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11 results on '"Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)"'

1. Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues

Author

Alexandre Martinière, Yvon Jaillais, Matthieu Pierre Platre, Marcelo Nollmann, Claire Burny, Vincent Bayle, Jean-Bernard Fiche, Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut für Populationsgenetik [Vienna], Veterinärmedizinische Universität Wien, Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-18-CE13-0025,caLIPSO,Mécanismes du pattern lipidique du réseau trans-Golgien (trans-Golgi Network) et rôles dans le tri des protéines, la polarité cellulaire et le développement des plantes(2018), and ANR-19-CE20-0008,CellOsmo,Nanodomain membranaire: un role dans la signalisation osmotique(2019)

Subjects
Fluorescence-lifetime imaging microscopy, Computer science, [SDV]Life Sciences [q-bio], single particle tracking, Arabidopsis, Total internal reflection microscopy, Fluorescence correlation spectroscopy, total internal reflection fluorescence microscopy, General Biochemistry, Genetics and Molecular Biology, Diffusion, molecular diffusion, 03 medical and health sciences, 0302 clinical medicine, Plant Cells, super-resolution microscopy, Microscopy, Photoactivated localization microscopy, 030304 developmental biology, 0303 health sciences, plants, Super-resolution microscopy, sptPALM, Optical Imaging, photo-activated localization microscopy, Rho GTPase, Membrane Proteins, Fluorescence recovery after photobleaching, root, Single Molecule Imaging, Autofluorescence, Spectrometry, Fluorescence, Microscopy, Fluorescence, single molecule localization microscopy, auxin, Biological system, 030217 neurology & neurosurgery, Fluorescence Recovery After Photobleaching
Abstract

Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3–4 h of imaging followed by 3–4 d of image processing and data analysis. This protocol describes how to perform single-particle tracking photoactivated localization microscopy (sptPALM) of membrane proteins in living plant tissues. The procedure covers all stages, from sample preparation to data analysis.

Published
2021

2. Insights into the activation mechanism of human estrogen-related receptor γ by environmental endocrine disruptors

Author

Erwan Thouennon, Vanessa Delfosse, Rémy Bailly, Pauline Blanc, Abdelhay Boulahtouf, Marina Grimaldi, Alessandro Barducci, William Bourguet, Patrick Balaguer, Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Transcriptional Activation, Estrogen-related receptor γ, Molecular Dynamics Simulation, Crystallography, X-Ray, Ligands, 03 medical and health sciences, Cellular and Molecular Neuroscience, 0302 clinical medicine, Phenols, Cell Line, Tumor, Humans, Benzhydryl Compounds, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Endocrine disruptors, Molecular Biology, 030304 developmental biology, Pharmacology, 0303 health sciences, Binding Sites, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Xenoestrogens, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Recombinant Proteins, Protein Structure, Tertiary, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Receptors, Estrogen, Thermodynamics, Molecular Medicine, Protein–ligand interaction, 030217 neurology & neurosurgery, Protein Binding
Abstract

International audience; The estrogen-related receptor γ (ERRγ, NR3B3) is a constitutively active nuclear receptor which has been proposed to act as a mediator of the low-dose effects of a number of environmental endocrine-disrupting chemicals (EDCs) such as the xenoestrogen bisphenol-A (BPA). To better characterize the ability of exogenous compounds to bind and activate ERRγ, we used a combination of cell-based, biochemical, structural and computational approaches. A purposely created stable cell line allowed for the determination of the EC50s for over 30 environmental ERRγ ligands, including previously unknown ones. Interestingly, affinity constants (Kds) of the most potent compounds measured by isothermal titration calorimetry were in the 50–500 nM range, in agreement with their receptor activation potencies. Crystallographic analysis of the interaction between the ERRγ ligand-binding domain (LBD) and compounds of the bisphenol, alkylphenol and naphthol families revealed a partially shared binding mode and minimal alterations of the receptor conformation upon ligand binding. Further biophysical characterizations coupled to molecular dynamics simulations suggested a mechanism through which ERRγ ligands would exhibit their agonistic properties by preserving the transcriptionally active form of the receptor while rigidifying some loop regions with associated functions. This unique mechanism contrasts with the classical one involving a ligand-induced repositioning and stabilization of the C-terminal activation helix H12.

