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1. Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

Author

Eric Oswald, Christian Chalut, Marie Penary, Patricia Martin, Maïwenn Olier, Jean-Philippe Nougayrède, Delphine Payros, Michèle Boury, Marc Audebert, Christophe Garcie, Ingrid Marcq, Sören Schubert, Giuseppe Magistro, Centre de Physiopathologie Toulouse Purpan (CPTP - U1043 INSERM - UMR5282 CNRS - UT3), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service de Bactériologie-Hygiène [CHU Toulouse] (Hôpital Purpan), Centre Hospitalier Universitaire de Toulouse, Neuro-Gastroentérologie et Nutrition (NGN), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole supérieure d'agriculture de Purpan (ESAP), ToxAlim (ToxAlim), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Institut de pharmacologie et de biologie structurale (IPBS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre de Physiopathologie Toulouse Purpan (CPTP), CHU Toulouse [Toulouse], Ecole supérieure d'agriculture de Purpan (ESAP)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Métabolisme et Xénobiotiques (ToxAlim-MeX), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), FEDER (MYCA), Centre de Physiopathologie de Toulouse Purpan (CPTP, Sepsis: the critical role of iron), French National Research Agency (ANR), Institut de Recherche en Santé Digestive (IRSD ), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Chalut, Christian, Service Bactériologie et hygiène [CHU Toulouse], Institut Fédératif de Biologie (IFB), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Pôle Biologie [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), and Institut National de la Recherche Agronomique (INRA)-Ecole supérieure d'agriculture de Purpan (ESAP)

Subjects
Siderophore, GENE-CLUSTER, Siderophores, Transferases (Other Substituted Phosphate Groups), HIGH-PATHOGENICITY ISLAND, COMPLETE GENOME SEQUENCE, PHOSPHOPANTETHEINYL TRANSFERASE, PHYLOGENETIC DISTRIBUTION, 4'-PHOSPHOPANTETHEINYL TRANSFERASE, IRON ACQUISITION, MAMMALIAN-CELLS, IN-VIVO, VIRULENCE, medicine.disease_cause, Yersiniabactin, chemistry.chemical_compound, Mice, Gene cluster, Biology (General), Escherichia coli Infections, ComputingMilieux_MISCELLANEOUS, Genetics, 0303 health sciences, Escherichia coli Proteins, Glycopeptides, Isoenzymes, Female, Research Article, Genomic Islands, QH301-705.5, Immunology, Virulence, Biology, Microbiology, Enterobactin, 03 medical and health sciences, Bacterial Proteins, Phenols, Virology, Sepsis, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Escherichia coli, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Gene, 030304 developmental biology, 030306 microbiology, RC581-607, Pathogenicity island, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Mice, Inbred C57BL, Thiazoles, chemistry, Polyketides, Mutation, Parasitology, Immunologic diseases. Allergy, Peptides, Gene Deletion, Mutagens
Abstract

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4′-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies., Author Summary The synthesis of numerous molecules involved in the virulence potential and fitness of pathogenic bacteria requires a particular enzyme family, i.e. phosphopantetheinyl transferases (PPTases). To date, the synthesis of a given bioactive metabolite was thought to require a specific PPTase. As PPTases are being investigated as promising targets for antibacterial development, we addressed the question of a possible functional interchangeability between PPTases in Escherichia coli. PPTases are known to be involved in the synthesis of low-molecular weight iron chelators (siderophores), and of a genotoxin named colibactin. Here we demonstrated interplay between the synthesis pathways leading to the production of siderophores and of colibactin. We showed that inactivation of both PPTases abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model. To our knowledge, this is the first demonstration of interplay between multiple PPTases-requiring pathways leading to the biosynthesis of functionally distinctive virulence factors, in a given microorganism. The extensive substrate specificity of PPTase ClbA could account for the co-selection and co-evolution of genomic islands encoding colibactin and yersiniabactin siderophore.

Published
2013
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2. Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity

Author

Maïwenn Olier, Christel Salvador-Cartier, Michèle Boury, Thomas Secher, Ulrich Dobrindt, Jean Fioramonti, Ingrid Marcq, Eric Gaultier, Marie Penary, Jean-Philippe Nougayrède, Valerie Bacquie, Eric Oswald, ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Centre de Physiopathologie Toulouse Purpan (CPTP), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Neuro-Gastroentérologie et Nutrition (NGN), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole supérieure d'agriculture de Purpan (ESAP), Centre de Physiopathologie de Toulouse Purpan, Institut National de la Recherche Agronomique (INRA), University of Münster, CHU Toulouse [Toulouse], non-profit Association F. Aupetit (AFA), ANR-09-MIEN-0005,COLIBACTIN,Conséquences sur la santé de la colonisation néonatale du tractus digestif par des entérobactéries produisant la Colibactine(2009), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Centre de Physiopathologie de Toulouse-Purpan (INSERM U563 - CNRS UMR1037), CHU Toulouse [Toulouse]-Institut Claudius Regaud-Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de lutte contre le cancer (CLCC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Physiopathologie Toulouse Purpan ex IFR 30 et IFR 150 (CPTP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire de Purpan (CHU Purpan), ANR-09-MIEN-005-01, AFA, Institut National de la Recherche Agronomique (INRA)-Ecole supérieure d'agriculture de Purpan (ESAP), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Jean-Philippe, Nougayrede, and MIE : Maladies infectieuses, immunité et environnement - Conséquences sur la santé de la colonisation néonatale du tractus digestif par des entérobactéries produisant la Colibactine - - COLIBACTIN2009 - ANR-09-MIEN-0005 - MIE - VALID

Subjects
Male, colitis, Mutant, medicine.disease_cause, law.invention, Probiotic, Gene Knockout Techniques, law, genotoxin, DSS, 0303 health sciences, Gastroenterology, Colibactin, 3. Good health, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, CD4(+) CD45RB(high) T-cell transfer model, probiotic, Research Paper, Microbiology (medical), DNA damage, Biology, Microbiology, Cell Line, 03 medical and health sciences, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Escherichia coli, Animals, Colitis, Rats, Wistar, Gene, 030304 developmental biology, 030306 microbiology, Probiotics, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Epithelial Cells, medicine.disease, biology.organism_classification, Inflammatory Bowel Diseases, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Rats, Disease Models, Animal, inflammation, Polyketides, CD4+ CD45RBhigh T-cell transfer model, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Genotoxicity, Bacteria, Mutagens
Abstract

International audience; Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Published
2012
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3. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways

Author

Jean-Philippe Nougayrède, C. Erec Stebbins, Ascel Samba-Louaka, Eric Oswald, Brenda A. Schulman, Claude Watrin, David M. Duda, Grégory Jubelin, Yun Hsu, Marie Penary, Rika Nobe, Frédéric Taieb, Unité de Microbiologie (MIC), Institut National de la Recherche Agronomique (INRA), Interactions hôtes-agents pathogènes [Toulouse] (IHAP), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Department of Structural Biology, Howard Hughes Medical Institute (HHMI)-St Jude Children's Research Hospital, Rockefeller University [New York], Institut de Recherche en Santé Digestive (IRSD ), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), St Jude Children's Research Hospital, Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), taieb, frederic, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and CHU Toulouse [Toulouse]

Subjects
Infectious Diseases/Gastrointestinal Infections, Cell, Cell Biology/Cell Growth and Division, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], NEDD8, Cell Biology/Cell Signaling, Infectious Diseases/Bacterial Infections, sécrétion, Ubiquitin, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], invertébré, Microbiology/Parasitology, Biology (General), Host cell nucleus, Cells, Cultured, Escherichia coli Infections, 0303 health sciences, ubiquitine, biology, Escherichia coli Proteins, 030302 biochemistry & molecular biology, Cell Cycle, Microbiology and Parasitology, Intracellular Signaling Peptides and Proteins, in vitro, Cell cycle, Microbiologie et Parasitologie, 3. Good health, Cell biology, Ubiquitin ligase, Protein Transport, medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Signal transduction, escherichia coli, bactérie pathogène, Cyclin-Dependent Kinase Inhibitor p27, Signal Transduction, Research Article, Infectious Diseases/Tropical and Travel-Associated Diseases, Cyclin-Dependent Kinase Inhibitor p21, NEDD8 Protein, QH301-705.5, Immunology, Blotting, Western, Microbiology, 03 medical and health sciences, Virology, Two-Hybrid System Techniques, Genetics, medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, Humans, Immunoprecipitation, [SDV.EE.SANT] Life Sciences [q-bio]/Ecology, environment/Health, Ubiquitins, Molecular Biology, Actin, 030304 developmental biology, Cell Nucleus, [SDV.EE.SANT]Life Sciences [q-bio]/Ecology, environment/Health, SKP Cullin F-Box Protein Ligases, Ubiquitination, [SDV.EE.IEO] Life Sciences [q-bio]/Ecology, environment/Symbiosis, RC581-607, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, vertébré, Actins, Rats, biology.protein, Parasitology, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Immunologic diseases. Allergy, [SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis
Abstract

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria., Author Summary Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.

Published
2010
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4. MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling

Author

Cyril Favard, Daniel Tovar, Barbara Lortal, Isabelle Lajoie-Mazenc, Marie Penary, Anne Pradines, Sophie Allart, Gilles Favre, Meryem Brihoum, Departement /u563 : Oncogenèse, Signalisation et Innovation thérapeutique, Centre de Physiopathologie Toulouse Purpan (CPTP), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Claudius Regaud, Institut Claude de Préval (ICP), 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, Institut FRESNEL (FRESNEL), Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), 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)-Université Toulouse III - Paul Sabatier (UT3), 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 Claudius Regaud, Université de Toulouse (UT)-Université de Toulouse (UT)-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), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Signal Transduction, MESH: Epidermal Growth Factor, RHOA, Receptor expression, RHOB, Endocytic cycle, Fluorescent Antibody Technique, MESH: Amino Acid Sequence, MESH: Base Sequence, Biochemistry, Epidermal growth factor, MESH: RNA, Small Interfering, MESH: Microscopy, Confocal, RNA, Small Interfering, rhoB GTP-Binding Protein, MESH: Fluorescent Antibody Technique, 0303 health sciences, MESH: Guanosine Triphosphate, Microscopy, Confocal, biology, 030302 biochemistry & molecular biology, Cell biology, ErbB Receptors, Endocytic vesicle, Guanosine Triphosphate, Microtubule-Associated Proteins, Protein Binding, Signal Transduction, MESH: Cell Line, Tumor, Endosome, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Molecular Sequence Data, MESH: Receptor, Epidermal Growth Factor, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 03 medical and health sciences, Cell Line, Tumor, MESH: Protein Binding, Humans, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, MESH: rhoB GTP-Binding Protein, MESH: Humans, MESH: Molecular Sequence Data, Base Sequence, Epidermal Growth Factor, Cell Biology, Actin cytoskeleton, MESH: Microtubule-Associated Proteins, biology.protein
Abstract

International audience; Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.

Published
2007
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5. Reversible inactivation of the transcriptional function of P53 protein by farnesylation

Author

Marie Penary, Anne Pradines, Danièle Berg, Gilles Favre, Antoine Casteignau, Mustapha Tohfe, Bettina Couderc, Departement /u563 : Oncogenèse, Signalisation et Innovation thérapeutique, Centre de Physiopathologie Toulouse Purpan (CPTP), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Claudius Regaud, FTI-277 was generously provided by S. Sebti, Moffitt Cancer Center, USF, Tampa, FL with the permission of the University of Pittsburgh. This work was supported by INSERM (F), a grant from the REGION MIDI-PYRENNES (F) and a grant from the University Paul Sabatier (F). We are grateful to the GVPN Network (Genethon, Evry) which provides us with the AdeGFP., and Maylin, Françoise

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, Apoptosis, MESH: Protein Structure, Tertiary, 0302 clinical medicine, Protein structure, MESH: Genetic Vectors, Enzyme Inhibitors, Cellular localization, 0303 health sciences, Farnesyl Transferase Inhibitor, MESH: Adenoviridae, Biochemistry, MESH: Recombinan, MESH: Enzyme Inhibitors, 030220 oncology & carcinogenesis, Biotechnology, Research Article, Transcriptional Activation, MESH: Cell Line, Tumor, MESH: Mutation, lcsh:Biotechnology, Recombinant Fusion Proteins, Genetic Vectors, MESH: Protein Isoprenylation, Protein Prenylation, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Adenoviridae, Proto-Oncogene Proteins p21(ras), 03 medical and health sciences, MESH: Proto-Oncogene Proteins p21(ras), Prenylation, [SDV.CAN] Life Sciences [q-bio]/Cancer, lcsh:TP248.13-248.65, MESH: Cell Proliferation, Cell Line, Tumor, Farnesyltranstransferase, Humans, MESH: Farnesyltranstransferase, 030304 developmental biology, Cell Proliferation, MESH: Humans, C-terminus, MESH: Apoptosis, Cell Membrane, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Protein Structure, Tertiary, Mutation, Protein prenylation, Lipid modification, Tumor Suppressor Protein p53, MESH: Cell Membrane
Abstract

Background The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function. Results We constructed an adenoviral vector coding a chimeric p53 protein by fusing p53 sequence with the 21 COOH term amino acids sequence of H-Ras. Indeed, the translation products of Ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a CAAX motif present at the C terminus of Ras proteins. The chimeric p53HRCaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. The farnesylated form of p53 resided mainly in the cytosol, where it is non-functional. Farnesyl transferase inhibitors (FTIs) specifically inhibited farnesyl isoprenoid lipid modification of proteins. Following treatment of the cells with an FTI, p53HRCaax underwent translocation into the nucleus where it retained transcription factor activity. Shifting p53 into the nucleus resulted in the induction of p21waf1/CIP1 and Bax transcription, cell growth arrest, caspase activation and apoptosis. Conclusion Artificial protein farnesylation impaired the transcriptional activity of p53. This could be prevented by Farnesyl transferase inhibition. These data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor.

Published
2005
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