Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

The yeast Kes1p/Osh4p protein functions as a sterol/PI(4)P exchanger between lipid membranes, which suggests the possibility of creating a sterol gradient via phosphoinositide metabolism

α-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding

Two membrane curvature–sensing molecules with opposite chemistries are targeted to distinct vesicle classes through direct interaction with different lipid environments

La reconnaissance de la courbure membranaire par ArfGAP1

The COPI coat drives the budding of small vesicle from Golgi apparatus. After being recruited by membrane-bound Arf1-GTP, the COPI coat subunits (Coatomer) polymerise, and shape mechanically the lipid membrane into a spherical structure. Then, GTP hydrolysis in Arf1 is required for the disassembly of the COPI coat, but this reaction must be finely tuned to permit coat disassembly only after vesicle formation. ArfGAP1 is a cytosolic protein that associates in dynamic manner with the Golgi apparatus, and controls the cycling of COPI coat on membranes by catalyzing GTP hydrolysis on Arf1. Results from our lab indicated that the activity of ArfGAP1 is highly sensitive to membrane curvature. Thus a negative feed-back loop was suggested, where COPI assembly drives membrane curvature, which in turn prepares the coat for disassembly by recruiting ArfGAP1, thereby promoting GTP hydrolysis in Arf1.During my thesis, I found that two motifs in the central region of ArfGAP1 act as lipid-packing sensors and help anchor ArfGAP1 at the surface of highly curved membrane. These “ALPS” motifs, which are not structured in solution, adopt an amphipathic helical structure on curved membranes. These helices contrast from classical membrane-adsorbing helices in the abundance of serine and threonine residues and the paucity of charged residues in their polar faces. Because these residues cannot make electrostatic interaction with lipid polar heads, the adsorption of ALPS motifs is driven solely by the insertion of hydrophobic residues between lipids and is thus strongly favored by the creation of lipid packing defects such as those introduced by high membrane curvature.La formation d'un bourgeon vésiculaire repose sur une machinerie complexe qui déforme, par une action mécanique, une membrane plane en une membrane courbée. Le manteau COPI, responsable de ce phénomène dans le Golgi, se polymérise latéralement à la surface de la membrane, sous le contrôle de la petite protéine G Arf1 activée. Suite à la formation de la vésicule, Arf1 revient à l'état inactif par hydrolyse de son GTP et se dissocie de la membrane, provoquant alors le désassemblage du manteau. Cette réaction d'hydrolyse doit être finement régulée, pour ne pas intervenir trop tôt et compromettre l'assemblage du manteau. Notre laboratoire a révélé que l'activité de la protéine ArfGAP1, responsable de la désactivation d'Arf1, est hypersensible à la courbure membranaire. Ceci permettrait à ArfGAP1 de réguler de manière spatio-temporelle l'état du manteau COPI en couplant son désassemblage à la courbure membranaire qu'il a lui-même induite.Au cours de ma thèse, j'ai montré que la dépendance d'ArfGAP1 à la courbure s'explique par la présence dans cette protéine de deux motifs « ALPS », qui se replient en hélices alpha lors de leur adsorption membranaire. Ce sont des hélices amphipathiques atypiques car, si elles possèdent une face hydrophobe classique, elles ont une face polaire riche en sérine et thréonine et pauvre en résidus chargés. Puisque ces résidus hydroxylés ne peuvent interagir avec les têtes polaires des lipides, la liaison d'un motif ALPS ne repose que sur l'insertion de ses résidus hydrophobes entre les lipides, ce qui est favorisé par l'écartement lipidique induit par la courbure membranaire, mais plus difficile sur membrane plane où les lipides sont plus compactés

