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

Publications de l’équipe


  • M. David, C. Lebrun, T. Duguet, F. Talmont, R. Beech, S. Orlowski, F. André, R. K. Prichard, et A. Lespine, « Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13 », International Journal for Parasitology. Drugs and Drug Resistance, vol. 8, nᵒ 1, p. 145-157, avr. 2018.
    Résumé : Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.
    Mots-clés : ABC transporters, B3S, Haemonchus contortus, Homology modeling, Ivermectin, LPSM, LSOD, Nematode, P-glycoprotein.


  • A. M. Acuña, C. Lemaire, R. van Grondelle, B. Robert, et I. H. M. van Stokkum, « Energy transfer and trapping in Synechococcus WH 7803 », Photosynthesis Research, oct. 2017.
    Résumé : Excitation energy transfer (EET) and trapping in Synechococcus WH 7803 whole cells and isolated photosystem I (PSI) complexes have been studied by time-resolved emission spectroscopy at room temperature (RT) and at 77 K. With the help of global and target analysis, the pathways of EET and the charge separation dynamics have been identified. Energy absorbed in the phycobilisome (PB) rods by the abundant phycoerythrin (PE) is funneled to phycocyanin (PC645) and from there to the core that contains allophycocyanin (APC660 and APC680). Intra-PB EET rates have been estimated to range from 11 to 68/ns. It was estimated that at RT, the terminal emitter of the phycobilisome, APC680, transfers its energy at a rate of 90/ns to PSI and at a rate of 50/ns to PSII. At 77 K, the redshifted Chl a states in the PSI core were heterogeneous, with maximum emission at 697 and 707 nm. In 72% of the PSI complexes, the bulk Chl a in equilibrium with F697 decayed with a main trapping lifetime of 39 ps.
    Mots-clés : B3S, Excitation energy transfer, Global analysis, LBMS, Light harvesting, LPSM, Target analysis.

  • 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.

  • 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 », in Heterologous Gene Expression in E.coli, vol. 1586, N. A. Burgess-Brown, Éd. New York, NY: Springer New York, 2017, p. 181-195.

  • 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.

  • G. Gaibelet, F. Tercé, S. Allart, C. Lebrun, X. Collet, N. Jamin, et S. Orlowski, « Fluorescent probes for detecting cholesterol-rich ordered membrane microdomains: entangled relationships between structural analogies in the membrane and functional homologies in the cell », AIMS Biophysics, vol. 4, nᵒ 1, p. 121-151, 2017.

  • L. Negroni, M. Zivy, et C. Lemaire, « Mass Spectrometry of Mitochondrial Membrane Protein Complexes », in Membrane Protein Structure and Function Characterization, vol. 1635, J. - J. Lacapere, Éd. New York, NY: Springer New York, 2017, p. 233-246.

  • 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.

  • F. Muzzopappa, A. Wilson, V. Yogarajah, S. Cot, F. Perreau, C. Montigny, C. Bourcier de Carbon, et D. Kirilovsky, « The paralogs to the C-terminal domain of the cyanobacterial OCP are carotenoid donors to HCPs », Plant Physiology, sept. 2017.
    Résumé : The photoactive Orange Carotenoid Protein photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain (NTD) is the active part of the protein and the C-terminal domain (CTD) regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins or HCPs), paralogs of NTD-OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized and proved to be very good singlet oxygen quenchers. When synthesized in E. coli or Synechocystis PCC 6803, CTDHs form dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable. HCPs need the presence of CTDH to become holo-proteins. We propose that in cyanobacteria the CTDHs are carotenoid donors to HCPs.
    Mots-clés : B3S, LPSM, MROP.


  • 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.

  • S. Bakari, M. Lembrouk, L. Sourd, F. Ousalem, F. André, S. Orlowski, M. Delaforge, et A. Frelet-Barrand, « Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1 », Molecular Biotechnology, vol. 58, nᵒ 4, p. 299-310, 2016.
    Mots-clés : B3S, DBG, LPSM, LSOD, PEPS.

  • T. Barbot, C. Montigny, P. Decottignies, M. le Maire, C. Jaxel, N. Jamin, et V. Beswick, « Functional and Structural Insights into Sarcolipin, a Regulator of the Sarco-Endoplasmic Reticulum Ca2+-ATPases », in Regulation of Ca2+-ATPases,V-ATPases and F-ATPases, S. Chakraborti et N. S. Dhalla, Éd. Cham: Springer International Publishing, 2016, p. 153-186.

  • 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.

  • A. Rodríguez-Banqueri, E. Errasti-Murugarren, P. Bartoccioni, L. Kowalczyk, A. Perálvarez-Marín, M. Palacín, et J. L. Vázquez-Ibar, « Stabilization of a prokaryotic LAT transporter by random mutagenesis », The Journal of General Physiology, vol. 147, nᵒ 4, p. 353-368, avr. 2016.
    Résumé : The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.
    Mots-clés : Amino Acid Substitution, Amino Acid Transport Systems, B3S, Bacillus subtilis, Bacterial Proteins, LPSM, Protein Stability.


  • 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, nov. 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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