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Accueil > Départements > Biochimie, Biophysique et Biologie Structurale > Francis HARAUX : Laboratoire des Protéines et Systèmes Membranaires

Publications de l’équipe

2017



  • H. Azouaoui, C. Montigny, T. Dieudonné, P. Champeil, A. Jacquot, J. L. Vázquez-Ibar, P. Le Maréchal, J. Ulstrup, M. - R. Ash, J. A. Lyons, P. Nissen, et G. Lenoir, « A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N- and C-terminal Extensions », Journal of Biological Chemistry, p. jbc.M116.751487, mars 2017.
    Mots-clés : autophosphorylation, B3S, Cdc50 protein, Flippase, inhibition mechanism, limited proteolysis, lipid-protein interaction, LPSM, phosphatidylserine, phosphoinositide.


  • V. Chaptal, F. Delolme, A. Kilburg, S. Magnard, C. Montigny, M. Picard, C. Prier, L. Monticelli, O. Bornert, M. Agez, S. Ravaud, C. Orelle, R. Wagner, A. Jawhari, I. Broutin, E. Pebay-Peyroula, J. - M. Jault, H. R. Kaback, M. le Maire, et P. Falson, « Quantification of Detergents Complexed with Membrane Proteins », Scientific Reports, vol. 7, p. 41751, févr. 2017.

  • E. Errasti-Murugarren, A. Rodríguez-Banqueri, et J. L. Vázquez-Ibar, « Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter », Methods in Molecular Biology (Clifton, N.J.), vol. 1586, p. 181-195, 2017.
    Résumé : Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.
    Mots-clés : B3S, FSEC, Heterologous expression, LAT, LPSM, Membrane Transport Proteins, Split GFP, SteT.


  • C. Montigny, T. Dieudonné, S. Orlowski, J. L. Vázquez-Ibar, C. Gauron, D. Georgin, S. Lund, M. le Maire, J. V. Møller, P. Champeil, et G. Lenoir, « Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics », PLOS ONE, vol. 12, nᵒ 1, p. e0170481, janv. 2017.

  • L. Negroni, M. Zivy, et C. Lemaire, « Mass Spectrometry of Mitochondrial Membrane Protein Complexes », Methods in Molecular Biology (Clifton, N.J.), vol. 1635, p. 233-246, 2017.
    Résumé : The ATP production (oxidative phosphorylation) involves five complexes embedded in the inner membrane of mitochondria. The yeast Saccharomyces cerevisiae is mainly used as a model for the study of oxidative phosphorylation; mutants are easy to produce and are still viable due to their ability to grow using the fermentation pathway. Here, we present a process for analyzing mitochondrial respiratory complexes using native electrophoresis (BN-PAGE) coupled to LC-MS/MS. BN-PAGE (1) permits the separation of functional respiratory complexes, thus allowing in-gel activity detection of most of the respiratory complexes and (2) provides convenient samples for bottom-up proteomics. Combining BN-PAGE and LC-MS/MS leads to the identification of the subunit composition of membrane complexes and offers the possibility of highlighting potential interacting proteins.
    Mots-clés : B3S, BN-PAGE, LPSM, Mass Spectrometry, OXPHOS, Respiratory complexes, S. cerevisiae.

  • M. Renvoisé, L. Bonhomme, M. Davanture, M. Zivy, et C. Lemaire, « Phosphoproteomic Analysis of Isolated Mitochondria in Yeast », Methods in Molecular Biology (Clifton, N.J.), vol. 1636, p. 283-299, 2017.
    Résumé : Mitochondria play a central role in cellular energy metabolism and cell death. Deregulation of mitochondrial functions is associated with several human pathologies (neurodegenerative diseases, neuromuscular diseases, type II diabetes, obesity, cancer). The steadily increasing number of identified mitochondrial phosphoproteins, kinases, and phosphatases in recent years suggests that reversible protein phosphorylation plays an important part in the control of mitochondrial processes. In addition, many mitochondrial phosphoproteins probably still remain to be identified, considering that 30% of proteins are expected to be phosphorylated in eukaryotes. In this chapter, we describe two procedures for the analysis of the mitochondrial phosphoproteome. The first one is a qualitative method that combines blue native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-BN/SDS-PAGE) and specific phosphoprotein staining. The second one is a quantitative approach that associates mitochondrial peptide labeling, phosphopeptide enrichment, and mass spectrometry.
    Mots-clés : B3S, LPSM, Mitochondria, Phosphoproteome, Saccharomyces cerevisiae.