Published
2019

3. 1H, 13C and 15N backbone chemical shift assignments of camelid single-domain antibodies against active state µ-opioid receptor

Author

Hélène Déméné, Yinshan Yang, Sébastien Granier, Remy Sounier, Joanna Hagelberger, KUHN-BERAUD, Sandrine, Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Stereochemistry, [SDV]Life Sciences [q-bio], [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biochemistry, Article, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Single domain, Receptor, G protein-coupled receptor, biology, Chemistry, Chemical shift, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, G protein Coupled Receptor, Combinatorial chemistry, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, Membrane protein, Camelid antibody, Nanobody, biology.protein, µ opioid receptor, Antibody, 030217 neurology & neurosurgery, Single-Chain Antibodies
Abstract

International audience; Nanobodies are single chain antibodies that have become a highly valuable and versatile tool for biomolecular and therapeutic research. One application field is the stabilization of active states of flexible proteins, among which G-protein coupled receptors (GPCRs) represent a very important class of membrane proteins. Here we present the backbone and side-chain assignment of the 1H, 13C and 15N resonances of Nb33 and Nb39, two nanobodies that recognize and stabilize the μ-opioid receptor (μOR) to opioids in its active agonist-bound conformation. In addition, we present a comparison of their secondary structures as derived from NMR chemical shifts.

Published
2017

4. The mechanism of force transmission at bacterial focal adhesion complexes

Author

Leon Espinosa, Jean-Bernard Fiche, Erkin Kuru, Mélanie Sotes, Michael S. Van Nieuwenhze, Adrien Ducret, Laura M. Faure, L. Aravind, Yves V. Brun, Julie Tréguier, Salim T. Islam, Sébastien Lhospice, Tâm Mignot, Olivier Theodoly, Vivek Anantharaman, Marcelo Nollmann, Jennifer Luciano, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Microbiologie de la Méditerranée (IMM), Adhésion et Inflammation (LAI), Assistance Publique - Hôpitaux de Marseille (APHM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Immunologie [Hôpital de la Conception - APHM], Assistance Publique - Hôpitaux de Marseille (APHM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital de la Conception [CHU - APHM] (LA CONCEPTION)-Centre National de la Recherche Scientifique (CNRS), National Center for Biotechnology Information (NCBI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
bacterial cell envelope, 0301 basic medicine, Myxococcus xanthus, Rotation, Gliding motility, Applied Microbiology, 030106 microbiology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Flagellum, Microbiology, Article, molecular motors, Bacterial Adhesion, Focal adhesion, 03 medical and health sciences, Bacterial Proteins, Cell Movement, Genetics, Molecular motor, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, Focal Adhesions, Multidisciplinary, biology, Molecular Motor Proteins, Periplasmic space, Adhesion, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, adhesion, 030104 developmental biology, motility, [SDE]Environmental Sciences, Periplasm, [SDV.IMM]Life Sciences [q-bio]/Immunology, proton channel, Bacterial outer membrane, Bacterial Outer Membrane Proteins
Abstract

Cell motility is a central function of living cells, as it empowers colonization of new environmental niches, cooperation, and development of multicellular organisms. This process is achieved by complex yet precise energy-consuming machineries in both eukaryotes and bacteria. Bacteria move on surfaces using extracellular appendages such as flagella and pili but also by a less-understood process called gliding motility. During this process, rod-shaped bacteria move smoothly along their long axis without any visible morphological changes besides occasional bending. For this reason, the molecular mechanism of gliding motility and its origin have long remained a complete mystery. An important breakthrough in the understanding of gliding motility came from single cell and genetic studies in the delta-proteobacterium Myxococcus xanthus. These early studies revealed, for the first time, the existence of bacterial Focal Adhesion complexes (FA). FAs are formed at the bacterial pole and rapidly move towards the opposite cell pole. Their attachment to the underlying surface is linked to cell propulsion, in a process similar to the rearward translocation of actomyosin complexes in Apicomplexans. The protein machinery that forms at FAs was shown to contain up to seventeen proteins predicted to localize in all layers of the bacterial cell envelope, the cytosolic face, the inner membrane (IM), the periplasmic space and the outer membrane (OM). Among these proteins, a proton-gated channel at the inner membrane was identified as the molecular motor. Thus, thrust generation requires the transduction of traction forces generated at the inner membrane through the cell envelope beyond the rigid barrier of the bacterial peptidoglycan.

