Descriptor: "MESH: Phosphorylation" - Searchworks@Jio Institute Digital Library Search Results

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643 results on '"MESH: Phosphorylation"'

1. Rare pathogenic variants in WNK3 cause X-linked intellectual disability

Author: Sébastien Küry, Jinwei Zhang, Thomas Besnard, Alfonso Caro-Llopis, Xue Zeng, Stephanie M. Robert, Sunday S. Josiah, Emre Kiziltug, Anne-Sophie Denommé-Pichon, Benjamin Cogné, Adam J. Kundishora, Le T. Hao, Hong Li, Roger E. Stevenson, Raymond J. Louie, Wallid Deb, Erin Torti, Virginie Vignard, Kirsty McWalter, F. Lucy Raymond, Farrah Rajabi, Emmanuelle Ranza, Detelina Grozeva, Stephanie A. Coury, Xavier Blanc, Elise Brischoux-Boucher, Boris Keren, Katrin Õunap, Karit Reinson, Pilvi Ilves, Ingrid M. Wentzensen, Eileen E. Barr, Solveig Heide Guihard, Perrine Charles, Eleanor G. Seaby, Kristin G. Monaghan, Marlène Rio, Yolande van Bever, Marjon van Slegtenhorst, Wendy K. Chung, Ashley Wilson, Delphine Quinquis, Flora Bréhéret, Kyle Retterer, Pierre Lindenbaum, Emmanuel Scalais, Lindsay Rhodes, Katrien Stouffs, Elaine M. Pereira, Sara M. Berger, Sarah S. Milla, Ankita B. Jaykumar, Melanie H. Cobb, Shreyas Panchagnula, Phan Q. Duy, Marie Vincent, Sandra Mercier, Brigitte Gilbert-Dussardier, Xavier Le Guillou, Séverine Audebert-Bellanger, Sylvie Odent, Sébastien Schmitt, Pierre Boisseau, Dominique Bonneau, Annick Toutain, Estelle Colin, Laurent Pasquier, Richard Redon, Arjan Bouman, Jill. A. Rosenfeld, Michael J. Friez, Helena Pérez-Peña, Syed Raza Akhtar Rizvi, Shozeb Haider, Stylianos E. Antonarakis, Charles E. Schwartz, Francisco Martínez, Stéphane Bézieau, Kristopher T. Kahle, Bertrand Isidor, Clinical Genetics, Clinical sciences, Medical Genetics, Reproduction and Genetics, Centre hospitalier universitaire de Nantes (CHU Nantes), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ)-Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ), University of Exeter, MitoVasc - Physiopathologie Cardiovasculaire et Mitochondriale (MITOVASC), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), The Greenwood Genetic Center, GeneDx [Gaithersburg, MD, USA], Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Centre hospitalier universitaire de Poitiers (CHU Poitiers), Centre Hospitalier Régional Universitaire de Brest (CHRU Brest), CHU Pontchaillou [Rennes], Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Genève = University of Geneva (UNIGE), Yale School of Medicine [New Haven, Connecticut] (YSM), This work was granted by the French network of University Hospitals HUGO ('Hôpitaux Universitaires du Grand Ouest'), the French Ministry of Health, and and the Health Regional Agencies from Poitou-Charentes (represented by Frédérique Allaire), Bretagne, Pays de la Loire, and Centre-Val de Loire (HUGODIMS, 2013, RC14_0107). W.K.C. was supported by grants from Simons Foundation Autism Research Initiative, United
Subjects: MESH: Symporters, Exome sequencing, Male, KCC2, Mutation, Missense, MESH: Catalytic Domain, Neurodevelopmental disease, Protein Serine-Threonine Kinases, X-linked intellectual disability, MESH: Brain, WNK3, SDG 3 - Good Health and Well-being, Loss of Function Mutation, Catalytic Domain, MESH: Mental Retardation, X-Linked, Humans, Phosphorylation, MESH: Hemizygote, Genetics (clinical), Hemizygote, MESH: Mutation, Missense, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, MESH: Phosphorylation, Symporters, Brain, MESH: Loss of Function Mutation, MESH: Protein Serine-Threonine Kinases, MESH: Male, Mental Retardation, X-Linked, Maternal Inheritance, MESH: Maternal Inheritance
Abstract: PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had identifier with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked identifier with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.
Published: 2022

