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512 results on '"MESH: Cell Membrane"'

1. Nicotinic acetylcholine receptor and chronic obstructive pulmonary disease

Author: Valérian Dormoy, Stéphanie Pons, Philippe Gosset, Uwe Maskos, Pathologies Pulmonaires et Plasticité Cellulaire - UMR-S 1250 (P3CELL), Université de Reims Champagne-Ardenne (URCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV), Neurobiologie intégrative des Systèmes cholinergiques / Integrative Neurobiology of Cholinergic Systems (NISC), Institut Pasteur [Paris] (IP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), and Sciences, EDP
Subjects: [SDV] Life Sciences [q-bio], MESH: Humans, [SDV]Life Sciences [q-bio], MESH: Receptors, Nicotinic, MESH: Pulmonary Disease, Chronic Obstructive, General Medicine, General Biochemistry, Genetics and Molecular Biology, MESH: Cell Membrane
Abstract: International audience; No abstract available
Published: 2022

2. Nicotinic acetylcholine receptor and chronic obstructive pulmonary disease

Author: Dormoy, Valérian, Pons, Stéphanie, Gosset, Philippe, Maskos, Uwe, Pathologies Pulmonaires et Plasticité Cellulaire - UMR-S 1250 (P3CELL), Université de Reims Champagne-Ardenne (URCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA), Neurobiologie intégrative des Systèmes cholinergiques / Integrative Neurobiology of Cholinergic Systems (NISC), Institut Pasteur [Paris] (IP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Pulmonary Disease, Chronic Obstructive, MESH: Humans, [SDV]Life Sciences [q-bio], Cell Membrane, MESH: Receptors, Nicotinic, Humans, MESH: Pulmonary Disease, Chronic Obstructive, Receptors, Nicotinic, MESH: Cell Membrane
Abstract: International audience; No abstract available
Published: 2022

3. Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

Author: Patricia Flamant, Isabelle Mouyna, Samantha Gozel, Alix T. Coste, Dominique Sanglard, Frédéric Lamoth, Josie E. Parker, Daniel Bachmann, Marion Aruanno, Lausanne University Hospital, Université de Lausanne = University of Lausanne (UNIL), Aspergillus, Institut Pasteur [Paris] (IP), Swansea University, and This work was supported by the Swiss National Science Foundation (SNSF Ambizione-Score, grant number: PZ00P3 161140) and by the Santos-Suarez foundation.
Subjects: Azoles, Antifungal Agents, ATP-binding cassette transporter, Aspergillus fumigatus, chemistry.chemical_compound, MESH: HMGB1 Protein, Cytochrome P-450 Enzyme System, MESH: Reverse Transcriptase Polymerase Chain Reaction, MESH: Azoles, MESH: Ku Autoantigen, HMGB1 Protein, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, chemistry.chemical_classification, 0303 health sciences, Ergosterol, biology, MESH: Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, MESH: Voriconazole, General Medicine, isavuconazole milbemycine, Sterols, Infectious Diseases, MESH: CCAAT-Binding Factor, MESH: Cytochrome P-450 Enzyme System, MESH: ATP-Binding Cassette Transporters, MESH: Fungal Proteins, Efflux, MESH: Aspergillus fumigatus, efflux pumps, transporters, Real-Time Polymerase Chain Reaction, Gas Chromatography-Mass Spectrometry, MESH: Drug Resistance, Fungal, Microbiology, resistance, Fungal Proteins, 03 medical and health sciences, Drug Resistance, Fungal, Ku Autoantigen, Transcription factor, 030304 developmental biology, 030306 microbiology, MESH: Transcriptome, Cell Membrane, Transporter, MESH: Antifungal Agents, biology.organism_classification, posaconazole, Major facilitator superfamily, MESH: Gas Chromatography-Mass Spectrometry, CCAAT-Binding Factor, chemistry, MESH: Sterols, Azole, ATP-Binding Cassette Transporters, Voriconazole, erogsterol, Transcriptome, MESH: Cell Membrane
Abstract: Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.
Published: 2021

4. Allosteric modulation of ghrelin receptor signaling by lipids

Author: Louet, Maxime, Casiraghi, Marina, Damian, Marjorie, Costa, Mauricio GS, Renault, Pedro, Gomes, Antoniel AS, Batista, Paulo, M'Kadmi, Céline, Mary, Sophie, Cantel, Sonia, Denoyelle, Severine, Ben Haj Salah, Khoubaib, Perahia, David, Bisch, Paulo, Fehrentz, Jean-Alain, Catoire, Laurent, Floquet, Nicolas, Banères, Jean-Louis, Mustafá, Emilio, Cordisco González, Santiago, Wagner, Renaud, Schiöth, Helgi, Perelló, Mario, Raingo, Jesica, Gomes, Antoniel Augusto Severo, M’Kadmi, Céline, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Universidade Federal do Rio de Janeiro (UFRJ), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), ANR-17-CE11-0011,allosig,allostérie, dynamique conformationnelle et signalisation via les RCPG(2017), ANR-17-CE18-0022,GHScReen2,Biosenseurs originaux pour le criblage des ligands des Récepteurs Couplés aux Protéines G. Application au récepteur de la ghréline.(2017), and Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Phosphatidylinositol 4,5-Diphosphate, MESH: Signal Transduction, MESH: Fluorescence Resonance Energy Transfer, Protein Conformation, Lipid Bilayers, General Physics and Astronomy, MESH: Allosteric Regulation, MESH: Receptors, Ghrelin, MESH: Protein Conformation, 0302 clinical medicine, Fluorescence Resonance Energy Transfer, Receptors, Ghrelin, Receptor, MESH: Lipid Metabolism, 0303 health sciences, Multidisciplinary, Chemistry, MESH: Lipid Bilayers, digestive, oral, and skin physiology, MESH: G(M3) Ganglioside, Lipids, MESH: Phosphatidylinositol 4,5-Diphosphate, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Membrane, Ghrelin, lipids (amino acids, peptides, and proteins), medicine.symptom, Intracellular, Signal Transduction, Biophysical chemistry, MESH: Mutation, G protein, Science, Allosteric regulation, Mechanism of action, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Allosteric Regulation, medicine, G(M3) Ganglioside, Humans, Cysteine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, Binding Sites, MESH: Humans, Cell Membrane, General Chemistry, MESH: Cysteine, Lipid Metabolism, MESH: Lipids, MESH: Binding Sites, Mutation, Biophysics, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract: The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated., The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here authors demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects and observe PIP2 and GM3 induced shifts of the conformational equilibrium of GHSR away from its inactive state.
Published: 2021

5. Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner

Author: Meza-Torres, Jazmín, Lelek, Mickaël, Quereda, Juan, Sachse, Martin, Manina, Giulia, Ershov, Dmitry, Tinevez, Jean-Yves, Radoshevich, Lilliana, Maudet, Claire, Chaze, Thibault, Giai Gianetto, Quentin, Matondo, Mariette, Lecuit, Marc, Martin-Verstraete, Isabelle, Zimmer, Christophe, Bierne, Hélène, Dussurget, Olivier, Cossart, Pascale, Pizarro-Cerdá, Javier, Yersinia, Institut Pasteur [Paris] (IP), Interactions Bactéries-Cellules, Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC), Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universidad Cardenal Herrera-CEU (CEU-UCH), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Individualité microbienne et infection - Microbial Individuality and Infection, Hub d'analyse d'images - Image Analysis Hub (Platform) (IAH), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, University of Iowa [Iowa City], Biologie des Infections - Biology of Infection, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Service des Maladies infectieuses et tropicales [CHU Necker], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Pathogénèse des Bactéries Anaérobies / Pathogenesis of Bacterial Anaerobes (PBA (U-Pasteur_6)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Paris-Saclay, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This work received financial support from Institut Pasteur, Institut Carnot Pasteur Microbes et Santé (PasteurInnov Grant LLS-Bact), Région Ile-de-France (DIM-MALINF), and Fondation pour la Recherche Médicale (Grant FDT201904008152) (to J.M.-T.). J.J.Q. is supported by a 'Ramón y Cajal' contract from the Spanish Ministry of Science, Innovation and Universities (RYC-2018-024985-I). The Yersinia Research Unit is a member of the LabEX Integrative Biology of Emerging Infectious Diseases (ANR LBX-62 IBEID), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Bierne, Hélène, and Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID
Subjects: MESH: Cytoplasm, [SDV]Life Sciences [q-bio], microfluidic microscopy, MESH: Listeria monocytogenes, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, contact-dependent inhibition (CDI), [SDV] Life Sciences [q-bio], MESH: Bacteriocins, bacteriocin, MESH: Hemolysin Proteins, Listeria monocytogenes (Lm), MESH: Adenosine Triphosphate, Listeriolysin S (LLS), [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Cell Membrane
Abstract: International audience; Significance Listeria monocytogenes ( Lm ) is a bacterial pathogen that causes listeriosis, a foodborne disease characterized by gastroenteritis, meningitis, bacteremia, and abortions in pregnant women. The most severe human listeriosis outbreaks are associated with a subset of Lm hypervirulent clones that encode the bacteriocin Listeriolysin S (LLS), which modifies the gut microbiota and allows efficient Lm gut colonization and invasion of deeper organs. Our present work identifies the killing mechanism displayed by LLS to outcompete gut commensal bacteria, demonstrating that it induces membrane permeabilization and membrane depolarization of target bacteria. Moreover, we show that LLS is a thiazole/oxazole–modified microcin that displays a contact-dependent inhibition mechanism.
Published: 2021

6. Bacterial intracellularly active toxins: Membrane localisation of the active domain

Author: Michel R. Popoff, Arnaud Blondel, Carolina Varela-Chavez, Toxines bactériennes - Bacterial Toxins, Institut Pasteur [Paris], Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: rho GTP-Binding Proteins, Cytoplasm, Botulinum Toxins, [SDV]Life Sciences [q-bio], MESH: Catalytic Domain, Bacterial virulence factors, MESH: Botulinum Toxins, Cell membrane, chemistry.chemical_compound, Pasteurella multocida toxin, Catalytic Domain, Rho/Ras-GTPases, MESH: Animals, MARTX toxins, MESH: Bacterial Proteins, 0303 health sciences, biology, MESH: Phosphatidic Acids, Metalloendopeptidases, Phosphatidylserine, MESH: Phosphatidylserines, Cell biology, medicine.anatomical_structure, Intracellular, Gram-negative bacteria, Virulence Factors, Endosome, Bacterial Toxins, Neurotoxins, Immunology, Phosphatidic Acids, Virulence, MESH: Metalloendopeptidases, Phosphatidylserines, Microbiology, Legionella pneumophila, 03 medical and health sciences, Bacterial Proteins, Tetanus Toxin, Virology, medicine, Animals, Humans, large clostridial glucosylating toxins, MESH: Tetanus Toxin, phospholipids, 030304 developmental biology, MESH: Neurotoxins, MESH: Virulence Factors, MESH: Humans, 030306 microbiology, Synaptic vesicle membrane, MESH: Cytoplasm, biology.organism_classification, MESH: rho GTP-Binding Proteins, MESH: Legionella pneumophila, Cytosol, chemistry, MESH: Bacterial Toxins, cell membrane, MESH: Cell Membrane
Abstract: International audience; Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras-GTPases, certain virulence factors of Gram negative bacteria, Rho-GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G-proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four-helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine.
Published: 2020

7. Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor

Author: Jens Bohne, Daniel Todt, Steppich K, Sergej Franz, Angelina Malassa, Thomas Zillinger, Eike Steinmann, Xu S, Nicoletta Casartelli, Passos, Christine Goffinet, Winfried Barchet, Wachs As, Oliver Schwartz, Schilling H, Kathrin Sutter, Ulf Dittmer, Centre for Experimental and Clinical Infection Research [Hanover] (TWINCORE), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Institute of Clinical Chemistry and Clinical Pharmacology [Bonn, Allemagne], University of Bonn, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Hannover Medical School [Hannover] (MHH), Ruhr University Bochum (RUB), University of Duisburg-Essen, German Centre for Infection Research (DZIF), Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Berlin Institute of Health (BIH), Vânia Passos and Sergej Franz are supported by the Infection Biology International Ph.D. Program of the Hannover Biomedical Research School. Vânia Passos is supported by the GABBA Ph.D. Program and the Fundação para a Ciência e Tecnologia (FCT). Thomas Zillinger and Winfried Barchet acknowledge Bonfor and DZIF funding and German Research Foundation (Deutsche Forschungsgemeinschaft [DFG]) grants EXC1023: ImmunoSensation and CRCs 670 and 704. This work is supported by DFG funding to Christine Goffinet (Collaborative Research Centre SFB900, Microbial Persistence and its Control, project C8 and Priority Program 1923, Innate Sensing and Restriction of Retroviruses, GO2153/4 grant) and funding from the HZI and the Berlin Institute of Health (BIH) to Christine Goffinet., Universidade do Porto = University of Porto, Universität Bonn = University of Bonn, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universität Duisburg-Essen = University of Duisburg-Essen [Essen], and TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.
Subjects: MESH: Gene Editing, MESH: CRISPR-Cas Systems, T-Lymphocytes, Mutant, Medizin, HIV Infections, Endogeny, Jurkat cells, Epitope, MESH: HIV-1, MESH: Genotype, Gene Knockout Techniques, SERINC5, MESH: nef Gene Products, Human Immunodeficiency Virus, MESH: Anti-HIV Agents, Internalization, media_common, MESH: Gene Knockout Techniques, Gene Editing, 0303 health sciences, human immunodeficiency virus, 030302 biochemistry & molecular biology, virus diseases, MESH: HIV Infections, MESH: Gene Expression Regulation, Virus-Cell Interactions, 3. Good health, Cell biology, interferons, MESH: HEK293 Cells, Host-Pathogen Interactions, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Virion, MESH: Membrane Proteins, MESH: Interferon-alpha, Genotype, Anti-HIV Agents, media_common.quotation_subject, Immunology, Alpha interferon, Heterologous, Biology, Microbiology, Cell Line, 03 medical and health sciences, Virology, Nitriles, Humans, nef Gene Products, Human Immunodeficiency Virus, antiviral factor, CRISPR/Cas9, 030304 developmental biology, Nef, MESH: Humans, Cell Membrane, MESH: Host-Pathogen Interactions, Virion, Interferon-alpha, Membrane Proteins, Subcellular localization, MESH: Cell Line, HEK293 Cells, Pyrimidines, MESH: T-Lymphocytes, Gene Expression Regulation, Insect Science, HIV-1, Pyrazoles, CRISPR-Cas Systems, MESH: Pyrazoles, MESH: Cell Membrane
Abstract: SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions., When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface. IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.
Published: 2019

