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BBA - Molecular and Cell Biology of Lipids (v.1791, #1)
Influence of all- trans-retinoic acid on oxoglutarate carrier via retinoylation reaction by E. Cione; A. Pingitore; M. Perri; G. Genchi (pp. 3-7).
All- trans-retinoic acid (atRA), an activated metabolite of vitamin A, is incorporated covalently into proteins both in vivo and in vitro. AtRA reduced the transport activity of the oxoglutarate carrier (OGC) isolated from testes mitochondria to 58% of control via retinoylation reaction. Labeling of testes mitochondrial proteins with3HatRA demonstrated the binding of atRA to a 31.5 KDa protein. This protein was identified as OGC due to the competition for the labeling reaction with 2-oxoglutarate, the specific OGC substrate. The role of retinoylated proteins is currently being explored and here we have the first evidence that retinoic acids bind directly to OGC and inhibit its activity in rat testes mitochondria via retinoylation reaction. This study indicates the evidence of a specific interaction between atRA and OGC and establishes a novel mechanism for atRA action, which could influence the physiological biosynthesis of testosterone in situations such as retinoic acid treatment.
Keywords: Retinoic acid; 2-Oxoglutarate carrier; Retinoylation reaction
Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells by Haitao Wu; Sanae Ichikawa; Chiharu Tani; Beiwei Zhu; Mikiro Tada; Yasuaki Shimoishi; Yoshiyuki Murata; Yoshimasa Nakamura ⁎ (pp. 8-16).
Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.
Keywords: Abbreviations; DHA; docosahexaenoic acid; EPA; eicosapentaenoic acid; ERK; extracellular-regulated kinase; GAP-43; growth-associated protein-43; H; 2; DCF-DA; 2′,7′-dichlorofluorescin diacetate; PI3K; phosphatidyl inositol 3 kinase; PTPase; protein tyrosine phosphatase; PUFAs; polyunsaturated fatty acids; ROS; reactive oxygen species; (MAPK); mitogen-activated protein kinase; MEK; mitogen-activated protein kinase (MAPK)/ERK kinase; NAC; N; -acetylcysteineDocosahexaenoic acid; Neuritogenesis; Growth associated protein 43; Reactive oxygen species; Extracellular signal-regulated kinase
The dynamic of lipid oxidation in human myotubes by Michael Gaster (pp. 17-24).
Both endogenous and exogenous lipid levels may be regulators of total lipid oxidation in skeletal muscles. We studied the dynamics of lipid oxidation in human myotubes established from healthy, lean subjects exposed to acutely and chronically increased palmitate concentrations. The intramyocellular triacylglycerol content increased with chronic palmitate exposure. Both, ectopically increased intracellular and extracellular lipid levels were simultaneously oxidized and could partly suppress each other's oxidation. Overall, the highest acute palmitate treatments stimulated fatty acid oxidation whilst the highest chronic treatments decreased total lipid oxidation. Intracellular lipids showed a more complete oxidation than exogenous lipids. Endogenous lipids reduced insulin-mediated glucose oxidation. Thus, both endogenous and exogenous lipid concentrations regulated each other’s oxidation and total lipid oxidation in human myotubes. A reduced exogenous lipid oxidation, secondary to increased triacylglycerol levels, may redirect free fatty acids into esterification and oxidation from intracellular stores, thereby protecting myotubes from FFA lipotoxic effects.
Keywords: Abbreviations; FFA; Free fatty acids; PA; palmitateFuel selection; Human; Lipid oxidation; Myotube; Skeletal muscle
Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin by Veronica M.T. Lattanzio; Maristella Baronio; Aharon Oren; Nicholas J. Russell; Angela Corcelli (pp. 25-31).
The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Δ9cis and C18:1 Δ11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.
Keywords: Abbreviations; CL; cardiolipin or bisphosphatidylglycerol; ESI-MS; electrospray ionization mass spectrometry; HPLC; high performance liquid chromatography; GL; glycolipid; SL; sulfonolipid; PC; phosphatidylcholine; diMePE; dimethylphosphatidyl-ethanolamine; PG; phosphatidylglycerol; PS; phosphatidylserine; TLC; thin layer chromatography; FAME-GC; fatty acid methyl ester gas chromatography Salinibacter ruber; Bisphosphatidylglycerol; Cardiolipin; Sulfonolipids; Halophilic; Bacteroidetes
cDNA cloning and characterization of human and mouse Ca2+-independent phosphatidylethanolamine N-acyltransferases by Xing-Hua Jin; Toru Uyama; Jun Wang; Yasuo Okamoto; Takeharu Tonai; Natsuo Ueda (pp. 32-38).
The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca2+-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A1/A2-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca2+. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein–protein interaction.