2. Complex roles for reactive astrocytes in the triple transgenic mouse model of Alzheimer disease

Author: Martine Guillermier, Carole Escartin, Océane Guillemaud, Alexis-Pierre Bemelmans, Charlène Joséphine, Gilles Bonvento, Emmanuel Brouillet, Kelly Ceyzériat, Thomas Saint-Georges, Philippe Hantraye, Maria-Angeles Carrillo-de Sauvage, Karine Cambon, Sueva Bernier, Fanny Petit, Lucile Ben Haim, Anne-Sophie Hérard, Laboratoire des Maladies Neurodégénératives - UMR 9199 (LMN), Centre National de la Recherche Scientifique (CNRS)-Service MIRCEN (MIRCEN), Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), 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 Paris-Saclay-Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Service MIRCEN (MIRCEN), ANR-16-TERC-0016,DecodAstro,Decoder la complexité de la réactivité astrocytaire dans les maladies neurodégénératives(2016), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie François JACOB (JACOB), and 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)
Subjects: MESH: Signal Transduction, 0301 basic medicine, Aging, MESH: Hippocampus, JAK-STAT3 pathway, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Hippocampus, Hippocampal formation, MESH: Janus Kinase 2, 0302 clinical medicine, Neuroinflammation, MESH: Animals, SOCS3, Phosphorylation, 0303 health sciences, Chemistry, General Neuroscience, MESH: STAT3 Transcription Factor, MESH: tau Proteins, 3. Good health, medicine.anatomical_structure, Alzheimer disease, Alzheimer's disease, Signal Transduction, Astrocyte, STAT3 Transcription Factor, Genetically modified mouse, Elevated plus maze, Amyloid, MESH: Mice, Transgenic, Amyloidogenic Proteins, Mice, Transgenic, tau Proteins, Viral vector, 03 medical and health sciences, medicine, Animals, 030304 developmental biology, MESH: Amyloidogenic Proteins, MESH: Phosphorylation, Activator (genetics), Janus Kinase 2, medicine.disease, MESH: Astrocytes, Disease Models, Animal, 030104 developmental biology, Astrocytes, Reactive astrocytes, Neurology (clinical), Tau, MESH: Disease Models, Animal, Geriatrics and Gerontology, Neuroscience, MESH: Alzheimer Disease, 030217 neurology & neurosurgery, Developmental Biology
Abstract: In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction.We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops Tau hyperphosphorylation and aggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in the hippocampus of 3xTg-AD mice, did not significantly influence Tau phosphorylation or amyloid processing and deposition, at early, advanced and terminal stage of the disease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve short-term spatial memory in the Y maze but it reduced anxiety in the elevated plus maze. Our unique approach to specifically manipulate reactive astrocytes in situ show these cells may impact behavioral outcomes without influencing Tau or amyloid pathology.
Published: 2020

3. No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

Author: Sébastien Chamois, Claire Torchet, Rénette Saint-Fort, Julien Henri, Lionel Benard, Albertas Navickas, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes (LBMCE), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, RNA Stability, [SDV]Life Sciences [q-bio], General Physics and Astronomy, RNA decay, Ribosome, MESH: Saccharomyces cerevisiae Proteins, 0302 clinical medicine, Exoribonuclease, Phosphorylation, lcsh:Science, MESH: RNA Ligase (ATP), 0303 health sciences, Multidisciplinary, MRNA cleavage, Chemistry, MESH: RNA Stability, MESH: Saccharomyces cerevisiae, mRNA surveillance, Cell biology, MESH: Exoribonucleases, Saccharomyces cerevisiae Proteins, Science, Endoribonuclease, Saccharomyces cerevisiae, Cleavage (embryo), Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, 030304 developmental biology, MESH: RNA, Messenger, Messenger RNA, RNA quality control, MESH: Phosphorylation, MESH: RNA, Fungal, RNA Ligase (ATP), RNA, RNA, Fungal, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, 030104 developmental biology, Exoribonucleases, lcsh:Q, Ribosomes, MESH: Ribosomes, 030217 neurology & neurosurgery
Abstract: The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5′-hydroxylated RNA fragments requiring 5′-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs., In the No-Go decay mRNA surveillance pathway, mRNAs containing stalled ribosomes are cleaved by an endoribonuclease. Here the authors show the endonucleolytic cleavage on the artificial No-Go decay target mRNAs, revealing downstream decay process by Trl1 kinase and the 5′ to 3′ exonuclease Dxo1.
Published: 2020

