Publications by authors named "Isabelle Guillas"

17 Publications

  • Page 1 of 1

Slug, a Cancer-Related Transcription Factor, is Involved in Vascular Smooth Muscle Cell Transdifferentiation Induced by Platelet-Derived Growth Factor-BB During Atherosclerosis.

J Am Heart Assoc 2020 01 21;9(2):e014276. Epub 2020 Jan 21.

Institut de Biologie Paris-Seine (IBPS) Biological Adaptation and Ageing UMR 8256 Sorbonne Université Paris France.

Background Heart attacks and stroke often result from occlusive thrombi following the rupture of vulnerable atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) play a pivotal role in plaque vulnerability because of their switch towards a proinflammatory/macrophage-like phenotype when in the context of atherosclerosis. The prometastatic transcription factor Slug/Snail2 is a critical regulator of cell phenotypic transition. Here, we aimed to investigate the role of Slug in the transdifferentiation process of VSMCs occurring during atherogenesis. Methods and Results In rat and human primary aortic smooth muscle cells, Slug protein expression is strongly and rapidly increased by platelet-derived growth factor-BB (PDGF-BB). PDGF-BB increases Slug protein without affecting mRNA levels indicating that this growth factor stabilizes Slug protein. Immunocytochemistry and subcellular fractionation experiments reveal that PDGF-BB triggers a rapid accumulation of Slug in VSMC nuclei. Using pharmacological tools, we show that the PDGF-BB-dependent mechanism of Slug stabilization in VSMCs involves the extracellular signal-regulated kinase 1/2 pathway. Immunohistochemistry experiments on type V and type VI atherosclerotic lesions of human carotids show smooth muscle-specific myosin heavy chain-/Slug-positive cells surrounding the prothrombotic lipid core. In VSMCs, Slug siRNAs inhibit prostaglandin E2 secretion and prevent the inhibition of cholesterol efflux gene expression mediated by PDGF-BB, known to be involved in plaque vulnerability and/or thrombogenicity. Conclusions Our results highlight, for the first time, a role of Slug in aortic smooth muscle cell transdifferentiation and enable us to consider Slug as an actor playing a role in the atherosclerotic plaque progression towards a life-threatening phenotype. This also argues for common features between acute cardiovascular events and cancer.
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http://dx.doi.org/10.1161/JAHA.119.014276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033846PMC
January 2020

Free cholesterol transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis underlies the U-shape relationship between HDL-cholesterol and cardiovascular disease.

Eur J Prev Cardiol 2020 Oct 15;27(15):1606-1616. Epub 2019 Dec 15.

National Institute for Health and Medical Research (INSERM) UMR_S 1166, Faculty of Medicine Pitié-Salpétrière, Paris, France.

Background: Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk.

Methods: To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase.

Results: When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (-45%) and type 2 diabetes (-25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (-20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis.

Conclusions: Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.
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http://dx.doi.org/10.1177/2047487319894114DOI Listing
October 2020

Antioxidative activity of high-density lipoprotein (HDL): Mechanistic insights into potential clinical benefit.

BBA Clin 2017 Dec 19;8:66-77. Epub 2017 Aug 19.

National Institute for Health and Medical Research (INSERM), UMR ICAN 1166, University of Pierre and Marie Curie-Paris 6, AP-HP, Groupe hospitalier Pitié-Salpétrière, Paris F-75013, France.

Uptake of low-density lipoprotein (LDL) particles by macrophages represents a key step in the development of atherosclerotic plaques, leading to the foam cell formation. Chemical modification of LDL is however necessary to induce this process. Proatherogenic LDL modifications include aggregation, enzymatic digestion and oxidation. LDL oxidation by one-electron (free radicals) and two-electron oxidants dramatically increases LDL affinity to macrophage scavenger receptors, leading to rapid LDL uptake and fatty streak formation. Circulating high-density lipoprotein (HDL) particles, primarily small, dense, protein-rich HDL3, provide potent protection of LDL from oxidative damage by free radicals, resulting in the inhibition of the generation of pro-inflammatory oxidized lipids. HDL-mediated inactivation of lipid hydroperoxides involves their initial transfer from LDL to HDL and subsequent reduction to inactive hydroxides by redox-active Met residues of apolipoprotein A-I. Several HDL-associated enzymes are present at elevated concentrations in HDL3 relative to large, light HDL2 and can be involved in the inactivation of short-chain oxidized phospholipids. Therefore, HDL represents a multimolecular complex capable of acquiring and inactivating proatherogenic lipids. Antioxidative function of HDL can be impaired in several metabolic and inflammatory diseases. Structural and compositional anomalies in the HDL proteome and lipidome underlie such functional deficiency. Concomitant normalization of the metabolism, circulating levels, composition and biological activities of HDL particles, primarily those of small, dense HDL3, can constitute future therapeutic target.
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http://dx.doi.org/10.1016/j.bbacli.2017.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597817PMC
December 2017

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 Sep 18;1862(9):890-900. Epub 2017 May 18.