8. Cell Biology of T Cell Receptor Expression and Regulation

Author: Balbino Alarcón, Andrés Alcover, Vincenzo Di Bartolo, Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Centro de Biología Molecular Severo Ochoa [Madrid] (CBMSO), Universidad Autonoma de Madrid (UAM)-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Universidad Autónoma de Madrid (UAM)-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC)
Subjects: MESH: Signal Transduction, 0301 basic medicine, MESH: CD3 Complex, CD3 Complex, T-Lymphocytes, recycling, MESH: Structure-Activity Relationship, Immunology and Allergy, MESH: Animals, biology, MESH: Receptors, Antigen, T-Cell, MESH: Gene Expression Regulation, Endocytosis, 3. Good health, Cell biology, medicine.anatomical_structure, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, TCR-CD3, MESH: Endocytosis, Signal transduction, signaling, Signal Transduction, assembly, Endosome, T cell, Immunology, MESH: Immunomodulation, Receptors, Antigen, T-Cell, MESH: Proteolysis, Endosomes, Major histocompatibility complex, Immunomodulation, Structure-Activity Relationship, 03 medical and health sciences, conformational change, Antigen, medicine, Animals, Humans, MESH: Humans, Cell Membrane, T-cell receptor, MESH: Multiprotein Complexes, MESH: T-Lymphocytes, 030104 developmental biology, Gene Expression Regulation, MESH: Endosomes, Multiprotein Complexes, Proteolysis, MESH: Biomarkers, biology.protein, Biomarkers, MESH: Cell Membrane
Abstract: International audience; T cell receptors (TCRs) are protein complexes formed by six different polypeptides. In most T cells, TCRs are composed of αβ subunits displaying immunoglobulin-like variable domains that recognize peptide antigens associated with major histocompatibility complex molecules expressed on the surface of antigen-presenting cells. TCRαβ subunits are associated with the CD3 complex formed by the γ, δ, ε, and ζ subunits, which are invariable and ensure signal transduction. Here, we review how the expression and function of TCR complexes are orchestrated by several fine-tuned cellular processes that encompass (a) synthesis of the subunits and their correct assembly and expression at the plasma membrane as a single functional complex, (b) TCR membrane localization and dynamics at the plasma membrane and in endosomal compartments, (c) TCR signal transduction leading to T cell activation, and (d) TCR degradation. These processes balance each other to ensure efficient T cell responses to a variety of antigenic stimuli while preventing autoimmunity.
Published: 2018

9. Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

Author: Isabelle Richard, Chiara Fecchio, Roberta Sacchetto, Marcello Carotti, Elisa Bianchini, Michela Soardi, Sara F Henriques, Chiara Gomiero, Justine Marsolier, Dorianna Sandonà, Romeo Betto, Université Paris-Saclay, Universita degli Studi di Padova, CNR Institute of Neuroscience, National Research Council [Italy] (CNR), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Università degli Studi di Padova = University of Padua (Unipd), and École Pratique des Hautes Études (EPHE)
Subjects: 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, MESH: Muscle Contraction, Cystic Fibrosis Transmembrane Conductance Regulator, MESH: Proof of Concept Study, Missense mutation, Muscular dystrophy, Genetics (clinical), MESH: Cystic Fibrosis Transmembrane Conductance Regulator, MESH: Muscle, Skeletal, biology, Myogenesis, MESH: Sarcoglycanopathies, Articles, General Medicine, musculoskeletal system, 3. Good health, Cell biology, small molecules, Sarcoglycan, MESH: HEK293 Cells, Dystrophin, MESH: Muscle, Striated, Muscle Contraction, musculoskeletal diseases, congenital, hereditary, and neonatal diseases and abnormalities, MESH: Cell Line, Tumor, CFTR corrector, Mutation, Missense, Proof of Concept Study, 03 medical and health sciences, pharmacological treatment, Cell Line, Tumor, Sarcoglycans, sarcoglycan, Sarcoglycanopathies, Genetics, medicine, Humans, MESH: Sarcoglycans, Muscle, Skeletal, Molecular Biology, SGCA, MESH: Mutation, Missense, MESH: Humans, Sarcolemma, Cell Membrane, sarcoglycan,LGMD2D,CFTR corrector,myogenic cells,myotubes,small molecules,pharmacological treatment, Dystrophy, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, Muscle, Striated, HEK293 Cells, 030104 developmental biology, myotubes, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, biology.protein, LGMD2D, myogenic cells, MESH: Cell Membrane
Abstract: International audience; Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, β-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.
Published: 2018

10. Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

Author: Giulia Nigro, Jörg Vogel, Ines Rodrigues Lopes, Miguel Mano, Philippe J. Sansonetti, Caroline Tawk, Ana Eulalio, Clivia Lisowski, Carmen Aguilar, University of Würzburg, Pathogénie microbienne moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Center for Neuroscience and Cell Biology (CNC) (CNC), University of Coimbra [Portugal] (UC)-Neuroscience Research Domain, Collège de France - Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), Helmholtz-Zentrum für Infektionsforschung GmbH (HZI), This work was supported by grants from the Bavarian Ministry of Sciences, Research and the Arts in the framework of the Bavarian Molecular Biosystems Research Network (BioSysNet to J.V and A.E.), and the European Research Council (ERC) Advanced Grant (GA no. 339579 to P.S)., European Project: 339579,EC:FP7:ERC,ERC-2013-ADG,DECRYPT(2014), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Chaire Microbiologie et Maladies infectieuses, HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany., Nigro, Giulia, and Decrypting signals in the crypt. - DECRYPT - - EC:FP7:ERC2014-04-01 - 2019-03-31 - 339579 - VALID
Subjects: Salmonella typhimurium, 0301 basic medicine, MESH: Shigella flexneri, [SDV]Life Sciences [q-bio], medicine.disease_cause, Shigella flexneri, Salmonella, Shigella, MESH: Animals, Membrane & Intracellular Transport, acid sphingomyelinase, MESH: Stress, Physiological, Pathogen, Host cell membrane, Gene knockdown, General Neuroscience, Articles, Microbiology, Virology & Host Pathogen Interaction, Cell biology, [SDV] Life Sciences [q-bio], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Epithelial Cells, Salmonella Infections, MESH: Immunity, Innate, Acid sphingomyelinase, Intracellular, medicine.drug, MESH: Dysentery, Bacillary, Guinea Pigs, 030106 microbiology, host stress response, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Article, General Biochemistry, Genetics and Molecular Biology, MESH: Guinea Pigs, 03 medical and health sciences, Stress, Physiological, medicine, Animals, Permissive, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Molecular Biology, Dysentery, Bacillary, MESH: Salmonella Infections, General Immunology and Microbiology, Cell Membrane, Epithelial Cells, MESH: Salmonella typhimurium, biology.organism_classification, Immunity, Innate, 030104 developmental biology, membrane remodeling, MESH: Cell Membrane
Abstract: International audience; While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.
Published: 2018

11. High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

Author: Medina-Cano, Daniel, Ucuncu, Ekin, Nguyen, Lam Son, Nicouleau, Michael, Lipecka, Joanna, Bizot, Jean-Charles, Thiel, Christian, Foulquier, François, Lefort, Nathalie, Faivre-Sarrailh, Catherine, Colleaux, Laurence, Guerrera, Ida Chiara, Cantagrel, Vincent, Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de chimie et procédés pour l'énergie, l'environnement et la santé (ICPEES), Université de Strasbourg (UNISTRA)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), JRC Institute for Energy and Transport (IET), European Commission - Joint Research Centre [Petten], Institut de Neurobiologie de la Méditerranée [Aix-Marseille Université] (INMED - INSERM U1249), Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et nanosciences d'Alsace, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), PRA, Fanny, Mécanismes développementaux des anomalies structurelles cérébelleuses - - SCD-Mec2016 - ANR-16-CE12-0005 - AAPG2016 - VALID, ERA-NET rare disease research implementing IRDiRC objectives - E-Rare-3 - - H20202014-12-01 - 2019-11-30 - 643578 - VALID, Institut de psychiatrie et neurosciences (U894 / UMS 1266), Key-Obs, ANR-16-CE12-0005,SCD-Mec,Mécanismes développementaux des anomalies structurelles cérébelleuses(2016), European Project: 643578,H2020,H2020-HCO-2014,E-Rare-3(2014), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Centre de recherche en neurobiologie - neurophysiologie de Marseille (CRN2M), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Sorbonne Paris Cité (USPC), and Centre de Psychiatrie et Neurosciences (U894)
Subjects: Glycosylation, MESH: Immunoglobulins, [SDV]Life Sciences [q-bio], MESH: Neurons, N-glycosylation, MESH: Unfolded Protein Response, MESH: Mice, Knockout, neuroscience, Purkinje Cells, MESH: Axon Guidance, Neural Pathways, cell biology, MESH: Animals, Biology (General), Mice, Knockout, Neurons, neuronal migration, MESH: Proteomics, Srd5a3, Cell Differentiation, MESH: Glycosylation, MESH: Motor Activity, Axon Guidance, MESH: Reproducibility of Results, MESH: Cell Adhesion Molecules, Medicine, lipids (amino acids, peptides, and proteins), [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Membrane Proteins, Research Article, MESH: Cell Differentiation, MESH: Mutation, animal structures, cerebellum, QH301-705.5, Science, Induced Pluripotent Stem Cells, Immunoglobulins, MESH: Cytoplasmic Granules, Motor Activity, MESH: Induced Pluripotent Stem Cells, Cytoplasmic Granules, MESH: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase, MESH: Cell Adhesion, MESH: Purkinje Cells, proteomics, 3-Oxo-5-alpha-Steroid 4-Dehydrogenase, Polysaccharides, Animals, human, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], mouse, MESH: Neural Pathways, Cell Membrane, Membrane Proteins, Reproducibility of Results, cell adhesion, MESH: Cerebellum, carbohydrates (lipids), MESH: Polysaccharides, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, MESH: Gene Deletion, Protein N-glycosylation, Mutation, Unfolded Protein Response, Cell Adhesion Molecules, Gene Deletion, MESH: Cell Membrane, congenital disorders of glycosylation
Abstract: International audience; Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.
Published: 2018

12. Increased expression of ATP12A proton pump in cystic fibrosis airways

Author: Davide Tosi, Arianna Venturini, Luis J. V. Galietta, Alessandro Palleschi, Paolo Scudieri, Isabelle Sermet-Gaudelus, Gilles Crambert, Ilaria Musante, Pablo Martín-Vasallo, P. Morelli, Emanuela Caci, Gabrielle Planelles, Christine Walter, Centre de recherche Croissance et signalisation (UMR_S 845), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Espace et action, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Scudieri, Paolo, Musante, Ilaria, Caci, Emanuela, Venturini, Arianna, Morelli, Patrizia, Walter, Christine, Tosi, Davide, Palleschi, Alessandro, Martin-Vasallo, Pablo, Sermet-Gaudelus, Isabelle, Planelles, Gabrielle, Crambert, Gille, and Galietta, Luis Jv
Subjects: 0301 basic medicine, MESH: Hydrogen-Ion Concentration, Cystic Fibrosis, Pulmonology, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Genetic disease, Cystic fibrosis, MESH: Mice, Knockout, H(+)-K(+)-Exchanging ATPase, Mice, MESH: Ouabain, MESH: H(+)-K(+)-Exchanging ATPase, MESH: Animals, Ouabain, Cells, Cultured, MESH: Sodium-Potassium-Exchanging ATPase, Submucosal glands, Mice, Knockout, Cultured, Chemistry, MESH: Bronchi, Epithelial transport of ions and water, Genetic diseases, Animals, Bronchi, Cell Membrane, Colon, Goblet Cells, Humans, Hydrogen-Ion Concentration, Interleukin-4, Permeability, Potassium, Primary Cell Culture, Proton Pump Inhibitors, Sodium-Potassium-Exchanging ATPase, General Medicine, 3. Good health, Cytokine, MESH: Permeability, medicine.symptom, Research Article, MESH: Cells, Cultured, MESH: Proton Pump Inhibitors, MESH: Cystic Fibrosis, Cells, Knockout, Inflammation, MESH: Primary Cell Culture, 03 medical and health sciences, Downregulation and upregulation, medicine, Secretion, MESH: Colon, MESH: Mice, MESH: Humans, MESH: Interleukin-4, medicine.disease, Molecular biology, 030104 developmental biology, Cell culture, MESH: Potassium, Respiratory epithelium, MESH: Goblet Cells, MESH: Cell Membrane
Abstract: International audience; Proton secretion mediated by ATP12A protein on the surface of the airway epithelium may contribute to cystic fibrosis (CF) lung disease by favoring bacterial infection and airway obstruction. We studied ATP12A in fresh bronchial samples and in cultured epithelial cells. In vivo, ATP12A expression was found almost exclusively at the apical side of nonciliated cells of airway epithelium and in submucosal glands, with much higher expression in CF samples. This could be due to bacterial infection and inflammation, since treating cultured cells with bacterial supernatants or with IL-4 (a cytokine that induces goblet cell hyperplasia) increased the expression of ATP12A in nonciliated cells. This observation was associated with upregulation and translocation of ATP1B1 protein from the basal to apical epithelial side, where it colocalizes with ATP12A. ATP12A function was evaluated by measuring the pH of the apical fluid in cultured epithelia. Under resting conditions, CF epithelia showed more acidic values. This abnormality was minimized by inhibiting ATP12A with ouabain. Following treatment with IL-4, ATP12A function was markedly increased, as indicated by strong acidification occurring under bicarbonate-free conditions. Our study reveals potentially novel aspects of ATP12A and remarks its importance as a possible therapeutic target in CF and other respiratory diseases.
Published: 2018

13. In situ and high-resolution Cryo-EM structure of the Type VI secretion membrane complex

Author: Riccardo Pellarin, Victoria Schmidt, Yassine Cherrak, Laureen Logger, Rémi Fronzes, Romain Kooger, Chiara Rapisarda, Eric Durand, Eric Cascales, Martin Pilhofer, Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Institute of Biochemistry [ETH Zürich], Department of Biology [ETH Zürich] (D-BIOL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), European Institute of Chemistry and Biology, Université Sciences et Technologies - Bordeaux 1, This work was funded by the Centre National de la Recherche Scientifique, the Aix‐Marseille Université, and grants from the Agence Nationale de la Recherche (ANR‐14‐CE14‐0006‐02, ANR‐17‐CE11‐0039‐01) and the Fondation pour la Recherche Médicale (DEQ20180339165) to EC. ED was supported by the INSERM and an EMBO short‐term fellowship (ASTF 417 – 2015). YC is supported by a Doctoral school PhD fellowship from the FRM (ECO20160736014). VS is supported by a post‐doctoral fellowship from the association Espoir contre la Mucoviscidose. LL was supported by a fourth year PhD fellowship from the FRM (FDT20160435498). MP is funded by the European Research Council, the Swiss National Science Foundation and the Helmut Horten Foundation. RF and CR were supported by IDEX Bordeaux through a 'chaire d'excellence' to RF., ANR-17-CE11-0039,T6-PLATFORM,Une approche multidisciplinaire et intégrative pour comprendre l'assemblage, la structure et la dynamique d'une plateforme de queue contractile(2017), ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Sciences et Technologies - Bordeaux 1 (UB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich)-Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich), Bioinformatique structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: In situ, MESH: Protein Structure, Quaternary, Cryo-electron microscopy, [SDV]Life Sciences [q-bio], MESH: Type VI Secretion Systems, Single particle analysis, MESH: Escherichia coli Proteins, law.invention, 03 medical and health sciences, law, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Bacterial Secretion Systems, 030304 developmental biology, Type VI secretion system, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, 030306 microbiology, Chemistry, Negative stain, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Membrane, Biophysics, MESH: Cryoelectron Microscopy, MESH: Membrane Proteins, Electron microscope, MESH: Models, Molecular, MESH: Cell Membrane
Abstract: Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. The T6SS is composed of a baseplate that contains a spike onto which an inner tube is built, surrounded by a contractile sheath. Unlike phages that are released to and act in the extracellular medium, the T6SS is an intracellular machine inserted in the bacterial membranes by a trans-envelope complex. This membrane complex (MC) comprises three proteins: TssJ, TssL and TssM. We previously reported the low-resolution negative stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography analysis of the T6SS MC confirmed the 5-fold symmetry in situ and identified the regions of the structure that insert into the bacterial membranes. A high resolution model obtained by single particle cryo-electron microscopy reveals its global architecture and highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, a 11-residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we revisit the model on the mechanism of action of the MC during T6SS assembly and function.
Published: 2018