4. Phosphorylated tau181 in plasma as a potential biomarker for Alzheimer’s disease in adults with Down syndrome

Author: Kaj Blennow, Sylvain Lehmann, Nicholas J. Ashton, Jordi Pegueroles, Rafael Blesa, Maria Florencia Iulita, Alberto Lleó, Miren Altuna, Valle Camacho, Henrik Zetterberg, Maria Carmona-Iragui, Daniel Alcolea, Diana Garzón, Susana Fernández, Juan Lantero-Rodriguez, Santiago Medrano-Martorell, Juan Fortea, Jordi Clarimón, Thomas K. Karikari, Olivia Belbin, Alexandre Bejanin, Laura Videla, Sílvia Valldeneu, Bessy Benejam, Isabel Barroeta, Victor Montal, Hospital de la Santa Creu i Sant Pau, Centro de Investigacion Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III [Madrid] (ISC), Universitat Autònoma de Barcelona (UAB), Sahlgrenska Academy at University of Gothenburg [Göteborg], Sahlgrenska University Hospital [Gothenburg], UK Dementia Research Institute (UK DRI), University College of London [London] (UCL), Institute of Neurology [London], Fundació Catalana de Síndrome de Down [Barcelona], University of Gothenburg (GU), King‘s College London, IMIM-Hospital del Mar, Generalitat de Catalunya, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Institut des Neurosciences de Montpellier (INM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Cellules Souches, Plasticité Cellulaire, Médecine Régénératrice et Immunothérapies (IRMB), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), and Herrada, Anthony
Subjects: Male, 0301 basic medicine, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, General Physics and Astronomy, MESH: Cognition, Disease, Gastroenterology, Cognition, 0302 clinical medicine, Neurofilament Proteins, Medicine, Phosphorylation, MESH: Neurofilament Proteins, Cerebral Cortex, education.field_of_study, MESH: Middle Aged, Multidisciplinary, Area under the curve, Middle Aged, Alzheimer's disease, MESH: Amyloid beta-Peptides, MESH: tau Proteins, Area Under Curve, Disease Progression, Biomarker (medicine), MESH: Disease Progression, Female, medicine.symptom, Adult, medicine.medical_specialty, Down syndrome, Science, Population, tau Proteins, MESH: Atrophy, Predictive markers, Asymptomatic, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, MESH: Cross-Sectional Studies, Atrophy, Alzheimer Disease, Internal medicine, Humans, Dementia, education, MESH: Humans, Amyloid beta-Peptides, MESH: Phosphorylation, business.industry, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, MESH: Adult, MESH: Down Syndrome, General Chemistry, medicine.disease, MESH: Cerebral Cortex, MESH: Male, Cross-Sectional Studies, 030104 developmental biology, MESH: Biomarkers, MESH: Area Under Curve, Down Syndrome, business, MESH: Female, MESH: Alzheimer Disease, Biomarkers, 030217 neurology & neurosurgery
Abstract: Plasma tau phosphorylated at threonine 181 (p-tau181) predicts Alzheimer’s disease (AD) pathology with high accuracy in the general population. In this study, we investigated plasma p-tau181 as a biomarker of AD in individuals with Down syndrome (DS). We included 366 adults with DS (240 asymptomatic, 43 prodromal AD, 83 AD dementia) and 44 euploid cognitively normal controls. We measured plasma p-tau181 with a Single molecule array (Simoa) assay. We examined the diagnostic performance of p-tau181 for the detection of AD and the relationship with other fluid and imaging biomarkers. Plasma p-tau181 concentration showed an area under the curve of 0.80 [95% CI 0.73–0.87] and 0.92 [95% CI 0.89–0.95] for the discrimination between asymptomatic individuals versus those in the prodromal and dementia groups, respectively. Plasma p-tau181 correlated with atrophy and hypometabolism in temporoparietal regions. Our findings indicate that plasma p-tau181 concentration can be useful to detect AD in DS., Plasma tau phosphorylated at threonine 181 (p-tau181) predicts Alzheimer’s disease (AD) pathology. Here, the authors investigated whether plasma ptau181 could be a potential biomarker of AD in individuals with Down syndrome (DS) and find plasma p-tau181 can detect AD in DS adults.
Published: 2021

5. G protein‐coupled receptors can control the Hippo/YAP pathway through Gq signaling

Author: Zindel, Diana, Mensat, Patrick, Vol, Claire, Homayed, Zeinab, Charrier-Savournin, Fabienne, Trinquet, Eric, Banères, Jean-Louis, Pin, Jean-Philippe, Pannequin, Julie, Roux, Thomas, Dupuis, Elodie, Prézeau, Laurent, Charrier‐Savournin, Fabienne, Banères, Jean‐Louis, Pin, Jean‐Philippe, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Guerineau, Nathalie C., Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Cisbio Bioassays, and Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Cellular differentiation, [SDV]Life Sciences [q-bio], Hippo pathway, Cell Cycle Proteins, MESH: Receptors, G-Protein-Coupled, inverse agonist, Biochemistry, Receptors, G-Protein-Coupled, 0302 clinical medicine, Phosphorylation, Receptor, Tissue homeostasis, Chemistry, Ghrelin receptor, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], MESH: Transcription Factors, MESH: Gene Expression Regulation, Cell biology, [SDV] Life Sciences [q-bio], MESH: GTP-Binding Protein alpha Subunits, Gq-G11, MESH: HEK293 Cells, Biotechnology, Cell signaling, G protein, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: GTP-Binding Protein alpha Subunits, G12-G13, Protein Serine-Threonine Kinases, GTP-Binding Protein alpha Subunits, G12-G13, MESH: Protein-Serine-Threonine Kinases, 03 medical and health sciences, MESH: Cell Cycle Proteins, MESH: Activating Transcription Factor 6, Genetics, Humans, Hippo Signaling Pathway, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, constitutive activity, G protein-coupled receptor, Hippo signaling pathway, MESH: Humans, MESH: Phosphorylation, fungi, Activating Transcription Factor 6, HEK293 Cells, 030104 developmental biology, Gene Expression Regulation, GTP-Binding Protein alpha Subunits, Gq-G11, cellular signaling, 030217 neurology & neurosurgery, Transcription Factors
Abstract: International audience; The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.
Published: 2021