Published
2016

5. Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis

Author

Gergely N. Nagy, Rachel Cerdan, Yinshan Yang, François Hoh, Jean-François Guichou, Henri Vial, Fanni Hajdú, Beáta G. Vértessy, Ewelina Guca, Lívia Marton, Richard Izrael, Department of Biochemistry and Molecular Biology [Debrecen, Hungary], University of Debrecen [Hungary], Ningxia Medical College, Dynamique moléculaire des interactions membranaires (DMIM), Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 2 - Sciences et Techniques (UM2), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Dynamique des interactions membranaires normales et pathologiques (DIMNP), and Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Plasmodium falciparum, Cytidylyltransferase, lcsh:Medicine, Ligands, Catalysis, Article, Substrate Specificity, Antimalarials, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Catalytic Domain, Lipid biosynthesis, parasitic diseases, Animals, Humans, Amino Acid Sequence, Choline-Phosphate Cytidylyltransferase, Malaria, Falciparum, Threonine, lcsh:Science, Phosphocholine, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Lipogenesis, lcsh:R, Active site, biology.organism_classification, Lipids, 3. Good health, Kinetics, 030104 developmental biology, Biochemistry, chemistry, Membrane biogenesis, biology.protein, lcsh:Q, Protein Binding
Abstract

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.

Published
2018

6. Protein–ligand structure guided by backbone and side-chain proton chemical shift perturbations

Author

Clémentine Aguirre, Isabelle Krimm, Olivier Cala, Tim ten Brink, Jean-François Guichou, Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Proton, Stereochemistry, Ligands, 010402 general chemistry, 01 natural sciences, Biochemistry, chemistry.chemical_compound, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Amide, Side chain, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Nuclear Magnetic Resonance, Biomolecular, Spectroscopy, Carbon Isotopes, 010405 organic chemistry, Ligand, Peroxiredoxins, 0104 chemical sciences, Molecular Docking Simulation, NMR spectra database, Crystallography, chemistry, Docking (molecular), Protons, Protein target, Protein ligand
Abstract

The fragment-based drug design approach consists of screening libraries of fragment-like ligands, to identify hits that typically bind the protein target with weak affinity ( $$100\,\upmu \hbox {M}$$ –5 mM). The determination of the protein–fragment complex 3D structure constitutes a crucial step for uncovering the key interactions responsible for the protein–ligand recognition, and for growing the initial fragment into potent active compounds. The vast majority of fragments are aromatic compounds that induce chemical shift perturbations (CSP) on protein NMR spectra. These experimental CSPs can be quantitatively used to guide the ligand docking, through the comparison between experimental CSPs and CSP back-calculation based on the ring current effect. Here we implemented the CSP back-calculation into the scoring function of the program PLANTS. We compare the results obtained with CSPs measured either on amide or aliphatic protons of the human peroxiredoxin 5. We show that the different kinds of protons lead to different results for resolving the 3D structures of protein–fragment complexes, with the best results obtained with the $$\hbox {H}_{\alpha }$$ protons.