14. Organization of GPI-anchored proteins at the cell surface and its physiopathological relevance

Author: Simona Paladino, Stéphanie Lebreton, Chiara Zurzolo, Trafic membranaire et Pathogénèse, Institut Pasteur [Paris], Università degli studi di Napoli Federico II, CEINGE - Biotecnologie Avanzate, This work is supported by Institut Pasteur-Institut Curie PIC3i, and by research grants from Agence Nationale de la Recherche [ANR-16-CE16–0019-01] and Equipe Fondation Recherche Medicale 2014 [DEQ20140329557] to C.Z., We would like to thank Dr. N. Sauvonnet (Institut Pasteur) for fruitful discussion., ANR-16-CE16-0019,Neurotunn,Role des nanotubes membranaires dans la propagation d'agrégats protéiques impliqués dans les maladie neurodégénératives(2016), Lebreton, Stéphanie, Zurzolo, Chiara, Paladino, Simona, Institut Pasteur [Paris] (IP), and University of Naples Federico II = Università degli studi di Napoli Federico II
Subjects: 0301 basic medicine, MESH: Signal Transduction, MESH: Neoplasm Proteins, actin cytoskeleton, membrane domain, Cell, Oligosaccharides, Biochemistry, Oligosaccharide, MESH: Neurodegenerative Diseases, Cell membrane, Neoplasms, MESH: Animals, MESH: Neoplasms, Plasma membrane organization, Chemistry, Neurodegenerative Diseases, GPI-Linked Protein, membrane organization, GPI-anchored proteins, Neoplasm Proteins, Cell biology, medicine.anatomical_structure, membrane domains, lipids (amino acids, peptides, and proteins), Signal transduction, protein trafficking and sorting, Human, Signal Transduction, clustering, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, GPI-Linked Proteins, MESH: Cell Adhesion, Neoplasm Protein, 03 medical and health sciences, Cell Adhesion, Extracellular, medicine, Animals, Humans, Cell adhesion, Molecular Biology, GPI-anchored protein, MESH: Humans, Neurodegenerative Disease, epithelial cell polarity, Animal, Cell Membrane, cholesterol, Actin cytoskeleton, carbohydrates (lipids), 030104 developmental biology, Membrane protein, MESH: Protein Processing, Post-Translational, Neoplasm, MESH: GPI-Linked Proteins, Protein Processing, Post-Translational, MESH: Oligosaccharides, MESH: Cell Membrane
Abstract: International audience; Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrPC, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.
Published: 2018

15. Biogenesis and structure of a type VI secretion baseplate

Author: Fabrice Allain, Yassine Cherrak, Christian Malosse, Rémi Fronzes, Guillaume Bouvier, Martial Rey, Benjamin Bardiaux, Julia Chamot-Rooke, Chiara Rapisarda, Riccardo Pellarin, Eric Durand, Eric Cascales, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Microbiologie fondamentale et pathogénicité (Bordeaux), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the CNRS, the Aix-Marseille Université, the Institut Pasteur and the INSERM, and by grants from the Agence Nationale de la Recherche (grants no. ANR-14-CE14-0006 and ANR-17-CE11-0039 to E.C., and no. ANR-11-EQPX-008 to J.C.R.). Work of Y.C. was supported by an 'Ecole Doctorale' PhD fellowship from the 'Fondation pour la Recherche Médicale' (grant no. FRM-ECO20160736014). R.F. and C.R. were supported by IDEX Bordeaux through a 'chaire d’excellence' to R.F. We acknowledge the European Synchrotron Radiation Facility for provision of beamtime on CM01 and thank G. Effantin and E. Kandiah for assistance. F.A. thanks the French Institute of Bioinformatics (IFB, grant no. ANR-11-INBS-0013) for financial support., We thank members of the E.C. and R.F. research groups, and J. Sturgis for helpful discussions and support, M. Weigt and C. Feinauer for useful discussion on evolutionary covariance, T. Huynh and G. Canet for assistance with the computer clusters at the Institut Pasteur and Institut Européen de Chimie et Biologie (IECB), respectively, A. Kosta (Plateforme de microscopie, Institut de Microbiologie de la Méditerranée (IMM), Marseille, France) for providing access to the IMM EM facility and for checking the quality of samples by negative stain EM, A. Bezault for support at the cryo-EM facility at IECB and T. Doan and L. Espinosa for their helpful support with the fluorescence microscopy device and analysis. We thank M. Nilges (Institut Pasteur) for access to the computer cluster of the ERC project 'BayCellS'., ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), ANR-17-CE11-0039,T6-PLATFORM,Une approche multidisciplinaire et intégrative pour comprendre l'assemblage, la structure et la dynamique d'une plateforme de queue contractile(2017), ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), ANR-11-INBS-0013,IFB (ex Renabi-IFB),Institut français de bioinformatique(2011), Bioinformatique structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], ANR-11-EQPX-0008/11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), ANR-11-INBS-0013/11-INBS-0013,ReNaBi-IFB,Institut français de bioinformatique(2011), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Protein Conformation, alpha-Helical, Protein Conformation, [SDV]Life Sciences [q-bio], MESH: Type VI Secretion Systems, MESH: Escherichia coli Proteins, Applied Microbiology and Biotechnology, Cell membrane, 0302 clinical medicine, Protein structure, Bacterial secretion, MESH: Protein Conformation, Bacteriophages, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, MESH: Escherichia coli, Escherichia coli Proteins, Type VI Secretion Systems, Cell biology, medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Cryoelectron Microscopy, Cell envelope, MESH: Models, Molecular, Microbiology (medical), Immunology, Microbiology, 03 medical and health sciences, Genetics, medicine, Escherichia coli, Secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Interaction Domains and Motifs, MESH: Bacteriophages, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, Type VI secretion system, MESH: Protein Conformation, alpha-Helical, MESH: Protein Interaction Domains and Motifs, Cell Membrane, Cryoelectron Microscopy, Cell Biology, Bacterial pathogenesis, MESH: Multiprotein Complexes, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Multiprotein Complexes, 030217 neurology & neurosurgery, Function (biology), Biogenesis, MESH: Cell Membrane
Abstract: Comment in Type VI secretion system baseplate. [Nat Microbiol. 2018]; International audience; To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and eukaryotic cells. The T6SS comprises two functional subcomplexes: a bacteriophage-related tail structure anchored to the cell envelope by a membrane complex. As in other contractile injection systems, the tail is composed of an inner tube wrapped by a sheath and built on the baseplate. In the T6SS, the baseplate is not only the tail assembly platform, but also docks the tail to the membrane complex and hence serves as an evolutionary adaptor. Here we define the biogenesis pathway and report the cryo-electron microscopy (cryo-EM) structure of the wedge protein complex of the T6SS from enteroaggregative Escherichia coli (EAEC). Using an integrative approach, we unveil the molecular architecture of the whole T6SS baseplate and its interaction with the tail sheath, offering detailed insights into its biogenesis and function. We discuss architectural and mechanistic similarities but also reveal key differences with the T4 phage and Mu phage baseplates.
Published: 2018

16. Pore-forming activity of the $Pseudomonas\ aeruginosa$ type III secretion system translocon alters the host epigenome

Author: Charlotte Lombardi, Alain Filloux, Laurent Dortet, François Cretin, Andréa Dessen, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Vaincre la Mucoviscidose RF20140501133, ANR project PRP1.4 T3SS, European Project: 654909,H2020,H2020-MSCA-IF-2014,T6SS-PSEUDO-LIP(2016), Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Imperial College London, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Medical Research Council (MRC)
Subjects: 0301 basic medicine, EFFECTOR TARGETS, NF-KAPPA-B, Moths, medicine.disease_cause, Applied Microbiology and Biotechnology, Virulence factor, Epigenesis, Genetic, Type three secretion system, Histones, INFECTION, Type III Secretion Systems, MESH: Animals, MESH: Epigenesis, Genetic, PHOSPHORYLATION, MESH: Bacterial Proteins, Host cell membrane, MESH: Histones, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, MESH: Moths, EPITHELIAL-CELLS, Translocon, Cell biology, Histone Code, Larva, Host-Pathogen Interactions, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Life Sciences & Biomedicine, Microbiology (medical), GENES, Immunology, Microbiology, 03 medical and health sciences, MESH: Type III Secretion Systems, Bacterial Proteins, Genetics, medicine, Animals, Humans, Secretion, HISTONE H3, PROTEIN PHOSPHATASES, Antigens, Bacterial, Science & Technology, MESH: Humans, Cell Membrane, MESH: Host-Pathogen Interactions, Cell Biology, Epigenome, biochemical phenomena, metabolism, and nutrition, MAPK, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, MESH: Histone Code, A549 Cells, MESH: HeLa Cells, bacteria, MESH: A549 Cells, VI SECRETION, MESH: Larva, MESH: Antigens, Bacterial, HeLa Cells, MESH: Cell Membrane
Abstract: Recent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS ‘needle’ delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB–PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion. The pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon serves as a virulence factor to induce host epigenome modification and promote infection.
Published: 2018

17. Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells

Author: Chopard, Christophe, Tong, Phuoc Bao Viet, Toth, Petra, Schatz, Malvina, Yezdi, Hocine, Debaisieux, Solène, Mettling, Clément, Gross, Antoine, Pugniere, Martine, Tu, Annie, Strub, Jean Marc, Mesnard, Jean-Michel, Vitale, Nicolas, Beaumelle, Bruno, Institut des Neurosciences Cellulaires et Intégratives (INCI), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Biomatériaux et Bioingénierie (BB), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Matériaux et nanosciences d'Alsace, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Université de Strasbourg (UNISTRA), Institut Pluridisciplinaire Hubert Curien (IPHC), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Gross, Antoine, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Équipe 'Rythme, vie et mort de la rétine', Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique humaine (IGH), 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), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Matériaux et nanosciences d'Alsace, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Spectrométrie de Masse BioOrganique, Département des Sciences Analytiques, Institut Pluridisciplinaire Hubert Curien (LSMBO-DSA-IPHC), Département Recherches Subatomiques (DRS-IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Département Interactions Physique, Chimie et Vivant (DIPCV-IPHC), Département Ecologie, Physiologie et Ethologie (DEPE-IPHC), Laboratoire de spectrométrie de masse BioOrganique, Institut Pluridisciplinaire Hubert Curien (IPHC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Université de Strasbourg (UNISTRA)-Institut des Neurosciences Cellulaires et Intégratives (INCI)-Centre National de la Recherche Scientifique (CNRS), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Microbiologie et Pathologie Cellulaire Infectieuse, Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM), Département Neurotransmission et sécrétion neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives (INCI)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)
Subjects: Phosphatidylinositol 4,5-Diphosphate, MESH: Rats, Science, Lipoylation, MESH: Lipoylation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Acyltransferases, PC12 Cells, MESH: Animals, Newborn, Article, MESH: HIV-1, Jurkat Cells, Mice, MESH: Mice, Inbred C57BL, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], MESH: Jurkat Cells, MESH: PC12 Cells, MESH: Protein Binding, MESH: RAW 264.7 Cells, Animals, Humans, MESH: Animals, lcsh:Science, MESH: Mice, ComputingMilieux_MISCELLANEOUS, MESH: tat Gene Products, Human Immunodeficiency Virus, MESH: Humans, MESH: Cyclophilin A, Cell Membrane, MESH: Phosphatidylinositol 4,5-Diphosphate, Rats, Mice, Inbred C57BL, HEK293 Cells, RAW 264.7 Cells, Animals, Newborn, MESH: HEK293 Cells, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, HIV-1, lcsh:Q, tat Gene Products, Human Immunodeficiency Virus, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Cyclophilin A, Acyltransferases, MESH: Cell Membrane, Protein Binding
Abstract: Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P2. It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P2. In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P2-dependent membrane traffic such as phagocytosis and neurosecretion., It is not clear whether and how incoming HIV-1 Tat accumulates in uninfected cells. Here, Chopard et al. show that, in uninfected cells, incoming Tat is palmitoylated on Cys31 by DHHC-20, which increases its affinity for PI(4,5)P2 and results in its accumulation at the plasma membrane.
Published: 2018

18. Simultaneous Determination of Protein Structure and Dynamics Using Cryo-Electron Microscopy

Author: Massimiliano Bonomi, Riccardo Pellarin, Michele Vendruscolo, Department of Chemistry [Cambridge, UK], University of Cambridge [UK] (CAM), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Time Factors, Protein Conformation, Cryo-electron microscopy, Biophysics, Biology, Molecular Dynamics Simulation, Bioinformatics, 01 natural sciences, Molecular dynamics, 03 medical and health sciences, MESH: Protein Conformation, Protein structure, 0103 physical sciences, Microscopy, MESH: Molecular Dynamics Simulation, MESH: Proteins, 030304 developmental biology, Physics, 0303 health sciences, 010304 chemical physics, Cell Membrane, Cryoelectron Microscopy, MESH: Time Factors, Dynamics (mechanics), Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 030104 developmental biology, Proteins metabolism, Thermodynamics, MESH: Cryoelectron Microscopy, MESH: Thermodynamics, Biological system, MESH: Cell Membrane
Abstract: International audience; Cryo-electron microscopy is rapidly emerging as a powerful technique to determine the structures of complex macromolecular systems elusive to other techniques. Because many of these systems are highly dynamical, characterizing their movements is also a crucial step to unravel their biological functions. To achieve this goal, we report an integrative modeling approach to simultaneously determine structure and dynamics of macromolecular systems from cryo-electron microscopy density maps. By quantifying the level of noise in the data and dealing with their ensemble-averaged nature, this approach enables the integration of multiple sources of information to model ensembles of structures and infer their populations. We illustrate the method by characterizing structure and dynamics of the integral membrane receptor STRA6, thus providing insights into the mechanisms by which it interacts with retinol binding protein and translocates retinol across the membrane.
Published: 2017

19. A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling

Author: Sylviane Hoos, Chi Zhang, Gitte W. Haxholm, Patrick England, Birthe B. Kragelund, Isabelle Broutin, Florence Boutillon, Mads Nygaard, Marie Bernadet, Vincent Goffin, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université Paris Descartes - Paris 5 (UPD5), University of Copenhagen = Københavns Universitet (KU), Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Danish Cancer Society grants (to B. B. K.), Danish Research Councils for Health and Disease Grants 09-072179 and 12-125862 (to B. B. K.), the Novo Nordic Foundation (to B. B. K.), Ligue Contre le Cancer Grant RS11/75-26 (to I. B.), University Paris Descartes Grant 990 UMRS 845/2011/02 (to V. G. and I. B.), and the Agence Nationale de la Recherche Grant ANR-07-PCVI-0029 (to V. G.)., ANR-07-PCVI-0029,PROLACTUMOR,Processus physiopathologiques impliquant le récepteur de la prolactine : approches physico-chimiques dynamiques et structurales(2007), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University of Copenhagen = Københavns Universitet (UCPH), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), broutin, isabelle, and Physique et chimie du vivant - Processus physiopathologiques impliquant le récepteur de la prolactine : approches physico-chimiques dynamiques et structurales - - PROLACTUMOR2007 - ANR-07-PCVI-0029 - PCVI - VALID
Subjects: MESH: Signal Transduction, MAPK/ERK pathway, Magnetic Resonance Spectroscopy, Nuclear Magnetic Resonance (NMR), [SDV]Life Sciences [q-bio], Ligands, STAT Transcription Factor, Biochemistry, MESH: Circular Dichroism, MESH: Protein Structure, Tertiary, 0302 clinical medicine, STAT5 Transcription Factor, MESH: Ligands, Extracellular Signal-Regulated MAP Kinases, Receptor, MESH: Extracellular Signal-Regulated MAP Kinases, MESH: Cytokines, 0303 health sciences, MESH: Protein Multimerization, Circular Dichroism, Cell Surface Receptor, Cell biology, [SDV] Life Sciences [q-bio], Extracellular Signal-regulated Kinase (ERK), 030220 oncology & carcinogenesis, Mitogen-activated protein kinase, Cytokines, Signal transduction, Cytokine receptor, MESH: Spectrometry, Fluorescence, Protein Binding, Signal Transduction, MAP Kinase Signaling System, Receptors, Prolactin, Biology, Cell Line, 03 medical and health sciences, MESH: Receptors, Prolactin, Cell surface receptor, MESH: Cell Proliferation, Extracellular, Humans, MESH: Protein Binding, Cytokine, Molecular Biology, Cell Proliferation, 030304 developmental biology, MESH: Humans, MESH: MAP Kinase Signaling System, MESH: Magnetic Resonance Spectroscopy, MESH: STAT5 Transcription Factor, Prolactin receptor, Cell Membrane, Cell Biology, Protein Structure, Tertiary, MESH: Cell Line, Spectrometry, Fluorescence, biology.protein, Protein Multimerization, MESH: Cell Membrane
Abstract: Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination. Background: Discrimination of cytokine receptor signaling pathways is poorly understood. Results: Manipulation of extracellular prolactin receptor interface residues selectively affected activation of the MAPK pathway but not the STAT5 pathway. Conclusion: MAPK pathway activation correlated with apparent ligand·receptor complex lifetimes suggesting pathway-specific lifetime thresholds for activation. Significance: Discrimination of prolactin receptor signaling is controlled by the extracellular homodimer receptor interface.
Published: 2015

20. Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis

Author: Anne K. Kenworthy, Mijo Simunovic, Anne A. Schmidt, Valérie Chambon, Patricia Bassereau, Henri-François Renard, Christian Wunder, Cécile Sykes, Joël Lemière, Emmanuel Boucrot, Christophe Lamaze, Harvey T. McMahon, Maria Daniela Garcia-Castillo, Senthil Arumugam, Ludger Johannes, Bondidier, Martine, Chimie biologique des membranes et ciblage thérapeutique ( CBMCT - UMR 3666 / U1143 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut Curie-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Physico-Chimie-Curie ( PCC ), Centre National de la Recherche Scientifique ( CNRS ) -INSTITUT CURIE-Université Pierre et Marie Curie - Paris 6 ( UPMC ), Department of Chemistry, University of Chicago, Université Paris Diderot - Paris 7 ( UPD7 ), Institute of Structural and Molecular Biology, Birkbeck College, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine. Nashville, Vanderbilt University School of Medicine. Nashville-Vanderbilt University School of Medicine. Nashville, Institut Jacques Monod ( IJM ), Université Paris Diderot - Paris 7 ( UPD7 ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratory of Molecular Biology, Division of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK, Agence Nationale pour la Recherche : (ANR-09-BLAN-283, ANR-10-LBX-0038, ANR-11 BSV2 014 03, ANR-12-BSV5-0014), Indo-French Centre for the Promotion of Advanced Science (project no. 3803), Marie Curie Actions — Networks for Initial Training (FP7-PEOPLE-2010-ITN), Marie Curie International Reintegration Grant (FP7-RG-277078), European Research Council advanced grant (project 340485), Royal Society (RG120481), Fondation ARC pour la Recherche sur le Cancer (DEQ20120323737), National Institutes of Health (RO1 GM106720), Ligue contre le Cancer, Comité de Paris (RS08/75-89), Fondation ARC pour la Recherche sur le Cancer, AXA Research Funds, Biological Sciences Research Council, Chateaubriand fellowship, France and Chicago Collaborating in the Sciences grant, Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Physico-Chimie-Curie (PCC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), Vanderbilt University School of Medicine [Nashville], Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)
Subjects: MESH : Cell Line, MESH: Rats, MESH : Endocytosis, MESH : Cell Membrane, MESH: Shiga Toxin, Endocytic cycle, MESH : Actins, MESH : Dynamins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, MESH: Acyltransferases, Biology, MESH: Actins, Endocytosis, environment and public health, Clathrin, Cell membrane, 03 medical and health sciences, 0302 clinical medicine, medicine, BAR domain, MESH: Animals, MESH: Clathrin, Endophilin-A2, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, MESH: Cholera Toxin, 030304 developmental biology, Dynamin, 0303 health sciences, MESH: Humans, MESH : Clathrin, Multidisciplinary, MESH : Rats, MESH : Cholera Toxin, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, MESH : Humans, MESH: Cell Line, Cell biology, MESH: Dynamins, medicine.anatomical_structure, MESH : Shiga Toxin, MESH: Endocytosis, Amphiphysin, biology.protein, MESH : Animals, MESH : Acyltransferases, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract: International audience; During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.
Published: 2014

21. Ion Channels as Reporters of Membrane Receptor Function: Automated Analysis in Xenopus Oocytes

Author: Zlatomir Todorov, Michel Vivaudou, Gina Catalina Reyes-Mejia, Christophe Moreau, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Centre National de la Recherche Scientifique (CNRS), ANR-11-RPIB-0022,VenomPicoScreen,Découverte de principes actifs à partir de venins en utilisant des bicouches artificielles miniaturisées(2011), ANR-11-LABX-0015,ICST,Canaux ioniques d'intérêt thérapeutique(2011), European Project: NIH, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, MESH: Signal Transduction, G-protein-coupled receptor, Robot, Ion-channel-coupled receptor, Xenopus, MESH: Automation, Laboratory, MESH: Receptors, G-Protein-Coupled, MESH: Oocytes, 03 medical and health sciences, 0302 clinical medicine, MESH: Xenopus laevis, Two-electrode voltage clamp, MESH: Animals, G protein-coupled inwardly-rectifying potassium channel, Potassium channel, MESH: Electrophysiological Phenomena, MESH: Xenopus Proteins, Ion channel, G protein-coupled receptor, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, biology.organism_classification, Molecular biology, 030104 developmental biology, Second messenger system, Biophysics, Signal transduction, Xenopus oocyte, MESH: Female, 030217 neurology & neurosurgery, Intracellular, MESH: G Protein-Coupled Inwardly-Rectifying Potassium Channels, MESH: Cell Membrane
Abstract: International audience; G-protein-coupled receptors (GPCR) are the most widely used system of communication used by cells. They sense external signals and translate them into intracellular signals. The information is carried mechanically across the cell membrane, without perturbing its integrity. Agonist binding on the extracellular side causes a change in receptor conformation which propagates to the intracellular side and causes release of activated G-proteins, the first messengers of a variety of signaling cascades.Permitting access to powerful electrophysiological techniques, ion channels can be employed to monitor precisely the most proximal steps of GPCR signaling, receptor conformational changes, and G-protein release. The former is achieved by physical attachment of a potassium channel to the GPCR to create an Ion-Channel Coupled Receptor (ICCR). The latter is based on the use of G-protein-regulated potassium channels (GIRK). We describe here how these two systems may be used in the Xenopus oocyte heterologous system with a robotic system for increased throughput.
Published: 2017

22. Dynamic distinctions in the Na + /Ca 2+ exchanger adopting the inward- and outward-facing conformational states

Author: Bosmat Refaeli, Petr Man, Liat van Dijk, Moshe Giladi, Eric Forest, Daniel Khananshvili, Lior Almagor, Reuben Hiller, Department of Physiology and Pharmacology, Tel Aviv University [Tel Aviv], the BioCeV-Institute of Microbiology, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Centre National de la Recherche Scientifique (CNRS), Tel Aviv University (TAU), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, Methanococcus, Biochemistry, exchanger, MESH: Recombinant Proteins, MESH: Apoproteins, MESH: Protein Conformation, Calcium-binding protein, calcium transport, calcium-binding protein, MESH: Ligands, MESH: Sodium, membrane protein, MESH: Peptide Fragments, biology, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, MESH: Archaeal Proteins, MESH: Amino Acid Substitution, MESH: Deuterium Exchange Measurement, transporter, MESH: Calcium, hydrogen exchange mass spectrometry, Intracellular, MESH: Models, Molecular, MESH: Computational Biology, MESH: Mutation, membrane transport, 03 medical and health sciences, MESH: Protein Stability, Extracellular, sodium-calcium exchange, Molecular Biology, MESH: Protein Interaction Domains and Motifs, calcium, Cell Biology, Membrane transport, MESH: Cysteine, biology.organism_classification, MESH: Sodium-Calcium Exchanger, Crystallography, Cytosol, 030104 developmental biology, Membrane protein, MESH: Mutagenesis, Insertional, MESH: Binding Sites, Biophysics, MESH: Methanocaldococcus, Cysteine, MESH: Cell Membrane
Abstract: International audience; Na(+)/Ca(2+) exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na(+) or Ca(2+) specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.
Published: 2017

23. Polar N-terminal Residues Conserved in Type 2 Secretion Pseudopilins Determine Subunit Targeting and Membrane Extraction Steps during Fibre Assembly

Author: Ingrid Guilvout, Alexandra East, Guy Tran Van Nhieu, Olivera Francetic, Nathalie Nadeau, Peter J. Bond, Javier Santos-Moreno, Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution)), University of Cambridge [UK] (CAM), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Systèmes macromoléculaires et Signalisation, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Agency for science, technology and research [Singapore] (A*STAR), National University of Singapore (NUS), This work was funded by the ANR FiberSpace grant N°ANR-14-CE09-0004. Javier Santos-Moreno was funded by a fellowship from the Basque Government, and Alexandra East was supported by an EPSRC PhD studentship. The computational work benefited from use of the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/), provided by Dell Inc. using Strategic Research, Infrastructure Funding from the Higher Education Funding Council for England., We thank Evelyne Richet and Jenny-Lee Thomassin for insightful comments and critical reading of the manuscript. We thank Cesar Valencia and Jenny-Lee Thomassin for help with IF data processing and statistical analysis, Mariette Bonnet for help and advice, and Stéphane Romero for stimulating discussions about biological fibres. We are grateful to all members of the Laboratory of Intercellular Communication and Microbial Infections, the Laboratory for Macromolecular Systems and Signalling and of the Biochemstry of Macromolecular Interactions Unit for helpful discussions and friendly support. We thank Gouzel Karimova and Daniel Ladant for the strains, plasmids, and advice concerning the BAC2H analysis., ANR-14-CE09-0004,FiberSpace,Pili de type IV et pseudopili: structure, dynamique, assemblage et fonction moléculaire(2014), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Francetic, Olivera, and Appel à projets générique - Pili de type IV et pseudopili: structure, dynamique, assemblage et fonction moléculaire - - FiberSpace2014 - ANR-14-CE09-0004 - Appel à projets générique - VALID
Subjects: 0301 basic medicine, Protein subunit, 030106 microbiology, Mutant, membrane proteins, Flagellum, Molecular Dynamics Simulation, Pilus, 03 medical and health sciences, MESH: Mutant Proteins, Structural Biology, Type II Secretion Systems, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Protein Binding, MESH: Molecular Dynamics Simulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Conserved Sequence, Phospholipids, MESH: Phospholipids, MESH: Type II Secretion Systems, MESH: Conserved Sequence, biology, MESH: Protein Multimerization, Cell Membrane, immunofluorescence microscopy, molecular dynamics simulations, N-methylation, type 4 fibre assembly, MESH: Fimbriae Proteins, N-terminus, 030104 developmental biology, Membrane protein, Biochemistry, Chaperone (protein), Pilin, biology.protein, Biophysics, Mutant Proteins, Fimbriae Proteins, Protein Multimerization, Protein Binding, MESH: Cell Membrane
Abstract: International audience; Bacterial type 2 secretion systems (T2SS), type 4 pili, and archaeal flagella assemble fibres from initially membrane-embedded pseudopilin and pilin subunits. Fibre subunits are made as precursors with positively charged N-terminal anchors, whose cleavage via the prepilin peptidase, essential for pilin membrane extraction and assembly, is followed by N-methylation of the mature (pseudo)pilin N terminus. The conserved Glu residue at position 5 (E5) of mature (pseudo)pilins is essential for assembly. Unlike T4 pilins, where E5 residue substitutions also abolish N-methylation, the E5A variant of T2SS pseudopilin PulG remains N-methylated but is affected in interaction with the T2SS component PulM. Here, biochemical and functional analyses showed that the PulM interaction defect only partly accounts for the PulG(E5A) assembly defect. First, PulG(T2A) variant, equally defective in PulM interaction, remained partially functional. Furthermore, pseudopilus assembly defect of pulG(E5A) mutant was stronger than that of the pulM deletion mutant. To understand the dominant effect of E5A mutation, we used molecular dynamics simulations of PulG(E5A), methylated PulG(WT) (MePulG(WT)), and MePulG(E5A) variant in a model membrane. These simulations pointed to a key role for an intramolecular interaction between the pseudopilin N-terminal amine and E5 to limit polar interactions with membrane phospholipids. N-methylation of the N-terminal amine further limited its interactions with phospholipid head-groups to facilitate pseudopilin membrane escape. By binding to polar residues in the conserved N-terminal region of PulG, we propose that PulM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process.
Published: 2017

24. Emerging roles of MICAL family proteins - from actin oxidation to membrane trafficking during cytokinesis

Author: Guillaume Romet-Lemonne, Stéphane Frémont, Arnaud Echard, Anne Houdusse, Trafic membranaire et Division cellulaire - Membrane Traffic and Cell Division, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL), This work of our laboratories been supported by Institut Pasteur, Centre National de la Recherche Scientifique (CNRS), Fondation pour la Recherche Médicale (FRM) (Equipe FRM DEQ20120323707), Institut National Du Cancer (INCa) (2014-1-PLBIO-04-IP1), Agence Nationale de la Recherche (ANR) (AbCyStem, CytoSign) and the IXCORE Foundation to A.E., by grants from the CNRS and INCa (2014-1-PLBIO-04-ICR-1) to A.H. The A.H. team is part of by the Labex CelTisPhyBio 11-LBX-0038, which is part of the Initiative d’Excellence at PSL Research University (ANR-10-IDEX-0001-02 PSL)., ANR-15-CE13-0001,AbsCyStem,Régulation de l'abscission dans les cellules animales(2015), ANR-16-CE13-0004,CYTOSIGN,Polarité épithéliale et signalisation au cours de la cytodiérèse des cellules animales(2016), and ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010)
Subjects: 0301 basic medicine, MESH: Cytokinesis, MESH: Oxidation-Reduction, MESH: Protein Transport, Cell division, macromolecular substances, Membrane trafficking, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, MESH: Actins, ESCRT, 03 medical and health sciences, Abscission, Animals, Humans, Oxidoreduction, MESH: Animals, Cytoskeleton, Actin, Cytokinesis, MESH: Humans, MICAL, Cell Membrane, Cell Biology, Actin cytoskeleton, Actins, Cell biology, Protein Transport, 030104 developmental biology, Multigene Family, MESH: Multigene Family, Rab, Oxidation-Reduction, Rab GTPase, MESH: Cell Membrane
Abstract: Cytokinetic abscission is the terminal step of cell division, leading to the physical separation of the two daughter cells. The exact mechanism mediating the final scission of the intercellular bridge connecting the dividing cells is not fully understood, but requires the local constriction of endosomal sorting complex required for transport (ESCRT)-III-dependent helices, as well as remodelling of lipids and the cytoskeleton at the site of abscission. In particular, microtubules and actin filaments must be locally disassembled for successful abscission. However, the mechanism that actively removes actin during abscission is poorly understood. In this Commentary, we will focus on the latest findings regarding the emerging role of the MICAL family of oxidoreductases in F-actin disassembly and describe how Rab GTPases regulate their enzymatic activity. We will also discuss the recently reported role of MICAL1 in controlling F-actin clearance in the ESCRT-III-mediated step of cytokinetic abscission. In addition, we will highlight how two other members of the MICAL family (MICAL3 and MICAL-L1) contribute to cytokinesis by regulating membrane trafficking. Taken together, these findings establish the MICAL family as a key regulator of actin cytoskeleton dynamics and membrane trafficking during cell division.
Published: 2017

25. Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

Author: Sandra Cortès, Marie José Stasia, Jean-Luc Lenormand, Julie Brault, Guillaume Vaganay, Aline Le Roy, Université Grenoble Alpes - UFR Pharmacie (UGA UFRP), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Synthelis, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Centre National de la Recherche Scientifique (CNRS), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, Pharmaceutical Science, chronic granulomatous disease, Granulomatous Disease, Chronic, law.invention, Cell membrane, chemistry.chemical_compound, Chronic granulomatous disease, MESH: Granulomatous Disease, Chronic, International Journal of Nanomedicine, NADPH oxidase complex, law, protein therapy, Drug Discovery, MESH: Phagocytosis, Candida, Original Research, Oxidase test, NADPH oxidase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, MESH: Reactive Oxygen Species, Cell Differentiation, Genetic Diseases, X-Linked, General Medicine, MESH: Protein Subunits, 3. Good health, medicine.anatomical_structure, Child, Preschool, Recombinant DNA, Nicotinamide adenine dinucleotide phosphate, MESH: Proteolipids, MESH: Cell Differentiation, induced pluripotent stem cells, Proteolipids, Biophysics, Bioengineering, MESH: Induced Pluripotent Stem Cells, MESH: NADPH Oxidases, Biomaterials, 03 medical and health sciences, Phagocytosis, proteoliposomes, MESH: Candida, MESH: Genetic Diseases, X-Linked, medicine, Humans, MESH: Humans, Macrophages, MESH: Child, Preschool, Organic Chemistry, Cell Membrane, MESH: Macrophages, NADPH Oxidases, medicine.disease, Molecular biology, Protein Subunits, 030104 developmental biology, chemistry, Membrane protein, Immunology, biology.protein, Reactive Oxygen Species, MESH: Cell Membrane
Abstract: Julie Brault,1,2 Guillaume Vaganay,3 Aline Le Roy,4–6 Jean-Luc Lenormand,1 Sandra Cortes,3 Marie José Stasia1,2 1UMR CNRS 5525, University of Grenoble Alpes, Grenoble, France; 2CGD Diagnosis and Research Centre, University Hospital Centre of Grenoble Alpes, Grenoble, France; 3Synthelis SAS, La Tronche, France; 4IBS, University of Grenoble Alpes, Grenoble, France; 5CNRS, IBS, University Grenoble Alpes, Grenoble, France; 6CEA, IBS, University of Grenoble Alpes, Grenoble, France Abstract: Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochrome b558 composed of NOX2 and p22phox, the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22phox liposomes was estimated to be around 700nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47phox, p67phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22phox liposomes for 8h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments. Keywords: protein therapy, proteoliposomes, chronic granulomatous disease, NADPH oxidase, induced pluripotent stem cells, macrophages&nbsp
Published: 2017

26. Studying cytokinesis and midbody remnants using correlative light/scanning EM

Author: Frémont, Stéphane, Echard, A., Trafic membranaire et Division cellulaire - Membrane Traffic and Cell Division, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work has been supported by Institut Pasteur, CNRS, FRM (Equipe FRM DEQ20120323707), INCa (2014-1-PL BIO-04-IP1), iXCore foundation for Research, and ANR (AbsCyStem)., and ANR-15-CE13-0001,AbsCyStem,Régulation de l'abscission dans les cellules animales(2015)
Subjects: MESH: Cytokinesis, Abscission, Intercellular bridge, MESH: Humans, MESH: Microtubules, Cell Membrane, MESH: Microscopy, Electron, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microtubules, Microscopy, Electron, Live-cell imaging, MESH: HeLa Cells, MESH: Cytoskeleton, Humans, Microscopy, Phase-Contrast, Scanning electron microscopy, Cytoskeleton, MESH: Cell Membrane, MESH: Microscopy, Phase-Contrast, Cytokinesis, HeLa Cells
Abstract: International audience; Cytokinesis is an essential step of cell proliferation leading to the physical separation of the dividing cells. Cytokinesis relies on both large scale and local scale cell shape changes, and terminates with the final abscission cut that requires close apposition of the plasma membrane. While furrow ingression is a prominent feature of the early phase of cytokinesis and is easy to visualize in all models, from dividing eggs to culture cells, the later steps of cytokinesis until abscission can be much more difficult to visualize. One key issue is to combine live-cell imaging over several hours and detailed, structural analysis of the cell shape changes in 3D, in particular at the time of cytokinetic abscission. Here, we describe the methodologies that we recently developed for studying cytokinetic abscission in human culture cells using live-cell phase-contrast microscopy, combined with correlative scanning electron microscopy. This allows us to determine the membrane surface and underlying cytoskeleton of the intercellular bridge with unprecedented precision and to determine the fate of the midbody remnant after abscission.
Published: 2017

27. Secretory Vesicle Clustering in Fungal Filamentous Cells Does Not Require Directional Growth

Author: Agnese Seminara, Charles Puerner, Martine Bassilana, Robert A. Arkowitz, Patrícia M. Silva, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Institut de Physique de Nice (INPHYNI), Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA), ANR-15-IDEX-0001,UCA JEDI,Idex UCA JEDI (2016), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), ANR-15-IDEX-0001,UCA JEDI,Idex UCA JEDI(2015), Arkowitz, Robert, Idex UCA JEDI - - UCA JEDI2015 - ANR-15-IDEX-0001 - IDEX - VALID, 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), and COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)
Subjects: 0301 basic medicine, Light, MESH: Secretory Vesicles, [SDV]Life Sciences [q-bio], GTPase, CDC42, MESH: Optogenetics, 0302 clinical medicine, Models, Candida albicans, morphology, Cell polarity, polarity, Cdc42, cdc42 GTP-Binding Protein, lcsh:QH301-705.5, [SDV.BDD]Life Sciences [q-bio]/Development Biology, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Budding, MESH: Guanosine Triphosphate, Chemistry, Secretory Vesicle, Endocytosis, Cell biology, Actin Cytoskeleton, MESH: Endocytosis, MESH: Fungal Proteins, Guanosine Triphosphate, Protein subunit, Exocyst, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, membrane traffic, fungi, optogenetics, Cell Membrane, Fungal Proteins, Models, Biological, Optogenetics, Secretory Vesicles, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [SDV.BDD] Life Sciences [q-bio]/Development Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, MESH: cdc42 GTP-Binding Protein, Polarity (international relations), MESH: Candida albicans, MESH: Models, Biological, Biological, [SDV.MP.MYC] Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, MESH: Light, 030104 developmental biology, lcsh:Biology (General), MESH: Actin Cytoskeleton, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract: Summary: During symmetry breaking, the highly conserved Rho GTPase Cdc42 becomes stabilized at a defined site via an amplification process. However, little is known about how a new polarity site is established in an already asymmetric cell—a critical process in a changing environment. The human fungal pathogen Candida albicans switches from budding to filamentous growth in response to external cues, a transition controlled by Cdc42. Here, we have used optogenetic manipulation of cell polarity to reset growth in asymmetric filamentous C. albicans cells. We show that increasing the level of active Cdc42 on the plasma membrane results in disruption of the exocyst subunit Sec3 localization and a striking de novo clustering of secretory vesicles. This new cluster of secretory vesicles is highly dynamic, moving by hops and jumps, until a new growth site is established. Our results reveal that secretory vesicle clustering can occur in the absence of directional growth. : Silva et al. use light-dependent plasma membrane recruitment of active Cdc42 to reset polarity in asymmetric filamentous fungal cells. This transient increase in plasma membrane active Cdc42 disrupted membrane traffic and resulted in the formation of a striking de novo secretory vesicle cluster, in the absence of directional growth. Keywords: polarity, optogenetics, Cdc42, membrane traffic, morphology, fungi
Published: 2019

28. Rescue of Functional CFTR Channels in Cystic Fibrosis: A Dramatic Multivalent Effect Using Iminosugar Cluster-Based Correctors

Author: J. Bertrand, Terry D. Butters, Julien de Sousa, Romain Clément, Antoine Joosten, David Rodríguez-Lucena, Camille Decroocq, Philippe Compain, Caroline Norez, Frédéric Becq, Clément Boinot, Institut de Chimie Organique et Analytique (ICOA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Orléans (UO)-Institut de Chimie du CNRS (INC), Institut de Chimie Moléculaire de Reims - UMR 7312 (ICMR), SFR Condorcet, Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Gilbert-Laustriat : Biomolécules, Biotechnologie, Innovation Thérapeutique, Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Oxford Glycobiology Institute, University of Oxford [Oxford], Institut de physiologie et biologie cellulaires (IPBC), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: congenital, hereditary, and neonatal diseases and abnormalities, MESH: Mutation, Cystic Fibrosis, MESH: Cystic Fibrosis, [SDV]Life Sciences [q-bio], Iminosugar, Cystic Fibrosis Transmembrane Conductance Regulator, HL-60 Cells, MESH: Drug Design, medicine.disease_cause, 01 natural sciences, Biochemistry, Cystic fibrosis, 03 medical and health sciences, MESH: HL-60 Cells, Mutant protein, medicine, Humans, Mode of action, Molecular Biology, Gene, 030304 developmental biology, MESH: Cystic Fibrosis Transmembrane Conductance Regulator, 0303 health sciences, Mutation, MESH: Humans, biology, 010405 organic chemistry, Chemistry, Cell Membrane, Organic Chemistry, medicine.disease, Cystic fibrosis transmembrane conductance regulator, Imino Sugars, 3. Good health, 0104 chemical sciences, MESH: Imino Sugars, Drug Design, biology.protein, Molecular Medicine, Protein folding, MESH: Cell Membrane
Abstract: International audience; Cystic fibrosis is caused by a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. N-butyl 1-deoxynojirimycin (N-Bu DNJ), a clinical candidate for the treatment of cystic fibrosis, is able to act as a CFTR corrector by overcoming the processing defect of the mutant protein. To explore the potential of multivalency on CFTR correction activity, a library of twelve DNJ click clusters with valencies ranging from 3 to 14 were synthesized. Significantly, the trivalent analogues were found to be up to 225-fold more potent than N-Bu DNJ and up to 1000-fold more potent than the corresponding monovalent models. These results provide the first description of a multivalent effect for correcting protein folding defects in cells and should have application for the treatment of a number of protein folding disorders. Preliminary mechanistic studies indicated that CFTR correction activity enhancement was not due to a multivalent effect in ER-glucosidase inhibition or to a different mode of action of the multivalent iminosugars.
Published: 2013

29. Undressing the Fungal Cell Wall/Cell Membrane - the Antifungal Drug Targets

Author: Vishukumar Aimanianda, Rui Tada, Jean-Paul Latgé, Aspergillus, Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris]
Subjects: Antifungal Agents, Cell, Antifungal drug, Fungus, MESH: Drug Design, Polysaccharide, Microbiology, Cell wall, Cell membrane, 03 medical and health sciences, MESH: Cell Wall, MESH: Candida, Cell Wall, Polysaccharides, MESH: Molecular Targeted Therapy, Drug Discovery, Mechanical strength, medicine, Animals, Aspergillosis, Humans, MESH: Animals, MESH: Aspergillosis, Molecular Targeted Therapy, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Candida, 030304 developmental biology, Pharmacology, chemistry.chemical_classification, 0303 health sciences, Aspergillus, MESH: Humans, biology, 030306 microbiology, Cell Membrane, Candidiasis, MESH: Antifungal Agents, biology.organism_classification, MESH: Candidiasis, MESH: Polysaccharides, medicine.anatomical_structure, chemistry, MESH: Aspergillus, Drug Design, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH: Cell Membrane
Abstract: International audience; Being external, the fungal cell wall plays a crucial role in the fungal life. By covering the underneath cell, it offers mechanical strength and acts as a barrier, thus protecting the fungus from the hostile environment. Chemically, this cell wall is composed of different polysaccharides. Because of their specific composition, the fungal cell wall and its underlying plasma membrane are unique targets for the development of drugs against pathogenic fungal species. The objective of this review is to consolidate the current knowledge on the antifungal drugs targeting the cell wall and plasma membrane, mainly of Aspergillus and Candida species - the most prevalent fungal pathogens, and also to present challenges and questions conditioning the development of new antifungal drugs targeting the cell wall.
Published: 2013

30. The substrate specificity of the human ADP/ATP carrier AAC1

Author: Chris Chipot, François Dehez, Eva-Maria Krammer, John Mifsud, Eva Pebay-Peyroula, Edmund R.S. Kunji, Stéphanie Ravaud, Thomas, Frank, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Centre National de la Recherche Scientifique (CNRS), Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), MRC Mitochondrial Biology Unit, University of Cambridge [UK] (CAM), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Arylamine N-Acetyltransferase, ATPase, MESH: Adenine Nucleotides, Substrate Specificity, Adenosine Triphosphate, MESH: Adenosine Triphosphate, MESH: Molecular Dynamics Simulation, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], ATP synthase, biology, Adenine Nucleotides, MESH: Escherichia coli, 030302 biochemistry & molecular biology, Mitochondria, Adenosine Diphosphate, Isoenzymes, Lactococcus lactis, Protein Transport, Biochemistry, MESH: Isoenzymes, ATP–ADP translocase, MESH: Mitochondrial ADP, ATP Translocases, ATP synthase alpha/beta subunits, MESH: Protein Transport, Guanine, MESH: Mitochondria, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Biological Transport, Adenylate kinase, Photophosphorylation, Molecular Dynamics Simulation, 03 medical and health sciences, Escherichia coli, Humans, Molecular Biology, 030304 developmental biology, MESH: Guanine, Binding Sites, MESH: Humans, MESH: Adenosine Diphosphate, Chemiosmosis, Cell Membrane, Biological Transport, Cell Biology, Mitochondrial carrier, MESH: Arylamine N-Acetyltransferase, MESH: Binding Sites, MESH: Lactococcus lactis, biology.protein, MESH: Substrate Specificity, Mitochondrial ADP, ATP Translocases, MESH: Cell Membrane
Abstract: International audience; The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.
Published: 2012

31. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm

Author: Nora Mellouk, Jost Enninga, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), NM was supported by a fellowship from the Fondation pour la Recherche Medicale and a grant by the Region Ile de France (DIM-Malinf) to JE and Guy Tran Van Nhieu. JE is member of the LabEx consortium IBEID, and is supported by the Institut Pasteur CARNOT-MIE programme. JE also acknowledges support of an ERC starting grant for this work (Rupteffects, Nr. 261166)., We would like to thank members of the 'Dynamics of Host Pathogen Interactions (DIHP)' Research Unit and Guy Tran Van Nhieu (College de France) for fruitful discussions. We thank Allon Weiner for his contribution to the figure and his interest in the work, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 261166,EC:FP7:ERC,ERC-2010-StG_20091118,RUPTEFFECTS(2011), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Cytoplasm, lcsh:QR1-502, Vacuole, Review, medicine.disease_cause, MESH: Shigella, lcsh:Microbiology, Cytosol, MESH: Cytosol, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Type III Secretion Systems, Shigella, Intestinal Mucosa, Internalization, Host factor, media_common, Effector, membrane trafficking, Rab GTPases, Infectious Diseases, intracellular pathogens, MESH: Epithelial Cells, MESH: Intestinal Mucosa, Microbiology (medical), MESH: Dysentery, Bacillary, media_common.quotation_subject, 030106 microbiology, Immunology, vacuolar rupture, Biology, Microbiology, MESH: Vacuoles, 03 medical and health sciences, MESH: Type III Secretion Systems, medicine, Humans, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Dysentery, Bacillary, MESH: Humans, Intracellular parasite, MESH: Cytoplasm, Cell Membrane, Epithelial Cells, MESH: rab GTP-Binding Proteins, 030104 developmental biology, rab GTP-Binding Proteins, Vacuoles, Rab, MESH: Cell Membrane
Abstract: International audience; Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.
Published: 2016

