Nos tutelles


Nos partenaires

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

Publications de l’équipe


  • H. - J. Chang, P. Mayonove, A. Zavala, A. De Visch, P. Minard, M. Cohen-Gonsaud, et J. Bonnet, « A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria », ACS synthetic biology, vol. 7, nᵒ 1, p. 166-175, janv. 2018.
    Résumé : Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-binding domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.
    Mots-clés : B3S, MIP.

  • H. Safya, A. Mellouk, J. Legrand, S. M. Le Gall, M. Benbijja, C. Kanellopoulos-Langevin, J. M. Kanellopoulos, et P. Bobé, « Variations in Cellular Responses of Mouse T Cells to Adenosine-5'-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage », Frontiers in Immunology, vol. 9, p. 360, 2018.
    Résumé : A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5'-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlowTconvs, but not CD45RBhighTconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlowTconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44lowto effector/memory CD45RBlowCD44highstage. Maximum levels of upregulation are reached on recently activated CD69+naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+CD45RBhighCD44lowTconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44lowTconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlowTconvs, CD45RBlowFoxp3+Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.
    Mots-clés : B3S, CD39, CD62L shedding, CD73, cell death, MIP, P2X7, phosphatidyslerine exposure, pore formation, regulatory T lymphocyte.


  • 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, août 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), mai 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, mars 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.

  • R. Grzela, J. Nusbaum, S. Fieulaine, F. Lavecchia, M. Desmadril, N. Nhiri, A. Van Dorsselaer, S. Cianferani, E. Jacquet, T. Meinnel, et C. Giglione, « Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts », Biochimica Et Biophysica Acta, oct. 2017.
    Résumé : Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 specific activity to values similar to those of bacterial PDFs. We next show that Vp16PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.
    Mots-clés : AMIG, B3S, BDG, DBG, Enzyme mechanism, IMAPP, MIP, N-terminal methionine excision, Peptide deformylase, PF, PIM, PROMTI, Structure, Virus.

  • S. Hadpech, S. Nangola, K. Chupradit, K. Fanhchaksai, W. Furnon, A. Urvoas, M. Valerio-Lepiniec, P. Minard, P. Boulanger, S. - S. Hong, et C. Tayapiwatana, « Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation », Scientific Reports, vol. 7, nᵒ 1, p. 16335, nov. 2017.
    Résumé : A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (αRep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of αRep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions. Two strong αRep binders were isolated from the screen, αRep4E3 (32 kDa; 7 internal repeats) and αRep9A8 (28 kDa; 6 internal repeats). Their antiviral activity against HIV-1 was evaluated in VLP-producer cells and in human SupT1 cells challenged with HIV-1. Both αRep4E3 and αRep9A8 showed a modest but significant antiviral effects in all bioassays and cell systems tested. They did not prevent the proviral integration reaction, but negatively interfered with late steps of the HIV-1 life cycle: αRep4E3 blocked the viral genome packaging, whereas αRep9A8 altered both virus maturation and genome packaging. Interestingly, SupT1 cells stably expressing αRep9A8 acquired long-term resistance to HIV-1, implying that αRep proteins can act as antiviral restriction-like factors.
    Mots-clés : B3S, MIP.

  • J. Suurväli, P. Boudinot, J. Kanellopoulos, et S. Rüütel Boudinot, « P2X4: A fast and sensitive purinergic receptor », Biomedical Journal, vol. 40, nᵒ 5, p. 245-256, oct. 2017.
    Résumé : Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations), about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca2+-channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly characterized, more studies are needed as it is likely to be a potential therapeutic target in these multiple pathologies.
    Mots-clés : ATP, B3S, MIP, Neuropathic pain, P2X receptor, P2X4, Purinergic signaling.


  • 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, mars 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.


--- Exporter la sélection au format

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.

publié le , mis à jour le