2016


  • H. Azouaoui, C. Montigny, A. Jacquot, R. Barry, P. Champeil, et G. Lenoir, « Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex », Methods in Molecular Biology (Clifton, N.J.), vol. 1377, p. 37-55, 2016.
    Résumé : Structural and functional characterization of integral membrane proteins requires milligram amounts of purified sample. Unless the protein you are studying is abundant in native membranes, it will be critical to overexpress the protein of interest in a homologous or heterologous way, and in sufficient quantities for further purification. The situation may become even more complicated if you chose to investigate the structure and function of a complex of two or more membrane proteins. Here, we describe the overexpression of a yeast lipid flippase complex, namely the P4-ATPase Drs2p and its associated subunit Cdc50p, in a coordinated manner. Moreover, we can take advantage of the fact that P4-ATPases, like most other P-type ATPases, form an acid-stable phosphorylated intermediate, to verify that the expressed complex is functional.
    Mots-clés : B3S, Calcium-Transporting ATPases, Cdc50 protein, Co-expression, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Lipid transport, LPSM, Membrane protein, Membrane Proteins, Multiprotein Complexes, P4-ATPase, Phospholipids, Phosphorylation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Yeast.


  • P. Champeil, S. Orlowski, S. Babin, S. Lund, M. le Maire, J. Møller, G. Lenoir, et C. Montigny, « A robust method to screen detergents for membrane protein stabilization, revisited », Analytical Biochemistry, vol. 511, p. 31-35, 2016.
    Mots-clés : B3S, Detergent, Lipid, LMNG, LPSM, Membrane protein, SERCA1a, stability.


  • M. A. David, S. Orlowski, R. K. Prichard, S. Hashem, F. André, et A. Lespine, « In silico analysis of the binding of anthelmintics to Caenorhabditis elegans P-glycoprotein 1 », International Journal for Parasitology: Drugs and Drug Resistance, vol. 6, nᵒ 3, p. 299-313, 2016.


  • S. David-Bosne, M. V. Clausen, H. Poulsen, J. V. Møller, P. Nissen, et M. le Maire, « Reappraising the effects of artemisinin on the ATPase activity of PfATP6 and SERCA1a E255L expressed in Xenopus laevis oocytes », Nature Structural & Molecular Biology, vol. 23, nᵒ 1, p. 1-2, janv. 2016.


  • S. David-Bosne, M. V. Clausen, H. Poulsen, J. V. Møller, P. Nissen, et M. le Maire, « Erratum: Reappraising the effects of artemisinin on the ATPase activity of PfATP6 and SERCA1a E255L expressed in Xenopus laevis oocytes », Nature Structural & Molecular Biology, vol. 23, nᵒ 4, p. 358-358, avr. 2016.


  • N. Le Breton, T. Adrianaivomananjaona, G. Gerbaud, E. Etienne, E. Bisetto, A. Dautant, B. Guigliarelli, F. Haraux, M. Martinho, et V. Belle, « Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy », Biochimica et Biophysica Acta (BBA) - Bioenergetics, vol. 1857, nᵒ 1, p. 89-97, 2016.


  • C. Montigny, J. Lyons, P. Champeil, P. Nissen, et G. Lenoir, « On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport », Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1861, nᵒ 8, p. 767-783, 2016.

2015



  • P. Falson, B. Bartosch, K. Alsaleh, B. A. Tews, A. Loquet, Y. Ciczora, L. Riva, C. Montigny, C. Montpellier, G. Duverlie, E. - I. Pécheur, M. le Maire, F. - L. Cosset, J. Dubuisson, et F. Penin, « Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion », Journal of Virology, vol. 89, nᵒ 20, p. 10333-10346, oct. 2015.


  • G. Gaibelet, S. Allart, F. Tercé, V. Azalbert, J. Bertrand-Michel, S. Hamdi, X. Collet, et S. Orlowski, « Specific Cellular Incorporation of a Pyrene-Labelled Cholesterol: Lipoprotein-Mediated Delivery toward Ordered Intracellular Membranes », PLOS ONE, vol. 10, nᵒ 4, p. e0121563, avr. 2015.


  • L. Huynh, N. Perrot, V. Beswick, V. Rosilio, P. A. Curmi, A. Sanson, et N. Jamin, « Reply to “Comment on ‘Structural Properties of POPC Monolayers under Lateral Compression: Computer Simulations Analysis’” », Langmuir, vol. 31, nᵒ 2, p. 888-889, janv. 2015.


  • M. - F. Lecompte, G. Gaibelet, C. Lebrun, F. Tercé, X. Collet, et S. Orlowski, « Cholesterol and Sphingomyelin-Containing Model Condensed Lipid Monolayers: Heterogeneities Involving Ordered Microdomains Assessed by Two Cholesterol Derivatives », Langmuir, vol. 31, nᵒ 43, p. 11921-11931, nov. 2015.