32. HlyF Produced by Extraintestinal Pathogenic Escherichia coli Is a Virulence Factor That Regulates Outer Membrane Vesicle Biogenesis

Author: Emmanuelle Helloin, Sébastien Houle, Tetsuya Hayashi, Marie Penary, Patricia Martin, Gaëlle Porcheron, Kazunori Murase, Charles M. Dozois, Eric Oswald, Jean-Philippe Nougayrède, University of Miyazaki, Université Fédérale Toulouse Midi-Pyrénées, Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Laboratoire de Comportement, INRA-CNRS, 37380 Nouzilly, France, Institut National de la Recherche Agronomique (INRA), Department of Applied Chemistry, Osaka University [Osaka], CHU Toulouse [Toulouse], This work was supported by the French government, through the Investments for the Future program (ANR-11-EQPX-0003), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants-in-Aid for Young [B] 26860293 to K. M.), the National Science and Engineering Research Council of Canada (Discovery grant to C. M. D.), and a Canada Research Chair (to C. M. D.)., ANR-11-EQPX-0003/11-EQPX-0003,ANINFIMIP,Equipements plateforme animalerie infectieuse de haute-sécurité de Midi Pyrénées(2011), ANR-11-EQPX-0003,ANINFIMIP,Equipements plateforme animalerie infectieuse de haute-sécurité de Midi Pyrénées(2011), Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Courcelles, Michel, Equipements plateforme animalerie infectieuse de haute-sécurité de Midi Pyrénées - - ANINFIMIP2011 - ANR-11-EQPX-0003 - EQPX - VALID, Institut de Recherche en Santé Digestive (IRSD ), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], MESH: Escherichia coli Proteins, Vacuole, MESH: Virulence, medicine.disease_cause, Virulence factor, Immunology and Allergy, MESH: Animals, MESH: Phylogeny, Escherichia coli Infections, Phylogeny, Extraintestinal Pathogenic Escherichia coli, MESH: Gene Expression Regulation, Bacterial, biology, Virulence, MESH: Escherichia coli, Escherichia coli Proteins, pathogenesis, MESH: Chickens, OMV, 3. Good health, [SDV] Life Sciences [q-bio], MESH: Mutagenesis, Site-Directed, Infectious Diseases, Bacterial outer membrane, Hemolysin Factors, Virulence Factors, MESH: Poultry Diseases, MESH: Vacuoles, Microbiology, 03 medical and health sciences, medicine, Autophagy, Escherichia coli, Animals, Humans, MESH: Autophagy, Poultry Diseases, MESH: Virulence Factors, MESH: Escherichia coli Infections, MESH: Humans, Cell Membrane, Pathogenic bacteria, Gene Expression Regulation, Bacterial, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, HlyF, 030104 developmental biology, Vacuoles, MESH: Hemolysin Factors, Mutagenesis, Site-Directed, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Chickens, Bacteria, MESH: Cell Membrane
Abstract: International audience; Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.
Published: 2016

33. A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations

Author: Katrin Hörmann, Jacques Colinge, Leonhard X. Heinz, Keiryn L. Bennett, André C. Müller, Giulio Superti-Furga, Alexey Stukalov, Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), and CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)
Subjects: 0301 basic medicine, Proteomics, Proteome, plasma membrane, Biochemistry, Mass Spectrometry, Liquid chromatography–mass spectrometry, biotin, PIGS, silica beads, chemistry.chemical_classification, MESH: Proteomics, aminooxy-biotin, Anti-Bacterial Agents, MESH: Reproducibility of Results, MESH: Proteome, cell surface, shotgun proteomics, Biotinylation, MESH: Membrane Proteins, comparative analysis, MESH: Cell Line, Tumor, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, 03 medical and health sciences, Cell Line, Tumor, MESH: Anti-Bacterial Agents, Humans, MESH: Biotinylation, Shotgun proteomics, MESH: Tunicamycin, MESH: Mass Spectrometry, Chromatography, MESH: Humans, 030102 biochemistry & molecular biology, Elution, Proteomic Profiling, Cell Membrane, Membrane Proteins, Reproducibility of Results, General Chemistry, tunicamycin, Surface coating, 030104 developmental biology, chemistry, Glycoprotein, MESH: Chromatography, Liquid, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Chromatography, Liquid, MESH: Cell Membrane
Abstract: International audience; Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication, and account for more than two-thirds of known drug targets.1-8 In the past years, several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach.9-11 We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation, and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54% (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90% of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. Glycosylphosphatidylinositol (GPI)-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications.
Published: 2016

34. Ephrin-B1 Is a Novel Specific Component of the Lateral Membrane of the Cardiomyocyte and Is Essential for the Stability of Cardiac Tissue Architecture Cohesion

Author: Christophe Heymes, Michael D. Schneider, Céline Galés, Benjamin Honton, Denis Calise, Alexandre Dubrac, Christelle Coatrieux, Alice Davy, Etienne Dague, Christelle Cardin, Jean-Michel Senard, Marie-Hélène Seguelas, Fabien Despas, Bruno Payré, Marie-Bernadette Delisle, Céline Guilbeau-Frugier, Dina N. Arvanitis, Gael Genet, Caroline Dubroca, Pauline Marck, Marie-Françoise Altié, Atul Pathak, Cécile Nieto, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Service Anatomie et cytologie pathologiques [CHU Toulouse], Pôle Biologie [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Institut des Technologies Avancées en sciences du Vivant (ITAV), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), National Heart and Lung Institute, Imperial College London-British Heart Foundation, Centre de Microscopie Électronique Appliquée à la Biologie (CMEAB), Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de biologie du développement (CBD), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Service Pharmacologie Clinique [CHU Toulouse], Pôle Santé publique et médecine publique [CHU Toulouse], Simon, Marie Francoise, Service d'histopathologie, CHU Toulouse [Toulouse], Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre de Biologie Intégrative (CBI), and Laboratoire de pharmacologie médicale et clinique
Subjects: Male, Physiology, MESH: Myocytes, Cardiac, Cell Communication, Matrix (biology), MESH: Mice, Knockout, Extracellular matrix, Mice, MESH: Collagen, Myocyte, Myocytes, Cardiac, MESH: Animals, Cells, Cultured, Ultrasonography, Mice, Knockout, 0303 health sciences, MESH: Sarcomeres, 030302 biochemistry & molecular biology, Anatomy, Cell biology, medicine.anatomical_structure, MESH: Models, Animal, Models, Animal, Collagen, MESH: Endothelium, Vascular, MESH: Membrane Proteins, Cardiology and Cardiovascular Medicine, MESH: Cells, Cultured, Sarcomeres, animal structures, Ephrin-B1, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, 03 medical and health sciences, MESH: Mice, Inbred C57BL, MESH: Cell Communication, Dystroglycan, medicine, Animals, Ephrin, MESH: Mice, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, 030304 developmental biology, Pressure overload, Basement membrane, MESH: Ephrin-B1, Cell Membrane, Erythropoietin-producing hepatocellular (Eph) receptor, Membrane Proteins, MESH: Male, Mice, Inbred C57BL, biology.protein, Endothelium, Vascular, sense organs, MESH: Cell Membrane
Abstract: Rationale: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. Objective: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. Methods and Results: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. Conclusions: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.
Published: 2012

35. Regulating TRAIL Receptor-Induced Cell Death at the Membrane: A Deadly Discussion

Author: Sarah Shirley, Olivier Micheau, Alexandre Morizot, Lipides - Nutrition - Cancer (U866) (LNC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Bourgogne (UB)-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Centre Régional de Lutte contre le cancer Georges-François Leclerc [Dijon] (UNICANCER/CRLCC-CGFL), UNICANCER, This work is supported by grants of the Conseil Regional de Bourgogne, the INCa (Institut National du Cancer), Cancéropôle Grand-Est, ANR (Agence Nationale de la Recherche, ANR-06-JCJC-0103 and 07-PCV-0031), and the European Community (ApopTrain Marie Curie RTN) (O.M.). A.M., is supported by fellowships from the Ministry of Research and Education and the ARC (Association pour la Recherche sur le Cancer). S.S. is supported by the INCa., ANR-06-JCJC-0103,TRAILCHIM,TRAIL et chimiothérapies anticancéreuses(2006), Lipides - Nutrition - Cancer (U866) ( LNC ), Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ), Centre Régional de Lutte contre le cancer - Centre Georges-François Leclerc ( CRLCC - CGFL ), and ANR-06-JCJC-0103,TRAILCHIM,TRAIL et chimiothérapies anticancéreuses ( 2006 )
Subjects: MESH: Cell Death, MESH: Signal Transduction, Cancer Research, Apoptosis, TRAIL, MESH : Models, Biological, scaffold, Cell membrane, 0302 clinical medicine, Drug Discovery, MESH: Animals, Pharmacology (medical), Receptor, death effector domain, 0303 health sciences, Cell Death, General Medicine, TRAIL-R4, 3. Good health, Cell biology, medicine.anatomical_structure, Oncology, 030220 oncology & carcinogenesis, Signal transduction, MESH : Apoptosis Regulatory Proteins, Signal Transduction, Programmed cell death, c-FLIP, death domain, medicine.drug_class, MESH : Cell Membrane, Cancer therapy, Biology, Monoclonal antibody, Models, Biological, Article, 03 medical and health sciences, medicine, Animals, Humans, Chemotherapy, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Receptors, TNF-Related Apoptosis-Inducing Ligand, MESH : Receptors, TNF-Related Apoptosis-Inducing Ligand, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, MESH : Signal Transduction, MESH: Humans, MESH: Apoptosis Regulatory Proteins, MESH: Apoptosis, MESH : Humans, Cell Membrane, MESH: Models, Biological, DISC, Receptors, TNF-Related Apoptosis-Inducing Ligand, MESH : Cell Death, FADD, Cancer cell, MESH : Animals, Apoptosis Regulatory Proteins, MESH : Apoptosis, MESH: Cell Membrane
Abstract: Article Open access plus; International audience; The use of TRAIL/APO2L and monoclonal antibodies targeting TRAIL receptors for cancer therapy holds great promise, due to their ability to restore cancer cell sensitivity to apoptosis in association with conventional chemotherapeutic drugs in a large variety of tumors. TRAIL-induced cell death is tightly regulated right from the membrane and at the DISC (Death-Inducing Signaling Complex) level. The following patent and literature review aims to present and highlight recent findings of the deadly discussion that determines tumor cell fate upon TRAIL engagement.
Published: 2011

36. Actin-independent exclusion of CD95 by PI3K/AKT signalling: Implications for apoptosis

Author: Michel Castroviejo, Jean-François Moreau, Mathieu Pizon, Patrick Legembre, Sébastien Tauzin, Hariniaina Rampanarivo, Benjamin Chaigne-Delalande, Sophie Daburon, Patrick Moreau, Université Bordeaux Segalen - Bordeaux 2, Composantes innées de la réponse immunitaire et différenciation (CIRID), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Signalisation et Réponses aux Agents Infectieux et Chimiques (SeRAIC), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Laboratoire de biogenèse membranaire (LBM), CHU Bordeaux [Bordeaux], INCa (projets libres recherche biomédicale), Cancéropole GO, Région Bretagne, Rennes Métropole, Ligue Contre le Cancer (Comités d'Ille-et-Vilaine/Morbihan/Côtes d'Armor/Maine et Loire), University of Rennes-1, Ligue Contre Le Cancer, Université de Rennes (UR), Microbiologie Fondamentale et Pathogénicité (MFP), and Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Signal Transduction, Fas-Associated Death Domain Protein, MESH: Membrane Microdomains, Apoptosis, MESH: Flow Cytometry, PI3K, Jurkat Cells, Phosphatidylinositol 3-Kinases, 0302 clinical medicine, MESH: Jurkat Cells, MESH: Protein Kinase Inhibitors, Immunology and Allergy, FADD, Lipid raft, Phosphoinositide-3 Kinase Inhibitors, 0303 health sciences, biology, Signal transducing adaptor protein, MESH: Fas-Associated Death Domain Protein, Flow Cytometry, Fas receptor, MESH: Antigens, CD95, Cell biology, Caspases, 030220 oncology & carcinogenesis, CD95, [SDV.IMM]Life Sciences [q-bio]/Immunology, biological phenomena, cell phenomena, and immunity, Wortmannin, Signal Transduction, Fas Ligand Protein, MESH: Mutation, MESH: Cell Line, Tumor, Morpholines, Blotting, Western, Immunology, MESH: Morpholines, MESH: Actins, Cell Line, 03 medical and health sciences, Membrane Microdomains, Cell Line, Tumor, Humans, MESH: Blotting, Western, fas Receptor, Protein Kinase Inhibitors, Protein kinase B, Lipid rafts, PI3K/AKT/mTOR pathway, 030304 developmental biology, Death domain, MESH: Humans, MESH: Caspases, MESH: Proto-Oncogene Proteins c-akt, Akt, MESH: Apoptosis, Cell Membrane, MESH: Multiprotein Complexes, Actin cytoskeleton, Actins, MESH: Fas Ligand Protein, MESH: Cell Line, Androstadienes, MESH: Chromones, Chromones, MESH: Phosphatidylinositol 3-Kinases, Multiprotein Complexes, MESH: Androstadienes, Mutation, biology.protein, Proto-Oncogene Proteins c-akt, MESH: Cell Membrane
Abstract: International audience; The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.
Published: 2011

37. Cloning and functional characterization ofLjPLT4, a plasma membrane xylitol H+- symporter fromLotus japonicus

Author: Ourania I. Pavli, Katerina I. Kalliampakou, Haralabia Boleti, Michael K. Udvardi, Panagiotis Katinakis, Rémi Lemoine, Laurence Maurousset, Emmanouil Flemetakis, Evangelia D. Kouri, Agricultural University of Athens, Laboratoire de parasitologie moléculaire = Molecular Parasitology Laboratory [Athènes], Institut Pasteur Hellénique, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Equipe Physiologie Moléculaire du Transport de Sucres (PhyMoTS), Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), The Samuel Roberts Noble Foundation, and This work was co-funded by the European Social fund and Greek national resources – project PYTHAGORAS II (funding of research groups in Agricultural University of Athens) (EF), and by the bi-lateral French-Greek program, Platon (Molecular and biochemical characterization of monosaccharide transporters in root nodules) (RL and EF). The work was also supported, in part, by the Max Planck Society (MKU).
Subjects: MESH: Symporters, 0106 biological sciences, DNA, Complementary, MESH: Lotus, legumes, Lotus japonicus, Arabidopsis, Saccharomyces cerevisiae, Xylose, Xylitol, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Monosaccharide transport, monosaccharide transport, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, MESH: Arabidopsis, MESH: Cloning, Molecular, Cloning, Molecular, Molecular Biology, 030304 developmental biology, polyols, 0303 health sciences, Symporters, biology, Cell Membrane, MESH: Xylitol, food and beverages, Fructose, MESH: DNA, Complementary, Cell Biology, biology.organism_classification, MESH: Saccharomyces cerevisiae, Yeast, Plant Leaves, MESH: Plant Leaves, carbohydrates (lipids), xylitol, chemistry, Biochemistry, Symporter, Lotus, Sorbitol, MESH: Cell Membrane, 010606 plant biology & botany
Abstract: International audience; Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.
Published: 2011

