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BBA - Molecular and Cell Biology of Lipids (v.1771, #11)
The presence of a secretory phospholipase A2 in the nuclei of neuronal and glial cells of rat brain cortex by Vincenza Nardicchi; Lara Macchioni; Monica Ferrini; Gianfrancesco Goracci (pp. 1345-1352).
Confocal immunofluorescence analysis indicated a relatively high localization of group V secretory phospholipase A2 (GV) in the nuclei of cultured PC12 and U251 astrocytoma cells. Here, we report the biochemical evidence for the presence of a secretory PLA2 in the nuclei of neuronal and glial cells from rat brain cortex. Enzymic activity was determined using [3H]oleate labelled Escherichia coli membranes in intact nuclei and in their soluble fractions in which the specific activity was significantly more elevated. The treatment of soluble nuclear fractions with inhibitors of cytosolic Ca2+-dependent or Ca2+-independent phospholipases A2 was ineffective whereas DTT or Indoxam, a specific inhibitor of all isoforms of sPLA2, abolished enzyme activity. The enzyme was identified as group V secretory phospholipase A2 (GV) by Western blot analysis and its nucleoplasmic localization was demonstrated by CLSM.
Keywords: Abbreviations; PLA; 2; phospholipase A; 2; SNF; soluble nuclear fraction; AACOCF; 3; arachidonoyltrifluoromethyl ketone; MAFP; methyl arachidonoyl fluorophosphonateSecretory phospholipase A; 2; Rat brain cortex; Cell nuclei; Nuclear signalling
Human glycolipid transfer protein—Intracellular localization and effects on the sphingolipid synthesis by Jessica Tuuf; Peter Mattjus (pp. 1353-1363).
Glycolipid transfer proteins (GLTPs) are small proteins that specifically transfer glycolipids from one bilayer membrane to another in vitro. However, the precise biological function is still unknown. In this study the intracellular distribution of GLTP was determined. We have used several independent methods, including differential and discontinuous density gradient centrifugation, plasma membrane permeabilization and confocal microscopy imaging, and we demonstrate that GLTP has a cytosolic location. The GLTP is not located in the Golgi apparatus, endoplasmic reticulum, nucleus, lysosomes, mitochondria or peroxisomes in HeLa cells. We have also used a fluorescence resonance energy transfer assay to detect transfer of fluorescently labeled BODIPY-glucosylceramide in the cytosolic fraction from both wild-type and GLTP-overexpressing HeLa cells. Furthermore, we have studied de novo sphingolipid changes in cells overexpressing GLTP using sphinganine metabolic labeling. The results show a significant increase in the synthesis of glucosylceramide (GlcCer) and a decrease in the sphingomyelin (SM) synthesis. However, no changes were detected in the de novo sphingolipid synthesis in GLTP-knockdown cells compared to control cells. We propose that GLTP is not likely involved in the de novo synthesis of glycosphingolipids, but could rather have a role as a glycolipid sensor for the cellular levels of glucosylceramide.
Keywords: Abbreviations; DTT; dithiotreithol; ER; Endoplasmic Reticulum; FAPP-2; Phosphoinositol 4-phosphate adaptor protein-2; GalCer; Galactosylceramide; GLTP; Glycolipid transfer protein; GlcCer; Glucosylceramide; GSLs; Glycosphingolipids; LDH; Lactate dehydrogenase; LacCer; Lactosylceramide; SM; Sphingomyelin; PBS; Phosphate buffered saline; PMSF; phenylmethanesulphonylfluoride; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; RET; Resonance energy transfer; TLC; Thin-layer chromatographyGLTP; Glucosylceramide; Cytosolic localization; Confocal microscopy; Subcellular fractionation; RNAi
Lysophosphatidylcholine stimulates EGF receptor activation and mesangial cell proliferation: Regulatory role of Src and PKC by Babu V. Bassa; Jung W. Noh; Shobha H. Ganji; Mi-Kyung Shin; Daeyoung D. Roh; Vaijinath S. Kamanna (pp. 1364-1371).