Un marché d’échange de lipides

Le cholestérol est synthétisé dans le réticulum endoplasmique (RE) puis transporté vers les compartiments cellulaires dont la fonction en nécessite un taux élevé. Nous décrivons ici le mécanisme de transport du cholestérol du RE vers le réseau trans golgien (TGN) par la protéine OSBP (oxysterol binding protein). Celle-ci présente deux activités complémentaires : elle arrime les deux compartiments, RE et TGN, en formant un site de contact où les deux membranes sont à une vingtaine de nanomètres de distance ; puis elle échange le cholestérol du RE avec un lipide présent dans le TGN, le phosphatidylinositol 4-phosphate (PI4P). Dans le RE, le PI4P est hydrolysé, rendant le cycle d’échange irréversible. OSBP est donc au cœur d’un marché d’échange de lipides dans lequel un cholestérol transporté « coûte » un PI4P. Des molécules à activités antivirales ou anticancéreuses ont pour cible OSBP, suggérant une importance dans différents contextes physiopathologiques du cycle d’OSBP, dont les bases générales sont partagées par d’autres protéines transporteurs de lipides

Lipid exchange and signaling at ER–Golgi contact sites

Meticulous observations of the cell perinuclear region where the Golgi and the endoplasmic reticulum (ER) networks intermingle have revealed close contact sites of barely 20 nm between these two organelles. Recent studies demonstrate that molecular machineries, including lipid-transfer proteins, enriched in membrane contact sites between ER and trans-Golgi are capable of bridging membranes and exchanging key lipids such as sphingolipid precursors and cholesterol while bypassing the early secretory compartments. This occurs at the cost of an intense phosphoinositide turnover in order to prepare a membrane environment conducive to the signaling and trafficking functions of the trans-Golgi network. A tight control operates in the contact zone to adjust lipid transport and metabolism to the cellular needs

Improvement of the Performance of Targeted LC–MS Assays through Enrichment of Histidine-Containing Peptides

Mass spectrometric-based quantification using targeted methods has matured during the past decade and is now commonly used in proteomics. However, the reliability of protein quantification in complex matrixes using selected reaction monitoring is often impaired by interfering signals arising from coelution of nontargeted components. Sample preparation methods resulting in the reduction of the number of peptides present in the mixture minimizes this effect. One solution consists in the selective capture of peptides containing infrequent amino acids. The enrichment of histidine-containing peptides via immobilized metal-ion affinity chromatography loaded with Cu²⁺ ions (IMAC-Cu) was applied in a quantitative workflow and found to be a simple and cost effective method for the reduction of sample complexity with high recovery and selectivity. When applied to a series of depleted human plasma digests, the method decreased nonspecific signals, resulting in a more precise and robust protein quantification. The method was also shown to be an alternative to HSA/IgG depletion during plasma protein analysis. This method, used in conjunction with recent improvements in the instrument’s peak capacity, addresses a bottleneck generally encountered in quantitative proteomics studies by providing the robustness and throughput required for the analysis of large sample series without compromising the number of proteins monitored

Improvement of the Performance of Targeted LC–MS Assays through Enrichment of Histidine-Containing Peptides

Mass spectrometric-based quantification using targeted methods has matured during the past decade and is now commonly used in proteomics. However, the reliability of protein quantification in complex matrixes using selected reaction monitoring is often impaired by interfering signals arising from coelution of nontargeted components. Sample preparation methods resulting in the reduction of the number of peptides present in the mixture minimizes this effect. One solution consists in the selective capture of peptides containing infrequent amino acids. The enrichment of histidine-containing peptides via immobilized metal-ion affinity chromatography loaded with Cu²⁺ ions (IMAC-Cu) was applied in a quantitative workflow and found to be a simple and cost effective method for the reduction of sample complexity with high recovery and selectivity. When applied to a series of depleted human plasma digests, the method decreased nonspecific signals, resulting in a more precise and robust protein quantification. The method was also shown to be an alternative to HSA/IgG depletion during plasma protein analysis. This method, used in conjunction with recent improvements in the instrument’s peak capacity, addresses a bottleneck generally encountered in quantitative proteomics studies by providing the robustness and throughput required for the analysis of large sample series without compromising the number of proteins monitored

: A Tale of Resemblance and Contrast Between VAP Proteins

When considering the vesicle-associated membrane protein-associated protein (VAP) family, major receptors at the surface of the endoplasmic reticulum (ER), it appears that VAP-A and VAP-B paralogs largely overlap in structure and function, and that specific features to distinguish these two proteins hardly exist or are poorly documented. Here, we question the degree of redundancy between VAP-A and VAP-B: is one simply a backup plan, in case of loss of function of one of the two genes, or are there molecular and functional divergences that would explain their maintenance during evolution

Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP

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