38. Cdc42 localization and cell polarity depend on membrane traffic

Author: Sandrine Etienne-Manneville, Naël Osmani, Philippe Chavrier, Florent Peglion, Polarité cellulaire, Migration et cancer, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Morphogénèse et Signalisation Cellulaires, Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), and Polarité et Migration Cellulaires
Subjects: MESH: ADP-Ribosylation Factors, rac1 GTP-Binding Protein, MESH: Adenomatous Polyposis Coli Protein, Cell division, Cellular differentiation, Cell leading edge, Golgi Apparatus, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], CDC42, MESH: Centrosome, Microtubules, Cell membrane, Cell Movement, MESH: RNA, Small Interfering, Cell polarity, MESH: Guanine Nucleotide Exchange Factors, Guanine Nucleotide Exchange Factors, MESH: Animals, MESH: rab5 GTP-Binding Proteins, RNA, Small Interfering, cdc42 GTP-Binding Protein, MESH: Cell Movement, Cells, Cultured, Protein Kinase C, Research Articles, MESH: ADP-Ribosylation Factor 6, MESH: Microtubules, ADP-Ribosylation Factors, MESH: Cell Surface Extensions, Cell Polarity, MESH: Rats, Inbred Strains, Cell biology, Protein Transport, medicine.anatomical_structure, Cdc42 GTP-Binding Protein, MESH: Cell Polarity, biological phenomena, cell phenomena, and immunity, MESH: Cells, Cultured, MESH: Protein Transport, MESH: Rats, Adenomatous Polyposis Coli Protein, MESH: Carrier Proteins, Endosomes, macromolecular substances, Cell Surface Extension, Biology, Models, Biological, MESH: Golgi Apparatus, MESH: Rho Guanine Nucleotide Exchange Factors, Report, medicine, MESH: Transport Vesicles, Animals, Transport Vesicles, MESH: Adaptor Proteins, Signal Transducing, Adaptor Proteins, Signal Transducing, rab5 GTP-Binding Proteins, Centrosome, Wound Healing, MESH: cdc42 GTP-Binding Protein, MESH: rac1 GTP-Binding Protein, Cell Membrane, MESH: Models, Biological, Rats, Inbred Strains, Cell Biology, MESH: Protein Kinase C, Rats, MESH: Astrocytes, MESH: Wound Healing, MESH: Endosomes, ADP-Ribosylation Factor 6, Astrocytes, Cell Surface Extensions, Carrier Proteins, Rho Guanine Nucleotide Exchange Factors, MESH: Cell Membrane
Abstract: Arf6-dependent membrane dynamics concentrates active Cdc42 at the leading edge of migrating cells., Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6–aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.
Published: 2010

39. A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability

Author: Nicolas Bayan, Marielle Tropis, Célia de Sousa-d'Auria, Christine Houssin, Cécile Labarre, Xavier Meniche, Mamadou Daffé, Christophe Salmeron, Roland Bou Raad, Mohamed Chami, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Magnetic Resonance Spectroscopy, Physiology and Metabolism, Blotting, Western, Biology, Galactans, Microbiology, Corynebacterium glutamicum, Cell membrane, 03 medical and health sciences, Bacterial Proteins, Microscopy, Electron, Transmission, Arabinogalactan, medicine, MESH: Blotting, Western, Outer membrane efflux proteins, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Bacterial Proteins, Molecular Biology, 030304 developmental biology, 0303 health sciences, MESH: Magnetic Resonance Spectroscopy, 030306 microbiology, Cell Membrane, Cryoelectron Microscopy, MESH: Corynebacterium glutamicum, MESH: Galactans, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, medicine.anatomical_structure, Mycolate outer membrane, Biochemistry, Porin, MESH: Microscopy, Electron, Transmission, Electrophoresis, Polyacrylamide Gel, MESH: Cryoelectron Microscopy, Cell envelope, Bacterial outer membrane, MESH: Cell Membrane, MESH: Electrophoresis, Polyacrylamide Gel
Abstract: Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum . The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum . AftB catalyzes the transfer of Ara f (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Ara f residues. Here we show that Δ aftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a Δ aftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum , but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae , and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the Δ aftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum .
Published: 2010

40. Adaptation beyond the Stress Response: Cell Structure Dynamics and Population Heterogeneity in Staphylococcus aureus

Author: Tarek Msadek, Ryosuke L. Ohniwa, Kunio Takeyasu, Yoshikazu Tanaka, Toshiko Ohta, Kazuya Morikawa, Université de Tsukuba = University of Tsukuba, Hokkaido University [Sapporo, Japan], Kyoto University [Kyoto], Biologie des Bactéries pathogènes à Gram-positif, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Kyoto University, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Nasal Cavity, Adaptive potential, Plant Science, medicine.disease_cause, Fight-or-flight response, Drug Resistance, Multiple, Bacterial, MESH: Staphylococcus aureus, MESH: Genetic Variation, MESH: Stress, Physiological, membrane, Cross Infection, 0303 health sciences, education.field_of_study, General Medicine, Staphylococcal Infections, Adaptation, Physiological, 3. Good health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Staphylococcus aureus, Cell structure, Nasal Cavity, DNA, Bacterial, nucleoid, Population, MESH: Staphylococcal Infections, Soil Science, Biology, Microbiology, Bacterial genetics, 03 medical and health sciences, Stress, Physiological, medicine, Population Heterogeneity, Humans, education, Ecology, Evolution, Behavior and Systematics, population heterogeneity, 030304 developmental biology, MESH: Humans, 030306 microbiology, Cell Membrane, Genetic Variation, MESH: Cross Infection, MESH: Drug Resistance, Multiple, Bacterial, MESH: Adaptation, Physiological, MESH: DNA, Bacterial, variant, Evolutionary biology, Adaptation, MESH: Cell Membrane
Abstract: International audience; Staphylococcus aureus, a major opportunistic pathogen responsible for a broad spectrum of infections, naturally inhabits the human nasal cavity in about 30% of the population. The unique adaptive potential displayed by S. aureus has made it one of the major causes of nosocomial infections today, emphasized by the rapid emergence of multiple antibiotic-resistant strains over the past few decades. The uncanny ability to adapt to harsh environments is essential for staphylococcal persistence in infections or as a commensal, and a growing body of evidence has revealed critical roles in this process for cellular structural dynamics, and population heterogeneity. These two exciting areas of research are now being explored to identify new molecular mechanisms governing these adaptational strategies.
Published: 2010

41. HIV-1 Tat is unconventionally secreted through the plasma membrane

Author: Solène Debaisieux, Bruno Beaumelle, Fabienne Rayne, Anne Bonhoure, Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Institut de Recherche en Infectiologie de Montpellier (IRIM), 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: CD4-Positive T-Lymphocytes, Cell type, Glycosylation, Time Factors, Cell, Biology, Twin-arginine translocation pathway, Jurkat Cells, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), MESH: Jurkat Cells, medicine, Humans, Secretion, 030304 developmental biology, MESH: tat Gene Products, Human Immunodeficiency Virus, 0303 health sciences, MESH: Humans, Endoplasmic reticulum, MESH: Time Factors, Cell Membrane, Temperature, MESH: CD4-Positive T-Lymphocytes, Cell Biology, General Medicine, Transfection, MESH: Glycosylation, Molecular biology, MESH: Temperature, 3. Good health, medicine.anatomical_structure, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Microsome, tat Gene Products, Human Immunodeficiency Virus, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract: International audience; The Tat protein is required for efficient HIV-1 (human immunodeficiency virus type 1) transcription. Moreover, Tat is secreted by infected cells, and circulating Tat can affect several cell types, thereby contributing to HIV-1 pathogenesis. We monitored Tat secretion by transfected CD4+ T-cells. A Tat chimaera carrying an N-glycosylation site did not become glycosylated when expressed in cells, while the chimaera was glycosylated when mechanically introduced into purified microsomes. These data indicate that secreted Tat does not transit through the endoplasmic reticulum. The use of pharmacological inhibitors indicated that the Tat secretion pathway is unusual compared with previously identified unconventional secretion routes and does not involve intracellular organelles. Moreover, cell incubation at 16 degrees C inhibited Tat secretion and caused its accumulation at the plasma membrane, suggesting that secretion takes place at this level.
Published: 2010

42. Targeting of Murine Leukemia Virus Gag to the Plasma Membrane Is Mediated by PI(4,5)P 2 /PS and a Polybasic Region in the Matrix

Author: Jean-Luc Darlix, Jamil S. Saad, Christian Roy, Michael F. Summers, Catherine Picart, Delphine Muriaux, Franceline Juillard, Elise Hamard-Peron, Philippe Roingeard, Dynamique des interactions membranaires normales et pathologiques (DIMNP), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), 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), Virologie humaine, École normale supérieure de Lyon (ENS de Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), inconnu, Inconnu, Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université Montpellier 1 (UM1), and École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Phosphatidylinositol 4,5-Diphosphate, viruses, Immunology, Mutant, Gene Products, gag, Mutagenesis (molecular biology technique), Phosphatidylserines, Virus Replication, Microbiology, Virus, Cell membrane, 03 medical and health sciences, Virology, Murine leukemia virus, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, MESH: Mutagenesis, 030304 developmental biology, Gammaretrovirus, 0303 health sciences, Binding Sites, biology, Structure and Assembly, Cell Membrane, MESH: Virus Replication, 030302 biochemistry & molecular biology, MESH: Leukemia Virus, Murine, MESH: Phosphatidylserines, biology.organism_classification, Molecular biology, MESH: Phosphatidylinositol 4,5-Diphosphate, 3. Good health, Leukemia Virus, Murine, Retroviridae, MESH: Retroviridae, medicine.anatomical_structure, MESH: Binding Sites, Viral replication, Mutagenesis, MESH: Gene Products, gag, Insect Science, MESH: Cell Membrane
Abstract: Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P 2 only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P 2 depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P 2 appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P 2 binding cleft.
Published: 2010

43. On Some Aspects of the Thermodynamic of Membrane Recycling Mediated by Fluid Phase Endocytosis: Evaluation of Published Data and Perspectives

Author: Laurent Counillon, Neil Foster, Alain Pluen, Dominique Lagadic-Gossmann, Ali Mobasheri, Cyril Rauch, Paul T. Loughna, School of Veterinary Medicine and Science, University of Nottingham, UK (UON), School of Pharmacy and Pharmaceutical Science, University of Manchester [Manchester], Vienna Institute for International Economic Studies, Signalisation et Réponses aux Agents Infectieux et Chimiques (SeRAIC), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), CNRS FRE3093 Transport ionique aspects normaux et pathologiques, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Université de Rennes (UR), Transport ionique aspects normaux et pathologiques, Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Coated Vesicles, Hydrostatic pressure, 030204 cardiovascular system & hematology, Biochemistry, Aminophospholipid translocase, Cell membrane, Osmotic pressure, Cytosol, 0302 clinical medicine, MESH: Cytosol, Phospholipids, 0303 health sciences, MESH: Kinetics, Chemistry, MESH: Energy Metabolism, General Medicine, Membrane budding, MESH: Fluorescent Dyes, Endocytosis, Cell biology, MESH: Reproducibility of Results, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Phospholipid, MESH: Staining and Labeling, medicine.anatomical_structure, MESH: Endocytosis, Thermodynamics, MESH: Thermodynamics, MESH: Pressure, Coated Vesicles, Biophysics, Aquaporin, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Models, Biological, Exocytosis, 03 medical and health sciences, Pressure, medicine, Fluorescent Dyes, 030304 developmental biology, MESH: Phospholipids, Staining and Labeling, Cell Membrane, MESH: Models, Biological, Reproducibility of Results, Cell Biology, Kinetics, Tonicity, Energy Metabolism, MESH: Cell Membrane
Abstract: International audience; The theoretical and experimental description of fluid phase endocytosis (FPE) requires an asymmetry in phospholipid number between the two leaflets of the cell membrane, which provides the biomechanical torque needed to generate membrane budding. Although the motor force behind FPE is defined, its kinetic has yet to be determined. Based on a body of evidences suggesting that the mean surface tension is unlikely to be involved in endocytosis we decided to determine whether the cytosolic hydrostatic pressure could be involved, by considering a constant energy exchanged between the cytosol and the cell membrane. The theory is compared to existing experimental data obtained from FPE kinetic studies in living cells where altered phospholipid asymmetry or changes in the extracellular osmotic pressure have been investigated. The model demonstrates that FPE is dependent on the influx and efflux of vesicular volumes (i.e. vesicular volumes recycling) rather than the membrane tension of cells. We conclude that: (i) a relationship exists between membrane lipid number asymmetry and resting cytosolic pressure and (ii) the validity of Laplace's law is limited to cells incubated in a definite hypotonic regime. Finally, we discuss how the model could help clarifying elusive observations obtained from different fields and including: (a) the non-canonical shuttling of aquaporin in cells, (b) the relationship between high blood pressure and inflammation and (c) the mechanosensitivity of the sodium/proton exchanger.
Published: 2009

44. Ximelagatran increases membrane fluidity and changes membrane lipid composition in primary human hepatocytes

Author: Kim Ekroos, Gun-Britt Forsberg, Jan Oscarsson, Luz Lefeuvre-Orfila, Dominique Lagadic-Gossmann, Tommy B. Andersson, Mary Rissel, Odile Sergent, Signalisation et Réponses aux Agents Infectieux et Chimiques (SeRAIC), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-IFR140, Zora Biosciences, Station commune de Recherches en Ichtyophysiologie, Biodiversité et Environnement (SCRIBE), Institut National de la Recherche Agronomique (INRA)-IFR140, Détoxication et réparation tissulaire, Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-IFR140-Institut National de la Santé et de la Recherche Médicale (INSERM), AstraZeneca, Department of Physiology and Pharmacology, Karolinska Institutet [Stockholm], Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut National de la Recherche Agronomique (INRA), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Rennes (UR), and Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Benzylamines, medicine.medical_specialty, Ximelagatran, Membrane lipids, Phospholipid, MESH: Anticoagulants, 030204 cardiovascular system & hematology, Toxicology, Cell Line, MESH: Hepatocytes, Cell membrane, Membrane Lipids, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Internal medicine, Phosphatidylcholine, medicine, Membrane fluidity, Humans, 030304 developmental biology, Phosphatidylethanolamine, 0303 health sciences, MESH: Benzylamines, MESH: Humans, Chemistry, Cell Membrane, Lipid composition, Anticoagulants, General Medicine, MESH: Azetidines, MESH: Cell Line, 3. Good health, Endocrinology, medicine.anatomical_structure, Membrane, Biochemistry, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Hepatocytes, Tacrine, Azetidines, Drug induced liver injury, MESH: Membrane Lipids, MESH: Membrane Fluidity, MESH: Cell Membrane, MESH: Tacrine, medicine.drug
Abstract: International audience; Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to a potential risk of severe liver injury. Increased rates of liver enzyme elevations had been observed during clinical trials of chronic use. Despite intensive preclinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown. The aim of this study was to investigate whether ximelagatran has an effect on the plasma membrane fluidity and the membrane lipid composition which may be important for the cell integrity. After 1h exposure of primary human hepatocytes with 10 or 100 microM ximelagatran, a significant elevation of membrane fluidity was observed. This elevation was maintained at 24h, but diminished at 48 h exposure. As changed membrane lipid composition could influence membrane fluidities, changes in membrane lipid profiles were also studied. After 1h exposure, the phosphatidylcholine/phosphatidylethanolamine molar ratio decreased, whereas the total cholesterol/phospholipid molar ratio decreased after a 48 h exposure. The change in membrane fluidity and lipid composition in human hepatocytes exposed to ximelagatran might indicate changes in plasma membrane properties that in susceptible subjects, could result in loss of membrane integrity and leakage of cellular proteins.
Published: 2009

45. Functional in vitro assembly of the integral membrane bacterial thermosensor DesK

Author: Mariana Martín, Diego de Mendoza, Pedro M. Alzari, Daniela Albanesi, Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Facultad de Ciencias Bioquımicas y Farmaceuticas [Rosario] (FBIOyF), Universidad Nacional de Rosario [Santa Fe], Biochimie Structurale, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was supported by grants from the Institut Pasteur (France), Agence Nationale de la Recherche (ANR, France, contract N. ANR-06-PCVI-0009-01), ECOS France-Argentine (Action A05B02), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET, Argentina) and Agencia de Promoción Científica y Tecnológica (FONCYT, Argentina). D.A. is a Marie Curie Incoming International Fellow (European Commission). M.M. is a fellow from Agencia de Promoción Científica y Tecnológica (FONCYT, Argentina) and D. de M. is a Career Investigator from CONICET and an International Fellow from Howard Hughes Medical Institute., ANR-06-PCVI-0009,ReguLipids+,Molecular mechanisms of signaling in fatty acid synthesis of Gram-positive bacteria(2006), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Signal Transduction, Bacillus subtilis, Cell-free protein expression, medicine.disease_cause, MESH: Protein Structure, Tertiary, Protein phosphorylation, MESH: Bacterial Proteins, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, Temperature, MESH: Temperature, Transmembrane protein, Membrane, Biochemistry, MESH: Histidine Kinase, MESH: Membrane Proteins, Signal Transduction, Biotechnology, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Membrane lipids, Detergents, education, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Histidine kinase, 03 medical and health sciences, Bacterial Proteins, Liposome insertion, [CHIM.CRIS]Chemical Sciences/Cristallography, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Protein Kinases, Escherichia coli, 030304 developmental biology, Cell Membrane, Membrane Proteins, MESH: Bacillus subtilis, biology.organism_classification, Protein Structure, Tertiary, Membrane protein, Liposomes, MESH: Liposomes, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Protein Kinases, MESH: Cell Membrane, MESH: Detergents
Abstract: International audience; The Bacillus subtilis DesK histidine kinase (HK) is an integral membrane thermosensor that forms part of a regulatory circuit which controls the physical state of membrane lipids. In the pursuit of biochemical and structural approaches to study lipid fluidity-dependent DesK thermosensing, we found that standard expression methods failed to produce enough amounts of a fully functional protein. Here, we describe a high-yield purification method based in an Escherichia coliin vitro transcription-translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 degrees C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.
Published: 2009

46. Catalytic Activity and Inhibition of Wegener Antigen Proteinase 3 on the Cell Surface of Human Polymorphonuclear Neutrophils

Author: Francis Gauthier, Alexandre Gauthier, Sylvie Attucci, Brice Korkmaz, Marie-Lise Jourdan, Jérôme Jaillet, Pathologies Respiratoires : Protéolyse et Aérosolthérapie, Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Nutrition, croissance et cancer (U 1069) (N2C), and Nocchi, Estelle
Subjects: MESH: Enzyme Stability, Neutrophils, Plasma protein binding, MESH: Neutrophils, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Biochemistry, Neutrophil Activation, Amino Acid Chloromethyl Ketones, Cell membrane, 0302 clinical medicine, Proteinase 3, Enzyme Stability, MESH: Myeloblastin, 0303 health sciences, Enzyme Catalysis and Regulation, MESH: Neutrophil Activation, biology, Chemistry, MESH: Wegener Granulomatosis, Elastase, Elafin, 3. Good health, Cell biology, MESH: Leukocyte Elastase, medicine.anatomical_structure, 030220 oncology & carcinogenesis, medicine.symptom, Antibody, Protein Binding, Myeloblastin, Inflammation, 03 medical and health sciences, medicine, Humans, MESH: Protein Binding, Molecular Biology, 030304 developmental biology, Serine protease, MESH: Humans, Cell Membrane, Granulomatosis with Polyangiitis, MESH: Amino Acid Chloromethyl Ketones, Cell Biology, MESH: alpha 1-Antitrypsin, MESH: Elafin, alpha 1-Antitrypsin, biology.protein, [SDV.MHEP.PSR] Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Leukocyte Elastase, MESH: Cell Membrane
Abstract: International audience; Proteinase 3 (Pr3), the main target of anti-neutrophil cytoplasmic antibodies, is a neutrophil serine protease that may be constitutively expressed at the surface of quiescent circulating neutrophils. This raises the question of the simultaneous presence in the circulation of constitutive membrane-bound Pr3 (mPr3) and its plasma inhibitor alpha1-protease inhibitor (alpha1-Pi). We have looked at the fate of constitutive mPr3 at the surface of circulating blood neutrophils and of induced mPr3 on triggered neutrophils. We found significant Pr3 activity at the surface of activated neutrophils but not at the surface of quiescent neutrophils whatever the constitutive expression. This suggests that constitutive mPr3 is enzymatically inactive or its active site is not accessible to the substrate. Supporting this conclusion, we have not been able to demonstrate any interaction between constitutive mPr3 and alpha1-Pi, whereas induced mPr3 is cleared from the cell surface when activated cells are incubated with this inhibitor. But, unlike membrane-bound elastase that is also cleared from the surface of activated cells, mPr3 remained bound to the membrane when inhibited by elafin or by a low molecular weight chloromethyl ketone inhibitor, which shows that it binds more tightly to the neutrophil membrane. mPr3 may thus be present at the surface of circulating neutrophils in an environment replete with alpha1-Pi. The permanent presence of inactive Pr3 at the surface of quiescent neutrophils may explain why Pr3 is a major target of anti-neutrophil cytoplasmic antibodies, whose binding activates neutrophils and triggers inflammation, as in Wegener granulomatosis.
Published: 2009

47. Heme uptake across the outer membrane as revealed by crystal structures of the receptor–hemophore complex

Author: Anne Lecroisey, Kay Diederichs, Stefanie Krieg, Nadia Izadi-Pruneyre, Wolfram Welte, Cécile Wandersman, Philippe Delepelaire, Frédéric Huché, Fachbereich Biologie, University of Konstanz, Membranes bactériennes, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), 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), Résonance Magnétique Nucléaire des Biomolécules, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, MESH: Protein Structure, Secondary, Crystallography, X-Ray, Ligands, 01 natural sciences, Protein Structure, Secondary, Cell membrane, MESH: Apoproteins, chemistry.chemical_compound, MESH: Ligands, membrane protein, Receptor, MESH: Bacterial Proteins, Heme, Serratia marcescens, MESH: Receptors, Cell Surface, 0303 health sciences, Multidisciplinary, Chemistry, Biological Sciences, Cell biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, medicine.anatomical_structure, MESH: Heme, MESH: Membrane Proteins, Bacterial outer membrane, MESH: Models, Molecular, Hemeproteins, Heme binding, Surface Properties, MESH: Biological Transport, membrane transport, Receptors, Cell Surface, MESH: Carrier Proteins, Calorimetry, 010402 general chemistry, Heme-Binding Proteins, 03 medical and health sciences, Bacterial Proteins, MESH: Serratia marcescens, ddc:570, medicine, MESH: Calorimetry, 030304 developmental biology, MESH: Surface Properties, MESH: Hemeproteins, iron uptake, protein complex, Cell Membrane, Membrane Proteins, Biological Transport, Membrane transport, MESH: Crystallography, X-Ray, heme binding, 0104 chemical sciences, Membrane protein, Biophysics, Apoproteins, Carrier Proteins, MESH: Cell Membrane
Abstract: Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 Å from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor.
Published: 2009

48. Expression of the GABAA receptor associated protein Gec1 is circadian and dependent upon the cellular clock machinery in GnRH secreting GnV-3 cells

Author: François P. Pralong, Annick Fraichard, Micheline Glauser, Virginie Mansuy, Pierre Yves Risold, Laboratoire d'Histologie, embryologie et cytogénétique, Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), and Estrogènes, Expression génique et pathologies du Système Nerveux Central - UFC (E2SNC / ESTROGENES)
Subjects: MESH: Period Circadian Proteins, Serum, Circadian clock, MESH: Neurons, Biochemistry, GABAA-rho receptor, Gonadotropin-Releasing Hormone, 0302 clinical medicine, Endocrinology, GABA receptor, MESH: Gonadotropin-Releasing Hormone, MESH: Animals, MESH: Receptors, GABA-A, Cells, Cultured, Neurons, 0303 health sciences, GABAA receptor, Intracellular Signaling Peptides and Proteins, MESH: Serum, Period Circadian Proteins, MESH: Gene Expression Regulation, Cell biology, Circadian Rhythm, CLOCK, GABAergic, MESH: Forskolin, hormones, hormone substitutes, and hormone antagonists, PER1, MESH: Cells, Cultured, medicine.medical_specialty, endocrine system, MESH: Rats, MESH: Biological Clocks, MESH: Carrier Proteins, Biology, 03 medical and health sciences, Biological Clocks, Internal medicine, MESH: Intracellular Signaling Peptides and Proteins, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Circadian rhythm, MESH: Circadian Rhythm, Horses, RNA, Messenger, MESH: Horses, Molecular Biology, 030304 developmental biology, MESH: RNA, Messenger, Cell Membrane, Colforsin, Receptors, GABA-A, Rats, Gene Expression Regulation, Carrier Proteins, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract: International audience; The timely regulation of gonadotropin-releasing hormone (GnRH) secretion requires a GABAergic signal. We hypothesized that GEC1, a protein promoting the transport of GABA(A) receptors, could represent a circadian effector in GnRH neurons. First, we demonstrated that gec1 is co-expressed with the GABA(A) receptor in hypothalamic rat GnRH neurons. We also confirmed that the clock genes per1, cry1 and bmal1 are expressed and oscillate in GnRH secreting GnV-3 cells. Then we could show that gec1 is expressed in GnV-3 cells, and oscillates in a manner temporally related to the oscillations of the clock transcription factors. Furthermore, we could demonstrate that these oscillations depend upon Per1 expression. Finally, we observed that GABA(A) receptor levels at the GnV-3 cell membrane are timely modulated following serum shock. Together, these data demonstrate that gec1 expression is dependent upon the circadian clock machinery in GnRH-expressing neurons, and suggest for the first time that the level of GABA(A) receptor at the cell membrane may be under timely regulation. Overall, they provide a potential mechanism for the circadian regulation of GnRH secretion by GABA, and may also be relevant to the general understanding of circadian rhythms.
Published: 2009
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49. TWEAK is expressed at the cell surface of monocytes during multiple sclerosis

Author: Jean Pelletier, L. Feuillet, Roselyne Rance, Linda C. Burkly, Kyungmin Hahm, José Boucraut, Michel Khrestchatisky, Sophie Desplat-Jégo, Rita Creidy, Irina Malikova, Neurobiologie des interactions cellulaires et neurophysiopathologie - NICN (NICN), Université de la Méditerranée - Aix-Marseille 2-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en neurobiologie - neurophysiologie de Marseille (CRN2M), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de résonance magnétique biologique et médicale (CRMBM), Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de la Méditerranée - Aix-Marseille 2, and Assistance Publique - Hôpitaux de Marseille (APHM)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, medicine.medical_treatment, MESH: Monocytes, Monocytes, Receptors, Tumor Necrosis Factor, 0302 clinical medicine, Immunology and Allergy, Lymphocytes, Receptor, Cytokine TWEAK, MESH: Aged, 0303 health sciences, MESH: Middle Aged, Experimental autoimmune encephalomyelitis, MESH: Tumor Necrosis Factors, Middle Aged, 3. Good health, Cytokine, TWEAK Receptor, MESH: Young Adult, Tumor Necrosis Factors, Female, medicine.symptom, Antibody, Adult, Multiple Sclerosis, Adolescent, Immunology, Inflammation, Biology, Peripheral blood mononuclear cell, Young Adult, 03 medical and health sciences, medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Aged, 030304 developmental biology, MESH: Adolescent, MESH: Humans, Multiple sclerosis, Cell Membrane, MESH: Adult, MESH: Multiple Sclerosis, Cell Biology, MESH: Receptors, Tumor Necrosis Factor, medicine.disease, MESH: Male, biology.protein, MESH: Lymphocytes, MESH: Female, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract: The TNF superfamily ligand, TNF-like weak inducer of apoptosis (TWEAK), regulates cellular responses ranging from proliferation to cell death in a manner highly dependent on the cell type and the microenvironmental context. We have shown previously that treatment of experimental autoimmune encephalomyelitis mice after the priming phase with neutralizing anti-TWEAK antibodies results in a reduction in the severity of the disease and leukocyte infiltration. To further characterize TWEAK/fibroblast growth factor-inducible 14-kDa protein (Fn14) involvement during multiple sclerosis (MS), we evaluated in MS patients and controls: TWEAK and Fn14 expression on PBMC and soluble TWEAK concentration in serum and cerebrospinal fluid (CSF). Thirty-six consecutive patients were enrolled, including 11 patients with relapsing-remitting MS, 11 with a clinical isolated syndrome suggestive of MS (CISSMS), and 14 controls with non-MS diseases. Intracellular TWEAK could be observed in lymphocytes and/or monocytes in all groups of patients. None of the 36 patients displayed TWEAK expression at the cell surface of lymphocytes. In contrast, 12 out of the 36 patients were positive for membrane TWEAK expression on their monocytes. Among these patients, eight were from the CISSMS group. Fn14 was not detected in PBMC. The soluble form of TWEAK is detectable in serum and CSF of patients, and TWEAK concentrations were not statistically different between the disease groups. We demonstrated for the first time that TWEAK is expressed at the cell surface of monocytes during MS, especially in the CISSMS group. Our results support the proposal that TWEAK could be a target for antibody therapy in MS.
Published: 2008

50. Identification and characterization of a new Leishmania major specific 3′nucleotidase/nuclease protein

Author: Yvan Boublik, Mehdi Chenik, Hechmi Louzir, Yosser Ben Achour-Chenik, Ahlem Aamouri, Inès Lakhal-Naouar, Abdellatif Fattoum, Mounira Meddeb, Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP), Plateforme de Production des Protéines Recombinantes, Université de Montpellier (UM), Département d'Histologie et de Cytogénétique - Institut Pasteur de Tunis, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)
Subjects: [SDV]Life Sciences [q-bio], Protozoan Proteins, MESH: Amino Acid Sequence, MESH: Nucleotidases, Biochemistry, Leishmania mexicana, MESH: Leishmania major, 0302 clinical medicine, Nucleotidases, purine scavenging pathway, MESH: Animals, Leishmania major, MESH: Phylogeny, MESH: Protozoan Proteins, Peptide sequence, Phylogeny, Leishmania, 0303 health sciences, 3 ` nucleotidase/nuclease, biology, medicine.diagnostic_test, MESH: Purines, MESH: Gene Expression Regulation, Molecular Sequence Data, 030231 tropical medicine, sandfly, Biophysics, survival, 03 medical and health sciences, Western blot, Nucleotidase, medicine, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Amino Acid Sequence, Molecular Biology, Gene, 030304 developmental biology, Nuclease, MESH: Molecular Sequence Data, Cell Membrane, Cell Biology, biology.organism_classification, Molecular biology, Gene Expression Regulation, Purines, biology.protein, MESH: Cell Membrane
Abstract: International audience; We report the characterization of a new Leishmania major gene, lmaj3'nt/nu, encoding a 382 amino acids protein, Lmaj3'NT/NU, that belongs to the 3'nucleotidase/nuclease family. Interestingly, sequence and phylogenetic analysis show that this protein is Leishmania major specific and thus constitutes a new 3'nucleotidase/nuclease subgroup. Lmaj3'NT/NU displays nuclease enzymatic activity and Western blot analysis shows that it is exclusively expressed in promastigotes. Immunofluorescence microscopy using a specific anti-Lmaj3'NT/NU shows that the protein has a plasma membrane localization. Surprisingly, contrary to the previously described Leishmania mexicana 3'NT/NU, lmaj3'nt/nu is not up-regulated when parasites are cultured under purine starvation conditions. Together, these findings suggest Lmaj3'NT/NU may constitute a new important compound of the L. major purine scavenging pathway and could be involved in sandfly parasite survival and colonization.
Published: 2008

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