National Institute for Health and Medical Research (INSERM), INSERM UMR 1166 ICAN, Paris, France; University of Pierre and Marie Curie-Paris 6, Paris, France; AP-HP, Groupe Hospitalier Pitié Salpétrière, Paris, France. Electronic address:

Aims: High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised.

Methods: Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.

Results: rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.

Conclusions: Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics. Non-standard abbreviations and acronyms: AAPH, 2,2'-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.
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http://dx.doi.org/10.1016/j.bbalip.2017.05.010DOI Listing
September 2017

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress.

Plant Cell Environ 2015 Dec 16;38(12):2688-97. Epub 2015 Jul 16.

UFR 927, Sorbonne Universités, UPMC Univ Paris 06, F-75252, Paris, France.

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress.
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http://dx.doi.org/10.1111/pce.12578DOI Listing
December 2015

Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

J Biol Chem 2014 Jun 14;289(26):18466-77. Epub 2014 May 14.

From the Institut National de la Recherche Agronomique UMR1392, Institut d'Ecologie et des Sciences de l'Environnement, Université Pierre et Marie Curie (UPMC), Bât A 7ème Etage Case 237, 7 Quai St.-Bernard, 75252 Paris, France,

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.
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http://dx.doi.org/10.1074/jbc.M114.562769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140250PMC
June 2014

Nitric oxide-sphingolipid interplays in plant signalling: a new enigma from the Sphinx?

Front Plant Sci 2013 Sep 12;4:341. Epub 2013 Sep 12.

UR 5, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Université Pierre et Marie Curie - Paris 6 Paris, France ; EAC 7180, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Centre National de la Recherche Scientifique Paris, France.

Nitric oxide (NO) emerged as one of the major signaling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of NO into complex signaling networks. Beside the close relationships between NO and calcium or phosphatidic acid signaling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids (SLs). This mini-review summarizes our current knowledge of the influence NO and SLs might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although SL-NO interplays are common features in signaling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.
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http://dx.doi.org/10.3389/fpls.2013.00341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770979PMC
September 2013

Phosphatidylinositol 4,5-bisphosphate is required for invasive growth in Saccharomyces cerevisiae.

J Cell Sci 2013 Aug 18;126(Pt 16):3602-14. Epub 2013 Jun 18.

Université Nice - Sophia Antipolis, Institute of Biology Valrose, 06108 Nice Cedex 2, France.

Phosphatidylinositol phosphates are important regulators of processes such as the cytoskeleton organization, membrane trafficking and gene transcription, which are all crucial for polarized cell growth. In particular, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has essential roles in polarized growth as well as in cellular responses to stress. In the yeast Saccharomyces cerevisiae, the sole phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) Mss4p is essential for generating plasma membrane PtdIns(4,5)P2. Here, we show that Mss4p is required for yeast invasive growth in low-nutrient conditions. We isolated specific mss4 mutants that were defective in cell elongation, induction of the Flo11p flocculin, adhesion and cell wall integrity. We show that mss4-f12 cells have reduced plasma membrane PtdIns(4,5)P2 levels as well as a defect in its polarized distribution, yet Mss4-f12p is catalytically active in vitro. In addition, the Mss4-f12 protein was defective in localizing to the plasma membrane. Furthermore, addition of cAMP, but not an activated MAPKKK allele, partially restored the invasive growth defect of mss4-f12 cells. Taken together, our results indicate that plasma membrane PtdIns(4,5)P2 is crucial for yeast invasive growth and suggest that this phospholipid functions upstream of the cAMP-dependent protein kinase A signaling pathway.
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http://dx.doi.org/10.1242/jcs.122606DOI Listing
August 2013

Long chain base changes triggered by a short exposure of Arabidopsis to low temperature are altered by AHb1 non-symbiotic haemoglobin overexpression.

Plant Physiol Biochem 2013 Feb 5;63:191-5. Epub 2012 Dec 5.

