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Accueil > Départements > Biochimie, Biophysique et Biologie Structurale > Philippe MINARD : Modélisation et Ingénierie des Protéines

Publications de l’équipe

2017


  • A. Chevrel, A. Mesneau, D. Sanchez, L. Celma, S. Quevillon-Cheruel, A. Cavagnino, S. Nessler, I. Li de la Sierra-Gallay, H. van Tilbeurgh, P. Minard, M. Valerio-Lepiniec, et A. Urvoas, « Alpha Repeat proteins (αRep) as expression and crystallization helpers », Journal of Structural Biology, 2017.
    Résumé : We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric protein corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.
    Mots-clés : artificial repeat proteins, B3S, Crystallization helper, FAAM, Fusion protein, MIP, Protein complementation assay, Protein library.

  • T. Di Meo, W. Ghattas, C. Herrero, C. Velours, P. Minard, J. - P. Mahy, R. Ricoux, et A. Urvoas, « αRep A3: A versatile artificial scaffold for metalloenzyme design », Chemistry (Weinheim an Der Bergstrasse, Germany), 2017.
    Résumé : αRep is a new family of artificial proteins based on a thermostable alpha-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to receive metal complexes and thus appears as suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen and changed independently into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine of each protein variant and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was bound specifically by the two biohybrids with two different binding modes and, in addition, the holo biohybrid A3F119NPH was found to be able to catalyze enantioselectively the Diels-Alder (D-A) cycloaddition with up to 62% ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a new promising route for the design and production of new enantioselective biohybrids based on entirely artificial proteins issued from a highly diverse library.
    Mots-clés : artificial repeat proteins, B3S, Diels-Alder reaction, Enantioselective Catalysis, MIP, PF, PIM.

  • V. R. Figliuolo, L. E. B. Savio, H. Safya, H. Nanini, C. Bernardazzi, A. Abalo, H. S. P. de Souza, J. Kanellopoulos, P. Bobé, C. M. L. M. Coutinho, et R. Coutinho-Silva, « P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells », Biochimica Et Biophysica Acta, 2017.
    Résumé : P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RB(low). Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.
    Mots-clés : ATP, B3S, colitis, MIP, P2X7 receptor, regulatory T cells.

2016



  • M. Figueroa, M. Sleutel, M. Vandevenne, G. Parvizi, S. Attout, O. Jacquin, J. Vandenameele, A. W. Fischer, C. Damblon, E. Goormaghtigh, M. Valerio-Lepiniec, A. Urvoas, D. Durand, E. Pardon, J. Steyaert, P. Minard, D. Maes, J. Meiler, A. Matagne, J. A. Martial, et C. Van de Weerdt, « The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools », Journal of Structural Biology, vol. 195, nᵒ 1, p. 19-30, 2016.


  • M. Figueroa, J. Vandenameele, E. Goormaghtigh, M. Valerio-Lepiniec, P. Minard, A. Matagne, et C. Van de Weerdt, « Biophysical characterization data of the artificial protein Octarellin V.1 and binding test with its X-ray helpers », Data in Brief, vol. 8, p. 1221-1226, 2016.

  • W. Ghattas, L. Cotchico-Alonso, J. - D. Maréchal, A. Urvoas, M. Rousseau, J. - P. Mahy, et R. Ricoux, « Artificial Metalloenzymes with the Neocarzinostatin Scaffold: Toward a Biocatalyst for the Diels-Alder Reaction », Chembiochem: A European Journal of Chemical Biology, vol. 17, nᵒ 5, p. 433-440, 2016.
    Résumé : A copper(II) cofactor coupled to a testosterone anchor, copper(II)-(5-(Piperazin-1-yl)-1,10-phenanthroline)testosterone-17-hemisuccinamide (10) was synthesized and associated with a neocarzinostatin variant, NCS-3.24 (KD =3 μm), thus generating a new artificial metalloenzyme by following a "Trojan horse" strategy. Interestingly, the artificial enzyme was able to efficiently catalyze the Diels-Alder cyclization reaction of cyclopentadiene (1) with 2-azachalcone (2). In comparison with what was observed with cofactor 10 alone, the artificial enzymes favored formation of the exo products (endo/exo ratios of 84:16 and 62:38, respectively, after 12 h). Molecular modeling studies assigned the synergy between the copper complex and the testosterone (KD =13 μm) moieties in the binding of 10 to good van der Waals complementarity. Moreover, by pushing the modeling exercise to its limits, we hypothesize on the molecular grounds that are responsible for the observed selectivity.
    Mots-clés : artificial metalloenzymes, B3S, Biocatalysis, Carbon-13 Magnetic Resonance Spectroscopy, copper complexes, Cycloaddition Reaction, diels-alder cyclization reaction, Enzymes, Metalloproteins, MIP, Molecular Docking Simulation, molecular modeling, Proton Magnetic Resonance Spectroscopy, Spectrometry, Mass, Electrospray Ionization, Zinostatin.


  • K. L. Gurunatha, A. C. Fournier, A. Urvoas, M. Valerio-Lepiniec, V. Marchi, P. Minard, et E. Dujardin, « Nanoparticles Self-Assembly Driven by High Affinity Repeat Protein Pairing », ACS Nano, vol. 10, nᵒ 3, p. 3176-3185, mars 2016.

2015

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Publications principales avant 2015

-  Guellouz, A., Valerio-Lepiniec, M., Urvoas, A., Chevrel, A., Graille, M., Fourati-Kammoun, Z., Desmadril, M., van Tilbeurgh, H., and Minard, P. (2013). Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep). PloS one 8, e71512.
-  Urvoas, A., Valerio-Lepiniec, M., and Minard, P. (2012). Artificial proteins from combinatorial approaches. Trends in biotechnology 30, 512-520.
-  Nangola, S., Urvoas, A., Valerio-Lepiniec, M., Khamaikawin, W., Sakkhachornphop, S., Hong, S.-S., Boulanger, P., Minard, P., and Tayapiwatana, C. (2012). Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein. Retrovirology 9, 17.
-  Urvoas, A., Guellouz, A., Valerio-Lepiniec, M., Graille, M., Durand, D., Desravines, D.C., van Tilbeurgh, H., Desmadril, M., and Minard, P. (2010). Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (alphaRep) based on thermostable HEAT-like repeats. J Mol Biol 404, 307-327.
-  Drevelle, A., Urvoas, A., Hamida-Rebai, M.B., Van Vooren, G., Nicaise, M., Valerio-Lepiniec, M., Desmadril, M., Robert, C.H., and Minard, P. (2009). Disulfide bond substitution by directed evolution in an engineered binding protein. Chembiochem 10, 1349-1359.

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