Corinne Buré

Lipids of plant membrane rafts

Lipids tend to organize in mono or bilayer phases in a hydrophilic environment. While they have long been thought to be incapable of coherent lateral segregation, it is now clear that spontaneous assembly of these compounds can confer microdomain organization beyond spontaneous fluidity. Membrane raft microdomains have the ability to influence spatiotemporal organization of protein complexes, thereby allowing regulation of cellular processes. In this review, we aim at summarizing briefly: (i) the history of raft discovery in animals and plants, (ii) the main findings about structural and signalling plant lipids involved in raft segregation, (iii) imaging of plant membrane domains, and their biochemical purification through detergent-insoluble membranes, as well as the existing debate on the topic. We also discuss the potential involvement of rafts in the regulation of plant physiological processes, and further discuss the prospects of future research into plant membrane rafts.

Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry

The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.

RE-VISITING PLANT PLASMA MEMBRANE LIPIDS IN TOBACCO: A FOCUS ON SPHINGOLIPIDS

Glycosyl-Inositol-Phosphoryl-Ceramides are major lipids of the plasma membrane and form rafts in the outer leaflet, yet extend into the inner leaflet of the membrane through very long-chain fatty acids to interdigitate across the membrane. The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.

A single run LC-MS/MS method for phospholipidomics

Liquid chromatography coupled to tandem mass spectrometry has been compared to shotgun analysis with the objective of finding the best compromise for a single run analysis of whole cell phospholipids. Hydrophilic interaction liquid chromatography (HILIC), normal phase (NP), and reversed phase (RP) liquid chromatography were evaluated with reference phospholipids belonging to phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) classes. NP-HPLC- and RP-HPLC-ESI-MS/MS were applied to yeast phospholipidome analysis, using a wild-type strain and two strains defective for acyltransferases that are known to be involved in de novo phospholipid synthesis or phospholipid remodeling. The MRM mode was used for relative quantitation of individual compounds based on reference phospholipids bearing fatty acid chains with an odd number of carbon atoms. Combined LC-MS/MS was found superior to shotgun analysis, leading to a larger number of quantified species than shotgun analysis. Finally, RP-HPLC-MS/MS was the preferred method for its higher selectivity, robustness, and better repeatability.

Enzymatic synthesis of phosphatidylserine using bile salt mixed micelles.

Phosphatidylserine (PS) rich in polyunsaturated fatty acids of the n-3 series was obtained by enzymatic synthesis with phospholipase D (PLD) and a marine lipid extract as substrate. Synthesis was performed using mixed micelles composed of either sodium deoxycholate (SDC) or sodium cholate (SC). To limit the use of surfactant and to monitor the performance of PLD, the mixed micelles were characterized both in terms of bile salt/lipid molar ratio in the aggregates and of mean diameter. A fractional factorial experiment was selected to study the effect of pH, temperature, enzyme, L-serine concentrations, bile salt/lipid molar ratio and Ca(2+) content (in the case of SC only) on PS synthesis. The amount of L-serine was the main factor governing the equilibrium between transphosphatidylation and hydrolysis reaction. Increasing the bile salt/lipid molar ratio decreased PS synthesis yield. In contrast, pH (6.5-8) and temperature (35-45°C) did not affect PLD activity in the tested conditions. This statistical approach allowed determining a combination of parameters (pH, temperature, bile salt/lipid molar ratio, enzyme and alcohol acceptor concentrations) for PS synthesis. After 24 h, the transphosphatidylation reaction led to 57±2% and 56±3% of PS in the phospholipid mixtures with SDC and SC, respectively. In both cases, about 10% of phosphatidic acid was present as a side-product. On the whole, this work provided fundamental basis for a possible development of enzymatic PLD technology using food-grade emulsifiers to produce PS complying with industrial constraints for nutritional applications.