6. PERIOD-controlled deadenylation of the timeless transcript in the Drosophila circadian clock

Author: Eric Jacquet, Christian Papin, Prishila Ponien, Elisabeth Chélot, François Rouyer, Béatrice Martin, Brigitte Grima, Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), and Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Period Circadian Proteins, Transcription, Genetic, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV]Life Sciences [q-bio], Circadian clock, CLOCK Proteins, MESH: Down-Regulation, MESH: ARNTL Transcription Factors, 0302 clinical medicine, Drosophila Proteins, MESH: Animals, mRNA poly(A) tail, Phosphorylation, 0303 health sciences, Multidisciplinary, [SDV.NEU.PC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, ARNTL Transcription Factors, [SDV.NEU.SC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, CAF1/POP2, Period Circadian Proteins, Biological Sciences, MESH: Gene Expression Regulation, Circadian Rhythm, Cell biology, CLOCK, Drosophila melanogaster, MESH: Ribonucleases, MESH: Circadian Clocks, MESH: Drosophila Proteins, Timeless, Period (gene), Down-Regulation, Biology, MESH: Drosophila melanogaster, 03 medical and health sciences, Ribonucleases, Circadian Clocks, CCR4–NOT complex, clock genes, CCR4-NOT complex, Animals, RNA, Messenger, MESH: Circadian Rhythm, Gene, Transcription factor, MESH: RNA, Messenger, 030304 developmental biology, Messenger RNA, MESH: Phosphorylation, MESH: Transcription, Genetic, MESH: CLOCK Proteins, Gene Expression Regulation, circadian rhythms, 030217 neurology & neurosurgery
Abstract: Significance Circadian oscillators rely on transcriptional negative feedback loops. In Drosophila , the key transcriptional repressor PERIOD (PER) slowly accumulates during the night under the control of its partner TIMELESS (TIM). A large number of posttranslational mechanisms regulate PER and TIM stability, but no mechanisms affecting the stability of their transcripts have been described. mRNA stability depends on the length of the poly(A) tail. We show that a deadenylase, POP2, shortens tim mRNA poly(A) tail, thus decreasing tim mRNA and TIM protein levels. Moreover, POP2 activity on tim mRNA appears to be inhibited by PER itself. These results reveal polyadenylation control of a core clock gene transcript and suggest that the repressor of the feedback loop also acts as a posttranscriptional regulator.
Published: 2019

7. Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases

Author: Pierre Savatier, Nuria Montserrat, Laia Richart-Ginés, Camille Stephan-Otto Attolini, Raquel Bernad, Susana Prieto, Marta I. Sierra, Joaquín Pastor, Magdalena Zernicka-Goetz, Sonia Sánchez Martínez, Daniel Fisher, Javier Munoz, Sandrina Nóbrega-Pereira, Noelia Alcazar, Ana Martinez-Val, Gonzalo Gómez-López, Mario F. Fraga, Marta N. Shahbazi, Elisabeth Simboeck, Carmen Blanco-Aparicio, Elena Garreta, Osvaldo Graña-Castro, Alain Camasses, Irène Aksoy, Sagrario Ortega, Cian J. Lynch, Maribel Muñoz-Martin, Isabel A. Calvo, Agustín F. Fernández, Manuel Serrano, Carolina Tarantino, Spanish National Cancer Research Center (CNIO), Barcelona Institute of Science and Technology (BIST), University of Cambridge [UK] (CAM), Institute for Research in Biomedicine ( iBiMED), Universidade de Aveiro, Institut Curie [Paris], Institut cellule souche et cerveau (U846 Inserm - UCBL1), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Instituto Universitario de Oncología del Principado de Asturias [Oviedo, Spain] (IUOPA), Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Division of Biology and Biological Engineering [Pasadena, USA] (BBE), California Institute of Technology (CALTECH), Institució Catalana de Recerca i Estudis Avançats (ICREA), Centre National de la Recherche Scientifique (CNRS), Leverhulme Trust, Wellcome Trust, Generalitat de Catalunya, European Research Council, Fondation pour la Recherche Médicale, European Commission, Agence Nationale de la Recherche (France), Université de Lyon, Ministerio de Economía y Competitividad (España), Centro Nacional de Investigaciones Oncológicas (España), Institut National du Cancer (France), Ligue Nationale contre le Cancer (France), Asociación Española Contra el Cáncer, Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, Agencia Estatal de Investigación (España), Principado de Asturias, Liberbank, Obra Social Cajastur, Banco Santander, Fundación Botín, Fundación 'la Caixa', Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and KARLI, Mélanie
Subjects: MESH: Signal Transduction, Cellular differentiation, [SDV]Life Sciences [q-bio], ADN, RNA polymerase II, Stem cells, Mice, 0302 clinical medicine, MESH: DNA Methylation, MESH: Animals, Phosphorylation, Promoter Regions, Genetic, Induced pluripotent stem cell, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, biology, Kinase, Chemistry, Cell Differentiation, Cyclin-Dependent Kinases, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Enhancer Elements, Genetic, MESH: Cyclin-Dependent Kinases, 030220 oncology & carcinogenesis, MESH: Pluripotent Stem Cells, Female, RNA Polymerase II, Signal transduction, Cèl·lules mare, Metilació, Signal Transduction, Pluripotent Stem Cells, MESH: Cell Differentiation, Methylation, MESH: Cyclin-Dependent Kinase 8, 03 medical and health sciences, MESH: Promoter Regions, Genetic, Animals, Humans, Kinase activity, Enhancer, MESH: Mice, 030304 developmental biology, MESH: Humans, MESH: Phosphorylation, Cell Biology, DNA, DNA Methylation, Cyclin-Dependent Kinase 8, MESH: RNA Polymerase II, biology.protein, Cyclin-dependent kinase 8, MESH: Enhancer Elements, Genetic, MESH: Female
Abstract: Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF–MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF–MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity., M.N.S. was funded by a Leverhulme Trust early career fellowship. Work in the laboratory of M.Z.-G. was funded by the Wellcome Trust (098287/Z/12/Z) and the European Research Council (ERC) (669198). I.C. was funded by the Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and European Social Fund. I.A. and P.S. were supported by the Fondation pour la Recherche Medicale (DEQ20170336757), Infrastructure Nationale en Biologie et Santé INGESTEM (ANR-11-INBS-0009), IHU-B CESAME (ANR-10-IBHU-003), LabEx REVIVE (ANR-10-LABX-73), LabEx DEVweCAN (ANR-10-LABX-0061) and LabEx CORTEX (ANR-11-LABX-0042) of University of Lyon within the programme ‘Investissements d’Avenir’ (ANR-11-IDEX-0007). Research by J.P., S.M. and C.B.-A. was supported in part by a grant from the Spanish Ministry of Economy and Competitiveness (SAF2013-44267-R) and by the CNIO. Work in the laboratory of D.F. was funded by the Institut National du Cancer (PLBIO10-068 and PLBIO15-005) and the Ligue National Contre le Cancer (EL2018.LNCC/DF). Work in the laboratory of N.M. was funded by the ERC, under the European Union Horizon 2020 research and innovation programme (StG-2014–640525_REGMAMKID), the Spanish Association Against Cancer (AECC/LABAE16006), Carlos III Health Institute (Red TerCel, CardioCel, RD16/0011/0027), Ministry of Economy and Competitiveness (MINECO) projects SAF2017–89782-R, SAF2015–72617-EXP and RYC-2014–16242, and the CERCA/Government of Catalonia (2017 SGR 1306). Work in the laboratory of S.O. was funded by SAF2013–44866-R from MINECO Spain. Work in the laboratory of M.F.F. was funded by Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892, to M.F.F. and A.F.F.); the ISCIII-Subdireccion General de Evaluación y Fomento de la Investigación and Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131, to A.F.F.); IUOPA (to M.I.S.); and the Asturias Regional Government (GRUPIN14–052, to M.F.F.). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Work in the laboratory of M.S. was funded by the CNIO, the IRB and by grants from Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), Botin Foundation, Banco Santander (Santander Universities Global Division), laCaixa Foundation and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282).
Published: 2020