Published
2014

7. 1H, 15N and 13C Backbone resonance assignments of murine met-neuroglobin, free and in complex with cyanide

Author

Stephane Delbecq, Ewelina Guca, Yinshan Yang, Beatrice Vallone, Frédéric Allemand, Christian Roumestand, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Istituto di Biologia e Patologia Molecolari, CNR, and Dipartimento di Scienze Biochimiche, Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Vaccination Antiparasitaire : Laboratoire de Biologie Cellulaire et Moléculaire (LBCM), and Université Montpellier 1 (UM1)-Université de Montpellier (UM)

Subjects
Models, Molecular, mice, Hemeprotein, globins, Stereochemistry, [SDV]Life Sciences [q-bio], ligand binding, Cyanide, protein binding, Biochemistry, Protein Structure, Secondary, models, chemistry.chemical_compound, biomolecular, Structural Biology, hemic and lymphatic diseases, [CHIM]Chemical Sciences, molecular, Globin, nerve tissue proteins, protein structure, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, Chemistry, fungi, cyanides, heme orientation, neuroglobin, animals, models, molecular, protein structure, secondary, nuclear magnetic resonance, biomolecular, Resonance (chemistry), Diatomic molecule, 3. Good health, nuclear magnetic resonance, Lower affinity, Neuroglobin, secondary
Abstract

Neuroglobin is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. We report here the resonance assignment of murine met-Neuroglobine, free and in complex with cyanide.

Published
2014

8. EtpB Is a Pore-Forming Outer Membrane Protein Showing TpsB Protein Features Involved in the Two-Partner Secretion System

Author

Laurent Coquet, Maria Kondratova, Andrey V. Kajava, Nathalie Saint, Albano C. Meli, Virginie Molle, Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Polymères, biopolymères, membranes (PBM), Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), and Dubois, Frederic

Subjects
Models, Molecular, Physiology, [SDV]Life Sciences [q-bio], Lipid Bilayers, Molecular Sequence Data, Biophysics, Biology, medicine.disease_cause, Mass Spectrometry, Protein Structure, Secondary, 03 medical and health sciences, Enterotoxigenic Escherichia coli, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Extracellular, medicine, Computer Simulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Secretion, Amino Acid Sequence, Peptide sequence, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Sequence Homology, Amino Acid, 030306 microbiology, Escherichia coli Proteins, Cell Biology, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Transmembrane protein, 3. Good health, Electrophysiology, Bacterial adhesin, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, Electrophoresis, Polyacrylamide Gel, Bacterial outer membrane, Bacterial Outer Membrane Proteins
Abstract

International audience; Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane beta-barrel domain that is occluded by an N-terminal alpha-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a beta-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.

Published
2009

9. Influence of the transcription factor RORγt on the development of NKp46+ cell populations in gut and skin

Author

Carmelo Luci, Dan R. Littman, Ana Reynders, Céline Cognet, Esperanza Anguiano, Jean Hardwigsen, Eric Vivier, Ivaylo I. Ivanov, Marc Dalod, Elena Tomasello, Laurent Chiche, Damien Chaussabel, Lionel Chasson, Jacques Banchereau, Immunité muqueuse et vaccination, Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Rostov State University, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Hôpital de la Conception [CHU - APHM] (LA CONCEPTION), Baylor Institute for Immunology Research (BIIR), Baylor College of Medecine, New York University School of Medicine (NYU), The Kimmel Center for Biology and Medicine at the Skirball Institute and Howard Hughes Medical Institute, Hôpital de la Timone [CHU - APHM] (TIMONE), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
MESH: Interleukins, MESH: Antigens, CD3, MESH: Mice, Mutant Strains, MESH: Interferon-gamma, MESH: Natural Cytotoxicity Triggering Receptor 1, CD3, Immunology, Biology, Interleukin 22, 03 medical and health sciences, 0302 clinical medicine, Dermis, MESH: Mice, Inbred C57BL, medicine, Immunology and Allergy, MESH: Animals, MESH: Receptors, Thyroid Hormone, MESH: Lymphocyte Activation, MESH: Mice, 030304 developmental biology, MESH: Receptors, Retinoic Acid, 0303 health sciences, Lamina propria, MESH: Humans, Innate immune system, Lymphokine-activated killer cell, MESH: Transcription Factors, MESH: Dermis, Natural killer T cell, Cell biology, Lymphatic system, medicine.anatomical_structure, MESH: Natural Killer T-Cells, MESH: Intestinal Mucosa, biology.protein, MESH: Peyer's Patches, MESH: Cell Division, [SDV.IMM]Life Sciences [q-bio]/Immunology, 030215 immunology
Abstract