Keywords: N; -acylethanolamine; N; -acylphosphatidylethanolamine; Anandamide; Phospholipase A; 1; /A; 2; Phospholipid
Identification of cis-acting promoter sequences required for expression of the glycerol-3-phosphate acyltransferase 1 gene in mice by Masaki Yoshida; Nagakatsu Harada ⁎; Hironori Yamamoto; Yutaka Taketani; Tadahiko Nakagawa; Yunjie Yin; Atsushi Hattori; Tomoe Zenitani; Sayuri Hara; Haruka Yonemoto; Aki Nakamura; Masayuki Nakano; Kazuaki Mawatari; Kiyoshi Teshigawara; Hidekazu Arai; Toshio Hosaka; Akira Takahashi; Katsuhiko Yoshimoto; Yutaka Nakaya (pp. 39-52).
Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the “classical” sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/ db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.
Keywords: Abbreviations; GPAT1; glycerol-3-phosphate acyltransferase 1; USF-1; upstream stimulatory factor-1; SREBP-1; sterol regulatory element-binding protein-1; ORF; open reading frame; 5′-RACE; 5′-Rapid amplification of cDNA ends; siRNA; small interfering RNA; EMSA; Electrophoretic Mobility Shift Assay; ChIP; Chromatin immunoprecipitation; ELISA; Enzyme-Linked Immunosorbent Assay; DMEM; Dulbecco's modified Eagle's medium; PMSF; phenylmethylsulfonyl fluoride; SDS; sodium dodecyl sulfate; TAG; triacylglycerol; SRE; sterol regulatory element; ERR; estrogen receptor response element; LXR; liver X receptor; PPAR-α; peroxisome proliferator-activated receptor-α; RXR; retinoid X receptorGlycerol-3-phosphate acyltransferase; Promoter; Sterol regulatory element-binding protein-1; Upstream stimulating factor-1; Liver; Mouse
Development of a potent inhibitor of 2-arachidonoylglycerol hydrolysis with antinociceptive activity in vivo by Tiziana Bisogno; Giorgio Ortar; Stefania Petrosino; Enrico Morera; Enza Palazzo; Marianna Nalli; Sabatino Maione; Vincenzo Di Marzo (pp. 53-60).
Although inhibitors of the enzymatic hydrolysis of the endocannabinoid 2-arachidonoylglycerol are available, they are either rather weak in vitro (IC50>30 μM) or their selectivity towards other proteins of the endocannabinoid system has not been tested. Here we describe the synthesis and activity in vitro and in vivo of a tetrahydrolipstatin analogue, OMDM169, as a potent inhibitor of 2-AG hydrolysis, capable of enhancing 2-AG levels and of exerting analgesic activity via indirect activation of cannabinoid receptors. OMDM169 exhibited 0.13 μM50<0.41 μM towards 2-AG hydrolysing activities in COS-7 cells and rat cerebellum, and inhibited (IC50=0.89 μM) the human recombinant MAGL, whilst being inactive ( Ki>10 μM) at human CB1 and CB2 receptors. However, OMDM169 shared with tetrahydrolipstatin the capability of inhibiting the human pancreatic lipase (IC50=0.6 μM). OMDM169 inhibited fatty acid amide hydrolase and diacylglycerol lipase only at higher concentrations (IC50=3.0 and 2.8 μM, respectively), and, accordingly, it increased by ∼1.6-fold the levels of 2-AG, but not anandamide, in intact ionomycin-stimulated N18TG2 neuroblastoma cells. Acute intraperitoneal (i.p.) administration of OMDM169 to mice inhibited the second phase of the formalin-induced nocifensive response with an IC50 of ∼2.5 mg/kg, and concomitantly elevated 2-AG, but not anandamide, levels in the ipsilateral paw of formalin-treated mice. The antinociceptive effect of OMDM169 was antagonized by antagonists of CB1 and CB2 receptors, AM251 and AM630, respectively (1 mg/kg, i.p.). OMDM69 might represent a template for the development of selective and even more potent inhibitors of 2-AG hydrolysis.
Keywords: Endocannabinoid; Cannabinoid receptor; 2-arachidonoylglycerol; Anandamide; Monoacylglycerol lipase; Diacylglycerol lipase; Fatty acid amide hydrolase; Pain; Inhibitor
Lysophosphatidylcholine-induced apoptosis in H19-7 hippocampal progenitor cells is enhanced by the upregulation of Fas Ligand by Yuanjie Sun; Joo-Hyun Lee; Nam-Ho Kim; Chang-Wook Lee; Min-Ju Kim; Seung-Hyuk Kim; Sung-Oh Huh ⁎ (pp. 61-68).
Lysophospholipids regulate a wide array of biological processes including apoptosis and neutrophil migration. Fas/Apo-1 and its ligand (FasL) participate in neuronal cell apoptosis causing various neurological diseases. Here, we use hippocampal neuroprogenitor cells to investigate how lysophosphatidylcholine (LPC) induces apoptosis in H19-7 hippocampal progenitor cells via Fas/Fas ligand-mediated apoptotic signaling pathway. Exposed cells with LPC presented on apoptotic morphology, positive TUNEL staining, and DNA fragmentation. We found that the expression of FasL was increased after LPC treatment. Furthermore, LPC-induced H19-7 cell apoptosis was decreased by agonistic anti-FasL antibody. In addition to promotion of caspase cascade activity by LPC, the administration of the caspase inhibitor, DEVD-fmk, prevented H19-7 cell apoptosis. LPC also increased the activation of nuclear factor-κB (NF-κB), which in turn, significantly increased FasL mRNA level. The increase in FasL mRNA level by NF-κB transfection was significantly decreased in the presence of IκB-SR, a super-repressor of IκB. Taken together, these results demonstrate that LPC has the ability to induce apoptosis in H19-7 cells through the upregulation of FasL expression via NF-κB activation.
