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA2-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA2 inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA2-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA2-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.
Keywords: Phospholipase A2; Cyclooxygenase; Arachidonic acid; Eicosanoid; Lysophosphatidylcholine; Lipid mediator;

The fatty acid elongase-1 β-ketoacyl-CoA synthase, FAE1 KCS, a seed-specific elongase condensing enzyme from Arabidopsis, is involved in the production of eicosenoic (C20:1) and erucic (C22:1) acids. Alignment of the amino acid sequences of FAE1 KCS, KCS1, and five other putative elongase condensing enzymes (KCSs) revealed the presence of six conserved cysteine and four conserved histidine residues. Each of the conserved cysteine and histidine residues was individually converted by site-directed mutagenesis to both alanine and serine, and alanine and lysine respectively. After expression in yeast cells, the mutant enzymes were analyzed for their fatty acid elongase activity. Our results indicated that only cysteine 223 is an essential residue for enzyme activity, presumably for acyl chain transfer. All histidine substitutions resulted in complete loss of elongase activity. The loss of activity of these mutants was not due to their lower expression level since immunoblot analysis confirmed each was expressed to the same extent as the wild type FAE1 KCS.
Keywords: β-Ketoacyl-CoA synthase; Membrane-bound; Mutagenesis; Active site; Very long chain fatty acid;

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A2 (sPLA2) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75–80% of the total amount of synthesized group IIA sPLA2 was secreted. The synthesized group V sPLA2, however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA2. We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA2 with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA2 expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA2 show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA2 was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA2 was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.
Keywords: Group IIA secretory phospholipase A2; Group V secretory phospholipase A2; Expression; Localization; Secretion;

Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification by Jeffrey M Horn; Jason A Lehman; Gerald Alter; Joel Horwitz; Julian Gomez-Cambronero (97-110).
Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8–8.0 was determined. PIP2, EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-β-glucopyranoside, divalent cations, GTPγS and H2O2. The apparent K m for the butanol substrate was 0.1 mM and the V max was 6.0 nmol mg−1 h−1. Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of ∼90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme’s regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.
Keywords: Phospholipase D; Enzyme characterization; Neutrophil; Signal transduction; Phosphotyrosine;

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL2, an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL2 on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL2 at low concentrations (40 μg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL2 at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL2 has two potential roles in reverse cholesterol transport. In one, HDL2 is an acceptor of macrophage FC. In the other, more novel role, HDL2 increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL2 inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.
Keywords: Cholesteryl ester; Macrophage; High-density lipoprotein; Cholesterol transport; Acyl-coenzyme A:cholesterol acyltransferase;