8. The Global Phosphorylation Landscape of SARS-CoV-2 Infection

Author: Yiming Cai, Maya Modak, Sebastian Weigang, Emmie de Wit, Jean K. Lim, Alistair Dunham, Benjamin J. Polacco, Qiongyu Li, Svenja Ulferts, Gwendolyn M. Jang, Aurelien Dugourd, David E. Gordon, Jeffrey Z. Guo, Kirsten Obernier, Sophia Bouhaddou, Elizabeth R. Fischer, Anna Gaulton, Jason C.J. Chang, Bjoern Meyer, Diego Quintero, Julian Knerr, Trupti Patil, Emma J. Manners, Michael C. O’Neal, Monita Muralidharan, Joseph Hiatt, Ajda Rojc, James E. Melnyk, Tanja Kortemme, Benjamin R. tenOever, Thomas Vallet, Rémy Robinot, Cassandra Koh, Benjamin E. Nilsson-Payant, Ruth Hüttenhain, Saker Klippsten, Alicia L. Richards, Eloy Felix, Brian K. Shoichet, Beril Tutuncuoglu, Danielle L. Swaney, Veronica V. Rezelj, Jeffery R. Johnson, Margaret Soucheray, Marisa Goff, R. Dyche Mullins, Kris M. White, Erica Stevenson, Jyoti Batra, Christopher J.P. Mathy, Yuan Zhou, Minkyu Kim, Marco Vignuzzi, Claudia Hernandez-Armenta, Kevan M. Shokat, Julio Saez-Rodriguez, Jacqueline M. Fabius, Timothy McBride, Adolfo García-Sastre, Quang Dinh Tran, Alexandra Hardy, Elena Moreno, Alberto Valdeolivas, Mehdi Bouhaddou, Andrew R. Leach, Melanie Ott, Georg Kochs, Pedro Beltrao, Jiewei Xu, Robyn M. Kaake, Merve Cakir, Ying Shi, Nevan J. Krogan, Lisa Miorin, Danish Memon, David J. Broadhurst, Miguel Correa Marrero, Robert Grosse, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Quantitative Biosciences Institute [UC San Francisco, USA] (QBI), University of California [San Francisco] (UC San Francisco), University of California (UC)-University of California (UC), Gladstone Institutes [San Francisco], European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Howard Hughes Medical Institute (HHMI), University of Freiburg [Freiburg], Virus et Immunité - Virus and immunity (CNRS-UMR3569), Universität Heidelberg [Heidelberg] = Heidelberg University, Heidelberg University Hospital [Heidelberg], Zoic Labs [Culver City, CA], Rocky Mountain Laboratories, Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Centre for Integrative Biological Signalling Studies [Freiburg] (CIBSS), This research was funded by grants from the National Institutes of Health ( P50AI150476 , U19AI135990 , U19AI135972 , R01AI143292 , R01AI120694 , P01A1063302 , and R01AI122747 to N.J.K., 1R01CA221969 and 1R01CA244550 to K.M.S., R01GM133981 to D.L.S., 1F32CA236347-01 to J.E.M., U19AI118610 to J.R.J., and F32CA239333 to M.B.), Defense Advance Research Projects Agency HR0011-19-2-0020 (to N.J.K., A.G.S., and K.M.S.), by the Laboratory for Genomics Research (LGR) Excellence in Research Award (ERA) from the Innovative Genomics Institute at UC Berkeley (grant number 133122P ), by CRIP (Center for Research for Influenza Pathogenesis), a NIAID-supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract HHSN272201400008C ) (to A.G.S.), by supplements to NIAID grant U19AI135972 and DoD grant W81XWH-19-PRMRP-FPA (to A.G.S.), and by the generous support of the JPB Foundation , the Open Philanthropy Project (research grant 2020-215611 [5384] ), and other philanthropic donations (to A.G.S.), by the Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' grant ANR-10-LABX-62-IBEID (to M.V.), by the DFG under Germany's Excellence Strategy ( EXC-2189 , project ID 390939984 to R.G.), by a Starting Grant Award from the European Research Council ( ERC-2014-STG 638884 PhosFunc to P.B.), by the Federal Ministry of Education and Research (BMBF, Computational Life Sciences grant 031L0181B to J.S.R.), by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (to E.R.F. and E.D.W.), and by funding from F. Hoffmann-La Roche and Vir Biotechnology and gifts from The Ron Conway Family . K.M.S. is an investigator of the Howard Hughes Medical Institute., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 638884,H2020,ERC-2014-STG,PhosFunc(2015), Universität Heidelberg [Heidelberg], and Vaccine Research Institute (VRI)
Subjects: Proteomics, MAPK/ERK pathway, MESH: Angiotensin-Converting Enzyme 2, MESH: Casein Kinase II, PIKFYVE, 0302 clinical medicine, MESH: Chlorocebus aethiops, MESH: Protein Kinase Inhibitors, MESH: Animals, Casein Kinase II, Lung, 0303 health sciences, Kinase, MESH: Proteomics, Phosphoproteomics, antiviral, Spike Glycoprotein, Cyclin-Dependent Kinases, 3. Good health, MESH: HEK293 Cells, Spike Glycoprotein, Coronavirus, Phosphorylation, Infection, MESH: Pandemics, p38 mitogen-activated protein kinases, Pneumonia, Viral, MESH: Vero Cells, p38, Antiviral Agents, General Biochemistry, Genetics and Molecular Biology, Article, Betacoronavirus, 03 medical and health sciences, Biodefense, Humans, MESH: SARS-CoV-2, MESH: Humans, MESH: Phosphorylation, Prevention, MESH: Host-Pathogen Interactions, fungi, Receptor Protein-Tyrosine Kinases, AXL, Pneumonia, Virology, MAPK, Coronavirus, MESH: Peptidyl-Dipeptidase A, MESH: Pneumonia, Viral, MESH: Phosphatidylinositol 3-Kinases, A549 Cells, Vero cell, Drug Evaluation, 030217 neurology & neurosurgery, Developmental Biology, MESH: Coronavirus Infections, [SDV]Life Sciences [q-bio], viruses, CDK, Drug Evaluation, Preclinical, MESH: Spike Glycoprotein, Coronavirus, Medical and Health Sciences, p38 Mitogen-Activated Protein Kinases, Phosphatidylinositol 3-Kinases, Chlorocebus aethiops, MESH: COVID-19, Viral, Phosphoinositide-3 Kinase Inhibitors, mass spectrometry, biology, phosphoproteomics, Biological Sciences, Preclinical, MESH: Cyclin-Dependent Kinases, Infectious Diseases, Host-Pathogen Interactions, MESH: Betacoronavirus, MESH: Drug Evaluation, Preclinical, MESH: Receptor Protein-Tyrosine Kinases, MESH: Caco-2 Cells, Angiotensin-Converting Enzyme 2, Coronavirus Infections, MESH: Antiviral Agents, casein kinase II, Peptidyl-Dipeptidase A, Vaccine Related, Cyclin-dependent kinase, Proto-Oncogene Proteins, Animals, Pandemics, Protein Kinase Inhibitors, Vero Cells, MESH: Phosphoinositide-3 Kinase Inhibitors, 030304 developmental biology, SARS-CoV-2, COVID-19, Axl Receptor Tyrosine Kinase, MESH: p38 Mitogen-Activated Protein Kinases, MESH: Proto-Oncogene Proteins, Emerging Infectious Diseases, Good Health and Well Being, HEK293 Cells, biology.protein, MESH: A549 Cells, Caco-2 Cells
Abstract: Summary The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies., Graphical Abstract, Highlights • Phosphoproteomics analysis of SARS-CoV-2-infected cells uncovers signaling rewiring • Infection promotes host p38 MAPK cascade activity and shutdown of mitotic kinases • Infection stimulates CK2-containing filopodial protrusions with budding virus • Kinase activity analysis identifies potent antiviral drugs and compounds, Phosphoproteomics analysis of SARS-CoV-2-infected Vero E6 cells reveals host cellular pathways hijacked by viral infection, leading to the identification of small molecules that target dysregulated pathways and elicit potent antiviral efficacy.
Published: 2020