UPMC Univ Paris 06, UR 5, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, F-75005 Paris, France.

Long chain bases (LCB) are both precursors of complex sphingolipids (SL) and cellular signals in eukaryotic cells. Increasing evidence support a function for SL and/or LCBs in plant responses to environmental cues. In this study we analysed the impact of a short exposure to cold on the global LCB content and composition in Arabidopsis thaliana seedlings. We report that the total LCB amount significantly decreased after low temperature exposure. The decline was essentially due to reduction of t18:1 isomer content. On the other hand, chilling led to the increase of LCB content in a mutant over-expressing the non-symbiotic haemoglobin AHb1. Furthermore, this mutant was impaired in cold-dependent root growth inhibition and anthocyanin synthesis. As AHb1 is an element of nitric oxide turnover, our data suggest a possible link between nitric oxide, SL content and cold stress response.
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http://dx.doi.org/10.1016/j.plaphy.2012.11.020DOI Listing
February 2013

A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth.

J Cell Biol 2012 Aug 13;198(4):711-30. Epub 2012 Aug 13.

Institute of Biology Valrose, Université Nice - Sophia Antipolis, 06108 Nice Cedex 2, France.

Membrane lipids have been implicated in many critical cellular processes, yet little is known about the role of asymmetric lipid distribution in cell morphogenesis. The phosphoinositide bis-phosphate PI(4,5)P(2) is essential for polarized growth in a range of organisms. Although an asymmetric distribution of this phospholipid has been observed in some cells, long-range gradients of PI(4,5)P(2) have not been observed. Here, we show that in the human pathogenic fungus Candida albicans a steep, long-range gradient of PI(4,5)P(2) occurs concomitant with emergence of the hyphal filament. Both sufficient PI(4)P synthesis and the actin cytoskeleton are necessary for this steep PI(4,5)P(2) gradient. In contrast, neither microtubules nor asymmetrically localized mRNAs are critical. Our results indicate that a gradient of PI(4,5)P(2), crucial for filamentous growth, is generated and maintained by the filament tip-localized PI(4)P-5-kinase Mss4 and clearing of this lipid at the back of the cell. Furthermore, we propose that slow membrane diffusion of PI(4,5)P(2) contributes to the maintenance of such a gradient.
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http://dx.doi.org/10.1083/jcb.201203099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514036PMC
August 2012

Arabidopsis type-III phosphatidylinositol 4-kinases β1 and β2 are upstream of the phospholipase C pathway triggered by cold exposure.

Plant Cell Physiol 2012 Mar 8;53(3):565-76. Epub 2012 Feb 8.

CNRS, EAC7180, Physiologie Cellulaire et Moléculaire des Plantes, Paris and UPMC Univ Paris 06, UR5, Physiologie Cellulaire et Moléculaire des Plantes, Paris, France.

Phosphatidylinositol-4-phosphate (PtdIns4P) is the most abundant phosphoinositide in plants and the precursor of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)]. This lipid is the substrate of phosphoinositide-dependent phospholipase C (PI-PLC) that produces diacylglycerol (DAG) which can be phosphorylated to phosphatidic acid (PtdOH). In plants, it has been suggested that PtdIns4P may also be a direct substrate of PI-PLC. Whether PtdIns4P is the precursor of PtdIns(4,5)P(2) or a substrate of PI-PLC, its production by phosphatidylinositol-4-kinases (PI4Ks) is the first step in generating the phosphoinositides hydrolyzed by PI-PLC. PI4Ks can be divided into type-II and type-III. In plants, the identity of the PI4K upstream of PI-PLC is unknown. In Arabidopsis, cold triggers PI-PLC activation, resulting in PtdOH production which is paralleled by decreases in PtdIns4P and PtdIns(4,5)P(2). In suspension cells, both the PtdIns4P decrease and the PtdOH increase in response to cold were impaired by 30 μM wortmannin, a type-III PI4K inhibitor. Type-III PI4Ks include AtPI4KIIIα1, β1 and β2 isoforms. In this work we show that PtdOH resulting from the PI-PLC pathway is significantly lowered in a pi4kIIIβ1β2 double mutant exposed to cold stress. Such a decrease was not detected in single pi4kIIIβ1 and pi4kIIIβ2 mutants, indicating that AtPI4KIIIβ1 and AtPI4KIIIβ2 can both act upstream of the PI-PLC. Although several short-term to long-term responses to cold were unchanged in pi4kIIIβ1β2, cold induction of several genes was impaired in the double mutant and its germination was hypersensitive to chilling. We also provide evidence that de novo synthesis of PtdIns4P by PI4Ks occurs in parallel to PI-PLC activation.
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http://dx.doi.org/10.1093/pcp/pcs011DOI Listing
March 2012

Phytosphingosine-phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling.