Lysophosphatidylcholine (LPC), a major component of oxidized-low density lipoproteins (ox-LDL), modulates various pathobiological processes involved in vascular and glomerular diseases. Although several studies have shown increased plasma concentrations of ox-LDL as well as LPC in patients with renal disease, the role of LPC in mesangial cell proliferation and associated signaling mechanisms are not clearly understood. In this study, we have shown that LPC induced the phosphorylation of epidermal growth factor receptor (EGFR), as well as the p42/44 MAP kinases. LPC activated Src-kinase and protein kinase C (PKC), and both Src kinase inhibitor PP-2 and PKC inhibitor inhibited the activation of EGFR by LPC. LPC (5–25 μM) stimulated human mesangial cell proliferation by 4–5 fold. Preincubation of mesangial cells with the Src inhibitor (PP-2), or PKC inhibitor (bisindolylmaleimide GF109203-X), or EGF receptor kinase inhibitor (AG1478), or MEK inhibitor (PD98059) significantly inhibited LPC-mediated mesangial cell proliferation. The data suggest that LPC, by activating Src and PKC signaling pathways, stimulates EGF receptor transactivation and down-stream MAP kinase signaling resulting in mesangial hypercellularity, which is a characteristic feature of diverse renal diseases.
Keywords: Lysophosphatidylcholine; Low density lipoprotein; Mesangial cell proliferation; Signal transduction; EGF receptor; Glomerular disease
Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues by Lihang Yao; Katie Jenkins; Paul S. Horn; M. Hayden Lichtenberg; Laura A. Woollett (pp. 1372-1379).
The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only marginally in fetal livers, fetal bodies, placentas, and yolk sacs when cholesterol concentrations were increased. To begin to elucidate the mechanism responsible for the blunted response of sterol synthesis rates in fetal tissues to exogenous cholesterol, the ratio of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) to Insig-1 was measured in these same tissues since the ratio of SCAP to the Insigs can impact SREBP processing. The fetal tissues had anywhere from a 2- to 6-fold greater ratio of SCAP to Insig-1 than did the adult liver, suggesting constitutive processing of the SREBPs. As expected, the level of mature, nuclear SREBP-2 was not different in the fetal tissues with different levels of cholesterol whereas it was different in adult livers. These findings indicate that the suppression of sterol synthesis to exogenous sterol is blunted in developing tissues and the lack of response appears to be mediated at least partly through relative levels of Insigs and SCAP.
Keywords: Abbreviations; SLOS; Smith–Lemli–Opitz syndrome; DHCR7; 3β-hydroxysterol Δ; 7; -reductase; HMGR; HMG-CoA reductase; SREBP; sterol regulatory element binding protein; SCAP; SREBP cleavage-activating protein; DPS; digitonin-precipitable sterols; Shh; Sonic hedgehogCholesterol; Smith–Lemli–Opitz; HMG-CoA reductase; Fetus; SREBP-2; IUGR
Effects of bile acids on expression of the human apical sodium dependent bile acid transporter gene by William C. Duane; Wendy Xiong; Jennifer Wolvers (pp. 1380-1388).
Using a luciferase reporter assay in both LMH cells and Caco2 cells we found that certain bile acids including unconjugated deoxycholic and others transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) at concentrations ranging from 20 to 300 μM. Confirming this effect, addition of deoxycholic acid to fresh human ileal biopsies caused an approximate 40% increase in endogenous ASBT mRNA production. Promoter deletion analysis indicated the effect of bile acids was mediated by a response element located in the downstream half of the 5′-UTR, a region known to contain a retinoic acid (RXR/RAR) response element and an activated protein-1 (AP-1) response element. Site-directed mutagenesis of the RAR/RXR response element actually enhanced response to deoxycholic acid. Site-directed mutagenesis of the downstream AP-1 response element reduced activation by deoxycholic acid while deletion of this response element completely eliminated this response. The epidermal growth factor (EGF) receptor inhibitor, AG1478, completely eliminated the response to bile acid while the mitogen-activated protein extracellular signal-regulated kinase cascade (MEK) inhibitor, U0126, partially inhibited the response to bile acid. These studies demonstrate that certain bile acids stimulate ASBT gene expression acting on the down-stream AP-1 response element via the EGF receptor and MEK cascade.