  • P. Nicole, P. Couvineau, N. Jamin, T. Voisin, et A. Couvineau, « Crucial role of the orexin-B C-terminus in the induction of OX1 receptor-mediated apoptosis: analysis by alanine scanning, molecular modelling and site-directed mutagenesis », British Journal of Pharmacology, vol. 172, nᵒ 21, p. 5211-5223, 2015.
    Résumé : BACKGROUND AND PURPOSE: Orexins (A and B) are hypothalamic peptides that interact with OX1 and OX2 receptors and are involved in the sleep/wake cycle. We previously demonstrated that OX1 receptors are highly expressed in colon cancer tumours and colonic cancer cell lines where orexins induce apoptosis and inhibit tumour growth in preclinical animal models. The present study explored the structure-function relationships of orexin-B and OX1 receptors. EXPERIMENTAL APPROACH: The contribution of all orexin-B residues in orexin-B-induced apoptosis was investigated by alanine scanning. To determine which OX1 receptor domains are involved in orexin-B binding and apoptosis, a 3D model of OX1 receptor docked to the orexin-B C-terminus (AA-20-28) was developed. Substitution of residues present in OX1 receptor transmembrane (TM) domains by site-directed mutagenesis was performed. KEY RESULTS: Alanine substitution of orexin-B residues, L(11) , L(15) , A(22) , G(24) , I(25) , L(26) and M(28) , altered orexin-B's binding affinity. Substitution of these residues and of the Q(16) , A(17) , S(18) , N(20) and T(27) residues inhibited apoptosis in CHO-S-OX1 receptor cells. The K(120) , P(123) , Y(124) , N(318) , K(321) , F(340) , T(341) , H(344) and W(345) residues localized in TM2, TM3, TM6 and TM7 of OX1 receptors were shown to play a role in orexin-B recognition and orexin-B/OX1 receptor-induced apoptosis. CONCLUSIONS AND IMPLICATIONS: The C-terminus of orexin-B (i) plays an important role in its pro-apoptotic effect; and (ii) interacts with some residues localized in the OX1 receptor TM. This study defines the structure-function relationship for orexin-B recognition by human OX1 receptors and orexin-B/OX1 receptor-induced apoptosis, an important step for the future development of new agonist molecules.
    Mots-clés : Alanine, Animals, Apoptosis, B3S, CHO Cells, Cricetinae, Cricetulus, Humans, LPSM, Models, Molecular, Mutagenesis, Site-Directed, Orexin Receptors, Orexins, Structure-Activity Relationship.
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Publications Principales avant 2015

- Huynh L, Perrot N, Beswick V, Rosilio V, Curmi PA, Jamin N. (2014) Structural properties of POPC monolayers under lateral compression : computer simulations analysis. Langmuir 30 : 564-573
Renvoisé M, Bonhomme L, Davanture M, Valot B, Zivy M, Lemaire C. (2014) Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiae. J proteomics 25, 140-150
- Wu Q, Andrianaivomananjaona T, Tetaud E, Corvest V, Haraux F (2014) Interactions involved in grasping and locking of the inhibitory peptide IF1 by mitochondrial ATP synthase. Biochim Biophys Acta 1837 : 761-772
- Berrier C, Pozza A, de Lacroix de Lavalette A, Chardonnet S, Mesneau A, Jaxel C, le Maire M, Ghazi A (2013) The purified mechanosensitive channel TREK-1 is directly sensitive to membrane tension. J Biol Chem 288 : 27307-27314
- Clausen JD, Bublitz M, Arnou B, Montigny C, Jaxel C, Møller JV, Nissen P, Andersen JP, le Maire M. (2013) SERCA mutant E309Q binds two Ca(2+) ions but adopts a catalytically incompetent conformation. EMBO J 32, 3231-3243
- David-Bosne S, Florent I, Lund-Winther AM, Bondo-Hansen J, Buch-Pedersen M, Machillot P, le Maire M, Jaxel C (2013) Antimalarial screening via large-scale purification of PfATP6 and in vitro studies. FEBS J 280 : 5419-5429
- Galvagnion C, Montaville P, Coïc YM, Jamin N (2013) Production and initial structural characterization of the TM4TM5 helix-loop-helix domain of the translocator protein. J Peptide Sci 19 : 102-109
- Roux M, Sternin E, Bonnet V, Fajolles C, Djedaíni-Pilard F (2013) Dynamic lipid lateral segregation driven by lauryl cyclodextrin interactions at the membrane surface. Langmuir 29 : 3677-3687
- Jacquot A , Montigny C, Hennrich H, Barry R, le Maire M, Jaxel C, Holthuis J, Champeil P, Lenoir G (2012) Phosphatidylserine stimulation of Drs2p•Cdc50p lipid translocase dephosphorylation is controlled by phosphatidylinositol-4-phosphate. J Biol Chem 287 : 13249-13261
- Beswick V, Isvoran A, Nédellec P, Sanson A, Jamin N (2011) Membrane Interface Composition Drives the Structure and the Tilt of the Single Transmembrane Helix Protein PMP1 : MD Studies. Biophys J 100 : 1660-1667
- de Foresta B, Vincent M, Garrigos M, Gallay J (2011) Transverse and tangential orientation of predicted transmembrane fragments 4 and 10 from the human multidrug resistance protein (hMRP1/ABCC1) in membrane mimics. Eur Biophys J 40 : 1043-1060
- Cardi D, Pozza A, Arnou B, Marchal E, Clausen JD, Andersen JP, Krishna S, Møller JV, le Maire M, Jaxel C (2010) Purified E255L mutant SERCA1a and purified PfATP6 are sensitive to SERCA-type inhibitors but insensitive to artemisinins. J Biol Chem 285 : 26406-26416

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