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Cell polarity is achieved by regulators such as small G proteins, exocyst members and phosphoinositides, with the latter playing a key role when bound to the exocyst proteins Sec3p and Exo70p, and Rho GTPases. This ensures asymmetric growth via the routing of proteins and lipids to the cell surface using actin cables. Previously, using a yeast mutant for a lysophosphatidylinositol acyl transferase encoded by the PSI1 gene, we demonstrated the role of stearic acid in the acyl chain of phosphoinositides in cytoskeletal organization and secretion. Here, we use a genetic approach to characterize the effect on late steps of the secretory pathway. The constitutive overexpression of PSI1 in mutants affecting kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane, which is essential for the function of the Cdc42p module. Decreasing the levels of stearic acid-containing phosphoinositides modifies the environment of the actors involved in the control of late steps in the secretory pathway. This leads to decreased interactions between Exo70p and Sec3p, with Cdc42p, Rho1p and Rho3p, because of disruption of the GTP/GDP ratio of at least Rho1p and Rho3p GTPases, thereby preventing activation of the exocyst

Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells

Cardiolipin (CL) optimizes diverse mitochondrial processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase Tafazzin (TAZ). Loss-of-function mutations in the TAZ gene are responsible for the Barth syndrome (BTHS), a rare X-linked cardiomyopathy, presumably because of a diminished OXPHOS capacity. Herein we show that a partial inhibition of cytosolic protein synthesis, either chemically with the use of cycloheximide or by specific genetic mutations, fully restores biogenesis and the activity of the oxidative phosphorylation system in a yeast BTHS model (taz1Δ). Interestingly, the defaults in CL were not suppressed, indicating that they are not primarily responsible for the OXPHOS deficiency in taz1Δ yeast. Low concentrations of cycloheximide in the picomolar range were beneficial to TAZ-deficient HeLa cells, as evidenced by the recovery of a good proliferative capacity. These findings reveal that a diminished capacity of CL remodeling deficient cells to preserve protein homeostasis is likely an important factor contributing to the pathogenesis of BTHS. This in turn, identifies cytosolic translation as a potential therapeutic target for the treatment of this disease

Shotgun lipidomics and mass spectrometry imaging unveil diversity and dynamics in Gammarus fossarum lipid composition

Sentinel species are playing an indispensable role in monitoring environmental pollution in aquatic ecosystems. Many pollutants found in water prove to be endocrine disrupting chemicals that could cause disruptions in lipid homeostasis in aquatic species. A comprehensive profiling of the lipidome of these species is thus an essential step toward understanding the mechanism of toxicity induced by pollutants. Both the composition and spatial distribution of lipids in freshwater crustacean Gammarus fossarum were extensively examined herein. The baseline lipidome of gammarids of different sex and reproductive stages was established by high throughput shotgun lipidomics. Spatial lipid mapping by high resolution mass spectrometry imaging led to the discovery of sulfate-based lipids in hepato-pancreas and their accumulation in mature oocytes. A diverse and dynamic lipid composition in G. fossarum was uncovered, which deepens our understanding of the biochemical changes during development and which could serve as a reference for future ecotoxicological studies.Approches Protéomique et Lipidomique pour la compréhension des mécanismes moléculaires de toxicité en lien avec l'altération du métabolisme lipidique chez l'espèce sentinelle Gammarus fossarum durant le cycle de reproductio

Caractérisation de lysolipide acyltransférases chez S. cerevisiae - Apport de la Spectrométrie de Masse

En plus de leurs propriétés structurales comme constituants majeurs des membranes biologiques des cellules, les lipides jouent de nombreux rôles dans la signalisation cellulaire, le stockage d énergie et le transport de protéines. Leurs importances biologiques ont mené à une augmentation accrue des méthodes analytiques pour la caractérisation d espèces moléculaires uniques. De récents progrès en spectrométrie de masse ont amené à la caractérisation et à la quantification des espèces moléculaires des lipides dans des extraits lipidiques bruts (Han and Gross, 2005; Murphy et al., 2001). Par exemple, les espèces moléculaires de phospholipides peuvent être identifiées spécifiquement par leur tête polaire, la nature de leurs chaînes d acide gras et leur positionnement au niveau du squelette glycérol.In addition to their structural properties as main constituents of biological membranes, lipids play a multitude of roles such as in cell signalling, energy storage, and protein transport. Their biological importance has led to an increasing focus on analytical methods for the characterisation of their individual molecular species. Improvements in mass spectrometric technology has provided a great advantage for the characterisation and quantification of molecular lipid species in total lipid extracts (Han and Gross, 2005; Murphy et al., 2001). For instance, phospholipid molecular species can be identified on the basis of a characteristic fragment of the lipid class, the nature of the acyl chains and their positions on the glycerol backbone.A method allowing the quantitative profiling of the yeast lipidome was developed in a recent study using automated shotgun infusion strategy (Ejsing et al., 2009). We applied this method to characterise several lysophospholipid acyltransferase yeast mutants produced using reverse-genetics. These enzymes are involved in essential biological processes like de novo synthesis or remodelling of the phospholipid membrane component (Testet et al., 2005; Le Guedard et al., 2009). The comparative analysis of phospholipid molecular species from the wild-type strain and the corresponding deletion mutants has allowed us to identify lipid compositional changes, and has given us significant indications about the in vivo function of the encoded lysophospholipid acyltransferases.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

Identification et caractérisation de quatre lysolipide acyltransférases de S. cerevisiae (études in silico, in vitro et in vivo)

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Utilisation des enzymes en microemulsions : applications aux processus d'esterification

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

A single run LC-MS/MS method for phospholipidomics

International audienc

Overexpression of mitochondrial oxodicarboxylate carrier (ODC1) preserves oxidative phosphorylation in a yeast model of Barth syndrome

Cardiolipin (CL) is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS), presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1.) of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1. yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS

Overexpression of mitochondrial oxodicarboxylate carrier (ODC1) preserves oxidative phosphorylation in a yeast model of Barth syndrome

Cardiolipin (CL) is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS), presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1.) of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1. yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS