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605 results on '"MESH: Gene Expression"'

1. Gene expression of circadian genes and CIART in bipolar disorder: A preliminary case-control study

Author

Cindie Courtin, Cynthia Marie-Claire, Gregory Gross, Vincent Hennion, Emeline Mundwiller, Justine Guégan, Manon Meyrel, Frank Bellivier, Bruno Etain, Etain, Bruno, Optimisation thérapeutique en Neuropsychopharmacologie (OPTeN (UMR_S_1144 / U1144)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Pôle Hospitalo-Universitaire de Psychiatrie d'Adultes et d'Addictologie du Grand Nancy (PHUPAA), Département de Psychiatrie et de Médecine Addictologique [AP-HP HU Saint-Louis, Lariboisière, Fernand Widal] (HUSLS-LRB-FW), Hôpitaux Universitaires Saint-Louis, Lariboisière, Fernand-Widal, Plateforme iGenSeq [Paris], Institut du Cerveau = Paris Brain Institute (ICM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Pharmacology, MESH: Humans, MESH: Gene Expression, Bipolar disorder, [SDV.MHEP.PSM] Life Sciences [q-bio]/Human health and pathology/Psychiatrics and mental health, Circadian, RNA sequencing, MESH: Case-Control Studies, Polymerase chain reaction, MESH: ARNTL Transcription Factors, CIART, MESH: Bipolar Disorder, [SDV.MHEP.PSM]Life Sciences [q-bio]/Human health and pathology/Psychiatrics and mental health, Gene expression, MESH: Circadian Rhythm, Biological Psychiatry
Abstract

International audience; Based on the observed circadian rhythms disruptions and sleep abnormalities in bipolar disorders (BD), a chronobiological model has been proposed suggesting that core clock genes play a central role in the vulnerability to the disorder. In this context, the analysis of circadian genes expression levels is particularly relevant, however studies focused on the whole set of core clock genes are scarce. We compared the levels of expression of 19 circadian genes (including the recently described circadian repressor (CIART)) in 37 euthymic individuals with BD and 20 healthy controls (HC), using data obtained by RNA sequencing of lymphoblastoid cell lines and validated the results using RT-qPCR. RNA sequencing data showed that CIART gene expression was correlated with those of ARNTL, ARNTL2, DBP, PER2 and TIMELESS. Data from RNA sequencing showed that the level of expression of four circadian genes (ARNTL, ARNTL2, BHLHE41 and CIART) discriminated individuals with BD from HC. We replicated this result using RT-qPCR for ARNTL and CIART. This study suggests that an imbalance between activation/repression of the transcription within the circadian system in individuals with BD as compared to HC and as such opens avenues for further research in larger independent samples combining both expression and epigenetic analyses.

Published
2022

2. The gut environment regulates bacterial gene expression which modulates susceptibility to bacteriophage infection

Author

Marta Lourenço, Lorenzo Chaffringeon, Quentin Lamy-Besnier, Marie Titécat, Thierry Pédron, Odile Sismeiro, Rachel Legendre, Hugo Varet, Jean-Yves Coppée, Marion Bérard, Luisa De Sordi, Laurent Debarbieux, Bactériophage, bactérie, hôte - Bacteriophage, bacterium, host, 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), Collège Doctoral, Sorbonne Université (SU), Centre de Recherche Saint-Antoine (CRSA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Paris Center for Microbiome Medicine (FHU PaCeMM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institute for Translational Research in Inflammation - U 1286 (INFINITE (Ex-Liric)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Biomics (plateforme technologique), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Transcriptome et Epigénome (PF2), Institut Pasteur [Paris] (IP), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Animalerie Centrale (plateforme) - Central Animal Facility (platform) (C2RA), M.L. is funded as part of the Pasteur-Paris University (PPU) International PhD Program. M.L. is funded by Institut Carnot Pasteur Maladies Infectieuses (ANR 11-CARN 017-01). L.D.S. is funded by a Roux-Cantarini Fellowship from the Institut Pasteur (Paris, France). L.C. is funded by a PhD Fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche, France, École Doctorale 394. Q.L.-B. is funded by École Doctorale FIRE - Programme Bettencourt. M.T. received a fellowship from Fondation DigestScience (Lille, France). The transcriptome and epigenome Platform is part of the France Génomique consortium (ANR10-NBS-09-08)., We thank Jorge Moura de Sousa for writing the customized R script for the pairwise comparisons between lists of genes from the different samples and for critically reading the manuscript. We thank Dwayne Roach and Anne Chevallereau for valuable discussions. We thank the Genetics of Biofilms team (Jean-Marc Ghigo) of Institut Pasteur and in particular Anne-Aurélie Lopes for training on the biofilm assays and Christophe Beloin for access to the ASKA plasmids collection. We thank the members of the Centre for Gnotobiology Platform of the Institut Pasteur (Thierry Angélique, Eddie Maranghi, Martine Jacob, Jérome Toutain, and Marisa Gabriela Lopez Dieguez) for their help with the animal work., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Varet, Hugo, and Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID

Subjects
intestinal microbiota, MESH: Gene Expression, [SDV]Life Sciences [q-bio], Gene Expression, enteric pathogens, Microbiology, auto-inducer, flagellum, Mice, transcriptomics, Virology, Escherichia coli, Animals, Bacteriophages, MESH: Animals, MESH: Bacteriophages, MESH: Mice, Bacteria, MESH: Escherichia coli, lipopolysaccharide, [SDV] Life Sciences [q-bio], MESH: Bacteria, motility, Genes, Bacterial, Parasitology, biofilms, MESH: Genes, Bacterial, gene regulation
Abstract

International audience; Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.

Published
2022

3. Cytoplasmic FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and inhibitory synaptic defects

Author

Scekic-Zahirovic, Jelena, Sanjuan-Ruiz, Inmaculada, Kan, Vanessa, Megat, Salim, De Rossi, Pierre, Dieterlé, Stéphane, Cassel, Raphaelle, Jamet, Marguerite, Kessler, Pascal, Wiesner, Diana, Tzeplaeff, Laura, Demais, Valérie, Sahadevan, Sonu, Hembach, Katharina M, Muller, Hans-Peter, Picchiarelli, Gina, Mishra, Nibha, Antonucci, Stefano, Dirrig-Grosch, Sylvie, Kassubek, Jan, Rasche, Volker, Ludolph, Albert, Boutillier, Anne-Laurence, Roselli, Francesco, Polymenidou, Magdalini, Lagier-Tourenne, Clotilde, Liebscher, Sabine, Dupuis, Luc, Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ludwig-Maximilians University [Munich] (LMU), Universität Zürich [Zürich] = University of Zurich (UZH), Laboratoire de neurosciences cognitives et adaptatives (LNCA), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), CRBS - Centre de Recherche en Biomédecine de Strasbourg (Inserm UMS38/UNISTRA), University of Ulm (UUlm), Deutsches Zentrum für Neurodegenerative Erkrankungen [Ulm] (DZNE), German Research Center for Neurodegenerative Diseases - Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Centre de Neurochimie, Harvard Medical School [Boston] (HMS), Harvard University [Cambridge], Munich Cluster for systems neurology [Munich] (SyNergy), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Ludwig-Maximilians-Universität München (LMU), Université de Strasbourg Inserm Faculté de médecine, Klinikum der Universitat Munchen, Ludwig-Maximilians-Universität München (LMU), University hospital of Zurich [Zurich], Universität Ulm - Ulm University [Ulm, Allemagne], Massachusetts Institute of Technology (MIT), Ludwig-Maximilians University Hospital (LMU Munich), ANR-16-CE92-0031,EpiFUS,Rôle de FUS dans la régulation des modifications épigénétiques : conséquences pour la sclérose latérale amyotrophique et la démence frontotemporale(2016), ANR-16-CE16-0015,ToFU,Interactions entre TAU, FUS et TDP-43 dans les maladies neurodégénératives(2016), ANR-19-CE17-0016,SPREADALS,Propagation et toxicité des facteurs pathogéniques de la sclérose latérale amyotrophique(2019), Technische Universität München [München] (TUM)-Ludwig-Maximilians-Universität München (LMU), Anne Laurence, Boutillier, Rôle de FUS dans la régulation des modifications épigénétiques : conséquences pour la sclérose latérale amyotrophique et la démence frontotemporale - - EpiFUS2016 - ANR-16-CE92-0031 - AAPG2016 - VALID, Interactions entre TAU, FUS et TDP-43 dans les maladies neurodégénératives - - ToFU2016 - ANR-16-CE16-0015 - AAPG2016 - VALID, Propagation et toxicité des facteurs pathogéniques de la sclérose latérale amyotrophique - - SPREADALS2019 - ANR-19-CE17-0016 - AAPG2019 - VALID, University of Zurich, Liebscher, Sabine, Dupuis, Luc, Centre de Recherche en Biomédecine de Strasbourg (CRBS), and Dieterle, Stéphane

Subjects
Male, Cytoplasm, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Gene Expression, Synaptic Transmission, MESH: Synapses, Mice, MESH: Animals, Gene Knock-In Techniques, MESH: Amyotrophic Lateral Sclerosis, MESH: Gene Knock-In Techniques, metabolism [RNA-Binding Protein FUS], Motor Neurons, MESH: RNA-Binding Protein FUS, FUS protein, mouse, 3100 General Physics and Astronomy, [SDV] Life Sciences [q-bio], genetics [Amyotrophic Lateral Sclerosis], Phenotype, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Female, ddc:500, MESH: Motor Neurons, MESH: Mutation, MESH: Gene Expression, Science, 610 Medicine & health, 1600 General Chemistry, Genetics and Molecular Biology, MESH: Phenotype, Article, 1300 General Biochemistry, Genetics and Molecular Biology, MESH: Mice, Inbred C57BL, MESH: Synaptic Transmission, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Mice, metabolism [Cytoplasm], metabolism [Amyotrophic Lateral Sclerosis], MESH: Cytoplasm, Amyotrophic Lateral Sclerosis, General Chemistry, metabolism [Motor Neurons], metabolism [Synapses], MESH: Male, Mice, Inbred C57BL, physiology [Synaptic Transmission], General Biochemistry, Mutation, Synapses, RNA-Binding Protein FUS, 11493 Department of Quantitative Biomedicine, genetics [RNA-Binding Protein FUS], MESH: Female
Abstract

Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These results indicate that FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, likely relevant also for other neurodegenerative diseases characterized by FUS mislocalization., Mutations in the RNA binding protein FUS are associated with ALS. Here the authors show that in FUS knock-in mice there is a progressive increase in neuronal activity in the frontal cortex which is associated with altered synaptic gene expression.

Published
2021

4. MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair

Author

de Krijger, Inge, Föhr, Bastian, Pérez, Santiago Hernández, Vincendeau, Estelle, Serrat, Judit, Thouin, Alexander Marc, Susvirkar, Vivek, Lescale, Chloé, Paniagua, Inés, Hoekman, Liesbeth, Kaur, Simranjeet, Altelaar, Maarten, Deriano, Ludovic, Faesen, Alex C, Jacobs, Jacqueline J L, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie du système immunitaire (Inserm U1223), Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University [Utrecht], This work was supported by project grant 10999/2017-1 from the Dutch Cancer Society (KWF) to J.J.L.J., by the Institut Pasteur to L.D., by the Institut National Du Cancer (INCA_13852) to L.D., by the Ligue Nationale Contre le Cancer (Equipe labellisée 2019) to L.D. The Proteomics Facility of the Netherlands Cancer Institute was supported by the X-omics Initiative, partially funded by the Dutch Research Council (NWO)(project 184.034.019). E.V. received funding from Université de Paris, Sorbonne Paris Cité. A.C.F. is grateful for generous core funding by the Max Planck Society., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC), Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], and Lescale, Chloé

Subjects
DNA Repair, Chemistry(all), ATPase, MESH: DNA Breaks, Double-Stranded, Gene Expression, General Physics and Astronomy, Cell Cycle Proteins, [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biochemistry, Piperazines, MESH: Recombinant Proteins, Mice, chemistry.chemical_compound, 0302 clinical medicine, DNA Breaks, Double-Stranded, MESH: Animals, 0303 health sciences, Multidisciplinary, biology, Chemistry, MESH: Protein Multimerization, DNA damage and repair, MESH: DNA, Recombinant Proteins, 3. Good health, Cell biology, DNA-Binding Proteins, MESH: ATPases Associated with Diverse Cellular Activities, MESH: HEK293 Cells, Mad2 Proteins, MESH: Phthalazines, MESH: Mad2 Proteins, Protein Binding, MESH: Cell Line, Tumor, MESH: Gene Expression, DNA repair, Science, Physics and Astronomy(all), Article, Chromosomes, General Biochemistry, Genetics and Molecular Biology, Cell Line, 03 medical and health sciences, MESH: Cell Cycle Proteins, Cell Line, Tumor, Animals, Humans, MESH: Protein Binding, Protein Interaction Domains and Motifs, MESH: Mice, 030304 developmental biology, MESH: Protein Interaction Domains and Motifs, [SDV.GEN]Life Sciences [q-bio]/Genetics, Binding Sites, MESH: Humans, TRIP13, Biochemistry, Genetics and Molecular Biology(all), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, General Chemistry, DNA Repair Pathway, Fibroblasts, MESH: Cell Line, HEK293 Cells, MESH: Binding Sites, MESH: Cisplatin, MESH: Fibroblasts, MESH: HeLa Cells, biology.protein, ATPases Associated with Diverse Cellular Activities, Phthalazines, Cisplatin, Protein Multimerization, 030217 neurology & neurosurgery, Function (biology), MESH: DNA-Binding Proteins, HeLa Cells, Genetics and Molecular Biology(all)
Abstract

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity., MAD2L2 — a member of the shieldin complex — is known to play important roles in DNA repair. Here the authors demonstrate how MAD2L2 dimerization mediated through SHLD2 participates in shieldin assembly and function.

Published
2021

5. Gene expression analysis in EBV-infected ataxia-telangiectasia cell lines by RNA-sequencing reveals protein synthesis defect and immune abnormalities

Author

Felipe Suarez, Moussab Tatfi, Kenzo-Hugo Hillion, Emeline Perthame, Marie-Agnès Dillies, Olivier Hermine, Hervé Ménager, Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), This work was supported in part by grants from Fondation ARC pour la recherche sur le cancer (France), Fondation ATEurope (France) and Le Centre de Référence Déficits Immunitaires Héréditaires (CEREDIH, France)., and Ménager, Hervé

Subjects
0301 basic medicine, Epstein-Barr Virus Infections, Herpesvirus 4, Human, Lymphoma, Gene Expression, medicine.disease_cause, MESH: Ataxia Telangiectasia, 0302 clinical medicine, Transcription (biology), hemic and lymphatic diseases, Gene expression, MESH: Sequence Analysis, RNA, Pharmacology (medical), Genetics (clinical), [INFO.INFO-BI] Computer Science [cs]/Bioinformatics [q-bio.QM], MESH: Epstein-Barr Virus Infections, General Medicine, 030220 oncology & carcinogenesis, Medicine, MESH: Epstein-Barr Virus Nuclear Antigens, Oncovirus, MESH: Gene Expression, Biology, Virus, Cell Line, Ataxia Telangiectasia, 03 medical and health sciences, Immune system, MESH: RNA, EBV, medicine, Humans, Gene, MESH: Humans, Sequence Analysis, RNA, Research, MESH: Herpesvirus 4, Human, medicine.disease, MESH: Cell Line, 030104 developmental biology, Epstein-Barr Virus Nuclear Antigens, ATM, Ataxia-telangiectasia, Cancer research, RNA, Ataxia, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], RNA-seq, Carcinogenesis
Abstract

Background Epstein–Barr virus (EBV) targets B-cells where it establishes a latent infection. EBV can transform B-cells in vitro and is recognized as an oncogenic virus, especially in the setting of immune compromise. Indeed, immunodeficient patients may fail to control chronic EBV infection, leading to the development EBV-driven lymphoid malignancies. Ataxia telangiectasia (AT) is a primary immune deficiency caused by mutations in the ATM gene, involved in the repair of double-strand breaks. Patients with AT are at high risk of developing cancers, mostly B-cell lymphoid malignancies, most of which being EBV-related. Aside from immune deficiency secondary to AT, loss of ATM function could also hinder the control of the virus within B-cells, favoring lymphomagenesis in AT patients. Results We used RNA sequencing on lymphoblastoid cell lines derived from patients with AT and healthy donors to analyze and compare both cellular and viral gene expression. We found numerous deregulated signaling pathways involving transcription, translation, oncogenesis and immune regulation. Specifically, the translational defect was confirmed in vitro, suggesting that the pathogenesis of AT may also involve a ribosomal defect. Concomitant analysis of viral gene expression did not reveal significant differential gene expression, however, analysis of EBV interactome suggests that the viral latency genes EBNA-3A, EBNA-3C and LMP1 may be disrupted in LCL from AT patients. Conclusion Our data support the notion that ATM deficiency deregulates cellular gene expression possibly disrupting interactions with EBV latent genes, promoting the oncogenic potential of the virus. These preliminary findings provide a new step towards the understanding of EBV regulation and of AT pathogenesis.

Published
2021

6. CD116+ fetal precursors migrate to the perinatal lung and give rise to human alveolar macrophages

Author

Evren, Elza, Ringqvist, Emma, Doisne, Jean-Marc, Thaller, Anna, Sleiers, Natalie, Flavell, Richard, Di Santo, James, Willinger, Tim, Karolinska Institutet [Stockholm], Immunité Innée - Innate Immunity, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Sorbonne Paris Cité (USPC), Department of Immunobiology [New Haven, CT, USA] (Yale School of Medicine), Yale University [New Haven], Howard Hughes Medical Institute (HHMI), This work was supported by a faculty-funded career position at Karolinska Institutet (2-1060/2018), a Karolinska Institutet Research Foundation Grant (2020-01438), a Junior Investigator and Junior Project Research Grant from the Center for Innovative Medicine financed by Region Stockholm (2-538/2014, 20190152), as well as project grants from the Swedish Research Council (2015-02413, 2019-01099), the Swedish Heart-Lung Foundation (20190198), Petrus och Augusta Hedlunds Stiftelse (M-2021-1568), and the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation (C19PI:G:012) to T. Willinger. E. Evren was supported by a scholarship from the Royal Swedish Academy of Sciences (BS2021-0030). A. Thaller was supported by the European Union’s Horizon 2020 research and innovation program (H2020 Marie Skłodowska-Curie Actions grant 765104). R.A. Flavell is an investigator of the Howard Hughes Medical Institute., and European Project: 765104,MATURE-NK

Subjects
MESH: Cell Differentiation, MESH: Macrophages, Alveolar, MESH: Gene Expression, MESH: Immunophenotyping, MESH: Mice, Transgenic, Genes, myb, [SDV]Life Sciences [q-bio], Innate Immunity and Inflammation, Gene Expression, Mice, Transgenic, MESH: Stem Cells, Article, Immunophenotyping, Mice, Fetus, MESH: Receptors, Granulocyte-Macrophage Colony-Stimulating Factor, Cell Movement, Macrophages, Alveolar, Animals, Humans, MESH: Animals, MESH: Lung, MESH: Mice, MESH: Cell Movement, Lung, MESH: Genes, myb, MESH: Humans, Stem Cells, MESH: Fetus, MESH: Immunohistochemistry, Cell Differentiation, Mucosal Immunology, respiratory system, Immunohistochemistry, Hematopoiesis, Liver, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor, MESH: Biomarkers, Biomarkers, MESH: Liver
Abstract

Evren et al. identify CD116+ fetal liver cells as precursors of human alveolar macrophages in early life and determine the impact of cell origin on lung macrophage identity and function in the human context with a unique in vivo model., Despite their importance in lung health and disease, it remains unknown how human alveolar macrophages develop early in life. Here we define the ontogeny of human alveolar macrophages from embryonic progenitors in vivo, using a humanized mouse model expressing human cytokines (MISTRG mice). We identified alveolar macrophage progenitors in human fetal liver that expressed the GM-CSF receptor CD116 and the transcription factor MYB. Transplantation experiments in MISTRG mice established a precursor–product relationship between CD34−CD116+ fetal liver cells and human alveolar macrophages in vivo. Moreover, we discovered circulating CD116+CD64−CD115+ macrophage precursors that migrated from the liver to the lung. Similar precursors were present in human fetal lung and expressed the chemokine receptor CX3CR1. Fetal CD116+CD64− macrophage precursors had a proliferative gene signature, outcompeted adult precursors in occupying the perinatal alveolar niche, and developed into functional alveolar macrophages. The discovery of the fetal alveolar macrophage progenitor advances our understanding of human macrophage origin and ontogeny., Graphical Abstract

Published
2021

7. Cynomolgus macaque IL37 polymorphism and control of SIV infection

Author

Henri-Jean Garchon, Nicolas Tchitchek, Delphine Desjardins, Roger Le Grand, Bruno Vaslin, Takashi Shiina, Nicolas Congy-Jolivet, Nathalie Dereuddre-Bosquet, Brigitte Autran, Ioannis Theodorou, Olivier Lambotte, Antoine Blancher, Alice Aarnink, Shingo Suzuki, Tokai University, Japan, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Department of Neurology, André Mignot Hospital, University of Versailles, St-Quentin-en-Yvelines, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Sorbonne Université (SU), Université Pierre et Marie Curie - Paris 6 (UPMC), Tokai University, Laboratoire d'Immunogénétique Moléculaire (LIMT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, CHU Toulouse [Toulouse], Université de Versailles Saint-Quentin-en-Yvelines - UFR Sciences de la santé Simone Veil (UVSQ Santé), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Hôpital Ambroise Paré [AP-HP], Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Infectious Diseases Models for Innovative Therapies (IDMIT), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Service d'Immunologie [CHU Pitié-Salpétrière], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Centre de Physiopathologie Toulouse Purpan (CPTP), 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), ANR-11-INBS-0008,IDMIT,Infrastructure nationale pour la modélisation des maladies infectieuses humaines(2011), ANR-10-EQPX-0002,FlowCyTech,Plateforme de phénotypage en Mass cytométrie pour l'analyse multiparamétrique de biomarqueurs complexes de l'efficacité de nouvelles stratégies thérapeutiques ou vaccinales(2010), Université de Toulouse (UT)-Université de Toulouse (UT), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Fédératif de Biologie (IFB) - Hôpital Purpan, Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse], Centre d'investigation clinique de Toulouse (CIC 1436), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Institut des Maladies Emergentes et des Thérapies Innovantes (IMETI)

Subjects
MESH: Histocompatibility Antigens Class I / immunology, Male, 0301 basic medicine, Candidate gene, [SDV]Life Sciences [q-bio], Simian Acquired Immunodeficiency Syndrome, Gene Expression, lcsh:Medicine, MESH: Base Sequence, MESH: Linear Models, Exon, 0302 clinical medicine, Polymorphism (computer science), Gene expression, Odds Ratio, MESH: Animals, lcsh:Science, MESH: High-Throughput Nucleotide Sequencing, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, MESH: Host-Pathogen Interactions / immunology, High-Throughput Nucleotide Sequencing, Exons, Viral Load, MESH: Interleukin-1 / genetics, 3. Good health, MESH: Histocompatibility Antigens Class I / genetics, MESH: Interferon-Stimulated Gene Factor 3, gamma Subunit / immunology, Host-Pathogen Interactions, [SDV.IMM]Life Sciences [q-bio]/Immunology, Simian Immunodeficiency Virus, MESH: Interleukin-1 / immunology, HIV infections, MESH: Host-Pathogen Interactions / genetics, MESH: Gene Expression, Single-nucleotide polymorphism, Biology, MESH: Genetic Loci, Polymorphism, Single Nucleotide, MESH: Interferon-Stimulated Gene Factor 3, gamma Subunit / genetics, Article, 03 medical and health sciences, Immunogenetics, Animals, Gene, Base Sequence, Histocompatibility Antigens Class I, Haplotype, lcsh:R, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Haplotypes, Odds ratio, Virology, Interferon-Stimulated Gene Factor 3, gamma Subunit, Macaca fascicularis, 030104 developmental biology, Haplotypes, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Genetic Loci, Linear Models, lcsh:Q, MESH: Exons, Sequence Alignment, 030217 neurology & neurosurgery, Interleukin-1
Abstract

The association between gene polymorphisms and plasma virus load at the set point (SP-PVL) was investigated in Mauritian macaques inoculated with SIV. Among 44 macaques inoculated with 50 AID50, six individuals were selected: three with SP-PVL among the highest and three with SP-PVL among the lowest. The exons of 390 candidate genes of these six animals were sequenced. Twelve non-synonymous single nucleotide polymorphisms (NS-SNPs) lying in nine genes potentially associated with PVL were genotyped in 23 animals. Three NS-SNPs with probabilities of association with PVL less than 0.05 were genotyped in a total of 44 animals. One NS-SNP lying in exon 1 of the IL37 gene displayed a significant association (p = 3.33 × 10−4) and a strong odds ratio (19.52). Multiple linear regression modeling revealed three significant predictors of SP-PVL, including the IL37 exon 1 NS-SNP (p = 0.0004) and the MHC Class IB haplotypes M2 (p = 0.0007) and M6 (p = 0.0013). These three factors in conjunction explained 48% of the PVL variance (p = 4.8 × 10−6). The potential role of IL37 in the control of SIV infection is discussed.

Published
2019

8. The steroid derivative 6-aminocholestanol inhibits the DEAD-box helicase eIF4A (LieIF4A) from the Trypanosomatid parasite Leishmania by perturbing the RNA and ATP binding sites

Author

N. Kyle Tanner, Mourad Barhoumi, Emna Harigua-Souiai, Arnaud Blondel, Josette Banroques, Yosser Zina Abdelkrim, Ikram Guizani, Expression Génétique Microbienne (EGM (UMR_8261 / FRE_3630)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP), Faculté des Sciences de Bizerte [Université de Carthage], Université de Carthage - University of Carthage, Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Centre National de la Recherche Scientifique, France, by the HelicaRN [2010 BLAN 1503 01] and HeliDEAD grants [ANR-13-BSV8-0009-01] from the Agence Nationale de la Recherche, France, and by the Initiative d'Excellence program from the French State [Grant DYNAMO, ANR-11-LABX-0011-01] to NKT. This work received financial support from the Pasteur Institute Transversal Research Program (grant PTR426), France, and partially from the Ministry of Higher Education and Research in Tunisia (LR11IPT04 & LR16IPT04) to IG. EHS received support from the UNESCO-L'Oréal, 'For Women in Science,' international fellowship and the Institut Pasteur International Network (Calmette and Yersin programme)., We thank Patrick Linder, Centre Médical Universitaire, Geneva, Switzerland, for the eIF4AIMus clone. The 6-aminocholestanol was a gift from Dr. Leila el Kihel, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), UFR des Sciences Pharmaceutiques, Université de Caen de Basse-Normandie, France, for the minimal costs of shipping fees. We thank Damien Monet for his assistance in the cavity generation and analyses., ANR-13-BSV8-0009,HeliDEAD,Les ARN hélicases à boite DEAD: quels sont leurs rôles, leurs partenaires, leurs substrats ARN et comment elles fonctionnent.(2013), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Faculté des Sciences de Bizerte, Bioinformatique structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), ANR-11-LABX-0011/11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), and Institut de biologie physico-chimique (IBPC)

Subjects
Hippuristanol, 0301 basic medicine, Antifungal Agents, MESH: Sequence Homology, Amino Acid, ATPase, Protozoan Proteins, Gene Expression, MESH: Protein Structure, Secondary, MESH: Amino Acid Sequence, Protein Structure, Secondary, MESH: Eukaryotic Initiation Factor-4A, MESH: Recombinant Proteins, Mice, chemistry.chemical_compound, Adenosine Triphosphate, MESH: Cholesterol, MESH: Genetic Vectors, MESH: Adenosine Triphosphate, MESH: Animals, MESH: Trypanocidal Agents, Cloning, Molecular, Leishmania infantum, MESH: Protozoan Proteins, Conserved Sequence, Ergosterol, MESH: Conserved Sequence, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, Trypanocidal Agents, RNA Helicase A, Recombinant Proteins, MESH: Leishmania infantum, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Cholesterol, Biochemistry, Translation-initiation factor, MESH: Drug Repositioning, Protein Binding, RNA helicase, MESH: Gene Expression, Genetic Vectors, MESH: Sequence Alignment, MESH: Binding, Competitive, Biology, Binding, Competitive, Drug design, 03 medical and health sciences, Escherichia coli, Animals, Humans, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Interaction Domains and Motifs, MESH: Cloning, Molecular, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding site, MESH: Mice, Molecular Biology, MESH: Protein Interaction Domains and Motifs, Binding Sites, MESH: Humans, Sequence Homology, Amino Acid, MESH: RNA, Helminth, 030102 biochemistry & molecular biology, Drug Repositioning, RNA, Helicase, MESH: Antifungal Agents, Kinetics, 030104 developmental biology, MESH: Binding Sites, chemistry, eIF4A, Eukaryotic Initiation Factor-4A, Nucleic acid, biology.protein, Parasitology, RNA, Helminth, Sequence Alignment
Abstract

International audience; The antifungal agent 6-aminocholestanol targets the production of ergosterol, which is the principle sterol in many fungi and protozoans; ergosterol serves many of the same roles as cholesterol in animals. We found that it also is an effective inhibitor of the translation-initiation factor eIF4AI from mouse (eIF4AIMus) and the Trypanosomatid parasite Leishmania (LieIF4A). The eIF4A proteins belong to the DEAD-box family of RNA helicases, which are ATP-dependent RNA-binding proteins and RNA-dependent ATPases. DEAD-box proteins contain a commonly-shared core structure consisting of two linked domains with structural homology to that of recombinant protein A (RecA) and that contain conserved motifs that are involved in RNA and ATP binding, and in the enzymatic activity. The compound inhibits both the ATPase and helicase activities by perturbing ATP and RNA binding, and it is capable of binding other proteins containing nucleic acid-binding sites as well. We undertook kinetic analyses and found that the Leishmania LieIF4A protein binds 6-aminocholestanol with a higher apparent affinity than for ATP, although multiple binding sites were probably involved. Competition experiments with the individual RecA-like domains indicate that the primary binding sites are on RecA-like domain 1, and they include a cavity that we previously identified by molecular modeling of LieIF4A that involve conserved RNA-binding motifs. The compound affects the mammalian and Leishmania proteins differently, which indicates the binding sites and affinities are not the same. Thus, it is possible to develop drugs that target DEAD-box proteins from different organisms even when they are implicated in the same biological process.

Published
2018

9. Influenza A(H5N1) viruses with A(H9N2) single gene (matrix or PB1) reassortment isolated from Cambodian live bird markets

Author

Paul F. Horwood, Andrew R. Greenhill, Aeron C. Hurt, Sokhoun Yann, San Sorn, Songha Tok, Davun Holl, Annika Suttie, Srey Viseth Horm, Yi-Mo Deng, Erik A. Karlsson, Philippe Dussart, Sothyra Tum, Ian G. Barr, Unité de Virologie / Virology Unit [Phnom Penh], Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Federation University [Churchill, Australia], The Peter Doherty Institute for Infection and Immunity [Melbourne], The Royal Melbourne Hospital-University of Melbourne, Ministry of Agriculture, Forestry and Fisheries [Cambodia], James Cook University (JCU), and This publication is the result of work conducted under a cooperative agreement with the Office of the Assistant Secretary for Preparedness and Response in the U.S. Department of Health and Human Services (HHS), grant number IDSEP140020-01-00. Its contents and conclusions are solely the responsibility of the authors and do not represent the official views of HHS. The study was also funded, in part, by the US Agency for International Development (grant No. AID-442-G-14-00005). Annika Suttie is funded by an Australian Government Research Training Program Scholarship and a Faculty of Science and Technology Research Scholarship from Federation University. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.

Subjects
0301 basic medicine, animal diseases, viruses, Reassortment, Gene Expression, medicine.disease_cause, A(H5N1), MESH: Ducks, Influenza A Virus, H9N2 Subtype, MESH: Animals, MESH: Phylogeny, Clade, Phylogeny, 2. Zero hunger, Live bird markets, MESH: Chickens, virus diseases, 3. Good health, MESH: Reassortant Viruses, Ducks, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Epidemiological Monitoring, Cambodia, Reassortant Viruses, MESH: Gene Expression, MESH: Influenza A Virus, H5N1 Subtype, Single gene, MESH: Poultry Diseases, Biology, Viral Matrix Proteins, Viral Proteins, 03 medical and health sciences, MESH: Influenza in Birds, Phylogenetics, Virology, medicine, Animals, Gene, Poultry Diseases, MESH: Viral Matrix Proteins, Viral matrix protein, Influenza A Virus, H5N1 Subtype, MESH: Cambodia, MESH: Viral Proteins, Influenza, Influenza A virus subtype H5N1, MESH: Influenza A Virus, H9N2 Subtype, 030104 developmental biology, Influenza in Birds, MESH: Epidemiological Monitoring, Chickens, A(H9N2)
Abstract

International audience; Live bird market surveillance for avian influenza viruses in Cambodia in 2015 has led to the detection of two 7:1 reassortant influenza A(H5N1) clade 2.3.2.1c viruses. These reassortant strains, designated A/duck/Cambodia/ Z564W35M1/2015 and A/chicken/Cambodia/Z850W49M1/2015, both contained a single gene (PB1 and matrix gene, respectively) from concurrently circulating A(H9N2) influenza viruses. All other viral genes from both isolates clustered with A(H5N1) clade 2.3.2.1 viruses. Continued and prolonged co-circulation of influenza A(H5N1) and A(H9N2) viruses in Cambodian live bird markets may present a risk for the emergence of novel influenza reassortant viruses with negative agricultural and/or public health implications.

Published
2018

10. The impact of genetic diversity on gene essentiality within the E. coli species

Author

Rousset, François, Caballero, José, Piastra-Facon, Florence, Fernández-Rodríguez, Jesús, Clermont, Olivier, Denamur, Erick, Rocha, Eduardo, Bikard, David, Biologie de Synthèse - Synthetic biology, Institut Pasteur [Paris] (IP), Collège Doctoral, Sorbonne Université (SU), Eligo Bioscience, Infection, Anti-microbiens, Modélisation, Evolution (IAME (UMR_S_1137 / U1137)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Université Sorbonne Paris Nord, AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Génomique évolutive des Microbes / Microbial Evolutionary Genomics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 677823), by the French Government’s Investissement d’Avenir programme and by Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID). F.R. is supported by a doctoral scholarship from Ecole Normale Supérieure. E.D. was partially supported by the ‘Fondation pour la Recherche Médicale’ (Equipe FRM 2016, grant no. DEQ20161136698). E.P.C.R. was partially supported by the ‘Fondation pour la Recherche Médicale’ (Equipe FRM EQU201903007835)., We thank B. Dupuy for sharing the use of the anaerobic chamber, as well as O. Tenaillon, P.-A. Kaminsky, G. Sezonov, A. Danchin and A. Calvo-Villamañan for useful discussions. We thank the P2M platform (Institut Pasteur, Paris, France) for genome sequencing and V. Briolat from the Biomics platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA. We also thank J. Rodríguez-Beltrán and S. Brisse for providing a strain of Citrobacter freundii and Klebsiella pneumoniae, respectively, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 677823,H2020,ERC-2015-STG,CRISPAIR(2016), Bikard, David, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Study of the interplay between CRISPR interference and DNA repair pathways towards the development of novel CRISPR tools - CRISPAIR - - H20202016-03-01 - 2021-02-28 - 677823 - VALID, Institut Pasteur [Paris], Collège doctoral [Sorbonne universités], Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)-Université Sorbonne Paris Nord, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Gene Expression, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: DNA Transposable Elements, MESH: Genetic Fitness, MESH: Escherichia coli, MESH: Species Specificity, MESH: Genetic Variation, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Genome, Bacterial, MESH: Phylogeny, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: Genes, Essential
Abstract

International audience; Bacteria from the same species can differ widely in their gene content. In E. coli, the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. While recent advances in bacterial genomics have unraveled genes required for fitness in various experimental conditions at the genome scale, most studies have focused on single model strains. As a result, the impact of this genetic diversity on core processes of the bacterial cell remains largely under-investigated. Here, we developed a new CRISPR interference platform for highthroughput gene repression that is compatible with most E. coli isolates and closely-related species. We applied it to assess the importance of ~3,400 nearly ubiquitous genes in 3 growth conditions in 18 representative E. coli strains spanning most common phylogroups and lifestyles of the species. Our screens revealed extensive variations in gene essentiality between strains and conditions. Investigation of the genetic determinants for these variations highlighted the importance of epistatic interactions with mobile genetic elements. In particular, we showed how prophage-encoded defense systems against phage infection can trigger the essentiality of persistent genes that are usually nonessential. This study provides new insights into the evolvability of gene essentiality and argues for the importance of studying various isolates from the same species under diverse conditions.

Published
2021

11. Combined gene expression and chromatin immunoprecipitation from a single mouse hippocampus

Author

Pierre Trifilieff, Paolo Sassone-Corsi, Marie-Pierre Moisan, Jean Christophe Helbling, Kenichiro Kinouchi, Nutrition et Neurobiologie intégrée (NutriNeuro), Université de Bordeaux (UB)-Institut Polytechnique de Bordeaux-Ecole nationale supérieure de chimie, biologie et physique-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Center for Epigenetics and Metabolism [Irvine, CA, États-Unis], Institut National de la Santé et de la Recherche Médicale (INSERM)-University of California [Irvine] (UCI), and University of California-University of California

Subjects
MESH: Gene Expression, MESH: Hippocampus, hippocampus, Transgene, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, ChIP, Health Informatics, Biology, General Biochemistry, Genetics and Molecular Biology, MESH: Chromatin, 03 medical and health sciences, 0302 clinical medicine, Gene expression, MESH: Animals, Epigenetics, MESH: Epigenesis, Genetic, General Pharmacology, Toxicology and Pharmaceutics, Transcription factor, MESH: Mice, mouse, 030304 developmental biology, Regulation of gene expression, MESH: Chromatin Immunoprecipitation, 0303 health sciences, General Immunology and Microbiology, General Neuroscience, Chromatin, Cell biology, Medical Laboratory Technology, gene expression, RNA extraction, Chromatin immunoprecipitation, 030217 neurology & neurosurgery, epigenetic
Abstract

International audience; All neuronal cells hold the same genetic information but vary by their structural and functional plasticity depending on the brain area and environmental influences. Such variability involves specific gene regulation, which is driven by transcription factors (TFs). In the field of neuroscience, epigenetics is the main mechanism that has been investigated to understand the dynamic modulation of gene expression by behavioral responses, stress responses, memory processes, etc. Nowadays, gene expression analyzed by real-time quantitative PCR and TF binding estimated by chromatin immunoprecipitation (ChIP) enables one to dissect this regulation. Because of the wide range of transgenic models, as well as cost-effective aspects, mouse models are widely used neuroscience. Thus, we have set up a protocol that allows extraction of both RNA for gene expression analysis and chromatin for ChIP experiment from a single mouse hippocampus. Using such protocols, information regarding gene expression and regulatory molecular mechanisms from the same animal can be integrated and correlated with neurobiological and behavioral outcomes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Hippocampus isolation from mouse brain Basic Protocol 2: RNA extraction and gene expression analysis from a mouse half hippocampus Basic Protocol 3: ChIP from one hemisphere side mouse hippocampus.

Published
2021

12. Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70–Hsp40 Substrates

Author

Andrija Finka, Manfredo Quadroni, Pierre Goloubinoff, Bruno Fauvet, Pierre Genevaux, Anne-Marie Cirinesi, Marie-Pierre Castanié-Cornet, Université de Lausanne (UNIL), University of Zadar, Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en pharmacologie - santé (CRPS), and Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Mycobacterium tuberculosis, SecB, medicine.medical_treatment, Mutant, ClpX, MESH: Escherichia coli Proteins, MESH: Protein Isoforms, medicine.disease_cause, Biochemistry, DnaJ, DnaK, MESH: Recombinant Proteins, 0302 clinical medicine, MESH: Genetic Vectors, MESH: Endopeptidase Clp, Molecular Biosciences, Toxin-antitoxin, Biology (General), MESH: Bacterial Proteins, Original Research, 0303 health sciences, MESH: Gene Expression Regulation, Bacterial, biology, Chemistry, MESH: Escherichia coli, HslV, Hsp90, MESH: ATPases Associated with Diverse Cellular Activities, MESH: Bacterial Genome, MESH: Molecular Chaperones, Proteases, MESH: Gene Expression, HtpG, chaperones, proteostasis, QH301-705.5, MESH: Proteolysis, Biochemistry, Genetics and Molecular Biology (miscellaneous), 03 medical and health sciences, HigB1-HigA1, chaperones, DnaK, DnaJ, proteostasis, HslV, HtpG, Heat shock protein, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, MESH: Cloning, Molecular, Molecular Biology, Escherichia coli, 030304 developmental biology, Protease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Hsp70, Proteostasis, biology.protein, ClpC1, 030217 neurology & neurosurgery, MESH: Toxin-Antitoxin Systems
Abstract

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70–Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease.

Published
2021

13. A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans

Author

Henry Houlden, Thomas Haaf, Pratishtha Varshney, Christian Beetz, Hamid Galehdari, Lucy A Dunbar, Alireza Sedaghat, Richard J.H. Smith, Michael R. Bowl, Aziz El-Amraoui, Kevin T. Booth, David Murphy, Neda Mazaheri, Sandrine Vitry, Kumar N. Alagramam, Ben Fowler, Shruthi VijayKumar, Aboulfazl Rad, Hela Azaiez, Cassidy Petree, Barbara Vona, Sheng-Jia Lin, Gholamreza Shariati, Reza Maroofian, Franz Rüschendorf, Gaurav K. Varshney, EL-AMRAOUI, Aziz, ECLAIRER LA SURDITÉ : UNE APPROCHE HOLISTIQUE DU SYNDROME D'USHER - - LIGHT4DEAF2015 - ANR-15-RHUS-0001 - RHUS - VALID, Surdité d'apparition tardive et progressive: de la physiopathologie à la thérapie - - HearInNoise2017 - ANR-17-CE16-0017 - AAPG2017 - VALID, Julius-Maximilians-Universität Würzburg (JMU), Eberhard Karls Universität Tübingen = Eberhard Karls University of Tuebingen, Shahid Chamran University of Ahvaz (SCU), Oklahoma Medical Research Foundation (OMRF), MRC Harwell Institute [UK], University College of London [London] (UCL), University of Iowa [Iowa City], Harvard Medical School [Boston] (HMS), Déficits Sensoriels Progressifs, Pathophysiologie et Thérapie / Progressive Sensory Disorders, Pathophysiology and Therapy (DSP), Sorbonne Université (SU)-Institut de l'Audition [Paris] (IDA), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Max Delbrück Center for Molecular Medicine [Berlin] (MDC), Helmholtz-Gemeinschaft = Helmholtz Association, CENTOGENE AG, Case Western Reserve University [Cleveland], Ahvaz Jundishapur University of Medical Sciences (AJUMS), Narges Medical Genetics and Prenatal Diagnostics, Open Access funding enabled and organized by Projekt DEAL. This work was supported by Intramural Funding (fortüne) at the University of Tübingen (2545-1-0 to BV), the Ministry of Science, Research and Art Baden-Württemberg (to BV), the Medical Research Council (MC_UP_1503/2 to MRB), ANR light4deaf (ANR-15-RHUS-0001), HearInNoise (ANR-17-CE16-0017), LHW-stiftung to AE), and a grant from NIH/COBRE GM103636 (Project 3), the Presbyterian Health Foundation (PHF) Grant to GKV. This study was funded in part by NIDCDs R01s DC002842 and DC012049 to RJS and T32 GM007748 to KTB. LAD is a Medical Research Council DPhil student (1774724)., We would like to extend our gratitude to the family for their participation. We thank Dr. Caroline Lekszas, Dr. Daniel Liedtke, and Dr. Indrajit Nanda from the Institute of Human Genetics at the University of Würzburg for their technical expertise., ANR-15-RHUS-0001,LIGHT4DEAF,ECLAIRER LA SURDITÉ : UNE APPROCHE HOLISTIQUE DU SYNDROME D'USHER(2015), ANR-17-CE16-0017,HearInNoise,Surdité d'apparition tardive et progressive: de la physiopathologie à la thérapie(2017), Julius-Maximilians-Universität Würzburg [Wurtzbourg, Allemagne] (JMU), Carver College of Medicine, University of Iowa, Institut de l'Audition [Paris] (IDA), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne Université - Faculté de Médecine (SU FM), Sorbonne Université (SU), Institute of Neurology - UCL/Queen Square [London, UK] (IN-UCL-QS), Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), University Hospitals of Cleveland, This work was supported by Intramural Funding (fortüne) at the University of Tübingen (2545-1-0 to B.V.), the Ministry of Science, Research and Art Baden-Württemberg (to B.V.), the Medical Research Council (MC_UP_1503/2 to M.R.B), ANR light4deaf (ANR-15-RHUS-0001), HearInNoise (ANR-17-CE16-0017), LHW-stiftung (to A.E.), and a grant from NIH/COBRE GM103636 (Project 3), the Presbyterian Health Foundation (PHF) Grant to G.K.V. This study was funded in part by NIDCDs R01s DC002842 and DC012049 to R.J.S and T32 GM007748 to K.T.B. L.A.D is a Medical Research Council DPhil student (1774724)., Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), and Déficits Sensoriels Progressifs, Pathophysiologie et Thérapie / Progressive Sensory Disorders, PathoPhysiology and Therapy (DSP)

Subjects
Male, Tetraspanins, [SDV]Life Sciences [q-bio], Gene Expression, MESH: Base Sequence, Consanguinity, Mice, 0302 clinical medicine, Missense mutation, MESH: Animals, Zebrafish, Genetics (clinical), Exome sequencing, Original Investigation, Genetics, 0303 health sciences, Chromosome Mapping, Disease gene identification, MESH: Amino Acid Substitution, Stop codon, Pedigree, [SDV] Life Sciences [q-bio], Female, Sensorineural hearing loss, MESH: Membrane Proteins, Chromosomes, Human, Pair 4, medicine.symptom, Adult, MESH: Chromosomes, Human, Pair 4, MESH: Gene Expression, Hearing loss, MESH: Pedigree, Hearing Loss, Sensorineural, Genes, Recessive, Locus (genetics), Biology, MESH: Hair Cells, Auditory, Inner, 03 medical and health sciences, MESH: Whole Exome Sequencing, Exome Sequencing, medicine, otorhinolaryngologic diseases, Animals, Humans, Point Mutation, ddc:610, MESH: Zebrafish, MESH: Mice, Alleles, MESH: Genes, Recessive, 030304 developmental biology, MESH: Point Mutation, MESH: Consanguinity, Hair Cells, Auditory, Inner, MESH: Humans, Base Sequence, MESH: Alleles, Intron, Membrane Proteins, MESH: Adult, medicine.disease, MESH: Male, Amino Acid Substitution, MESH: Hearing Loss, Sensorineural, Cardiovascular and Metabolic Diseases, MESH: Chromosome Mapping, MESH: Tetraspanins, MESH: Female, 030217 neurology & neurosurgery
Abstract

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

Published
2021

14. Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases

Author

Thomas C. Evans, Andrew F. Gardner, Ece Alpaslan, Kelly M Zatopek, Ludovic Sauguet, New England Biolabs, Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Agence Nationale de la Recherche [ANR-17-CE11-0005-01], Institut Pasteur, New England Biolabs. Funding foropen access charge: New England Biolabs., ANR-17-CE11-0005,ARCHAPOL,Spécificités structurales et potentiel biotechnologique de la PolD, une ADN polymérase atypique d'archée(2017), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Conformation, alpha-Helical, MESH: Genome, Archaeal, Ribonucleotide, MESH: Gene Expression Regulation, Archaeal, DNA polymerase, AcademicSubjects/SCI00010, MESH: Sequence Homology, Amino Acid, Gene Expression, MESH: Catalytic Domain, MESH: DNA Replication, DNA-Directed DNA Polymerase, MESH: Amino Acid Sequence, Genome Integrity, Repair and Replication, Substrate Specificity, MESH: Recombinant Proteins, chemistry.chemical_compound, MESH: Histidine, 0302 clinical medicine, Genome, Archaeal, RNA polymerase, Catalytic Domain, MESH: DNA-Directed DNA Polymerase, Cloning, Molecular, Polymerase, 0303 health sciences, biology, MESH: Kinetics, MESH: DNA, Archaeal, MESH: Archaeal Proteins, Recombinant Proteins, Thermococcus, DNA, Archaeal, Biochemistry, MESH: Protein Conformation, beta-Strand, MESH: Models, Molecular, Protein Binding, DNA Replication, MESH: Mutation, MESH: Gene Expression, Archaeal Proteins, MESH: Sequence Alignment, 03 medical and health sciences, Genetics, MESH: Protein Binding, Histidine, Protein Interaction Domains and Motifs, MESH: Cloning, Molecular, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Binding site, 030304 developmental biology, MESH: Protein Conformation, alpha-Helical, MESH: Protein Interaction Domains and Motifs, Binding Sites, Sequence Homology, Amino Acid, DNA replication, RNA, Ribonucleotides, Kinetics, MESH: Ribonucleotides, chemistry, MESH: Binding Sites, MESH: Thermococcus, Mutation, biology.protein, Protein Conformation, beta-Strand, MESH: Substrate Specificity, Gene Expression Regulation, Archaeal, Sequence Alignment, 030217 neurology & neurosurgery, DNA
Abstract

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2′ modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

Published
2020

15. Transmissible cancer influences immune gene expression in an endangered marsupial, the Tasmanian devil (Sarcophilus harrisii)

Author

Nynke Raven, Marcel Klaassen, Thomas Madsen, Frédéric Thomas, Rodrigo K. Hamede, Beata Ujvari, Deakin University [Burwood], Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM), Centre de Recherches Ecologiques et Evolutives sur le Cancer (MIVEGEC-CREEC), Processus Écologiques et Évolutifs au sein des Communautés (PEEC), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM), Deakin University [Waurn Ponds], ANR TRANSCAN, Grant/Award Number: ANR- 18- CE35- 0009, Centre National de la Recherche Scientifique, ARC Linkage, Grant/Award Number: LP170101105, Deakin SEBE, Grant/Award Number: RGS_2019, ARC Decra, Grant/Award Number: DE170101116, The University of Tasmania Foundation, Deakin CIE, Holsworth Wildlife Research Endowment, and ANR-18-CE35-0009,TRANSCAN,ECOLOGIE ET EVOLUTION DES CANCERS TRANSMISSIBLES(2018)

Subjects
Life history trade-off, MESH: Gene Expression, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, Gene Expression, Conservation physiology, [SDV.CAN]Life Sciences [q-bio]/Cancer, Animals, Wild, MESH: Marsupialia, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, MESH: Facial Neoplasms, Immune system, Marsupialia, Genetics, Animals, MESH: Animals, MESH: Animals, Wild, Seasons, Host-parasite interactions, Facial Neoplasms, MESH: Seasons, Ecology, Evolution, Behavior and Systematics, Cancer
Abstract

Understanding the effects of wildlife diseases on populations requires insight into local environmental conditions, host defence mechanisms, host life-history trade-offs, pathogen population dynamics, and their interactions. The survival of Tasmanian devils (Sarcophilus harrisii) is challenged by a novel, fitness limiting pathogen, Tasmanian devil facial tumour disease (DFTD), a clonally transmissible, contagious cancer. In order to understand the devils' capacity to respond to DFTD, it is crucial to gain information on factors influencing the devils' immune system. By using RT-qPCR, we investigated how DFTD infection in association with intrinsic (sex and age) and environmental (season) factors influences the expression of 10 immune genes in Tasmanian devil blood. Our study showed that the expression of immune genes (both innate and adaptive) differed across seasons, a pattern that was altered when infected with DFTD. The expression of immunogbulins IgE and IgM:IgG showed downregulation in colder months in DFTD infected animals. We also observed strong positive association between the expression of an innate immune gene, CD16, and DFTD infection. Our results demonstrate that sampling across seasons, age groups and environmental conditions are beneficial when deciphering the complex ecoevolutionary interactions of not only conventional host-parasite systems, but also of host and diseases with high mortality rates, such as transmissible cancers.

Published
2020

16. Differential activity and selectivity of N‐terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors

Author

Carmen Gallego, Françoise Bachelerie, Françoise Baleux, Erika Cecon, Agnieszka Jaracz-Ros, Pasquale Cutolo, Angélique Levoye, Irina Kufareva, Guillaume Bernadat, Martin Gustavsson, Tracy M. Handel, Inflammation, microbiome, immunosurveillance (MI2), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Biomolécules : Conception, Isolement, Synthèse (BioCIS), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-CY Cergy Paris Université (CY), Skaggs School of Pharmacy and Pharmaceutical Sciences [San Diego], University of California [San Diego] (UC San Diego), University of California-University of California, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Chimie des Biomolécules - Chemistry of Biomolecules, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Paris-Centre de Recherche Cardiovasculaire (PARCC (UMR_S 970/ U970)), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Université Sorbonne Paris Nord, This work was supported by grants from Ensemble Contre le SIDA (SIDACTION), the Institut National de la Santé et de la Recherche Médicale (INSERM), GIS-Network for Rare Diseases and the Agence Nationale de Recherches sur le SIDA (ANRS). We thank members of the Dr. F. Arenzana-Seisdedos laboratory (Laboratoire de Pathogénie Virale, Institut Pasteur, Paris, France) and Dr. R. Jockers (Institut Cochin, Paris, France), who provided critical discussion. We also thank E. Trinquet, N. Gregor and F. Maurin (CisBio International, Marcoule, France) for technical support in HTRF assays. AL was supported by Roux postdoctoral fellowship from Pasteur Institute. I.K. and T.M.H. are supported by NIH R01 grants AI118985 and GM117424. C.G. was beneficiary of a fellowship from Marie Skłodowska-Curie Innovative Training Networks (ITN-ETN) 'ONCOgenic Receptor Network of Excellence and Training' (MSCA-ITN-2014-ETN) and from Fondation pour la Recherche Médicale (FDT201805005700). A.J-R., P.C., C.G., and F. Bachelerie are members of the LabEx LERMIT supported by ANR grant (ANR-10-LABX-33) under the program 'Investissements d'Avenir' (ANR-11-IDEX-0003-01)., ANR-11-IDEX-0003,IPS,Idex Paris-Saclay(2011), ANR-10-LABX-0033,LERMIT,Research Laboratory on Drugs and Therapeutic Innovation(2010), European Project: 641833,H2020,H2020-MSCA-ITN-2014,ONCORNET(2015), University of California (UC)-University of California (UC), Université Paris Descartes - Paris 5 (UPD5)-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), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Baleux, Françoise, Idex Paris-Saclay - - IPS2011 - ANR-11-IDEX-0003 - IDEX - VALID, Research Laboratory on Drugs and Therapeutic Innovation - - LERMIT2010 - ANR-10-LABX-0033 - LABX - VALID, and ONCOgenic Receptor Network of Excellence and Training - ONCORNET - - H20202015-01-01 - 2018-12-31 - 641833 - VALID

Subjects
0301 basic medicine, Protein Conformation, alpha-Helical, ACKR3, Chemokine, Benzylamines, Protein Conformation, [SDV]Life Sciences [q-bio], Gene Expression, pluridimensional efficacy Receptors, MESH: Amino Acid Sequence, Cyclams, CXCR4, MESH: Recombinant Proteins, Chemokine receptor, 0302 clinical medicine, Heterocyclic Compounds, GPCR signaling, Receptors, Cyclic AMP, 2.1 Biological and endogenous factors, Immunology and Allergy, MESH: Molecular Dynamics Simulation, Aetiology, CXCR, Receptor, beta-Arrestins, MESH: Cyclic AMP, biology, MESH: Receptors, CXCR, CXCL12, Recombinant Proteins, Cell biology, [SDV] Life Sciences [q-bio], pluridimensional efficacy, 030220 oncology & carcinogenesis, MESH: HEK293 Cells, embryonic structures, MESH: Oligopeptides, MESH: Protein Conformation, beta-Strand, biological phenomena, cell phenomena, and immunity, MESH: Chemokine CXCL12, Selectivity, chemokine variants, MESH: Chemokine CXCL11, Oligopeptides, Biotechnology, Protein Binding, Agonist, Receptors, CXCR4, MESH: Mutation, MESH: Gene Expression, MESH: Heterocyclic Compounds, medicine.drug_class, Signal Transduction and Genes, 1.1 Normal biological development and functioning, Immunology, Molecular Dynamics Simulation, Article, MESH: Receptors, CXCR4, GPCR Signaling, Vaccine Related, 03 medical and health sciences, MESH: beta-Arrestins, Underpinning research, Biodefense, medicine, Humans, MESH: Protein Binding, Protein Interaction Domains and Motifs, Amino Acid Sequence, Receptors, CXCR, MESH: Protein Conformation, alpha-Helical, MESH: Protein Interaction Domains and Motifs, Binding Sites, MESH: Humans, Prevention, alpha-Helical, Cell Biology, biological factors, Chemokine CXCL12, Chemokine CXCL11, Emerging Infectious Diseases, 030104 developmental biology, HEK293 Cells, MESH: Binding Sites, Mutation, biology.protein, beta-Strand, Protein Conformation, beta-Strand, Biochemistry and Cell Biology
Abstract

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors’ TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2–4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

Published
2020

17. Does Gene Expression Noise Play a Functional Role in Plants?

Author

Sandra Cortijo, James C. W. Locke, Sainsbury Laboratory Cambridge University (SLCU), and University of Cambridge [UK] (CAM)

Subjects
0106 biological sciences, 0301 basic medicine, MESH: Gene Expression, [SDV]Life Sciences [q-bio], Population, MESH: Plants, Gene Expression, Plant Science, Computational biology, Biology, risk spreading, 01 natural sciences, 03 medical and health sciences, Stress, Physiological, Gene expression, transcriptional noise, medicine, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, education, MESH: Stress, Physiological, education.field_of_study, stochasticity, single-cell RNA-seq, Mechanism (biology), fungi, time-lapse microscopy, food and beverages, Plants, medicine.disease, Phenotype, Noise, Multicellular organism, 030104 developmental biology, Function (biology), Transcriptional noise, 010606 plant biology & botany
Abstract

International audience; Gene expression in individual cells can be surprisingly noisy. In unicellular organisms this noise can be functional; for example, by allowing a subfraction of the population to prepare for environmental stress. The role of gene expression noise in multicellular organisms has, however, remained unclear. In this review, we discuss how new techniques are revealing an unexpected level of variability in gene expression between and within genetically identical plants. We describe recent progress as well as speculate on the function of transcriptional noise as a mechanism for generating functional phenotypic diversity in plants.

Published
2020

18. Genes expression profiling of alveolar macrophages exposed to non-functionalized, anionic and cationic multi-walled carbon nanotubes shows three different mechanisms of toxicity

Author

Bertrand H. Rihn, Zahra Doumandji, Jaafar Ghanbaja, Olivier Joubert, Reuben Mercier, Hilary Cassidy, Luc Ferrari, Mélanie M Leroux, David Matallanas, Sara Nahle, Institut Jean Lamour (IJL), and Université de Lorraine (UL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, MESH: Macrophages, Alveolar, [SDV]Life Sciences [q-bio], Pharmaceutical Science, Medicine (miscellaneous), Gene Expression, 02 engineering and technology, Applied Microbiology and Biotechnology, Gene expression, Nanotechnology, MESH: Animals, Cytotoxicity, 0303 health sciences, Chemistry, MESH: NF-E2-Related Factor 2, MESH: Proteomics, 021001 nanoscience & nanotechnology, lcsh:R855-855.5, MESH: Cell Survival, Ribosomal protein s6, MESH: Nanotubes, Carbon, Molecular Medicine, Signal transduction, 0210 nano-technology, MESH: L-Lactate Dehydrogenase, lcsh:Medical technology, MESH: Gene Expression, MESH: Rats, Cell Survival, NF-E2-Related Factor 2, lcsh:Biotechnology, Ubiquitin-Protein Ligases, Biomedical Engineering, Bioengineering, Cell Line, 03 medical and health sciences, MESH: Gene Expression Profiling, MESH: Nanostructures, Downregulation and upregulation, Ribosomal protein, lcsh:TP248.13-248.65, Cations, Macrophages, Alveolar, Autophagy, Initiation factor, MESH: Autophagy, Animals, MESH: Particle Size, Particle Size, MESH: Cations, 030304 developmental biology, MESH: DNA Damage, L-Lactate Dehydrogenase, Nanotubes, Carbon, Research, Gene Expression Profiling, MESH: Ubiquitin-Protein Ligases, MESH: Cell Line, Nanostructures, Rats, Biophysics, DNA Damage
Abstract

Functionalized multi-walled carbon nanotubes (MWCNT) have become the focus of increased research interest, particularly in their application as tools in different areas, such as the biomedical field. Despite the benefits associated with functionalization of MWCNT, particularly in overcoming issues relating to solubility, several studies have demonstrated that these functionalized nanoparticles display different toxicity profiles. For this study, we aim to compare NR8383 cells responses to three well-characterized MWCNT with varying functional groups. This study employed cytotoxicity assays, transcriptomics and proteomics to assess their toxicity using NR8383 rat alveolar macrophages as an in vitro model. The study findings indicated that all MWCNT altered ribosomal protein translation, cytoskeleton arrangement and induced pro-inflammatory response. Only functionalized MWCNT alter mTOR signaling pathway in conjunction with increased Lamtor gene expression. Furthermore, the type of functionalization was also important, with cationic MWCNT activating the transcription factor EB and inducing autophagy while the anionic MWCNT altering eukaryotic translation initiation factor 4 (EIF4) and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) signaling pathway as well as upregulation Tlr2 gene expression. This study proposes that MWCNT toxicity mechanisms are functionalization dependent and provides evidence that inflammatory response is a key event of carbon nanotubes toxicity.

Published
2020

19. Mouse APOBEC1 cytidine deaminase can induce somatic mutations in chromosomal DNA

Author

Pierre Khalfi, Valérie Thiers, Jean-Pierre Vartanian, Noémie Berry, Wenjuan Jiao, Vincent Caval, Rodolphe Suspène, Emmanuelle Pitré, Simon Wain-Hobson, Rétrovirologie moléculaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), Université Sorbonne Paris Cité (USPC), ED 515 - Complexité du vivant, Sorbonne Université (SU), This work was supported by grants from the Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). NB and EP were supported by Allocations de Recherche du Ministère de la Recherche while PK was supported by La Ligue contre le Cancer. WJ was supported by China Scholarship Council., We would like to thank Yu Wei for mouse samples., Rétrovirologie moléculaire - Molecular Retrovirology, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: DNA Breaks, Double-Stranded, Gene Expression, MESH: Amino Acid Sequence, MESH: Base Sequence, medicine.disease_cause, Substrate Specificity, chemistry.chemical_compound, Mice, 0302 clinical medicine, DNA Breaks, Double-Stranded, MESH: Animals, APOBEC3A, MESH: Phylogeny, Phylogeny, Cancer, 0303 health sciences, Nuclear DNA, Cytidine, Cytidine deaminase, APOBEC1, Cell biology, MESH: DNA, Single-Stranded, RNA editing, 030220 oncology & carcinogenesis, Biotechnology, Research Article, MESH: Enzyme Activation, MESH: Gene Expression, lcsh:QH426-470, lcsh:Biotechnology, APOBEC-1 Deaminase, MESH: APOBEC-1 Deaminase/metabolism, DNA, Single-Stranded, Molecular cloning, Biology, MESH: APOBEC-1 Deaminase/chemistry, 03 medical and health sciences, Somatic mutations, lcsh:TP248.13-248.65, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, medicine, Animals, MESH: RNA Editing, Amino Acid Sequence, MESH: Mice, 030304 developmental biology, Base Sequence, MESH: Chromosomes, Mammalian/genetics, MESH: APOBEC-1 Deaminase/genetics, Chromosomes, Mammalian, Enzyme Activation, lcsh:Genetics, chemistry, Mutation, MESH: Mutation, MESH: Substrate Specificity, RNA Editing, Carcinogenesis, DNA
Abstract

BackgroundAPOBEC1 (A1) enzymes are cytidine deaminases involved in RNA editing. In addition to this activity, a few A1 enzymes have been shown to be active on single stranded DNA. As two human ssDNA cytidine deaminases APOBEC3A (A3A), APOBEC3B (A3B) and related enzymes across the spectrum of placental mammals have been shown to introduce somatic mutations into nuclear DNA of cancer genomes, we explored the mutagenic threat of A1 cytidine deaminases to chromosomal DNA.ResultsMolecular cloning and expression of various A1 enzymes reveal that the cow, pig, dog, rabbit and mouse A1 have an intracellular ssDNA substrate specificity. However, among all the enzymes studied, mouse A1 appears to be singular, being able to introduce somatic mutations into nuclear DNA with a clear 5’TpC editing context, and to deaminate 5-methylcytidine substituted DNA which are characteristic features of the cancer related mammalian A3A and A3B enzymes. However, mouse A1 activity fails to elicit formation of double stranded DNA breaks, suggesting that mouse A1 possess an attenuated nuclear DNA mutator phenotype reminiscent of human A3B.ConclusionsAt an experimental level mouse APOBEC1 is remarkable among 12 mammalian A1 enzymes in that it represents a source of somatic mutations in mouse genome, potentially fueling oncogenesis. While the orderRodentiais bereft of A3A and A3B like enzymes it seems that APOBEC1 may well substitute for it, albeit remaining much less active. This modifies the paradigm that APOBEC3 and AID enzymes are the sole endogenous mutator enzymes giving rise to off-target editing of mammalian genomes.

Published
2019

20. Genetic elimination of dopamine vesicular stocks in the nigrostriatal pathway replicates Parkinson’s disease motor symptoms without neuronal degeneration in adult mice

Author

Chloé Guinaudie, Léa C. Perret, Quentin Rainer, Bruno Giros, Alain Gratton, Luc Moquin, Elsa Isingrini, Research Center of the Douglas Mental Health University Institute, Physiopathologie des maladies psychiatriques = Pathophysiology of Psychiatric Disorders (NPS-07), Neurosciences Paris Seine (NPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Douglas Mental Health University Institute [Montréal], McGill University = Université McGill [Montréal, Canada], Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-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)-Centre National de la Recherche Scientifique (CNRS), Neuroscience Paris Seine (NPS), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Department of Psychiatry [Montreal, Quebec, Canada], McGill University- Douglas Mental Health Research Center [Montréal Quebec, Canada], Neuroscience Paris Seine ( NPS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), HAL-UPMC, Gestionnaire, and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-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)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS)

Subjects
Male, 0301 basic medicine, Pathology, MESH: Integrases, Parkinson's disease, Dopamine, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Gene Expression, Nigrostriatal pathway, lcsh:Medicine, MESH: Vesicular Monoamine Transport Proteins, MESH: Dependovirus, MESH: Eating, MESH: Corpus Striatum, Eating, Mice, 0302 clinical medicine, MESH: Dopaminergic Neurons, Monoaminergic, MESH: Animals, lcsh:Science, Multidisciplinary, Neurodegeneration, Dependovirus, 3. Good health, Substantia Nigra, medicine.anatomical_structure, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Synaptic Vesicles, MESH: Injections, Intraventricular, MESH: Parkinson Disease, Secondary, MESH: Drinking, medicine.drug, medicine.medical_specialty, MESH: Gene Expression, MESH: Mice, Transgenic, Drinking, MESH: Substantia Nigra, Mice, Transgenic, MESH: Dopamine, Biology, Article, MESH: Weight Loss, 03 medical and health sciences, Weight Loss, Genetic model, medicine, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Parkinson Disease, Secondary, MESH: Mice, Injections, Intraventricular, Integrases, Dopaminergic Neurons, lcsh:R, MESH: Synaptic Vesicles, medicine.disease, Corpus Striatum, MESH: Male, Vesicular monoamine transporter, 030104 developmental biology, Monoamine neurotransmitter, MESH: Gene Deletion, Vesicular Monoamine Transport Proteins, [ SDV.NEU ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], lcsh:Q, Neuroscience, Gene Deletion, 030217 neurology & neurosurgery
Abstract

International audience; The type 2 vesicular monoamine transporter (VMAT2), by regulating the storage of monoamines transmitters into synaptic vesicles, has a protective role against their cytoplasmic toxicity. Increasing evidence suggests that impairment of VMAT2 neuroprotection contributes to the pathogenesis of Parkinson's disease (PD). Several transgenic VMAT2 mice models have been developed, however these models lack specificity regarding the monoaminergic system targeting. To circumvent this limitation, we created VMAT2-KO mice specific to the dopamine (DA) nigrostriatal pathway to analyze VMAT2's involvement in DA depletion-induced motor features associated to PD and examine the relevance of DA toxicity in the pathogenesis of neurodegeneration. Adult VMAT2 floxed mice were injected in the substancia nigra (SN) with an adeno-associated virus (AAV) expressing the Cre-recombinase allowing VMAT2 removal in DA neurons of the nigrostriatal pathway solely. VMAT2 deletion in the SN induced both DA depletion exclusively in the dorsal striatum and motor dysfunction. At 16 weeks post-injection, motor symptoms were accompanied with a decreased in food and water consumption and weight loss. However, despite an accelerating death, degeneration of nigrostriatal neurons was not observed in this model during this time frame. This study highlights a non-cytotoxic role of DA in our genetic model of VMAT2 deletion exclusively in nigrostriatal neurons. Parkinson's disease (PD), is one of the most common neurodegenerative disorders affecting 1–2% of individuals older than 65 years old worldwide 1. PD is a progressive disorder clinically characterized by a large number of motor features in particular rest tremor, bradykinesia, rigidity and loss of postural reflexes 2,3. At the pathological level, processes leading to PD begin before the appearance of the typical motor symptoms, and by the time the disease is diagnosed about 70–80% of striatal dopamine (DA) is depleted and one third of substantia nigra (SN) DA neurons and striatal DA fibers are already lost 2,4–7. Understanding the biochemical markers relevant to the pathogenesis of neurodegeneration in PD remain essential. Oxidative stress contribution to PD neurodegeneration is currently well established 8. Post-mortem studies have shown an imbalance between reactive oxygen species (ROS) production and the antioxidant protective system resulting in oxidative stress in the SN of PD patients 9,10. Superoxide (O 2−), hydrogen peroxide (H 2 O 2), and the hydroxyl radical (OH •), three important ROS in mammalian cells, can damage macromolecules including nucleic acids, lipids and proteins, leading to DA neuron degeneration, and neuron network dysfunction, ultimately progressing to PD 11. For a better understanding of the oxidative stress mechanisms in PD, the MPTP

Published
2017

21. Hypoxia-induced human deoxyribonuclease I is a cellular restriction factor of hepatitis B virus

Author

Camille Hallez, Cristina M. Dorobantu, Mohamed S Bouzidi, Valérie Thiers, Vincent Lucansky, Jean-Pierre Vartanian, Xiongxiong Li, Simon Wain-Hobson, Raphael Gaudin, Rodolphe Suspène, Rétrovirologie moléculaire - Molecular Retrovirology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Sorbonne Université (SU), China National Biotec Group (CNBG), Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Viroclinics Biosciences B. V. [Rotterdam, Netherlands], Biomedical Center Martin [Slovaquie], Jessenius Medical Faculty [Commenius University, Slovaquie] (JFMED), Commenius University in Bratislava - Univerzita Komenského-Commenius University in Bratislava - Univerzita Komenského, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from the Institut Pasteur and Centre National de la Recherche Scientifique. C.H. and M.S.B. were supported by the Allocation de Recherche du Ministère de la Recherche and Ligue contre le Cancer, respectively. X.L. was supported by a stipend from the Lanzhou Institute of Biological Products Co., Ltd, subsidiary company of China National Biotec Group Company Limited and by a stipend from the Pasteur-Paris University International PhD program. V.L. was supported by the Fondation pour la Recherche Medicale (project no. ING20160435328). C.D. was supported by an EMBO Long Term Fellowship (EMBO ALTF 1428–2016). The study of patients was sponsored by the French National Agency for research on AIDS and hepatitis (ANRS). This work has been published within the framework of IdEx Université de Strasbourg and has received funding from the French State via the French National Research Agency (ANR) as part of the program ‘Investissements d’avenir’ to R.G. This work was supported by an ATIP-AVENIR starting grant to R.G., We would like to thank S. Urban for providing HepG2-NTCP cells, P. Pineau and A. Marchio for the cirrhotic liver samples, M.-L. Michel and S. Pol for serum samples, C. Neuveut, P. Moreau, P. Maillard and B. Quioc for HBV infection, Y. Jacob for the HIF-1α and HIF-1β plasmids, J. Krijnse-Locker and C. Schmitt for electron microscopy immunolabelling and V. Caval, N. Frampton and J. McKeating for helpful discussions., Rétrovirologie moléculaire, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Collège doctoral [Sorbonne universités], Vartanian, Jean-Pierre, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
Liver Cirrhosis, viruses, [SDV]Life Sciences [q-bio], MESH: Virus Replication*/drug effects, Gene Expression, medicine.disease_cause, Virus Replication, Applied Microbiology and Biotechnology, chemistry.chemical_compound, Hypoxia, ComputingMilieux_MISCELLANEOUS, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, MESH: Liver Cirrhosis/enzymology, Cytidine, Transfection, Cobalt, Hepatitis B, MESH: Cobalt/pharmacology, MESH: Deoxyribonuclease I/metabolism, Hepatitis B Core Antigens, 3. Good health, [SDV] Life Sciences [q-bio], Capsid, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hypoxia-Inducible Factor 1, MESH: Deoxyribonuclease I/genetics, Microbiology (medical), Hepatitis B virus, MESH: Mutation, MESH: Gene Expression, MESH: Hepatitis B/enzymology, MESH: Hypoxia-Inducible Factor 1/metabolism, Immunology, Biology, Microbiology, Gene Expression Regulation, Enzymologic, Cell Line, 03 medical and health sciences, Genetics, medicine, Deoxyribonuclease I, Humans, MESH: Gene Expression Regulation, Enzymologic/drug effects, 030304 developmental biology, MESH: Hypoxia*/chemically induced, MESH: Humans, 030306 microbiology, Virion, MESH: Virion/metabolism, MESH: Hepatitis B Core Antigens/metabolism, Cell Biology, medicine.disease, Molecular biology, In vitro, MESH: Cell Line, MESH: DNA, Viral/metabolism, chemistry, DNA, Viral, Mutation, DNA, MESH: Hepatitis B virus/physiology
Abstract

International audience; Numerous human APOBEC3 cytidine deaminases have proven to be, inter alia, host cell restriction factors for retroviruses and hepadnaviruses. Although they can bind to genomic RNA and become encapsidated, they are only catalytically active on single-stranded DNA. As there are many cellular deoxyribonucleases (DNases), we hypothesized that a parallel could be struck between APOBEC3 and DNases. For human hepatitis B virus (HBV), we show that DNase I can considerably reduce the virion genome copy number from a variety of transfected or infected cells. DNASE1 is overexpressed and encapsidated in HBV particles in vitro in hypoxic environments and in vivo in cirrhotic patient livers as well as in the serum of infected patients. The use of CoCl2 and dimethyloxalylglycine, mimetic agents used to induce hypoxia by inhibiting prolyl hydroxylase enzymes that stabilize hypoxia-inducible factor (HIF)-1α, showed that the formation of HIF-1α/HIF-1β heterodimers results in the induction of DNASE1. Indeed, transfection with HIF-1α and HIF-1β expression constructs upregulated DNASE1. These findings suggest that human DNase I can impact HBV replication through the catabolism of the DNA genome within the capsid. The activity of DNases in general may explain in part the high frequency of empty or 'light' hepatitis B virions observed in vivo.

Published
2019

22. Condensin-Mediated Chromosome Folding and Internal Telomeres Drive Dicentric Severing by Cytokinesis

Author

Natalja Barinova, Romain Koszul, Stéphane Marcand, Alice Deshayes, Claire Béneut, Karine Dubrana, Virginia Lopez, Agnès Thierry, Thomas Guerin, Luciana Lazar-Stefanita, Stabilité génétique, cellules souches et radiations (SGCSR (U_1274 / UMR_E_008)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Université Paris Cité (UPCité), Régulation spatiale des Génomes - Spatial Regulation of Genomes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by funding to S.M. from Fondation ARC, EDF, CEA Radiobiology call, DRF-Impulsion (4D-DSB-DIC), and ANR (DICENs-ANR-14-CE10-0021-01), to K.D. from the European Research Council under the Seventh Framework Program (FP7/2007 2013/ERC grant agreement 281287), and to R.K. from the European Research Council under the Horizon 2020 Program (ERC grant agreement 260822). T.M.G. was supported by a PhD fellowship from CEA, ANR, and a Fondation ARC young researcher grant., We thank Angela Taddei for lacI, lacI∗∗, and lacO array plasmids and suggestions, Frank Uhlmann and Thomas Kuilman for the G20 plasmid, Helle Ulrich for the AID tool kit, Didier Busso and Eléa Dizet (CIGEX platform) for the Rap1 sites plasmids, Pascale Lesage for the anti-Dps1 antibody, Rémi Montagne for assistance with the Hi-C data, Romain Le Bars (IMAGE-GIF platform) and Lamya Irbah (IRCM microscopy platform) for assistance with higher-resolution microscopy, Dan Throsby for text editing, and John Marko, Damien D’Amours, Sarah Lambert, François-Xavier Barre, Pablo Radicella, Eric Coïc, Laurent Maloisel, Paul-Henri Roméo, Mathias Toulouze, and Maoussi Lhuillier-Akakpo for fruitful discussions and suggestions., ANR-14-CE10-0021,DICENs,Prévention et résolution des chromosomes dicentriques(2014), European Project: 281287,EC:FP7:ERC,ERC-2011-StG_20101109,NDOGS(2012), European Project: 260822,EC:FP7:ERC,ERC-2010-StG_20091118,DICIG(2011), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Université de Paris (UP), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Condensin, Gene Expression, yeast, Shelterin Complex, MESH: Telomere-Binding Proteins, chemistry.chemical_compound, Chromosome Breakpoints, 0302 clinical medicine, MESH: Saccharomyces cerevisiae Proteins, Hi-C, MESH: Models, Genetic, DNA, Fungal, Adenosine Triphosphatases, 0303 health sciences, biology, SMC, MESH: Karyotype, MESH: Chromosome Breakpoints, MESH: Transcription Factors, Telomere, MESH: Saccharomyces cerevisiae, MESH: Chromosomes, Fungal, Cell biology, DNA-Binding Proteins, Chromosomes, Fungal, mutagenesis, lacI, MESH: Cytokinesis, Saccharomyces cerevisiae Proteins, MESH: Gene Expression, Saccharomyces cerevisiae, Karyotype, Telomere-Binding Proteins, 03 medical and health sciences, Dicentric chromosome, MESH: Adenosine Triphosphatases, Telophase, Molecular Biology, Mitosis, 030304 developmental biology, Cytokinesis, mitosis, Models, Genetic, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, MESH: Multiprotein Complexes, biology.organism_classification, telophase, abscission, MESH: DNA, Fungal, enzymes and coenzymes (carbohydrates), chemistry, Multiprotein Complexes, biology.protein, MESH: Telomere, 030217 neurology & neurosurgery, DNA, MESH: DNA-Binding Proteins, Transcription Factors, condensing
Abstract

International audience; In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.

Published
2019

23. Comparative Loss-of-Function Screens Reveal ABCE1 as an Essential Cellular Host Factor for Efficient Translation of Paramyxoviridae and Pneumoviridae

Author

Kristmundur Sigmundsson, Danielle E. Anderson, So Young Kim, Zhen Zhen Joanna Yeo, Sharon F. Jamison, Kristin Pfeffermann, James L. Pearson, Bevan Sawatsky, Mikhail Kovtun, Lin-Fa Wang, Pierre J. Talbot, Veronika von Messling, Mariano A. Garcia-Blanco, Yvonne Krebs, David L. Corcoran, Linda J. Rennick, W. Paul Duprex, Duke-NUS Medical School [Singapore], Paul-Ehrlich-Institute - Federal Institute for Vaccines and Biomedicines (EPI), Duke University [Durham], National Emerging Infectious Diseases Laboratories (NEIDL), Boston University [Boston] (BU), Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), The University of Texas Medical Branch (UTMB), This work was supported by grants CIHR MOP-66989 of the Canadian Institutes of Health Research, CFI 9488 Canada Foundation for Innovation, DZIF TTU Emerging Infections SFB1021 of the German Center for Infection Research, and intramural funding of the German Ministry of Health to V.V.M., NMRC/BNIG/2030/2015 (Singapore) to D.E.A., and USA NIH R01-AI089526 and R01-AI101431 to M.A.G.-B. and R01-AI099100 to W.P.D., and We thank members of our laboratories, past and present, for constructive comments throughout this project. We thank the Duke Functional Genomics Shared Resource for work related to the primary siRNA screens. We are grateful to Jaisri Lingappa for sharing the ABCE1 antibody and peptide sequence during our preliminary studies. We thank Ian Mendenhall for his assistance in the construction of the phylogenetic tree and Kevin Wittwer and Oliver Siering for technical assistance. We greatly appreciate the help provided by Robert Tampé, Ralph Wieneke, and Simon Trowitzsch with the phosphorimager and Andrea Maisner and Marc Ringel with the confocal microscope. We thank Stephan Becker for critical comments on the manuscript.

Subjects
MESH: Gene Expression, Paramyxoviridae, ABCE1, respiratory syncytial virus, [SDV]Life Sciences [q-bio], host factor, MESH: Pneumovirus/pathogenicity, RNA-binding protein, Computational biology, Microbiology, 03 medical and health sciences, Virology, MESH: RNA, Small Interfering, MESH: RNA-Binding Proteins/genetics, MESH: ATP-Binding Cassette Transporters/genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Pneumoviridae, RNAi screen, MESH: RNA, Messenger, 030304 developmental biology, Host factor, 0303 health sciences, MESH: Humans, biology, MESH: Paramyxoviridae/pathogenicity, 030302 biochemistry & molecular biology, Translation (biology), Pneumovirus, biology.organism_classification, QR1-502, MESH: Host Microbial Interactions/genetics, 3. Good health, Vesicular transport protein, biology.protein, MESH: A549 Cells, MESH: Computational Biology
Abstract

International audience; Paramyxoviruses and pneumoviruses have similar life cycles and share the respiratory tract as a point of entry. In comparative genome-scale siRNA screens with wild-type-derived measles, mumps, and respiratory syncytial viruses in A549 cells, a human lung adenocarcinoma cell line, we identified vesicular transport, RNA processing pathways, and translation as the top pathways required by all three viruses. As the top hit in the translation pathway, ABCE1, a member of the ATP-binding cassette transporters, was chosen for further study. We found that ABCE1 supports replication of all three viruses, confirming its importance for viruses of both families. More detailed characterization revealed that ABCE1 is specifically required for efficient viral but not general cellular protein synthesis, indicating that paramyxoviral and pneumoviral mRNAs exploit specific translation mechanisms. In addition to providing a novel overview of cellular proteins and pathways that impact these important pathogens, this study highlights the role of ABCE1 as a host factor required for efficient paramyxovirus and pneumovirus translation.IMPORTANCE The Paramyxoviridae and Pneumoviridae families include important human and animal pathogens. To identify common host factors, we performed genome-scale siRNA screens with wild-type-derived measles, mumps, and respiratory syncytial viruses in the same cell line. A comparative bioinformatics analysis yielded different members of the coatomer complex I, translation factors ABCE1 and eIF3A, and several RNA binding proteins as cellular proteins with proviral activity for all three viruses. A more detailed characterization of ABCE1 revealed its essential role for viral protein synthesis. Taken together, these data sets provide new insight into the interactions between paramyxoviruses and pneumoviruses and host cell proteins and constitute a starting point for the development of broadly effective antivirals.

Published
2019

24. Sorting of Phagocytic Cells Infected with Legionella pneumophila

Author

Carmen Buchrieser, Monica Rolando, Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Work in the CB laboratory is financed by the Institut Pasteur, the grant ANR-10-LABX-62-IBEID and the grant ANR-18-CE15-0005-01., Carmen Buchrieser, Hubert Hilbi, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-18-CE15-0005,LEGENhD,Deciphering Legionella pneumophila induced epigenetic modifications: characterization of a prokaryotic histone deacetylase(2018), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Single cell sorting, MESH: Gene Expression, MESH: Phagocytosis* / immunology, 030106 microbiology, Population, Cell, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Vacuole, Biology, Legionella pneumophila, Microbiology, MESH: Flow Cytometry* / methods, 03 medical and health sciences, MESH: Macrophages / metabolism, MESH: Phagocytes / immunology, medicine, MESH: Macrophages / microbiology, education, Pathogen, MESH: Legionnaires' Disease / immunology, education.field_of_study, MESH: Humans, Phagocytic cells, Effector, MESH: Genes, Reporter, MESH: Host-Pathogen Interactions, Fluorescent proteins, MESH: Phagocytes / microbiology, biology.organism_classification, MESH: Macrophages / immunology, MESH: Cell Line, 030104 developmental biology, medicine.anatomical_structure, Cell culture, MESH: Legionnaires' Disease / microbiology, MESH: Legionella pneumophila / physiology, Intracellular
Abstract

International audience; The ability of Legionella pneumophila to colonize host cells and to form a replicative vacuole depends on its ability to counteract the host cell response by secreting more than 300 effectors. The host cell responds to this bacterial invasion with extensive intracellular signaling to counteract the replication of the pathogen. When studying L. pneumophila infection in vitro, only a small proportion of the cell lines or primary cells used to analyze the host response are infected; the study of such a mixed cell population leads to unprecise results. In order to study the multitude of pathogen-induced phenotypic changes occurring in the host cell, the separation of infected from uninfected cells is a top priority. Here we describe a highly efficient FACS-derived protocol to separate cells infected with a L. pneumophila strain encoding a fluorescent protein. Indeed, the highly infected, homogenous cell population obtained after sorting is the best possible starting point for the studies of infection-induced effects.

Published
2019

25. The reaction mechanism of metallo-lactamases is tuned by the conformation of an active-site mobile loop

Author

Estefanía Giannini, Lisandro H. Otero, Antonela Rocio Palacios, Magdalena A. Taracila, Pedro M. Alzari, Sebastián Klinke, Christopher R. Bethel, Maria F. Mojica, Robert A. Bonomo, Leticia I. Llarrull, Alejandro J. Vila, Mojica, María Fernanda [0000-0002-1380-9824], 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], Department of Biochemistry, School of Medicine, Case Western Reserve University, Research Institute of University Hospitals of Cleveland-University Hospitals of Cleveland-Case Western Reserve University [Cleveland], Louis Stokes Cleveland Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Fundación Instituto Leloir [Buenos Aires], Plataforma de Biología Estructural y Metabolómica [Rosario] (PLABEM), Facultad de Ciencias Bioquımicas y Farmaceuticas [Rosario] (FBIOyF), Universidad Nacional de Rosario [Santa Fe], CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (CARES), A.R.P. and E.G. received doctoral fellowships from CONICET. M.F.M. received a doctoral scholarship from COLCIENCIAS. L.H.O., S.K., L.I.L., and A.J.V. are CONICET staff members. This study was supported by a grant from ANPCyT (A.J.V.), National Institutes of Health (NIH) grant R01 AI100560 (A.J.V. and R.A.B.), NIH grant R01 AI063517, NIH grant R01 AI072219 (R.A.B.), the ECOS-MinCyT collaborative project (A.J.V. and P.M.A.), the Cleveland Department of Veterans Affairs and Development (1I01BX001974 [R.A.B.]), and the Geriatric Research Education and Clinical Center (R.A.B.)., We thank Ahmed Haouz and Patrick Weber (Institut Pasteur) for help with the robot-driven crystallization screenings. We acknowledge the synchrotron sources Soleil (Saint-Aubin, France) and ESRF (Grenoble, France) for granting access to their facilities, and we thank their staff members for helpful assistance., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
antibiotic resistance, MESH: Ceftazidime, Enzyme mechanism, MESH: Sequence Homology, Amino Acid, Antibiotic resistance, MESH: beta-Lactamases, MESH: Amino Acid Sequence, Protein Engineering, Substrate Specificity, MESH: Recombinant Proteins, Pharmacology (medical), Catálisis, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, 3. Good health, enzyme structure, MESH: Protein Engineering, Zinc, METALLO-BETA-LACTAMASE, MESH: Models, Molecular, MESH: Piperacillin, MESH: Gene Expression, Stereochemistry, metallo-β-lactamase, MESH: Sequence Alignment, Reaction intermediate, beta-Lactam Resistance, Ciencias Biológicas, 03 medical and health sciences, MESH: Anti-Bacterial Agents, MESH: Protein Binding, Protein Interaction Domains and Motifs, MESH: Cloning, Molecular, Amino Acid Sequence, Pharmacology, MESH: Protein Conformation, alpha-Helical, MESH: Protein Interaction Domains and Motifs, 030306 microbiology, Active site, Substrate (chemistry), Meropenem, chemistry, Protein Conformation, beta-Strand, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Function (biology), Models, Molecular, Protein Conformation, alpha-Helical, Enzyme structure, Gene Expression, MESH: Catalytic Domain, Cefotaxime, Crystallography, X-Ray, Ceftazidime, MESH: Zinc, MESH: Meropenem, purl.org/becyt/ford/1 [https], MESH: Genetic Vectors, Catalytic Domain, Cloning, Molecular, Cefepime, MESH: Imipenem, biology, MESH: Kinetics, MESH: Cefepime, MESH: Cefotaxime, NEW DELHI METALLO-BETA-LACTAMASE, Recombinant Proteins, Anti-Bacterial Agents, Infectious Diseases, MESH: Protein Conformation, beta-Strand, CIENCIAS NATURALES Y EXACTAS, Protein Binding, Cristalografía por rayos X, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Genetic Vectors, Protonation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, beta-Lactamases, Mechanisms of Resistance, Hydrolase, [CHIM.CRIS]Chemical Sciences/Cristallography, Escherichia coli, enzyme mechanism, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, purl.org/becyt/ford/1.6 [https], 030304 developmental biology, Piperacillin, Sequence Homology, Amino Acid, MESH: Crystallography, X-Ray, Biofísica, MESH: beta-Lactam Resistance, Imipenem, Kinetics, Enzyme, New Delhi metallo-β-lactamase, biology.protein, MESH: Substrate Specificity, Sequence Alignment
Abstract

Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs. Fil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Mojica, María F.. Case Western Reserve University; Estados Unidos Fil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Alzari, Pedro M.. Institut Pasteur de Paris; Francia Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Published
2019

26. Sex Differences in Intestinal Carbohydrate Metabolism Promote Food Intake and Sperm Maturation

Author

Thomas Preat, Eva G.M. de Goeij, Pierre-Yves Plaçais, Pedro Gaspar, Chris Studd, Bruno Hudry, Dafni Hadjieconomou, Holger Kramer, Irene Miguel-Aliaga, Joao B. Mokochinski, Alessandro Mineo, MRC London Institute of Medical Sciences (LMC), 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), Laboratoire Plasticité du Cerveau Brain Plasticity (UMR 8249) (PdC), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), ANR-15-IDEX-0001,UCA JEDI,Idex UCA JEDI(2015), HUDRY, BRUNO, 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), Genes and Dynamics of Memory Systems [Paris], and Commission of the European Communities

Subjects
MESH: Signal Transduction, Male, MESH: Carbohydrate Metabolism, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV]Life Sciences [q-bio], ENERGY-METABOLISM, Physiology, Gene Expression, MESH: Eating, Eating, 0302 clinical medicine, FUNCTIONAL-CHARACTERIZATION, Intestinal mucosa, MITOCHONDRIAL CITRATE CARRIER, Testis, MESH: Janus Kinases, Drosophila Proteins, MESH: Animals, citrate, RNA-Seq, Intestinal Mucosa, 11 Medical and Health Sciences, ComputingMilieux_MISCELLANEOUS, 2. Zero hunger, 0303 health sciences, Sex Characteristics, biology, GERMLINE STEM-CELL, MESH: Testis, Sex reversal, [SDV] Life Sciences [q-bio], STAT Transcription Factors, medicine.anatomical_structure, Drosophila melanogaster, gender differences, MESH: Intestinal Mucosa, Carbohydrate Metabolism, Drosophila, Female, MESH: Sugars, Life Sciences & Biomedicine, MESH: Sex Characteristics, MESH: Sperm Maturation, Sex characteristics, Signal Transduction, Biochemistry & Molecular Biology, MESH: Citric Acid, MESH: Gene Expression, Gonad, MESH: Drosophila Proteins, testes, Carbohydrate metabolism, gonad, sperm, General Biochemistry, Genetics and Molecular Biology, Article, Citric Acid, MESH: Drosophila melanogaster, 03 medical and health sciences, SINGLE-CELL, medicine, Animals, MESH: RNA-Seq, Spermatogenesis, intestine, 030304 developmental biology, Janus Kinases, GENDER-DIFFERENCES, Science & Technology, PYRUVATE-DEHYDROGENASE, sexual dimorphisms, Cell Biology, 06 Biological Sciences, MESH: STAT Transcription Factors, biology.organism_classification, Sperm, MESH: Male, Sexual dimorphism, Sperm Maturation, SELF-RENEWAL, DROSOPHILA-MELANOGASTER, organ plasticity, Sugars, MESH: Female, ONCOMETABOLITE 2-HYDROXYGLUTARATE, 030217 neurology & neurosurgery, Developmental Biology
Abstract

Summary Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized., Graphical Abstract, Highlights • Intestinal carbohydrate metabolism is male-biased and region-specific • Testes masculinize gut sugar handling by promoting enterocyte JAK-STAT signaling • The male intestine secretes citrate to the adjacent testes • Gut-derived citrate promotes food intake and sperm maturation, Inter-organ communication couples diet with gamete production. The male gonad promotes sex differences in carbohydrate metabolism within an adjacent intestinal portion via JAK-STAT signalling. In response to this gonadal signal, gut-derived citrate controls food intake and sperm maturation.

Published
2018

27. Biallelic B3GALT6 mutations cause spondylodysplastic Ehlers–Danlos syndrome

Author

Xiaomeng Pang, Rolph Pfundt, Sofie Symoens, Fransiska Malfait, Marleen Simon, Brecht Guillemyn, Austin Larson, Saskia Bulk, Marion Gérard, Ariana Kariminejad, Sandrine Gulberti, Tim Van Damme, Kathryn C. Chatfield, Olivier Kaye, Delfien Syx, Riet De Rycke, Geneviève Pierquin, Christine E. M. de Die-Smulders, Anne Legrand, Sheela Nampoothiri, Sylvie Fournel-Gigleux, Center for Medical Genetics [Ghent], Ghent University Hospital, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Department of Plant Systems Biology, Flanders Institute for Biotechnology, Centre de Rhumatologie, Maastricht University Medical Centre (MUMC), Maastricht University [Maastricht], Department of Human Genetics, Radboud University Medical Center [Nijmegen], Kariminejad-Najmabadi Pathology& Genetics Center, Kariminejad-Najmabadi Pathology & Genetics Center, Amrita Institute of Medical Sciences and Research Center, Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Génétique Humaine, Université de Liège-CHU Liège, Deaprtment of Clinical Genetics, University Medical Center [Utrecht], Department of Pediatrics [san Diego], UC San Diego School of Medicine, 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), Service de Génétique [CHU Caen], Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Normandie Université (NU)-Normandie Université (NU), RS: GROW - R4 - Reproductive and Perinatal Medicine, MUMC+: DA KG Polikliniek (9), and Klinische Genetica

Subjects
Male, 0301 basic medicine, MESH: Ehlers-Danlos Syndrome, IMAGE, PROTEOGLYCAN, Gene Expression, DESBUQUOIS DYSPLASIA, medicine.disease_cause, MESH: Galactosyltransferases, chemistry.chemical_compound, MESH: Child, Child, MESH: High-Throughput Nucleotide Sequencing, Genetics (clinical), Exome sequencing, Mutation, biology, XYLT1 MUTATIONS, High-Throughput Nucleotide Sequencing, General Medicine, Heparan sulfate, Galactosyltransferases, Phenotype, MESH: Infant, 3. Good health, Child, Preschool, Female, SKELETAL DYSPLASIA, BONE FRAGILITY, Adult, MESH: Mutation, MESH: Gene Expression, [SDV.CAN]Life Sciences [q-bio]/Cancer, MESH: Phenotype, PATIENT, Collagen fibril organization, 03 medical and health sciences, All institutes and research themes of the Radboud University Medical Center, MESH: Whole Exome Sequencing, MESH: Enzyme Assays, Exome Sequencing, Genetics, medicine, Humans, Molecular Biology, Enzyme Assays, HEPARAN-SULFATE, Spondyloepimetaphyseal dysplasia, SPECTRUM, [SDV.GEN]Life Sciences [q-bio]/Genetics, Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7], MESH: Humans, MESH: Child, Preschool, Infant, MESH: Adult, medicine.disease, Molecular biology, B3GAT3 MUTATION, MESH: Male, 030104 developmental biology, Proteoglycan, chemistry, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Ehlers–Danlos syndrome, biology.protein, Ehlers-Danlos Syndrome, MESH: Female
Abstract

International audience; Proteoglycans are among the most abundant and structurally complex biomacromolecules and play critical roles in connective tissues. They are composed of a core protein onto which glycosaminoglycan (GAG) side chains are attached via a linker region. Biallelic mutations in B3GALT6, encoding one of the linker region glycosyltransferases, are known to cause either spondyloepimetaphyseal dysplasia (SEMD) or a severe pleiotropic form of Ehlers-Danlos syndromes (EDS). This study provides clinical, molecular and biochemical data on 12 patients with biallelic B3GALT6 mutations. Notably, all patients have features of both EDS and SEMD. In addition, some patients have severe and potential life-threatening complications such as aortic dilatation and aneurysm, cervical spine instability and respiratory insufficiency. Whole-exome sequencing, next generation panel sequencing and direct sequencing identified biallelic B3GALT6 mutations in all patients. We show that these mutations reduce the amount of β3GalT6 protein and lead to a complete loss of galactosyltransferase activity. In turn, this leads to deficient GAG synthesis, and ultrastructural abnormalities in collagen fibril organization. In conclusion, this study redefines the phenotype associated with B3GALT6 mutations on the basis of clinical, molecular and biochemical data in 12 patients, and provides an in-depth assessment of β3GalT6 activity and GAG synthesis to better understand this rare condition.

Published
2018

28. Neisseria meningitidisSerogroup X in Sub-Saharan Africa

Author

Alain Agnememel, Muhamed-Kheir Taha, Eva Hong, Viginia Nuñez-Samudio, Dario Giorgini, Ala-Eddine Deghmane, Institut Pasteur [Paris], This work was supported by the Institut Pasteur and by funding from the Fondation Total including a fellowship to A.A. This study made use of the Neisseria Multilocus Sequence Typing website (http://pubmlst.org/neisseria/), developed by Keith Jolley and sited at the University of Oxford, and the dynamic imaging facilities of Imagopole at the Institut Pasteur, Paris. The development of this site has been funded by the Wellcome Trust and European Union. This study also made use of the Meningitis Research Foundation Meningococcus Genome Library (http://www.meningitis.org/research/genome) developed by Public Health England, the Wellcome Trust Sanger Institute, and the University of Oxford., and Institut Pasteur [Paris] (IP)

Subjects
sub-Saharan Africa, 0301 basic medicine, Serotype, Neisseria meningitidis Serogroup X in Sub-Saharan Africa, Epidemiology, Gene Expression, lcsh:Medicine, MESH: Virulence, Neisseria meningitidis, MESH: Genome, Bacterial, medicine.disease_cause, Mice, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Genes, Reporter, MESH: Animals, MESH: Genetic Variation, MESH: Phylogeny, bacteria, Luciferases, MESH: High-Throughput Nucleotide Sequencing, Phylogeny, Mice, Inbred BALB C, Optical Imaging, MESH: Meningitis, Meningococcal, Dispatch, meningococci, High-Throughput Nucleotide Sequencing, meningitis, Europe, Infectious Diseases, whole-genome sequencing, Microbiology (medical), MESH: Operon, MESH: Gene Expression, Sub saharan, MESH: Mice, Transgenic, 030106 microbiology, MESH: Mice, Inbred BALB C, Virulence, Mice, Transgenic, Meningitis, Meningococcal, Biology, Serogroup, Meningococcal disease, MESH: Neisseria meningitidis, Neisseria meningitidis serogroup X, lcsh:Infectious and parasitic diseases, Microbiology, 03 medical and health sciences, Optical imaging, Operon, parasitic diseases, medicine, Animals, Humans, emergence, lcsh:RC109-216, MESH: Africa South of the Sahara, serogroup X, Serotyping, MESH: Mice, Africa South of the Sahara, Whole genome sequencing, MESH: Optical Imaging, MESH: Humans, MESH: Clone Cells, MESH: Genes, Reporter, lcsh:R, MESH: Serotyping, Genetic Variation, MESH: Serogroup, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Virology, Clone Cells, virulence, Disease Models, Animal, MESH: Luciferases, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, MESH: Europe, MESH: Disease Models, Animal, Genome, Bacterial
Abstract

International audience; The epidemiology of meningococcal disease varies by geography and time. Whole-genome sequencing of Neisseria meningitidis serogroup X isolates from sub-Saharan Africa and Europe showed that serogroup X emergence in sub-Saharan Africa resulted from expansion of particular variants within clonal complex 181. Virulence of these isolates in experimental mouse models was high.

Published
2016

29. Migration of Type III Secretion System Transcriptional Regulators Links Gene Expression to Secretion

Author

Blanca Sabarit, Michael Kokkinidis, Michalis Aivaliotis, Konstantina Psatha, Charalambos Pozidis, Dimitrios Anagnostou, Anastasia D. Gazi, Efstratios Mylonas, Nickolas J. Panopoulos, Carmen R. Beuzón, Spyridoula N. Charova, Institute of Molecular Biology and Biotechnology (IMBB-FORTH), Foundation for Research and Technology - Hellas (FORTH), University of Crete [Heraklion] (UOC), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Universidad de Málaga [Málaga] = University of Málaga [Málaga], This work was supported by the Pythagoras II program, the Onassis Public Benefit Foundation, the ESPA LS1 program (contract number 1808), and the EU-FP7 REGPOT InnovCrete program., European Project: 316223,EC:FP7:REGPOT,FP7-REGPOT-2012-2013-1,INNOVCRETE(2012), and Institut Pasteur [Paris]

Subjects
0301 basic medicine, MESH: Protein Transport, MESH: Gene Expression, Transcription, Genetic, MESH: Erwinia amylovora, Gene Expression, Pseudomonas syringae, Regulatory Sequences, Nucleic Acid, Microbiology, Type three secretion system, type III secretion system (T3SS), 03 medical and health sciences, chemistry.chemical_compound, MESH: Type III Secretion Systems, Virology, Gene expression, Type III Secretion Systems, Erwinia amylovora, MESH: Regulatory Sequences, Nucleic Acid, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Ternary complex, MESH: Pseudomonas syringae, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, MESH: Transcription, Genetic, Pseudomonas syringae pv. phaseolicola, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, gatekeeper complex, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, QR1-502, Cell biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Protein Transport, 030104 developmental biology, Cytoplasm, Chaperone (protein), [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, DNA, Research Article
Abstract

Many plant-pathogenic bacteria of considerable economic importance rely on type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. T3SS gene expression is regulated through the HrpG and HrpV proteins, while secretion is controlled by the gatekeeper HrpJ. A link between the two mechanisms was so far unknown. Here, we show that a mechanistic coupling exists between the expression and secretion cascades through the direct binding of the HrpG/HrpV heterodimer, acting as a T3SS chaperone, to HrpJ. The ternary complex is docked to the cytoplasmic side of the inner bacterial membrane and orchestrates intermediate substrate secretion, without affecting early substrate secretion. The anchoring of the ternary complex to the membranes potentially keeps HrpG/HrpV away from DNA. In their multiple roles as transcriptional regulators and gatekeeper chaperones, HrpV/HrpG provide along with HrpJ potentially attractive targets for antibacterial strategies., IMPORTANCE On the basis of scientific/economic importance, Pseudomonas syringae and Erwinia amylovora are considered among the top 10 plant-pathogenic bacteria in molecular plant pathology. Both employ type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. For Hrc-Hrp 1, no functional link was known between the key processes of T3SS gene expression and secretion. Here, we show that a mechanistic coupling exists between expression and secretion cascades, through formation of a ternary complex involving the T3SS proteins HrpG, HrpV, and HrpJ. Our results highlight the functional and structural properties of a hitherto-unknown complex which orchestrates intermediate T3SS substrate secretion and may lead to better pathogen control through novel targets for antibacterial strategies.

Published
2018

30. Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens

Author

Kunstmann, Sonja, Scheidt, Tom, Buchwald, Saskia, Helm, Alexandra, Mulard, Laurence A., Fruth, Angelika, Barbirz, Stefanie, University of Potsdam, Chimie des Biomolécules - Chemistry of Biomolecules, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Robert Koch Institute [Berlin] (RKI), This research was funded by Deutsche Forschungsgemeinschaft, grant number BA 4046/1-2 (Stefanie Barbirz) and the International Max Planck Research School on Multiscale Biosystems (Sonja Kunstmann)., We thank Nils Carlin (Scandinavian Biopharma) for providing us with S. flexneri LPS preparations. We are grateful to Catherine Guerreiro (Chemistry of Biomolecules Laboratory, Institut Pasteur, France) for her contribution to the chemical synthesis of S. flexneri 2a oligosaccharides. We thank Jörg Fettke for assistance with MALDI-TOF MS and CE-LIF. We thank Mandy Schietke and Melanie Anding for excellent technical assistance. We acknowledge the support of the Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of University of Potsdam., University of Potsdam = Universität Potsdam, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Gene Expression, MESH: Shigella flexneri, [SDV]Life Sciences [q-bio], lcsh:QR1-502, MESH: Protein Structure, Secondary, MESH: Biological Assay, lcsh:Microbiology, MESH: Bacterial Typing Techniques, Shigella flexneri, tailspike proteins, fluorescence sensor, MESH: Recombinant Proteins, ddc:05, bacteriophage, MESH: Genetic Vectors, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Protein Binding, MESH: Cloning, Molecular, ddc:610, MESH: Bacteriophages, MESH: Viral Tail Proteins, Institut für Biochemie und Biologie, microtiter plate assay, MESH: O Antigens, MESH: Carbohydrate Sequence, MESH: Escherichia coli, MESH: Oxadiazoles, MESH: Enzyme-Linked Immunosorbent Assay, MESH: Podoviridae, MESH: Serogroup, O-antigen, MESH: Fluorescent Dyes, serotyping, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, MESH: Staining and Labeling, 610 Medizin und Gesundheit, MESH: Models, Molecular
Abstract

International audience; Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.

Published
2018

31. Droplet digital PCR detects high rate of TP53 R249S mutants in cell-free DNA of middle African patients with hepatocellular carcinoma

Author

Aubin Béré, Pascal Pineau, Paul Jean Adrien Atangana, Dominique Noah Noah, Claudine Bekondi, Narcisse-Patrice Komas, Marie Amougou Atsama, Agnès Marchio, Richard Njouom, Serge-Magloire Camengo-Police, Organisation Nucléaire et Oncogenèse / Nuclear Organization and Oncogenesis, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Pasteur du Cameroun, Réseau International des Instituts Pasteur (RIIP), Institut Pasteur de Bangui, Hôpital Central de Yaoundé, Hôpital de l'Amitié [Bangui], This work was supported by Institut Pasteur International Network (Action Concertée Inter-Pasteurienne No17-2010, and Grant Dedonder), by the direction of Research Applications and Relations with the Industry (VALO Express), and by the French Ligue Nationale contre le Cancer (Equipe labélisée d’Anne Dejean)., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and MARCHIO, Agnes

Subjects
0301 basic medicine, Male, Aflatoxin B1, MESH: Mutation Rate, Hepatocellular carcinoma, Gene Expression, medicine.disease_cause, Gastroenterology, Polymerase Chain Reaction, Cell-free DNA, 0302 clinical medicine, MESH: Aged, 80 and over, Mutation Rate, MESH: Liver Neoplasms, Digital polymerase chain reaction, Cameroon, MESH: Tumor Suppressor Protein p53, MESH: Carcinoma, Hepatocellular, Aged, 80 and over, MESH: Aged, education.field_of_study, Hematology, MESH: Middle Aged, Liver Neoplasms, MESH: Aflatoxin B1, General Medicine, Middle Aged, Viral Load, MESH: Case-Control Studies, 3. Good health, Central African Republic, MESH: Hepatitis B virus, Cell-free fetal DNA, Droplet digital PCR, MESH: Central African Republic, 030220 oncology & carcinogenesis, Female, alpha-Fetoproteins, Liver cancer, MESH: Viral Load, Cell-Free Nucleic Acids, Adult, medicine.medical_specialty, Hepatitis B virus, Carcinoma, Hepatocellular, MESH: Gene Expression, Adolescent, MESH: Hepatitis B, Chronic, Population, [SDV.CAN]Life Sciences [q-bio]/Cancer, Malignancy, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Hepatitis B, Chronic, [SDV.CAN] Life Sciences [q-bio]/Cancer, Internal medicine, medicine, Humans, education, Aged, MESH: Adolescent, Hepatitis B Surface Antigens, MESH: Humans, business.industry, MESH: Adult, MESH: Polymerase Chain Reaction, MESH: Cameroon, medicine.disease, MESH: Male, MESH: DNA, Viral, MESH: Hepatitis B Surface Antigens, 030104 developmental biology, TP53 mutants, Case-Control Studies, DNA, Viral, MESH: Cell-Free Nucleic Acids, MESH: alpha-Fetoproteins, Tumor Suppressor Protein p53, business, MESH: Female
Abstract

International audience; Hepatocellular carcinoma (HCC) is still a major killing malignancy in sub-Saharan Africa. Lifelong intoxication with aflatoxin B1 is considered as one of the primary causes of this situation. The role of aflatoxin in HCC from a given population is commonly estimated through the prevalence of R249S mutation of TP53, a hallmark for previous exposure to the mycotoxin. However, the role of AFB1 is barely known in large part of Africa. We conducted a survey on circulating cell-free DNA from 149 patients with HCC and 213 control subjects with and without liver diseases from Cameroon and Central African Republic using droplet digital PCR technique. We observed a mutation prevalence of 24.8% (n = 37/149) in patients with tumor and 5.6% (n = 12/213) in controls (P = 2.2E-07). Patients with mutations usually displayed significantly increased circulating alpha-fetoprotein (AFP) values, high hepatitis B virus (HBV) DNA loads as well as worsened values of blood cells count. Interestingly, the fraction of droplets positive for R249S was significantly larger in patients with liver cancer (15.3 ± 3.7%) than in controls (0.5 ± 0.3%, P = 7.1E-04). Our survey indicates that AFB1 is instrumental for HCC development in Middle Africa and that droplet digital PCR might be used in the region both to diagnose HCC and to conduct public health surveys on populations at risk of chronic aflatoxin intoxication.

Published
2018

32. Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor

Author

Aline Desmyter, Horst Vogel, Lamia Mebarki, Takashi Tomizaki, Ruud Hovius, Romain Wyss, Sonja Minniberger, Xiao-Dan Li, Christophe Moreau, Lucie Peclinovska, Ghérici Hassaine, Cédric Deluz, Hugues Nury, Luigino Grasso, Henning Stahlberg, Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne (EPFL), Biozentrum, University of Basel (Unibas), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), 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)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Biomolecular Research, Paul Scherrer Institute (PSI), Biozentrum [Basel, Suisse], Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut de biologie structurale (IBS - UMR 5075 ), 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)-Centre National de la Recherche Scientifique (CNRS), 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 Lacapere, Jean-Jacques

Subjects
0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Gene Expression, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, law.invention, MESH: Recombinant Proteins, Mice, law, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Animals, Integral membrane protein, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein Stability, Peripheral membrane protein, MESH: Camelus, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Recombinant Proteins, [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], Biochemistry, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Recombinant DNA, MESH: Membrane Proteins, MESH: Cryoelectron Microscopy, Antibody, Camelus, MESH: Gene Expression, MESH: Receptors, Serotonin, 5-HT3, Cys-loop receptor, VHH, 5-HT3 receptor, Antibodies, Cell Line, 03 medical and health sciences, MESH: Protein Stability, Animals, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Mice, 5-HT receptor, MESH: Humans, MESH: Antibodies, Cryoelectron Microscopy, Membrane Proteins, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Lama antibody, MESH: Cell Line, 030104 developmental biology, Membrane protein, Cell culture, biology.protein, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Receptors, Serotonin, 5-HT3, Stable cell line
Abstract

International audience; There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

Published
2018

33. Rescue of GSDIII Phenotype with Gene Transfer Requires Liver- and Muscle-Targeted GDE Expression

Author

Federico Mingozzi, Patrice Vidal, Helena Costa Verdera, Bernard Gjata, Francesco Puzzo, Giuseppe Ronzitti, Laetitia van Wittenberghe, Giacomo P. Comi, Gilles Mithieux, Fanny Collaud, Sabrina Lucchiari, Pascal Laforêt, Edoardo Malfatti, Louisa Jauze, Monika Gjorgjieva, Fabienne Rajas, Marcelo Simon Sola, Solenne Marmier, Alban Vignaud, Serena Pagliarani, Severine Charles, Isabelle Richard, Pasqualina Colella, Université Pierre et Marie Curie - Paris 6 (UPMC), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), 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, Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Physiopatology and Transplantation, University of Milan (DEPT), Università degli Studi di Milano = University of Milan (UNIMI), IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, Généthon, Université Pierre et Marie Curie - Paris 6 - UFR de Médecine Pierre et Marie Curie (UPMC), Nutrition et cerveau (U1213, U855), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Pathophysiology and Transplantation, Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), U855, Nutrition et cerveau, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), ANR-17-CE18-0014,TRACeGSDIII,Optimisation translationnelle d'un vecteur AAV pour le traitement de GSDIII(2017), École pratique des hautes études (EPHE), Richard, Isabelle, Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut national de la santé et de la recherche médicale, Université d’Evry Val d’Essonne, Genethon (INSERM), University of Milan, Université de Lyon, Généthon, Evry, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Nutrition, diabète et cerveau, 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), GENETHON, Genethon, Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), and Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU)

Subjects
Blood Glucose, Male, 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, MESH: Glycogen, Gene Expression, MESH: Dependovirus, Glycogen storage disease type III, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Mice, Knockout, MESH: Hepatocytes, Mice, chemistry.chemical_compound, dual AAV vectors, MESH: Genetic Vectors, Drug Discovery, MESH: Animals, Vector (molecular biology), MESH: Glycogen Storage Disease Type III, MESH: Organ Specificity, Mice, Knockout, MESH: Muscle, Skeletal, Glycogen, Gene Transfer Techniques, Dependovirus, Phenotype, Cori disease, gene therapy, 3. Good health, Cell biology, Liver, Organ Specificity, Acid alpha-glucosidase, Molecular Medicine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Original Article, medicine.symptom, MESH: Enzyme Activation, MESH: Gene Expression, Transgene, Genetic Vectors, adeno-associated vector, MESH: Gene Transfer Techniques, Biology, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, MESH: Phenotype, 03 medical and health sciences, glycogenosis, Genetics, medicine, Animals, MESH: Glycogen Debranching Enzyme System, Muscle, Skeletal, Molecular Biology, MESH: Mice, glycogen storage disease type III, Pharmacology, MESH: Genetic Therapy, Muscle weakness, Glycogen Debranching Enzyme System, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Genetic Therapy, neuromuscular disease, medicine.disease, MESH: Male, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Enzyme Activation, Disease Models, Animal, 030104 developmental biology, chemistry, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, acid-alpha-glucosidase, Hepatocytes, MESH: Biomarkers, MESH: Blood Glucose, MESH: Disease Models, Animal, Biomarkers, MESH: Liver
Abstract

International audience; Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

Published
2018

34. Secretory lipase from the human pathogen Leishmania major: Heterologous expression in the yeast Pichia pastoris and biochemical characterization

Author

Madiha Bou Ali, Youssef Gargouri, Saoussen Ben Ayed, Aymen Bali, Yassine Ben Ali, Dhafer Laouini, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Université de Sfax - University of Sfax, Laboratoire de Transmission, Contrôle et Immunobiologie des Infections - Laboratory of Transmission, Control and Immunobiology of Infection (LR11IPT02), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), and The authors would like to thank Mr. Anouar Fendri, Professor of English, who kindly proofread this paper.

Subjects
0301 basic medicine, Models, Molecular, MESH: Leishmania major/genetics, MESH: Enzyme Stability, MESH: Hydrogen-Ion Concentration, Protein Conformation, Gene Expression, MESH: Amino Acid Sequence, MESH: Base Sequence, 01 natural sciences, Biochemistry, Pichia, MESH: Protein Conformation, Enzyme Stability, MESH: Pichia/genetics, Leishmania major, MESH: Lipase/antagonists & inhibitors, Cloning, Molecular, Enzyme Inhibitors, Leishmania, MESH: Lipase/metabolism, biology, Chemistry, Temperature, General Medicine, Hydrogen-Ion Concentration, MESH: Temperature, Parasite, MESH: Biocatalysis, MESH: Models, Molecular, MESH: Gene Expression, Leishmania donovani, Triacylglycerol lipase, MESH: Sequence Alignment, MESH: Leishmania major/enzymology, Pichia pastoris, 03 medical and health sciences, Humans, MESH: Cloning, Molecular, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Lipase, MESH: Humans, Base Sequence, MESH: Enzyme Inhibitors/pharmacology, 010401 analytical chemistry, MESH: Lipase/chemistry, biology.organism_classification, Yeast, 0104 chemical sciences, 030104 developmental biology, MESH: Lipase/genetics, biology.protein, Biocatalysis, Heterologous expression, Sequence Alignment
Abstract

International audience; Leishmaniasis is a parasitic reticuloendotheliosis whose pathogen is a zooflagellate belonging to the genus Leishmania transmitted by the bite of an infected phlebotome. Recently, a unique secretory lipase from the human pathogen Leishmania donovani Ldlip3 has been identified and characterized. This lipase has a high identity with a putative triacylglycerol lipase of Leishmania major (Lmlip2). In the present study, Lmlip2 was expressed in the eukaryotic heterologous expression system Pichia pastoris as tagged enzyme of 308 amino acids. Maximal protein production was reached after 2 days of fermentation. Optimal Lmlip2 lipase activity was measured using the pH stat technique at pH 8 at 26 °C using vinyl esters and triacylglycerols (true lipids) as substrates. Moreover, biochemical characterization of Lmlip2 contained in culture supernatant, illustrates that L. major secreted lipase is active and stable at low temperatures especially 26°and prefer neutral pH; concerning substrate specificityLmlip2 presents a preference for short chains lipid substrates vinyl esters such as VC2, VC3 and VC4 likewise, it is capable to hydrolyze long chain triacylglycerols like olive oil. Metal ions and surfactants tested in this study decrease Lmlip2 activity. Further studies are needed to clarify the relation between the lipase activity and the virulence. Thus, it could lead to the identification of novel targets to block cutaneous Leishmaniasis in human hosts.

Published
2018

35. A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing

Author

Raymond J. Pierce, Wolfgang Sippl, Julien Lancelot, Karin Schmidtkunz, Alokta Chakrabarti, Dina Robaa, Jelena Melesina, Tajith B. Shaik, Christophe Romier, Conrad V. Simoben, Manfred Jung, Srinivasaraghavan Kannan, Martin Marek, Romier, Christophe, Martin-Luther-Universität Halle Wittenberg (MLU), Albert-Ludwigs-Universität Freiburg, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), This work and the authors of this article received funding from the European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under Grant Agreements 241865 (SEtTReND) and 602080 (A-ParaDDisE). Further support was received by the Deutsche Forschungsgemeinschaft (Ju-295/13-1, SI-868/13-1). MM, TBS and CR are supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Université de Strasbourg., The authors acknowledge the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INBS-05 and of Instruct-ERIC. We wish to thank members of the ESRF-EMBL joint structural biology groups and the SOLEIL synchrotron for the use of their beamline facilities and for help during data collection. We are grateful to Pierre Legrand (SOLEIL) for his kind assistance for data processing, Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)

Subjects
0301 basic medicine, Pyrrolidines, MESH: Helminth Proteins/metabolism, MESH: Histone Deacetylases/metabolism, MESH: Histone Deacetylases/genetics, Pharmaceutical Science, MESH: Protein Structure, Secondary, Gene Expression, Apoptosis, Crystallography, X-Ray, Hydroxamic Acids, Molecular Docking Simulation, MESH: Helminth Proteins/antagonists & inhibitors, MESH: Chelating Agents/chemical synthesis, Protein Structure, Secondary, Analytical Chemistry, 0302 clinical medicine, MESH: Structure-Activity Relationship, MESH: Hydroxamic Acids/chemical synthesis, Drug Discovery, MESH: Zinc/metabolism, MESH: Animals, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Histone Deacetylase Inhibitors/pharmacology, Chelating Agents, Anthelmintics, biology, Chemistry, MESH: Anthelmintics/pharmacology, Helminth Proteins, Schistosoma mansoni, epigenetics, crystal structure, docking, histone deacetylase (HDAC) inhibitors, schistosomiasis, virtual screening, MESH: Histone Deacetylases/chemistry, 3. Good health, MESH: Schistosoma mansoni/drug effects, MESH: Pyrrolidines/pharmacology, Zinc, Biochemistry, Chemistry (miscellaneous), Molecular Medicine, Protein Binding, MESH: Apoptosis/drug effects, MESH: Gene Expression, In silico, 030231 tropical medicine, MESH: Zinc/chemistry, Article, Histone Deacetylases, lcsh:QD241-441, 03 medical and health sciences, Structure-Activity Relationship, lcsh:Organic chemistry, MESH: Molecular Docking Simulation, Structure–activity relationship, MESH: Protein Binding, Animals, MESH: Helminth Proteins/genetics, Protein Interaction Domains and Motifs, Physical and Theoretical Chemistry, MESH: Histone Deacetylase Inhibitors/chemical synthesis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Virtual screening, MESH: Protein Interaction Domains and Motifs, Binding Sites, Organic Chemistry, MESH: Hydroxamic Acids/pharmacology, HDAC8, MESH: Schistosoma mansoni/growth & development, Epigenome, biology.organism_classification, MESH: Crystallography, X-Ray, MESH: Anthelmintics/chemical synthesis, Histone Deacetylase Inhibitors, 030104 developmental biology, MESH: Binding Sites, Docking (molecular), MESH: Chelating Agents/pharmacology, MESH: Schistosoma mansoni/enzymology, MESH: Helminth Proteins/chemistry, MESH: Schistosoma mansoni/genetics, MESH: Pyrrolidines/chemical synthesis
Abstract

International audience; A promising means in the search of new small molecules for the treatment of schistosomiasis (amongst other parasitic ailments) is by targeting the parasitic epigenome. In the present study, a docking based virtual screening procedure using the crystal structure of histone deacetylase 8 from Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 values ranging from 4.4-20.3 µM against smHDAC8. These newly identified inhibitors were further tested against human histone deacetylases (hsHDAC1, 6 and 8), and were found also to be exerting interesting activity against them. In silico prediction of the docking pose of the compounds was confirmed by the resolved crystal structure of one of the identified hits. This confirmed these compounds were able to chelate the catalytic zinc ion in a bidentate fashion, whilst showing an inverted binding mode of the hydroxamate group when compared to the reported smHDAC8/hydroxamates crystal structures. Therefore, they can be considered as new potential scaffold for the development of new smHDAC8 inhibitors by further investigation of their structure-activity relationship.

Published
2018

36. Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart

Author

Philippe Valet, Benoit Lebas, Bernard Masri, Pierre Sicard, Barbara Garmy-Susini, Jerome Roncalli, Fransky Hantelys, Françoise Pujol, Oksana Kunduzova, Anne-Catherine Prats, Xavier Chaufour, Edith Renaud-Gabardos, Angelo Parini, Florence Tatin, Denis Calise, 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), Centre Hospitalier Universitaire de Toulouse, ANEXPLO / CREFRE, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-SANOFI Recherche-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aventis, Laboratoire de Physiopathologie et de Pharmacologie Cardiovasculaire Expérimentale (LPPCE), Université de Bourgogne (UB), Institut de médecine moléculaire de Rangueil (I2MR), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IFR150-Institut National de la Santé et de la Recherche Médicale (INSERM), Hormones, facteurs de croissance et physiopathologie vasculaire, IFR 31 Louis Bugnard (IFR 31), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Analytic and Computational Research, Inc. - Earth Sciences (ACRI-ST), Service de cardiologie [Toulouse], Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse], Institut National de la Santé et de la Recherche Médicale (INSERM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-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), Service Cardiologie [CHU Toulouse], Pôle Cardiovasculaire et Métabolique [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées- Institut Fédératif de Recherche Bio-médicale Institution (IFR150)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Masri, Bernard

Subjects
0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Angiogenesis, FGF2, Genetic enhancement, [SDV]Life Sciences [q-bio], Myocardial Ischemia, Gene Expression, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Bioinformatics, MESH: Gene Order, Mice, angiogenesis, Transduction, Genetic, IRES, MESH: Genetic Vectors, Gene Order, Drug Discovery, Gene Regulatory Networks, MESH: Animals, MESH: Endothelial Cells, Myocardial infarction, gene transfer, ComputingMilieux_MISCELLANEOUS, MESH: Gene Regulatory Networks, MESH: Lentivirus, Cardioprotection, Neovascularization, Pathologic, Gene Transfer Techniques, MESH: Transduction, Genetic, 3. Good health, Apelin, [SDV] Life Sciences [q-bio], apelin, MESH: Fibrosis, cardiovascular system, Molecular Medicine, MESH: Myocardial Ischemia, Original Article, Fibroblast Growth Factor 2, Cardiac function curve, MESH: Gene Expression, Genetic Vectors, lentivector, Cardiomegaly, Context (language use), MESH: Gene Transfer Techniques, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Sarcoplasmic Reticulum Calcium-Transporting ATPases, 03 medical and health sciences, MESH: Sarcoplasmic Reticulum Calcium-Transporting ATPases, combined therapy, Genetics, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Molecular Biology, MESH: Mice, Pharmacology, MESH: Genetic Therapy, MESH: Fibroblast Growth Factor 2, MESH: Transcriptome, Lentivirus, fibrosis, Endothelial Cells, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Genetic Therapy, medicine.disease, ischemic heart disease, Disease Models, Animal, SERCA2, 030104 developmental biology, MESH: Apelin, Heart failure, Immunology, MESH: Cardiomegaly, MESH: Disease Models, Animal, Transcriptome, MESH: Neovascularization, Pathologic
Abstract

International audience; Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

Published
2018

37. Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome

Author

Bruno Cavadas, Luísa Pereira, Fanny Koeth, Etienne Simon-Loriere, Marisa Oliveira, Isabelle Casademont, Worachart Lert-itthiporn, Prida Malasit, Orlando Anunciação, Prapat Suriyaphol, Marina Penova, Richard Paul, Fumihiko Matsuda, Chiea Chuen Khor, Ampaiwan Chuansumrit, Kanchana Tangnararatchakit, Anavaj Sakuntabhai, Verónica Fernandes, Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Siriraj Hospital, Mahidol University, Mahidol University [Bangkok], Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Pasteur Kyoto International Joint Research Unit for Integrative Vaccinomics [Kyoto, Japan], Institut Pasteur [Paris], Department of Pediatrics, Faculty of Medicine, Mahidol University [Bangkok]-Ramathibodi Hospital, National University of Singapore (NUS), French National Research Agency (ANR-10–INSB–04, Investments for the Future)., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Instituto de Investigação e Inovação em Saúde, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Universidade do Porto = University of Porto, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Universidade do Porto [Porto], Genome Institute of Singapore (GIS), Génétique fonctionnelle des maladies infectieuses - Functional Genetics of Infectious Diseases, Centre National de Génotypage (CNG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Male, Heredity, Basic Helix-Loop-Helix Transcription Factors / genetics, Thai People, Viral Nonstructural Proteins / genetics, MESH: Asia, Southeastern, Genome-wide association study, Genome, Viral / genetics, Dengue virus, MESH: Dengue Virus, Biochemistry, MESH: Genotype, 0302 clinical medicine, MESH: Basic Helix-Loop-Helix Transcription Factors, Odds Ratio, Ethnicities, Protein Phosphatase 2, MESH: Nerve Tissue Proteins, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, MESH: Asian Continental Ancestry Group, MESH: Protein Phosphatase 2, Genomics, Genomic Databases, Severe Dengue / ethnology, MESH: Infant, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 3. Good health, MESH: Repressor Proteins, MESH: Young Adult, Child, Preschool, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Metabolic Pathways, Carrier Proteins / genetics, MESH: Cell Nucleus, MESH: Gene Expression, Genotype, Bioinformatics, lcsh:RC955-962, Single-nucleotide polymorphism, MESH: Severe Dengue, MESH: Carrier Proteins, Serogroup, 03 medical and health sciences, Viral Proteins, Asian People, Sequence Motif Analysis, Cell Nucleus / virology, Genetic predisposition, Genetics, Xenobiotic Metabolism, Humans, MESH: Adolescent, MESH: Humans, [SDV.GEN.GPO]Life Sciences [q-bio]/Genetics/Populations and Evolution [q-bio.PE], Haplotype, MESH: Child, Preschool, Public Health, Environmental and Occupational Health, Biology and Life Sciences, Computational Biology, Infant, MESH: Adult, lcsh:RA1-1270, Dengue Virus, medicine.disease, Tropical Diseases, MESH: Sulfotransferases, MESH: Cell Line, 030104 developmental biology, Severe Dengue / genetics, MESH: Genome-Wide Association Study, MESH: Viral Nonstructural Proteins, Population Groupings, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Carrier Proteins, MESH: Female, 030217 neurology & neurosurgery, 0301 basic medicine, Viral Diseases, Gene Expression, Repressor Proteins / genetics, MESH: Dengue, Viral Nonstructural Proteins, medicine.disease_cause, Dengue fever, Dengue Fever, Geographical Locations, Dengue, Cohort Studies, Database and Informatics Methods, Medicine and Health Sciences, Basic Helix-Loop-Helix Transcription Factors, Nerve Tissue Proteins / genetics, MESH: Cohort Studies, Asia, Southeastern, Type C Phospholipases / genetics, lcsh:Public aspects of medicine, MESH: Genetic Predisposition to Disease, Thailand, Phenotype, Europe, Genetic Mapping, Infectious Diseases, Dengue Virus / genetics, Asian Continental Ancestry Group / genetics, Female, Sulfotransferases, MESH: Genome, Viral, MESH: Type C Phospholipases, Sequence Analysis, Research Article, Neglected Tropical Diseases, Adult, lcsh:Arctic medicine. Tropical medicine, Adolescent, Dengue / virology, Dengue / genetics, Nerve Tissue Proteins, Genome, Viral, Biology, Genetic Predisposition, Research and Analysis Methods, Cell Line, Young Adult, medicine, Viral Proteins / genetics, Genetic Predisposition to Disease, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Severe Dengue, MESH: Thailand, Cell Nucleus, Protein Phosphatase 2 / genetics, MESH: Serogroup, Genome Analysis, MESH: Viral Proteins, MESH: Male, MESH: Odds Ratio, Repressor Proteins, Biological Databases, Metabolism, Haplotypes, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Type C Phospholipases, People and Places, Genetics of Disease, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Genome-Wide Association Study
Abstract

Ethnic diversity has been long considered as one of the factors explaining why the severe forms of dengue are more prevalent in Southeast Asia than anywhere else. Here we take advantage of the admixed profile of Southeast Asians to perform coupled association-admixture analyses in Thai cohorts. For dengue shock syndrome (DSS), the significant haplotypes are located in genes coding for phospholipase C members (PLCB4 added to previously reported PLCE1), related to inflammation of blood vessels. For dengue fever (DF), we found evidence of significant association with CHST10, AHRR, PPP2R5E and GRIP1 genes, which participate in the xenobiotic metabolism signaling pathway. We conducted functional analyses for PPP2R5E, revealing by immunofluorescence imaging that the coded protein co-localizes with both DENV1 and DENV2 NS5 proteins. Interestingly, only DENV2-NS5 migrated to the nucleus, and a deletion of the predicted top-linking motif in NS5 abolished the nuclear transfer. These observations support the existence of differences between serotypes in their cellular dynamics, which may contribute to differential infection outcome risk. The contribution of the identified genes to the genetic risk render Southeast and Northeast Asian populations more susceptible to both phenotypes, while African populations are best protected against DSS and intermediately protected against DF, and Europeans the best protected against DF but the most susceptible against DSS., Author summary Dengue fever is endemic in tropical and subtropical areas of East Asia and America, but globalization and climate changes are introducing vector and virus to the naïve regions of Europe and North America. In this work we conducted a statistically robust, coupled association-admixture test in two dengue cohorts from Thailand (classical dengue fever, DF, and dengue shock syndrome, DSS) and a published Vietnamese (DSS only) cohort. We identified new candidate genes associated with DF risk and confirmed known gene family association with DSS risk. In DF, phosphatase control is crucial, including through binding to viral proteins, as we showed for PPP2R5E protein co-localization with DENV1 and DENV2-NS5 proteins within liver cells and differential cellular localizations along time. In DSS, cytokine dynamics, inflammation and activation of vascular endothelium cells are dominant features. The particular genetic risk conferred by these genes indicates that Southeast and Northeast Asians are highly susceptible to both phenotypes, while Africans are best protected against DSS, and Europeans best protected against DF but the most susceptible against DSS.

Published
2018

38. Stage-specific IFN-induced and IFN gene expression reveal convergence of type I and type II IFN and highlight their role in both acute and chronic stage of pathogenic SIV infection

Author

Nadia Echebli, Nicolas Tchitchek, Stéphanie Dupuy, Christine Bourgeois, Roger Le Grand, Benoit Favier, Nathalie Bosquet, Bruno Vaslin, Caroline Peireira Bittencourt Passaes, Rémi Cheynier, Timothée Bruel, Olivier Lambotte, Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie - Immunopathologie - Immunothérapie [CHU Pitié Salpêtrière] (I3), CHU Charles Foix [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Service de Médecine Interne - Immunologie Clinique [AP-HP Bicêtre], AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), This work was supported by the Agence Nationale de Recherches sur le SIDA et les hépatites virales (www.anrs.fr). NE, SD and CPBP benefited from ANRS post-doctoral fellowships. This work was also supported by Agence Nationale de Recherches sur le SIDA et les hépatites virales (ANRS) through research project grant 11360 attributed to BV, by the agence Nationale de la Recherche (http://www.agence-nationale-recherche.fr) 'Programme d’Investissements d’Avenir' (PIA) under Grant ANR-11-INBS-0008, that funds the Infectious Disease Models and Innovative Therapies (IDMIT, Fontenay-aux-Roses, France) infrastructure, and PIA grant ANR-10-EQPX-02-01 that funds the FlowCyTech facility (project investigator RLG)., ANR-11-INBS-0008,IDMIT,Infrastructure nationale pour la modélisation des maladies infectieuses humaines(2011), ANR-10-EQPX-0002,FlowCyTech,Plateforme de phénotypage en Mass cytométrie pour l'analyse multiparamétrique de biomarqueurs complexes de l'efficacité de nouvelles stratégies thérapeutiques ou vaccinales(2010), Passaes, Caroline, Infrastructures - Infrastructure nationale pour la modélisation des maladies infectieuses humaines - - IDMIT2011 - ANR-11-INBS-0008 - INBS - VALID, and Equipements d'excellence - Plateforme de phénotypage en Mass cytométrie pour l'analyse multiparamétrique de biomarqueurs complexes de l'efficacité de nouvelles stratégies thérapeutiques ou vaccinales - - FlowCyTech2010 - ANR-10-EQPX-0002 - EQPX - VALID

Subjects
0301 basic medicine, RNA viruses, MESH: Interferon Type I, [SDV]Life Sciences [q-bio], lcsh:Medicine, Gene Expression, Monkeys, Pathology and Laboratory Medicine, Biochemistry, Transcriptome, White Blood Cells, 0302 clinical medicine, Immunodeficiency Viruses, Interferon, Animal Cells, Gene expression, Medicine and Health Sciences, MESH: Animals, Cell Cycle and Cell Division, lcsh:Science, Genetic Interference, Mammals, Multidisciplinary, T Cells, Eukaryota, virus diseases, 3. Good health, [SDV] Life Sciences [q-bio], SIV, Medical Microbiology, Cell Processes, 030220 oncology & carcinogenesis, Viral Pathogens, Vertebrates, Viruses, MESH: Acute Disease, [SDV.IMM]Life Sciences [q-bio]/Immunology, Pathogens, Anatomy, Cellular Types, MESH: Simian Acquired Immunodeficiency Syndrome, Macaque, medicine.drug, Research Article, Primates, MESH: Gene Expression, [SDV.IMM] Life Sciences [q-bio]/Immunology, MESH: Interferon-gamma, Immune Cells, Immunology, MESH: Simian Immunodeficiency Virus, Biology, Peripheral blood mononuclear cell, Microbiology, Lymphatic System, 03 medical and health sciences, Downregulation and upregulation, Old World monkeys, Retroviruses, medicine, Genetics, Animals, Gene, Microbial Pathogens, Blood Cells, MESH: Transcriptome, MESH: Chronic Disease, lcsh:R, Lentivirus, MESH: Virus Replication, Organisms, Biology and Life Sciences, Proteins, Cell Biology, Chronic infection, 030104 developmental biology, Viral replication, MESH: Macaca fascicularis, Amniotes, lcsh:Q, Interferons, Lymph Nodes
Abstract

23 Feb 2018: Echebli N, Tchitchek N, Dupuy S, Bruel T, Passaes C, et al. (2018) Correction: Stage-specific IFN-induced and IFN gene expression reveal convergence of type I and type II IFN and highlight their role in both acute and chronic stage of pathogenic SIV infection. PLOS ONE 13(2): e0193629. https://doi.org/10.1371/journal.pone.0193629(corresponding to the second file attached); International audience; Interferons (IFNs) play a major role in controlling viral infections including HIV/SIV infections. Persistent up-regulation of interferon stimulated genes (ISGs) is associated with chronic immune activation and progression in SIV/HIV infections, but the respective contribution of different IFNs is unclear. We analyzed the expression of IFN genes and ISGs in tissues of SIV infected macaques to understand the respective roles of type I and type II IFNs. Both IFN types were induced in lymph nodes during early stage of primary infection and to some extent in rectal biopsies but not in PBMCs. Induction of Type II IFN expression persisted during the chronic phase, in contrast to undetectable induction of type I IFN expression. Global gene expression analysis with a major focus on ISGs revealed that at both acute and chronic infection phases most differentially expressed ISGs were inducible by both type I and type II IFNs and displayed the highest increases, indicating strong convergence and synergy between type I and type II IFNs. The analysis of functional signatures of ISG expression revealed temporal changes in IFN expression patterns identifying phase-specific ISGs. These results suggest that IFN-γ strongly contribute to shape ISG upregulation in addition to type I IFN.

Published
2018

39. Reverse-Transcriptase Inhibitors in the Aicardi–Goutières Syndrome

Author

Diana Rodriguez, Vincent Bondet, Alexandre Belot, Luis Seabra, Claude Cances, Isabelle Desguerre, Virginie Levrat, Diane Doummar, Elodie Henry, Marie Hully, Mathieu P Rodero, Isabelle Melki, Magalie Barth, Alba Llibre, Agathe Roubertie, François-Guillaume Debray, Jean-Marc Tréluyer, Michaela Semeraro, Valérie Jolaine, Nathalie Boddaert, Pierre Meyer, Christine Barnerias, Carolina Uggenti, Jinmi Baek, Gillian I. Rice, Flore Rozenberg, Darragh Duffy, Stéphane Blanche, Marie-Louise Frémond, Florence Renaldo, Naïm Bouazza, Hendy Abdoul, Leo A. H. Zeef, Mame N Sambe, Candice Meyzer, Yanick J. Crow, Marie-Christine Nougues, Alice Lepelley, Naoki Kitabayashi, University of Manchester [Manchester], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Necker - Enfants Malades [AP-HP], Université Paris Descartes - Paris 5 (UPD5), Institut Pasteur [Paris] (IP), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM), Université de Lyon, Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Université de Liège, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Annecy-Genevois [Saint-Julien-en-Genevois], AP-HP Hôpital universitaire Robert-Debré [Paris], Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Hôpital Robert Debré, Sorbonne Université (SU), University of Edinburgh, Supported by grants from the European Leukodystrophy Association (ELA 2012-008l1), the European Research Council (GA 309449 and 786142-E-T1IFNs), ERA-NET Neuron (MR/M501803/1), and the French National Research Agency (ANR-10-IAHU-01 and CE17001002). Medications were provided by GlaxoSmithKline and ViiV Healthcare., ANR-10-IAHU-0001,Imagine,Institut Hospitalo-Universitaire Imagine(2010), European Project: 309449,EC:FP7:ERC,ERC-2012-StG_20111109,T1-IFN(2013), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut Pasteur [Paris], CHU Toulouse [Toulouse], Vougny, Marie-Christine, Instituts Hospitalo-Universitaires - Institut Hospitalo-Universitaire Imagine - - Imagine2010 - ANR-10-IAHU-0001 - IAHU - VALID, and Definition and characterization of type I interferonopathies - T1-IFN - - EC:FP7:ERC2013-03-01 - 2018-02-28 - 309449 - VALID

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Gene Expression, Pilot Projects, Nervous System Malformations/drug therapy, Medicine, MESH: Interferons, ComputingMilieux_MISCELLANEOUS, Cerebrovascular Circulation/drug effects, MESH: Zidovudine, General Medicine, MESH: Cerebrovascular Circulation, 3. Good health, Drug Combinations, MESH: Dideoxynucleosides, Lamivudine, Cerebrovascular Circulation, Autoimmune Diseases of the Nervous System/drug therapy, Reverse Transcriptase Inhibitors, [SDV.IMM]Life Sciences [q-bio]/Immunology, France, Zidovudine, MESH: Lamivudine, Interferons/genetics, Reverse Transcriptase Inhibitors/therapeutic use, MESH: Gene Expression, [SDV.IMM] Life Sciences [q-bio]/Immunology, Zidovudine/therapeutic use, Encephalopathy, Gene Expression/drug effects, Nervous System Malformations, MESH: Nervous System Malformations, 03 medical and health sciences, Autoimmune Diseases of the Nervous System, Humans, Lamivudine/therapeutic use, MESH: Drug Combinations, MESH: Humans, business.industry, Dideoxynucleosides/therapeutic use, medicine.disease, MESH: Pilot Projects, Virology, Dideoxynucleosides, Reverse transcriptase, MESH: Autoimmune Diseases of the Nervous System, MESH: France, 030104 developmental biology, Nucleic acid, Aicardi–Goutières syndrome, Interferons, MESH: Reverse Transcriptase Inhibitors, business
Abstract

International audience; To the Editor:The Aicardi–Goutières syndrome is a genetic encephalopathy that is associated with childhood illness and death. The syndrome is hypothesized to be due to misidentification of self-derived nucleic acids as nonself and the subsequent induction of a type I interferon–mediated response that simulates an antiviral reaction.1 Endogenous retroelements, mobile genetic elements that can be transcribed to RNA and then to DNA by reverse transcription, constitute 40% of the human genome and represent a potential source of immunostimulatory nucleic acid in patients with this syndrome.2

Published
2018
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40. Functional reconstitution of cell-free synthesized purified K v channels

Author

Xavier Henry, Sandra Cortes, Michel De Waard, Beate Bersch, Béatrice Schaack, Stéphane Renauld, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-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)-Université Grenoble Alpes (UGA), Synthelis, Smartox Biotechnology, Université Joseph Fourier - Grenoble 1 (UJF), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), cell-free expression platform (PSB), ANR-11-RPIB-0022,VenomPicoScreen,Découverte de principes actifs à partir de venins en utilisant des bicouches artificielles miniaturisées(2011), Institut de biologie structurale [1992-2019] (IBS - UMR 5075 [1992-2019]), 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), Université de Bordeaux (UB), Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Université Joseph Fourier - Grenoble 1 ( UJF ), ANR : VenomPicoScreen,ANR-11-RPIB-022-04, Institut de biologie structurale (IBS - UMR 5075 ), Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)

Subjects
0301 basic medicine, MESH: Gene Expression, Proteoliposomes, MESH: Kv1.3 Potassium Channel, Biophysics, Ionophore, Synthetic membrane, MESH: Phosphatidylethanolamines, Biochemistry, MESH: Recombinant Proteins, DIB, 03 medical and health sciences, Valinomycin, chemistry.chemical_compound, 0302 clinical medicine, MESH: Genetic Vectors, K(v) channels, MESH: Membrane Potentials, MESH: Elapid Venoms, Membrane potential, Liposome, Chromatography, MESH: Humans, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, MESH: Escherichia coli, MESH: Valinomycin, MESH: Kv1.1 Potassium Channel, Cell Biology, MESH: Phosphatidylserines, MESH: Phosphatidylcholines, MESH: Fluorescent Dyes, Small molecule, Fluorescence, Oxonol VI, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Membrane, MESH: Isoxazoles, MESH: Subcellular Fractions, 030217 neurology & neurosurgery, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Proteolipids
Abstract

International audience; The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

Published
2017

41. Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem

Author

Christine Petit, Florent Delhommel, Benjamin Bardiaux, Julia Chamot-Rooke, Michael Nilges, Sébastien Brier, Florence Cordier, Amel Bahloul, Bertrand Raynal, Sylvie Nouaille, Nicolas Wolff, Baptiste Colcombet-Cazenave, Guillaume Bouvier, ED 515 - Complexité du vivant, Université Pierre et Marie Curie - Paris 6 (UPMC), Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Spectrométrie de Masse structurale et protéomique, Biophysique Moléculaire (Plate-forme), Chaire Génétique et physiologie cellulaire, Collège de France (CdF (institution)), Bioinformatique Structurale, Financial support from TGIR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged. We acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities, and we would like to thank the staff of the SWING and BM29 BioSAXS beamlines for assistance during the SAXS measurements. This work was supported by the Program Transversal de Recherche from the Institut Pasteur (PTR grant no. 483 to N.W.), the European Union (FP7-IDEAS-ERC 294809 to M.N.), the Ministère de l’Enseignement Supérieur et de la Recherche (grant no. 883/2013 to F.D), and the Comité Berthe Fouassier – Maladies de l’œil de la Fondation de France (no. 00071779 to F.D)., The authors are grateful to Bradley Worley for careful proofreading of the manuscript and to Muriel Delepierre for useful discussions. We thank Olivier Lequin, Patrick England, and Alain Chaffotte for their technical expertise., European Project: 294809,EC:FP7:ERC,ERC-2011-ADG_20110310,BAYCELLS(2012), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège de France - Chaire Génétique et physiologie cellulaire, Wolff, Nicolas, and A Bayesian Framework for Cellular Structural Biology - BAYCELLS - - EC:FP7:ERC2012-05-01 - 2017-04-30 - 294809 - VALID

Subjects
Protein Conformation, alpha-Helical, 0301 basic medicine, Scaffold protein, Protein Folding, MESH: Sequence Homology, Amino Acid, MESH: PDZ Domains, MESH: Nerve Tissue Proteins / genetics, Gene Expression, PDZ Domains, MESH: Amino Acid Sequence, MESH: Escherichia coli / genetics, 01 natural sciences, Mice, Transduction (genetics), Structural Biology, MESH: Hair Cells, Auditory / metabolism, MESH: Animals, MESH: Molecular Dynamics Simulation, MESH: Membrane Proteins / metabolism, Cloning, Molecular, MESH: Peptides / chemistry, MESH: Membrane Proteins / genetics, Tandem, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], SAXS, Recombinant Proteins, Thermodynamics, Stereocilia bundle, MESH: Protein Conformation, beta-Strand, MESH: Escherichia coli / metabolism, MESH: Thermodynamics, MESH: Hair Cells, Auditory / cytology, Usher syndrome, Protein Binding, MESH: Recombinant Proteins / chemistry, supramodule, MESH: Gene Expression, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Protein Folding, Genetic Vectors, PDZ domain, MESH: Sequence Alignment, Nerve Tissue Proteins, MESH: Nerve Tissue Proteins / chemistry, Molecular Dynamics Simulation, Biology, 010402 general chemistry, Closed conformation, MESH: Membrane Proteins / chemistry, Electric signal, 03 medical and health sciences, NMR spectroscopy, Hair Cells, Auditory, Escherichia coli, otorhinolaryngologic diseases, Animals, MESH: Protein Binding, MESH: Cloning, Molecular, Amino Acid Sequence, MESH: Genetic Vectors / chemistry, Molecular Biology, MESH: Mice, MESH: Nerve Tissue Proteins / metabolism, MESH: Protein Conformation, alpha-Helical, MESH: Peptides / chemical synthesis, Binding Sites, whirlin, Sequence Homology, Amino Acid, Stereocilia, Membrane Proteins, 0104 chemical sciences, Crystallography, 030104 developmental biology, MESH: Genetic Vectors / metabolism, MESH: Binding Sites, Biophysics, Protein Conformation, beta-Strand, sense organs, MESH: Recombinant Proteins / metabolism, Peptides, Sequence Alignment, MESH: Recombinant Proteins / genetics
Abstract

International audience; Hearing relies on the transduction of sound-evoked vibrations into electric signals, occurring in the stereocilia bundle of hair cells. The bundle is organized in a staircase pattern formed by rows of packed stereocilia. This architecture is pivotal to transduction and involves a network of scaffolding proteins with hitherto uncharacterized features. Key interactions in this network are mediated by PDZ domains. Here, we describe the architecture of the first two PDZ domains of whirlin, a protein involved in these assemblies and associated with congenital deaf-blindness. C-terminal hairpin extensions of the PDZ domains mediate the transient supramodular assembly, which improves the binding capacity of the first domain. We determined a detailed structural model of the closed conformation of the PDZ tandem and characterized its equilibrium with an ensemble of open conformations. The structural and dynamic behavior of this PDZ tandem provides key insights into the regulatory mechanisms involved in the hearing machinery.

Published
2017

42. Regulation of the interaction between the neuronal BIN1 isoform 1 and Tau proteins - role of the SH3 domain

Author

Isabelle Landrieu, François-Xavier Cantrelle, Yoann Sottejeau, Guy Lippens, Idir Malki, Jean-Charles Lambert, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP), The NMR facilities were funded by the Nord Region Council, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and Lille University. We acknowledge support from TGE RMN THC (FR-3050, France) and FRABio (Univ. Lille, CNRS, FR 3688). This study was supported by a grant from the LabEx (Laboratory of Excellence), DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease), by the French government funding agency Agence Nationale de la Recherche ANR-15-CE16-0002 BinAlz and by the Alzheimer's association (BFG-14-318355)., Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), CNRS, Université de Lille, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Facteurs de Risque et Déterminants Moléculaires des Maladies liées au Vieillissement - U 1167 [RID-AGE], and Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF]

Subjects
0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, Alzheimer’s disease, BIN1, nuclearmagnetic resonance spectroscopy, protein–protein interaction, SH3 domain, Tau, MESH: Sequence Homology, Amino Acid, MESH: Adaptor Proteins, Signal Transducing/metabolism, Amino Acid Motifs, MESH: Tumor Suppressor Proteins/genetics, Gene Expression, Peptide, Biochemistry, MESH: Amino Acid Motifs, 0302 clinical medicine, MESH: Adaptor Proteins, Signal Transducing/genetics, MESH: Nuclear Magnetic Resonance, Biomolecular, Protein Isoforms, Cloning, Molecular, MESH: tau Proteins/chemistry, chemistry.chemical_classification, Neurons, biology, MESH: Kinetics, Chemistry, Nuclear Proteins, MESH: Neurons/metabolism, Nuclear magnetic resonance spectroscopy, MESH: Peptides/genetics, MESH: Tumor Suppressor Proteins/chemistry, MESH: tau Proteins/metabolism, Recombinant Proteins, MESH: Tumor Suppressor Proteins/metabolism, MESH: Recombinant Proteins/chemistry, MESH: tau Proteins/genetics, MESH: Peptides/metabolism, Domain (ring theory), MESH: Protein Conformation, beta-Strand, MESH: Protein Isoforms/metabolism, MESH: Models, Molecular, MESH: Nuclear Proteins/genetics, Protein Binding, Gene isoform, MESH: Recombinant Proteins/metabolism, MESH: Gene Expression, MESH: Sequence Alignment, tau Proteins, MESH: Escherichia coli/genetics, Clathrin, Protein–protein interaction, MESH: Peptides/chemistry, 03 medical and health sciences, Escherichia coli, Humans, MESH: Protein Binding, Protein Interaction Domains and Motifs, MESH: Adaptor Proteins, Signal Transducing/chemistry, MESH: Recombinant Proteins/genetics, MESH: Cloning, Molecular, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Nuclear Proteins/metabolism, Molecular Biology, Gene, Nuclear Magnetic Resonance, Biomolecular, Adaptor Proteins, Signal Transducing, MESH: Protein Conformation, alpha-Helical, MESH: Protein Interaction Domains and Motifs, Binding Sites, MESH: Protein Isoforms/genetics, MESH: Humans, Sequence Homology, Amino Acid, Tumor Suppressor Proteins, Cell Biology, MESH: Nuclear Proteins/chemistry, Kinetics, MESH: Neurons/chemistry, 030104 developmental biology, MESH: Binding Sites, MESH: Protein Isoforms/chemistry, Biophysics, biology.protein, MESH: Escherichia coli/metabolism, Protein Conformation, beta-Strand, Peptides, Sequence Alignment, 030217 neurology & neurosurgery
Abstract

International audience; Bridging integrator 1 (bin1) gene is a genetic determinant of Alzheimer's disease (AD) and has been reported to modulate Alzheimer's pathogenesis through pathway(s) involving Tau. The functional impact of Tau/BIN1 interaction as well as the molecular details of this interaction are still not fully resolved. As a consequence, how BIN1 through its interaction with Tau affects AD risk is also still not determined. To progress in this understanding, interaction of Tau with two BIN1 isoforms was investigated using Nuclear Magnetic Resonance spectroscopy. 1H, 15N spectra showed that the C-terminal SH3 domain of BIN1 isoform 1 (BIN1Iso1) is not mobile in solution but locked with the core of the protein. In contrast, the SH3 domain of BIN1 isoform 9 (BIN1Iso9) behaves as an independent mobile domain. This reveals an equilibrium between close and open conformations for the SH3 domain. Interestingly, a 334–376 peptide from the clathrin and AP-2-binding domain (CLAP) domain of BIN1Iso1, which contains a SH3-binding site, is able to compete with BIN1-SH3 intramolecular interaction. For both BIN1 isoforms, the SH3 domain can interact with Tau(210–240) sequence. Tau(210–240) peptide can indeed displace the intramolecular interaction of the BIN1-SH3 of BIN1Iso1 and form a complex with the released domain. The measured Kd were in agreement with a stronger affinity of Tau peptide. Both CLAP and Tau peptides occupied the same surface on the BIN1-SH3 domain, showing that their interaction is mutually exclusive. These results emphasize an additional level of complexity in the regulation of the interaction between BIN1 and Tau dependent of the BIN1 isoforms.

Published
2017

43. Tissue kallikrein is required for the cardioprotective effect of Cyclosporin A in myocardial ischemia in the mouse

Author

C. Richer, M.-P. Vincent, G. Youcef, François Alhenc-Gelas, Anne Pizard, Elise Belaidi, M. Clemessy, L. Fazal, Ludovic Waeckel, G. Zadigue, Michel Ovize, 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), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National de la Recherche Agronomique (INRA), Défaillance Cardiovasculaire Aiguë et Chronique (DCAC), Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Centre d'investigation clinique [Nancy] (CIC), 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), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), CIC-Nancy, Institut Lorrain du Coeur et des Vaisseaux Louis Mathieu [Nancy]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), and Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy)

Subjects
Male, MESH: Signal Transduction, Myocardial Ischemia, Gene Expression, MESH: Myocytes, Cardiac, Mitochondrion, Pharmacology, MESH: Voltage-Dependent Anion Channel 1, Mitochondrial Membrane Transport Proteins, MESH: Mice, Knockout, MESH: Cyclosporine, Biochemistry, Mitochondria, Heart, Oxidative Phosphorylation, Cyclophilins, Mice, chemistry.chemical_compound, Cyclosporin a, Myocytes, Cardiac, MESH: Animals, Ischemic Postconditioning, Inner mitochondrial membrane, MESH: Heterozygote, Mice, Knockout, Cardioprotection, Tissue kallikrein, MPTP, Homozygote, MESH: Mitochondrial Membrane Transport Proteins, Mitochondria, Knockout mouse, Cyclosporine, cardiovascular system, Cardiac ischemia–reperfusion, MESH: Myocardial Ischemia, MESH: Mitochondria, Heart, Tissue Kallikreins, Cyclophilin D, Signal Transduction, MESH: Homozygote, Heterozygote, Cardiotonic Agents, MESH: Myocardium, MESH: Gene Expression, MESH: Rats, MESH: Tissue Kallikreins, Ischemia, Biology, MESH: Cyclophilins, MESH: Ischemic Postconditioning, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, MESH: Oxidative Phosphorylation, medicine, Animals, cardiovascular diseases, MESH: Mice, Mitochondrial Permeability Transition Pore, Myocardium, Voltage-Dependent Anion Channel 1, MESH: Cardiotonic Agents, medicine.disease, MESH: Male, Rats, Cyclosporin A, Mitochondrial permeability transition pore, chemistry
Abstract

International audience; Clinical and experimental studies suggest that pharmacological postconditioning with Cyclosporin A (CsA) reduces infarct size in cardiac ischemia and reperfusion. CsA interacts with Cyclophilin D (CypD) preventing opening of the mitochondrial permeability transition pore (mPTP). Tissue kallikrein (TK) and its products kinins are involved in cardioprotection in ischemia. CypD knockout mice are resistant to the cardioprotective effects of both CsA and kinins suggesting common mechanisms of action. Using TK gene knockout mice, we investigated whether the kallikrein-kinin system is involved in the cardioprotective effect of CsA. Homozygote and heterozygote TK deficient mice (TK(-/-), TK(+/-)) and wild type littermates (TK(+/+)) were subjected to cardiac ischemia-reperfusion with and without CsA postconditioning. CsA reduced infarct size in TK(+/+) mice but had no effect in TK(+/-) and TK(-/-) mice. Cardiac mitochondria isolated from TK(-/-) mice had indistinguishable basal oxidative phosphorylation and calcium retention capacity compared to TK(+/+) mice but were resistant to CsA inhibition of mPTP opening. TK activity was documented in mouse heart and rat cardiomyoblasts mitochondria. By proximity ligation assay TK was found in close proximity to the mitochondrial membrane proteins VDAC and Tom22, and CypD. Thus, partial or total deficiency in TK induces resistance to the infarct size reducing effect of CsA in cardiac ischemia in mice, suggesting that TK level is a critical factor for cardioprotection by CsA. TK is required for the mitochondrial action of CsA and may interact with CypD. Genetic variability in TK activity has been documented in man and may influence the cardioprotective effect of CsA.

Published
2015

44. Herpes Simplex Type 2 Encephalitis and Methotrexate Medication: A Fortuitous or Causative Association in a Patient with Spondyloarthritis?

Author

Monique Baccard-Longère, Ophélie Dos Santos, Olivier Epaulard, Jean-Paul Stahl, Raphaële Germi, Patrice Morand, Julien Lupo, Université Grenoble Alpes - UFR Pharmacie (UGA UFRP), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), 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), Université Grenoble Alpes - UFR Médecine (UGA UFRM), Laboratoire de virologie, Centre Hospitalo-Universitaire de Grenoble, Service de maladies infectieuses et tropicales, Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble, Department of infectious diseases, CHU Grenoble, 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
Male, 0301 basic medicine, medicine.medical_treatment, Acyclovir, Gene Expression, 030105 genetics & heredity, medicine.disease_cause, 0302 clinical medicine, Risk Factors, MESH: Risk Factors, Rare case, MESH: Acyclovir, Pharmacology (medical), HLA-B27 Antigen, MESH: Aged, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Encephalitis, Herpes Simplex, Immunosuppression, MESH: Spondylitis, Ankylosing, 3. Good health, MESH: Methotrexate, Infectious Diseases, MESH: Immunosuppressive Agents, Immunosuppressive Agents, Encephalitis, medicine.drug, MESH: Antiviral Agents, MESH: Contraindications, Drug, medicine.medical_specialty, MESH: Gene Expression, Severe disease, Antiviral Agents, 03 medical and health sciences, medicine, Humans, Spondylitis, Ankylosing, Risk factor, Aged, Pharmacology, MESH: Humans, business.industry, Contraindications, Drug, medicine.disease, Virology, Dermatology, MESH: Male, Methotrexate, Herpes simplex virus, Encephalitis, Herpes Simplex, MESH: HLA-B27 Antigen, business, 030217 neurology & neurosurgery
Abstract

International audience; It is unclear whether immunosuppression is a risk factor for herpes encephalitis. Herein, we describe a rare case of herpes simplex virus type 2 encephalitis in a patient treated with low-dose methotrexate for HLA-B27-associated spondyloarthritis. The patient was successfully treated with acyclovir but presented sequelae of encephalitis. Here we discuss the possible role of low-dose methotrexate therapy as a risk factor of neurological herpes reactivation and severe disease. The host-related and viral risk factors are also addressed.

Published
2016

45. Defining Membrane Protein Topology Using pho-lac Reporter Fusions

Author

Gouzel Karimova, Daniel Ladant, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institut Pasteur and Centre National de la Recherche Scientifique (CNRS UMR 3528, Biologie Structurale et Agents Infectieux)., Laure Journet, Eric Cascales, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, MESH: Enzyme Activation, MESH: Gene Expression, [SDV]Life Sciences [q-bio], Phosphatase, lac operon, β-galactosidase, MESH: Escherichia coli / genetics, MESH: Membrane Proteins / genetics, Topology, medicine.disease_cause, MESH: Gene Order, MESH: Membrane Proteins / chemistry, 03 medical and health sciences, Membrane proteins, medicine, MESH: Cloning, Molecular, MESH: Recombinant Fusion Proteins, Escherichia coli, Chemistry, MESH: Genes, Reporter, Dual reporter system, MESH: Plasmids / genetics, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Periplasmic space, MESH: Lac Operon, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Membrane protein, Cytoplasm, Membrane topology, Alkaline phosphatase, bacteria, MESH: Escherichia coli / metabolism, MESH: beta-Galactosidase / genetics
Abstract

International audience; Experimental determination of membrane protein topology can be achieved using various techniques. Here we present the pho-lac dual reporter system, a simple, convenient, and reliable tool to analyze the topology of membrane proteins in vivo. The system is based on the use of two topological markers with complementary properties, the Escherichia coli β-galactosidase LacZ, which is active in the cytoplasm, and the E. coli alkaline phosphatase PhoA, which is active in the bacterial periplasm. Specifically, in this pho-lac gene system, the reporter molecule is a chimera composed of the mature PhoA that is in frame with the β-galactosidase α-peptide, LacZα. Hence, when targeted to the periplasm, the PhoA-LacZα dual reporter displays high alkaline phosphatase activity but no β-galactosidase activity. Conversely, when located in the cytoplasm, PhoA-LacZα has no phosphatase activity but exhibits high β-galactosidase activity in E. coli cells expressing the ω fragment of LacZ, LacZω (via the α-complementation phenomenon). The dual nature of the PhoA- LacZα reporter allows a simple way to normalize both enzymatic activities to obtain readily interpretable information about the subcellular location of the fusion site between the membrane protein under study and the reporter. In addition, the PhoA-LacZα reporter permits utilization of dual-indicator agar plates to easily discriminate between colonies bearing cytoplasmic fusions, periplasmic fusions, or out-of-frame fusions. In total, the phoA-lacZα fusion reporter approach is a straightforward and rather inexpensive method of characterizing the topology of membrane proteins in vivo.

Published
2017

46. Modulation of Aneuploidy in In Vivo Environments and Its Impact on Gene Expression

Author

Jitka Myskova, Petr Volf, Franck Dumetz, Conor J. Meehan, G. De Muylder, Veronika Seblova, Mandy Sanders, Matthew Berriman, Giovanni Bussotti, Bart Cuypers, Manu Vanaerschot, Gerald F. Späth, J. R. Vermeesch, Pascale Pescher, Jean-Claude Dujardin, Malgorzata A. Domagalska, James Cotton, Hideo Imamura, Clinical sciences, Medical Genetics, Faculty of Sciences and Bioengineering Sciences, Faculty of Law and Criminology, Institute of Tropical Medicine [Antwerp] (ITM), The Wellcome Trust Sanger Institute [Cambridge], Charles University [Prague] (CU), Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Antwerp (UA), Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), This study was supported by Belgian Science Policy Office (TRIT, P7/41), Flemish Fund for Scientific Research (G.0.B81.12), and Department of Economy, Science and Innovation in Flanders ITM-SOFIB (SINGLE project, to J.C.D.). G.D. and B.C. were supported by the Research Foundation—Flanders (FWO) (grants 12Q8115N and 11O1614N, respectively). V.S., J.M. and P.V. were supported by Czech Science Foundation (project no. 13-07500S) and Charles University (UNCE 204017/2012). J.R.V. was supported by research grants from the KU Leuven (SymBioSys [PFV/10/016]) and the Hercules Foundation (ZW11-14). M.S., M.B., and J.A.C. were supported by the Wellcome Trust through the core support for the Wellcome Trust Sanger Institute (grant no. 098051). G.B., P.P., and G.F.S. were supported by Institut Pasteur strategic fund for the LeiSHield project (to G.F.S.). The funders had no role in study design, data collection and analysis, or preparation of the manuscript., and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]

Subjects
0301 basic medicine, MESH: Sequence Analysis, DNA, MESH: Gene Expression, [SDV]Life Sciences [q-bio], 030231 tropical medicine, Leishmania donovani, Aneuploidy, MESH: Cricetinae, gene dosage, Gene dosage, Microbiology, 03 medical and health sciences, MESH: Gene Expression Profiling, 0302 clinical medicine, MESH: Psychodidae, Virology, Gene expression, medicine, MESH: Aneuploidy, genomics, life cycle, MESH: Animals, aneuploidy, MESH: Environment, Gene, Biology, Genetics, Regulation of gene expression, Leishmania, MESH: Humans, MESH: Leishmania donovani, biology, MESH: Adaptation, Biological, Chromosome, Karyotype, MESH: Genome, Protozoan, medicine.disease, biology.organism_classification, QR1-502, Cell biology, 030104 developmental biology, Human medicine
Abstract

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania , a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro -cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro , aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness. IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania —a protozoan parasite that kills more than 30,000 people each year—is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo . We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

Published
2017

47. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

Author

Christian Cambillau, Christine Kellenberger, Alain Roussel, Aline Desmyter, Anaïs Gaubert, Jennifer Roche, Philippe Leone, Thi Trang Nhung Trinh, Yoan Duhoo, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Architecture et fonction des macromolécules biologiques ( AFMB ), and Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA )

Subjects
MESH: Sequence Homology, Amino Acid, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Plasma protein binding, MESH: Amino Acid Sequence, Biochemistry, MESH: Recombinant Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Animals, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide sequence, MESH: Bacterial Proteins, MESH : Porphyromonas gingivalis, MESH : Protein Conformation, alpha-Helical, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [ SDV.MHEP.ME ] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH : Amino Acid Sequence, MESH : Protein Binding, MESH: Camelus, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH : Sequence Homology, Amino Acid, MESH : Genetic Vectors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH : Crystallization, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Camelids, New World, MESH: Porphyromonas gingivalis, MESH: Models, Molecular, Camelus, MESH: Gene Expression, MESH : Cloning, Molecular, Biophysics, MESH: Sequence Alignment, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Single-Domain Antibodies, Microbiology, 03 medical and health sciences, Bacterial Proteins, Genetics, MESH: Protein Binding, Secretion, Molecular replacement, Protein Interaction Domains and Motifs, 5fwo, MESH: Cloning, Molecular, Amino Acid Sequence, Porphyromonas gingivalis, MESH: Protein Conformation, alpha-Helical, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, MESH: Protein Interaction Domains and Motifs, MESH : Molecular Chaperones, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Camelids, New World, MESH : Gene Expression, 030104 developmental biology, MESH: Binding Sites, Protein Conformation, beta-Strand, PorM, Molecular Chaperones, type IX secretion system, 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, MESH : Escherichia coli, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, MESH : Bacterial Secretion Systems, Research Communications, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Structural Biology, MESH: Genetic Vectors, 5lmj, MESH : Bacterial Proteins, Cloning, Molecular, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH : Protein Conformation, beta-Strand, MESH: Crystallization, 5lmw, MESH: Kinetics, MESH : Sequence Alignment, MESH : Camelus, Condensed Matter Physics, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH : Single-Domain Antibodies, Thermodynamics, MESH: Protein Conformation, beta-Strand, nb02, MESH : Kinetics, nb01, MESH: Thermodynamics, MESH: Molecular Chaperones, Crystallization, Camelids, New World, Protein Binding, crystallization chaperones, MESH : Recombinant Proteins, MESH : Models, Molecular, Genetic Vectors, Context (language use), Biology, MESH : Peptide Library, Peptide Library, Escherichia coli, Animals, nb130, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Peptide library, MESH : Thermodynamics, nb19, Binding Sites, Sequence Homology, Amino Acid, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Single-Domain Antibodies, biology.organism_classification, MESH: Crystallography, X-Ray, Kinetics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH : Animals, MESH: Peptide Library, MESH : Crystallography, X-Ray, camelid nanobodies, Sequence Alignment, 5lz0, MESH : Binding Sites, MESH : Protein Interaction Domains and Motifs, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

Published
2017

48. Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions

Author

Peter J. Lenting, Véronique Regnault, Olivier D. Christophe, Cécile V. Denis, Gabriel Aymé, Paulette Legendre, Amélie Harel, Vincent Muczynski, Cécile Loubière, Marc Vasse, Amine Bazaa, Delphine Borgel, Hémostase, Inflammation, Thrombose (HITH - U1176 Inserm - CHU Bicêtre), Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Inovarion SAS, Défaillance Cardiovasculaire Aiguë et Chronique (DCAC), Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Hôpital Foch [Suresnes], Service d'immuno-hématologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), REGNAULT, Véronique, AYME, Gabriel, and Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Vitamin K, [SDV]Life Sciences [q-bio], Gene Expression, MESH: Factor X, Plasma protein binding, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Mice, Knockout, Biochemistry, Mice, chemistry.chemical_compound, 0302 clinical medicine, MESH: RNA, Small Interfering, MESH: Animals, MESH: Nerve Tissue Proteins, RNA, Small Interfering, Internalization, Receptor, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Factor X Deficiency, MESH: Organ Specificity, media_common, Mice, Knockout, MESH: Kinetics, Chemistry, Factor X, Scavenger Receptors, Class A, Hematology, Endocytosis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV] Life Sciences [q-bio], C-Reactive Protein, Organ Specificity, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH: HEK293 Cells, MESH: Endocytosis, [SDV.IMM]Life Sciences [q-bio]/Immunology, [SDV.IMM.IMM] Life Sciences [q-bio]/Immunology/Immunotherapy, Protein Binding, [SDV.SP.MED] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, MESH: Gene Expression, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunoprecipitation, media_common.quotation_subject, Immunology, Nerve Tissue Proteins, MESH: Anticoagulants, MESH: Factor X Deficiency, [SDV.SP.PG] Life Sciences [q-bio]/Pharmaceutical sciences/Galenic pharmacology, Cell Line, 03 medical and health sciences, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, MESH: Mice, Inbred C57BL, MESH: C-Reactive Protein, MESH: Protein Binding, Animals, Humans, Scavenger receptor, MESH: Mice, MESH: Humans, Macrophages, HEK 293 cells, MESH: Macrophages, MESH: Vitamin K, Anticoagulants, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Cell Biology, Molecular biology, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, MESH: Scavenger Receptors, Class A, MESH: Cell Line, Mice, Inbred C57BL, Kinetics, [SDV.SP.PG]Life Sciences [q-bio]/Pharmaceutical sciences/Galenic pharmacology, HEK293 Cells, 030104 developmental biology, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, 030215 immunology
Abstract

International audience; Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 μM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K–dependent proteins was observed. Short hairpin RNA–mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI–deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti–vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI–mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.

Published
2017

49. Characterization of four endophytic fungi as potential consolidated bioprocessing hosts for conversion of lignocellulose into advanced biofuels

Author

Igor V. Grigoriev, Weihua Wu, John M. Gladden, Sirma Mihaltcheva, Kerrie Barry, Bernard Henrissat, Mary Bao Tran-Gyamfi, Mansi Chovatia, Alan Kuo, Erika Lindquist, Hope Hundley, Ryan W. Davis, Kurt LaButti, US Department of Energy Joint Genome Institute, U.S Department of Energy, U.S. Department of Energy (DOE)-U.S. Department of Energy (DOE), Dept Energy Great Lakes Bioenergy Res Ctr, University of California, Joint Genome Institute, United States Department of Energy, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), U.S. Department of Energy [Washington] (DOE)-U.S. Department of Energy [Washington] (DOE), University of California (UC), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), U.S. Department of Energy ( DOE ) -U.S. Department of Energy ( DOE ), Architecture et fonction des macromolécules biologiques ( AFMB ), and Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA )

Subjects
Ligninolytic enzymes, MESH : Polysaccharides, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Lignin, 7. Clean energy, Terpene, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH : Volatile Organic Compounds, Endophytes, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: Phylogeny, Phylogeny, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [ SDV.MHEP.ME ] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], General Medicine, MESH : Glycoside Hydrolases, Volatile organic compound, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, MESH: Genome, Fungal, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, CAZy, MESH: Sesquiterpenes, MESH: Fungal Proteins, Genome, Fungal, MESH: Monoterpenes, Sesquiterpenes, MESH: Gene Expression, Glycoside Hydrolases, 030106 microbiology, MESH: Alkyl and Aryl Transferases, MESH : Biofuels, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 03 medical and health sciences, Botany, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, Alkyl and Aryl Transferases, MESH : Genome, Fungal, MESH: Cellulase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Gene Expression, MESH : Monoterpenes, 030104 developmental biology, chemistry, Biofuels, MESH : Cellulases, 0301 basic medicine, MESH: Xylariales, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, MESH : Endophytes, MESH: Cellulases, Gene Expression, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Applied Microbiology and Biotechnology, Endophyte, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, chemistry.chemical_compound, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Cellulases, MESH : Fungal Proteins, 2. Zero hunger, Xylariales, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biofuel, Biotechnology, MESH : Sesquiterpenes, Cellulase, Biology, Plant use of endophytic fungi in defense, MESH: Endophytes, MESH: Volatile Organic Compounds, Fungal Proteins, Consolidated bioprocessing, Polysaccharides, MESH : Xylariales, MESH: Glycoside Hydrolases, Bioprocess, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH : Alkyl and Aryl Transferases, MESH: Biofuels, Volatile Organic Compounds, MESH : Lignin, MESH : Phylogeny, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, biology.organism_classification, MESH: Lignin, MESH: Polysaccharides, MESH : Cellulase, Monoterpenes, biology.protein, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

International audience; Recently, several endophytic fungi have been demonstrated to produce volatile organic compounds (VOCs) with properties similar to fossil fuels, called "mycodiesel," while growing on lignocellulosic plant and agricultural residues. The fact that endophytes are plant symbionts suggests that some may be able to produce lignocellulolytic enzymes, making them capable of both deconstructing lignocellulose and converting it into mycodiesel, two properties that indicate that these strains may be useful consolidated bioprocessing (CBP) hosts for the biofuel production. In this study, four endophytes Hypoxylon sp. CI4A, Hypoxylon sp. EC38, Hypoxylon sp. CO27, and Daldinia eschscholzii EC12 were selected and evaluated for their CBP potential. Analysis of their genomes indicates that these endophytes have a rich reservoir of biomass-deconstructing carbohydrate-active enzymes (CAZys), which includes enzymes active on both polysaccharides and lignin, as well as terpene synthases (TPSs), enzymes that may produce fuel-like molecules, suggesting that they do indeed have CBP potential. GC-MS analyses of their VOCs when grown on four representative lignocellulosic feedstocks revealed that these endophytes produce a wide spectrum of hydrocarbons, the majority of which are monoterpenes and sesquiterpenes, including some known biofuel candidates. Analysis of their cellulase activity when grown under the same conditions revealed that these endophytes actively produce endoglucanases, exoglucanases, and β-glucosidases. The richness of CAZymes as well as terpene synthases identified in these four endophytic fungi suggests that they are great candidates to pursue for development into platform CBP organisms.

Published
2017

50. Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis

Author

Alejandro Buschiazzo, Ariel E. Mechaly, Nicole Larrieux, Albert I. Ko, Fabiana San Martin, Elsio A. Wunder, Mathieu Picardeau, Felipe Trajtenberg, Molecular and structural microbiology / Microbiología Molecular y Estructural [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Yale School of Public Health (YSPH), Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP), Biologie des Spirochètes / Biology of Spirochetes, Institut Pasteur [Paris], Integrative Microbiology of Zoonotic Agents [Paris and Montevideo] (IMiZA), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris], Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ), Institut Pasteur [Paris] (IP), and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris] (IP)

Subjects
0301 basic medicine, MESH: Leptospira interrogans, MESH: Sequence Homology, Amino Acid, Protein Data Bank (RCSB PDB), Gene Expression, MESH: Amino Acid Sequence, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, MESH: Leptospira, MESH: Recombinant Proteins, Plasmid, Structural Biology, leptospirosis, spirochetes, Cloning, Molecular, Peptide sequence, MESH: Bacterial Proteins, Leptospira, biology, Chemistry, MESH: Escherichia coli, Condensed Matter Physics, Recombinant Proteins, motility, SAD phasing, flagella, Leptospira interrogans, Plasmids, MESH: Gene Expression, FcpA, Biophysics, MESH: Sequence Alignment, Sequence alignment, Flagellum, Leptospira biflexa, MESH: Flagella, Article, 03 medical and health sciences, Bacterial Proteins, MESH: Plasmids, Escherichia coli, Genetics, medicine, [CHIM.CRIS]Chemical Sciences/Cristallography, MESH: Cloning, Molecular, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Sequence Homology, Amino Acid, biology.organism_classification, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Sequence Alignment
Abstract

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genusLeptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenicLeptospiraparticularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA fromL. interroganswas purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic speciesL. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

Published
2017

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