New Phytol 2012 Apr 11;194(1):181-91. Epub 2012 Jan 11.

UPMC Univ Paris 06, UR 5, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, F-75252, Paris, France.

• Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling. • PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. • Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. • Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants.
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http://dx.doi.org/10.1111/j.1469-8137.2011.04017.xDOI Listing
April 2012

A matter of fat: interaction between nitric oxide and sphingolipid signaling in plant cold response.

Plant Signal Behav 2011 Jan 1;6(1):140-2. Epub 2011 Jan 1.

UPMC Université Paris 06, Unité de Recherche 5, Centre National de la Recherche Scientifique, Equipe d'Accueil Conventionnée 7180, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Paris, France.

We recently evidenced that plant response to cold stress includes a rapid formation of nitric oxide (NO) that participates in the control of cold-responsive gene expression. Unexpectedly we also shed light on a novel downstream element of NO signalling that is phosphosphingolipid (PS) metabolism. Indeed, two phosphosphingolipid species, phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) are specifically synthesized upon cold exposure. Manipulating NO levels by pharmacological or genetic means dramatically modified the cold-triggered synthesis of PHS-P and Cer-P, but did not affect the cold-responsive formation of phosphatidic acid (PtdOH), a ubiquitous lipid signal derived from phospholipid degradation. So far no crosstalk between NO and PS signalling had been reported in plants. How NO might modulate PS formation and whether this regulation might be extended to other physiological processes are further discussed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122028PMC
http://dx.doi.org/10.4161/psb.6.1.14280DOI Listing
January 2011

Nitric oxide participates in cold-responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana.

New Phytol 2011 Jan 11;189(2):415-27. Epub 2010 Oct 11.

UPMC Univ Paris 06, Unité de Recherche 5, Centre National de la Recherche Scientifique, Equipe d'Accueil Conventionnée 7180, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, F-75252 Paris Cedex 05, France.

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.
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http://dx.doi.org/10.1111/j.1469-8137.2010.03500.xDOI Listing
January 2011

Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides.

Mol Microbiol 2009 Mar 2;71(6):1523-37. Epub 2009 Feb 2.

Department of Medicine, University of Fribourg, Fribourg, Switzerland.

All mature Saccharomyces cerevisiae sphingolipids comprise inositolphosphorylceramides containing C26:0 or C24:0 fatty acids and either phytosphingosine or dihydrosphingosine. Here we analysed the lipid profile of lag1Delta lac1Delta mutants lacking acyl-CoA-dependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2Delta.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells, 2Delta.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1, yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303lag1Delta lac1Delta ypc1Delta ydc1Delta cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous inositolphosphorylsphingosides. Thus, data argue that inositolphosphorylceramides of yeast, the equivalent of mammalian sphingomyelins, are essential for growth. Data also clearly confirm that wild-type strains, when exposed to AbA, immediately stop growing because of ceramide intoxication, long before inositolphosphorylceramide levels become subcritical.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06628.xDOI Listing
March 2009

Incorporation of ceramides into Saccharomyces cerevisiae glycosylphosphatidylinositol-anchored proteins can be monitored in vitro.

Eukaryot Cell 2009 Mar 12;8(3):306-14. Epub 2008 Dec 12.

University of Fribourg, Department of Medicine, Ch. Du Musée 5, CH-1700 Fribourg, Switzerland.

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C(26:0) fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [(3)H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Delta or mcd4Delta mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [(3)H]DHS into anchors in vitro. Moreover, gup1Delta microsomes incorporate [(3)H]DHS into the same abnormal anchor lipids as gup1Delta cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [(3)H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C(26:0)-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [(3)H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.
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http://dx.doi.org/10.1128/EC.00257-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2653239PMC
March 2009

Human homologues of LAG1 reconstitute Acyl-CoA-dependent ceramide synthesis in yeast.

J Biol Chem 2003 Sep 17;278(39):37083-91. Epub 2003 Jul 17.

Department of Medicine, University of Fribourg, CH-1700 Fribourg, Switzerland.

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1delta lac1delta double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1delta lac1delta yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1delta lac1delta yeast mutants.
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http://dx.doi.org/10.1074/jbc.M307554200DOI Listing
September 2003