International audience; NKp46+CD3- natural killer lymphocytes isolated from blood, lymphoid organs, lung, liver and uterus can produce granule-dependent cytotoxicity and interferon-gamma. Here we identify in dermis, gut lamina propria and cryptopatches distinct populations of NKp46+CD3- cells with a diminished capacity to degranulate and produce interferon-gamma. In the gut, expression of the transcription factor RORgammat, which is involved in the development of lymphoid tissue-inducer cells, defined a previously unknown subset of NKp46+CD3- lymphocytes. Unlike RORgammat- lamina propria and dermis natural killer cells, gut RORgammat+NKp46+ cells produced interleukin 22. Our data show that lymphoid tissue-inducer cells and natural killer cells shared unanticipated similarities and emphasize the heterogeneity of NKp46+CD3- cells in innate immunity, lymphoid organization and local tissue repair.

Published
2008

10. Complete hydrolysis of myo-inositol hexakisphosphate by a novel phytase from Debaryomyces castellii CBS 2923

Author

Patrick Chemardin, Hélène Boze, Santiago Galvez, G. Moulin, Mélanie Ragon, André Aumelas, Ingénierie des Réactions Biologiques, Bio-productions (IR2B), Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Recherche Agronomique (INRA)

Subjects
Debaryomyces castellii, Phytic Acid, Applied Microbiology and Biotechnology, Phosphates, Substrate Specificity, 03 medical and health sciences, Hydrolysis, chemistry.chemical_compound, Enzyme Stability, HPIC, Inositol, Inositol phosphate, 030304 developmental biology, chemistry.chemical_classification, 6-Phytase, 0303 health sciences, Phytic acid, Chromatography, biology, 030306 microbiology, Chemistry, Hydrophilic interaction chromatography, Phytate degradation, Debaryomyces, Temperature, General Medicine, Hydrogen-Ion Concentration, Enzyme characterization, Phytase, biology.organism_classification, Phosphate, Yeast, NMR, Kinetics, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, Saccharomycetales, Inositol phosphates, Chromatography, Liquid, Biotechnology
Abstract

correspondance: boze@supagro.inra.fr; International audience; Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The Km value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P6) to inositol and inorganic phosphate. The sequence of Ins P6 hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P5, Ins (1,2,5,6) P4, Ins (1,2,6) P3, Ins (1,2) P2, Ins (1 or 2) P1, and inositol (notation 3/4/5/6/1 or 2).

Published
2008

11. Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

Author

Francois Stricher, André Ménez, Dorothée Missé, Christian Roumestand, Francisco Veas, Loïc Martin, Martine Pugnière, Paolo Lusso, Isabelle Freulon, Xavier Magne, Rafael Prado-Gotor, Claudio Vita, Francesca Sironi, Philippe Barthe, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), We thank the French Agence Nationale de Recherches sur le SIDA (ANRS) and Ensemble Contre le SIDA—SIDACTION for financial support., and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Proteomics, Quality Control, Phage display, Protein Conformation, INHIBITION, Biomedical Engineering, Bioengineering, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, HIV Antibodies, HIV Envelope Protein gp120, Applied Microbiology and Biotechnology, Neutralization, Virus, Epitope, Epitopes, 03 medical and health sciences, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, METHODE DE LUTTE, PROTEINE, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, SIDA, Molecular Mimicry, 030302 biochemistry & molecular biology, Virion, Rational design, Surface Plasmon Resonance, biology.organism_classification, Virology, 3. Good health, chemistry, CD4 Antigens, Lentivirus, HIV-1, biology.protein, VIRUS, VACCINATION, Molecular Medicine, Antibody, Peptides, Glycoprotein, HeLa Cells, ANTICORPS, Biotechnology
Abstract

International audience; The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.

Published
2002

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