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

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

Incorporation of ceramides into yeast glycosylphosphatidylinositol-anchored proteins can be monitored in vitro

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors the thus 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 having 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 [³H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, unable to make ceramide-based anchors in vivo, do not incorporate [³H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [³H]DHS into the same abnormal anchor lipids as gup1Δ 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 [³H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A or C26:0-Coenzyme A, particularly if microsomes are depleted of acyl-CoA. Thus, [³H]DHS cannot be incorporated into proteins in the absence of acyl-CoA

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

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

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

International audienceAlthough 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

Phosphoinositide-bis-phosphate is required for Saccharomyces cerevisiae invasive growth.

International audiencePhosphoinositide phosphates are important regulators of processes such as the cytoskeleton organization, membrane traffic and gene transcription, which are all critical for polarized cell growth. In particular PI(4,5)P2 plays essential roles in polarized growth as well as in cellular responses to stress. In the yeast Saccharomyces cerevisiae a sole PI(4)P-5-kinase, Mss4p is essential for generating plasma membrane PI(4,5)P2. Here we show that Mss4p is required for yeast invasive growth in low nutrient conditions. Specific mss4 mutants were isolated which are defective in cell elongation, induction of the Flo11p flocculin, adhesion and cell wall integrity. We show that mss4-f12 cells have reduced plasma membrane PI(4,5)P2 levels as well as a defect in the polarized distribution of this phospholipid, 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. Together our results indicate that plasma membrane PI(4,5)P2 is critical for yeast invasive growth and suggest that this phospholipid functions upstream of the cAMP-dependent protein kinase A signaling pathway

C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum (ER). When both genes are deleted, cells cannot transport glycosylphosphatidylinositol (GPI)-anchored proteins from the ER to the Golgi at a normal rate. Here we show that microsomes or detergent extracts from lag1Δlac1Δ double mutants lack an activity transferring C26 fatty acids from C26-coenzyme A onto dihydrosphingosine or phytosphingosine. As a consequence, in intact cells, the normal ceramides and inositolphosphorylceramides are drastically reduced. lag1Δlac1Δ cells compensate for the lack of normal sphingolipids by making increased amounts of C26 fatty acids, which become incorporated into glycerophospholipids. They also contain 20- to 25-fold more free long chain bases than wild type and accumulate very large amounts of abnormally polar ceramides. They make small amounts of abnormal mild base-resistant inositolphospholipids. The lipid remodelling of GPI-anchored proteins is severely compromised in lag1Δlac1Δ double mutants since only few and mostly abnormal ceramides are incorporated into the GPI anchors. The participation of Lag1p and Lac1p in ceramide synthesis may explain their role in determining longevity

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

International audienceAims: 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 characterized.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