Keywords: Abbreviations; ASBT; Apical sodium-dependent bile acid transporter; PCR; Polymerase chain reaction; FXR; Farnesoid X receptor; RAR/RXR; Retinoic acid receptor/retinoid X receptor; AP-1; Activated protein-1; PPAR; Peroxisome proliferation-activated; UTR; Untranslated region; CBS; Charcoal-stripped bovine calf serum; WME; Williams Medium E; MEM; Modified Eagle Media; GAPDH; Glyceraldehyde-3-phosphate dehydrogenase; RLA; Relative luciferase activity; PBS; Phosphate buffered saline; bp; Base pair; EGF; Epidermal growth factor; MAP; Mitogen-activated protein; ERK; Extracellular signal-regulated kinase; MEK; MAP/ERK kinaseIleum; Liver; Cholesterol; Activated Protein-1; Epidermal growth factor
Human group III phospholipase A2 suppresses adenovirus infection into host cells by Michiko Mitsuishi; Seiko Masuda; Ichiro Kudo; Makoto Murakami (pp. 1389-1396).
Of 10 mammalian secreted phospholipase A2 (sPLA2) enzymes identified to date, group V and X sPLA2s, which are two potent plasma membrane-acting sPLA2s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA2, which is structurally more similar to bee venom PLA2 than to other mammalian sPLA2s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA2 hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA2 domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA2 depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA2s target distinct phospholipid molecular species in cellular membranes.
Keywords: Abbreviations; PLA; 2; phospholipase A; 2; sPLA; 2; secreted PLA; 2; ESI/MS; electrospray ionization mass spectrometry; PC; phosphatidylcholine; PE; phosphatidylethanolamine; LPC; lysophosphatidylcholine; HIV; human immunodeficiency virusPhospholipase A; 2; Adenovirus; Plasma membrane; Lysophosphatidylcholine; Phosphatidylethanolamine; Mass spectrometry
Proteolytic activation and glycosylation of N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme involved in the endocannabinoid metabolism by Li-Ying Zhao; Kazuhito Tsuboi; Yasuo Okamoto; Shunichiro Nagahata; Natsuo Ueda (pp. 1397-1405).
N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme hydrolyzing bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine. Previously, we suggested that NAAA is glycosylated and proteolytically cleaved. Here, we investigated the mechanism and significance of the cleavage of human NAAA overexpressed in human embryonic kidney 293 cells. Western blotting with anti-NAAA antibody revealed that most of NAAA in the cell homogenate was the cleaved 30-kDa form. However, some of NAAA were released outside the cells and the extracellular enzyme was mostly the uncleaved 48-kDa form. When incubated at pH 4.5, the 48-kDa form was time-dependently converted to the 30-kDa form with concomitant increase in the N-palmitoylethanolamine-hydrolyzing activity. The purified 48-kDa form was also cleaved and activated. However, the cleavage did not proceed at pH 7.4 or in the presence of p-chloromercuribenzoic acid. The mutant C126S was resistant to the cleavage and remained inactive. These results suggested that this specific proteolysis is a self-catalyzed activation step. We next determined N-glycosylation sites of human NAAA by site-directed mutagenesis addressed to asparagine residues in six potential N-glycosylation sites. The results exhibited that Asn-37, Asn-107, Asn-309, and Asn-333 are actual N-glycosylation sites. The glycosylation appeared to play an important role in stabilizing the enzyme protein.
Keywords: Anandamide; Endocannabinoid; Glycosylation; Lipid mediator; Lipid metabolism; Lysosomal enzyme