Keywords: Abbreviations; LPC; lysophosphatidylcholine; FasL; Fas ligand; NF-κB; nuclear factor-kappa B; IκB; inhibitory factor kappa BLysophosphatidylcholine; Fas; FasL; NF-κB; Apoptosis; H19-7 cell
The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis by Beth Marbois; Peter Gin; Melissa Gulmezian; Catherine F. Clarke ⁎ (pp. 69-75).
Coenzyme Q is a redox active lipid essential for aerobic respiration. The Coq4 polypeptide is required for Q biosynthesis and growth on non-fermentable carbon sources, however its exact function in this pathway is not known. Here we probe the functional roles of Coq4p in a yeast Q biosynthetic polypeptide complex. A yeast coq4-1 mutant harboring an E226K substitution is unable to grow on nonfermentable carbon sources. The coq4-1 yeast mutant retains significant Coq3p O-methyltransferase activity, and mitochondria isolated from coq4-1 and coq4-2 (E121K) yeast point mutants contain normal steady state levels of Coq polypeptides, unlike the decreased levels of Coq polypeptides generally found in strains harboring coq gene deletions. Digitonin-solubilized mitochondrial extracts prepared from yeast coq4 point mutants show that Coq3p and Coq4 polypeptides no longer co-migrate as high molecular mass complexes by one- and two-dimensional Blue Native-PAGE. Similarly, gel filtration chromatography confirms that O-methyltransferase activity, Coq3p, Coq4p, and Coq7p migration are disorganized in the coq4-1 mutant mitochondria. The data suggest that Coq4p plays an essential role in organizing a Coq enzyme complex required for Q biosynthesis.
Keywords: Saccharomyces cerevisiae; Ubiquinone; Coenzyme Q; Mitochondria; Respiratory electron transport; Coq4
A novel peptide binding to the cytoplasmic domain of class A scavenger receptor reduces lipid uptake in THP-1 macrophages by Xiaohua Wang; Yuan Zheng; Yiming Xu; Jingjing Ben; Song Gao; Xudong Zhu; Yan Zhuang; Shen Yue; Hui Bai; Yaoyu Chen; Li Jiang; Yong Ji; Yong Xu; Leming Fan; Jiahao Sha; Zhigang He; Qi Chen ⁎ (pp. 76-83).
Class A scavenger receptor (SR-A) contributes primarily to lipid accumulation in cells. The cytoplasmic domain of SR-A (CSR-A) is responsible for internalization of the receptor-ligand complex into cells. In the present study we tried to reduce cellular uptake of acetylated low density lipoprotein (AcLDL) by inducing the interaction between the CSR-A and a novel peptide H11, which was screened from a phage-displayed peptide library. When H11 was fused with a cross membrane peptide TAT, the fusion peptide could enter cell efficiently. The peptide H11 inhibited the binding and uptake of DiI-AcLDL and attenuated lipid accumulation in the differentiated human acute monocytic leukemia cell line (THP-1) macrophages. Furthermore, the interaction of peptide H11 with the CSR-A inhibited the expression of SR-A protein as well as the phosphorylation of c- jun N-terminal kinase 2 (JNK2) in cells, which mediates cellular lipid accumulation-related signaling pathways. These results suggest that the CSR-A can be a potential target to prevent lipid accumulation in cells. The peptide H11 may be useful in regulating SR-A functions in macrophages.
Keywords: Abbreviations; AcLDL; acetyl-low-density lipoprotein; BCA; bicinchoninic acid; CE; cholesteryl ester; CSR-A; cytoplasmic domain of SR-A; DTT; dithiothreitol; GAPDH; glyceraldehyde 3-phosphage dehydrogenase; GST; glutathione S-transferase; HRP; horseradish peroxidase; HSP; heat shock protein; IPTG; isopropylthio-β-; d; -galactoside; JNK; c-; jun; N-terminal kinase; MTT; 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PDZ; PSD-95/Discs Large/ZO-1; pfu; plaque forming unit; PTK; protein tyrosine kinase; SR-A; class A macrophage scavenger receptor; THP-1; human acute monocytic leukemia cell lineClass A scavenger receptor (SR-A); Cytoplasmic domain; Foam cell; Phage peptide library; Acetylated low density lipoprotein (AcLDL); Atherosclerosis
Corrigendum to “Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates results in increased enzyme activity” [Biochim. Biophys. Acta 1761 (2006) 1498–1505]
by Erik Andersson ⁎; Frida Schain; Märta Svedling; Hans-Erik Claesson; Pontus K.A. Forsell (pp. 84-84).