Synthesis and analytical investigation of C-terminally modified peptide aldehydes and ketone: application to oxime ligation

C‐terminally modified peptides aldehyde (glycinal and alpha‐oxo aldehyde peptides) and ketone (pyruvic acid‐containing peptide) were synthesised to get new insights into the mechanism of acido‐catalysed oxime ligation. Their tetrahedral hydrated forms were investigated in solution and in the gas phase, using NMR and in‐source collision‐induced dissociation mass spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed that the first step of the oximation reaction was the limiting step for the pyruvic acid‐containing peptides because of the steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha‐oxo aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime reactions are performed in aqueous buffer. Copyright

Ion/molecule reactions with dimethyl ether and dimethyl-d6 ether: differentiation of four isomers contained in patchouli essential oil

In this study, we show that it is possible to differentiate four sesquiterpene isomers (C(15)H(24)) preliminarily separated by gas chromatography/mass spectrometry (GC/MS). Dimethyl ether is evaluated as a selective ionization reagent and the relative abundance of adducts formed with this reagent gas under positive chemical ionization conditions are compared and adduct ions are characterized using collision-induced dissociation. The mechanisms have been confirmed by achieving the same experiments with deuterated dimethyl ether. Copyright 2000 John Wiley & Sons, Ltd.

Liver Reptin/RUVBL2 controls glucose and lipid metabolism with opposite actions on mTORC1 and mTORC2 signalling

Objective The AAA+ ATPase Reptin is overexpressed in hepatocellular carcinoma and preclinical studies indicate that it could be a relevant therapeutic target. However, its physiological and pathophysiological roles in vivo remain unknown. This study aimed to determine the role of Reptin in mammalian adult liver. Design and results We generated an inducible liver-specific Reptin knockout (RepinLKO ) mouse model. Following Reptin invalidation, mice displayed decreased body and fat mass, hypoglycaemia and hypolipidaemia. This was associated with decreased hepatic mTOR protein abundance. Further experiments in primary hepatocytes demonstrated that Reptin maintains mTOR protein level through its ATPase activity. Unexpectedly, loss or inhibition of Reptin induced an opposite effect on mTORC1 and mTORC2 signalling, with: (1) strong inhibition of hepatic mTORC1 activity, likely responsible for the reduction of hepatocytes cell size, for decreased de novo lipogenesis and cholesterol transcriptional programmes and (2) enhancement of mTORC2 activity associated with inhibition of the gluconeogenesis transcriptional programme and hepatic glucose production. Consequently, the role of hepatic Reptin in the pathogenesis of insulin resistance (IR) and non-alcoholic fatty liver disease consecutive to a high-fat diet was investigated. We found that Reptin deletion completely rescued pathological phenotypes associated with IR, including glucose intolerance, hyperglycaemia, hyperlipidaemia and hepatic steatosis. Conclusion We show here that the AAA +ATPase Reptin is a regulator of mTOR signalling in the liver and global glucido-lipidic homeostasis. Inhibition of hepatic Reptin expression or activity represents a new therapeutic perspective for metabolic syndrome.

Contribution of the precursors and interplay of the pathways in the phospholipid metabolism of the malaria parasite

The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches.

Caveolae-mediated effects of TNF-α on human skeletal muscle cells

&NA; Chronic diseases are characterized by the production of pro‐inflammatory cytokines such than TNF‐&agr; and are frequently correlated with muscle wasting conditions. Among the pleiotropic effects of TNF‐&agr; within the cell, its binding to TNFR1 receptor has been shown to activate sphingomyelinases leading to the production of ceramides. Sphingomyelinases and TNF receptor have been localized within caveolae which are specialized RAFT enriched in cholesterol and sphingolipids. Because of their inverted omega shape, maintained by the oligomerization of specialized proteins, caveolins and cavins, caveolae serve as membrane reservoir therefore providing mechanical protection to plasma membranes. Although sphingolipids metabolites, caveolins and TNF‐&agr;/TNFR1 have been shown to independently interfere with muscle physiology, no data have clearly demonstrated their concerted action on muscle cell regeneration. In this context, our study aimed at studying the molecular mechanisms induced by TNF‐&agr; at the level of caveolae in LHCN‐M2 human muscle satellite cells. Here we showed that TNF‐&agr;‐induced production of ROS and nSMase activation requires caveolin. More strikingly, we have demonstrated that TNF‐&agr; induces the formation of additional caveolae at the plasma membrane of myoblasts. Furthermore, TNF‐&agr; prevents myoblast fusion suggesting that inflammation could modulate caveolae organization/function and satellite cell function. HighlightsTNF‐&agr; promotes the formation of additional caveolae at the plasma membrane of human myoblasts.Enhancement of nSMase activity and ROS production by TNF‐&agr; is caveolin1‐dependent.TNFR1 is localized in caveolae of human satellite cells.