9. Modulation of glycine receptor single-channel conductance by intracellular phosphorylation

Author: Gonzalo E. Yévenes, Gustavo Moraga-Cid, Hanns Ulrich Zeilhofer, Patricio A. Castro, Jorge Fuentealba, Luis G. Aguayo, Carola Muñoz-Montesino, Anggelo Sazo, Carlos F. Burgos, Cesar O. Lara, Pierre-Jean Corringer, Leonardo Guzmán, Ana M Marileo, Victoria P San Martín, Braulio Muñoz, University of Zurich, Moraga-Cid, Gustavo, Universidad de Concepción - University of Concepcion [Chile], Indiana University School of Medicine, Indiana University System, Universität Zürich [Zürich] = University of Zurich (UZH), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Récepteurs Canaux - Channel Receptors, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was supported by FONDECYT 1160851 and VRID N°219.033.111-INV (to G.M.-C), NIH R01AA025718 (to L.G.A) and by FONDECYT 1170252 (to G.E.Y). V. San Martin and A. Sazo were supported by the Graduate School Fellowship of the U. of Concepcion (MSc in Human Physiology, MSc in Neurobiology and PhD in Biological Sciences). C.O. Lara and A.M. Marileo were supported by CONICYT doctoral fellowships., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Signal Transduction, 0301 basic medicine, [SDV]Life Sciences [q-bio], [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Intracellular Space, lcsh:Medicine, 10050 Institute of Pharmacology and Toxicology, Receptors, Glycine, 0302 clinical medicine, Hyperekplexia, Phosphorylation, lcsh:Science, Receptor, Glycine receptor, Multidisciplinary, Chemistry, MESH: Amino Acid Substitution, MESH: Receptors, Glycine, MESH: Intracellular Space, MESH: Protein Domains, medicine.symptom, Intracellular, Signal Transduction, GLIC, Biophysics, 610 Medicine & health, Context (language use), MESH: Cyclic AMP-Dependent Protein Kinases, Article, 03 medical and health sciences, Protein Domains, MESH: Receptors, Prostaglandin E, EP2 Subtype, medicine, Humans, MESH: Excitatory Postsynaptic Potentials, Ion channel, 1000 Multidisciplinary, MESH: Humans, MESH: Phosphorylation, lcsh:R, Excitatory Postsynaptic Potentials, Receptors, Prostaglandin E, EP2 Subtype, Cyclic AMP-Dependent Protein Kinases, 030104 developmental biology, Amino Acid Substitution, MESH: Protein Processing, Post-Translational, 570 Life sciences, biology, lcsh:Q, Protein Processing, Post-Translational, Neuroscience, 030217 neurology & neurosurgery
Abstract: Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs., Scientific Reports, 10 (1), ISSN:2045-2322
Published: 2020

10. Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

Author: Ahmed Alshawi, Loranne Agius, Marc Foretz, Benoit Viollet, Silvia Marin, Natasha S. Adam-Primus, Shruti S. Chachra, Ziad H. Al-Oanzi, Tabassum Moonira, Marta Cascante, Catherine Arden, Brian E. Ford, Institut Cochin (IC UM3 (UMR 8104 / U1016)), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)
Subjects: 0301 basic medicine, Male, Phosphofructokinase-1, Hypoglucemic agents, AMP-Activated Protein Kinases, MESH: Metformin, MESH: Mice, Knockout, Biochemistry, MESH: Hepatocytes, chemistry.chemical_compound, Mice, Adenosine Triphosphate, MESH: Adenosine Triphosphate, Malalties cròniques, MESH: Animals, Glycolysis, MESH: AMP-Activated Protein Kinases, Phosphorylation, Antidiabètics, Mice, Knockout, Chemistry, MESH: Gluconeogenesis, MESH: Glycerolphosphate Dehydrogenase, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, glycolysis, MESH: Glucose-6-Phosphate, Metformin, 3. Good health, MESH: Glucose, phosphofructokinase, MESH: Glycolysis, Dihydroxyacetone, medicine.drug, Phosphofructokinase, medicine.medical_specialty, MESH: Rats, Allosteric regulation, Glucose-6-Phosphate, Glycerolphosphate Dehydrogenase, liver, 03 medical and health sciences, MESH: Phosphofructokinase-1, MESH: Mice, Inbred C57BL, Internal medicine, Rotenone, medicine, hepatocyte, Animals, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Rats, Wistar, MESH: Mice, Molecular Biology, glucose 6-phosphate, MESH: Dihydroxyacetone, MESH: Phosphorylation, 030102 biochemistry & molecular biology, Gluconeogenesis, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Fructose, MESH: Rats, Wistar, Cell Biology, Metabolism, MESH: Male, Rats, Mice, Inbred C57BL, 030104 developmental biology, Endocrinology, Glucose, Glucose 6-phosphate, Chronic diseases, Hepatocytes, MESH: Rotenone
Abstract: International audience; The chronic effects of metformin on liver gluconeogenesisinvolve repression of theG6pcgene, which is regulated by thecarbohydrate-response element–binding protein throughraised cellular intermediates of glucose metabolism. In thisstudy we determined the candidate mechanisms by which met-formin lowers glucose 6-phosphate (G6P) in mouse and rathepatocytes challenged with high glucose or gluconeogenic pre-cursors. Cell metformin loads in the therapeutic range loweredcell G6P but not ATP and decreasedG6pcmRNA at high glu-cose. The G6P lowering by metformin was mimicked by a com-plex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol)and by overexpression of mGPDH, which lowers glycerol3-phosphate and G6P and also mimics theG6pcrepression bymetformin. In contrast, direct allosteric activators of AMPK(A-769662, 991, and C-13) had opposite effects from metforminon glycolysis, gluconeogenesis, and cell G6P. The G6P loweringby metformin, which also occurs in hepatocytes from AMPKknockout mice, is best explained by allosteric regulation ofphosphofructokinase-1 and/or fructose bisphosphatase-1, assupported by increased metabolism of [3-3H]glucose relative to[2-3H]glucose; by an increase in the lactate m2/m1 isotopologratio from [1,2-13C2]glucose; by lowering of glycerol 3-phos-phate an allosteric inhibitor of phosphofructokinase-1; and bymarked G6P elevation by selective inhibition of phosphofruc-tokinase-1; but not by a more reduced cytoplasmic NADH/NADredox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glu-cose by stimulation of glycolysis by an AMP-activated proteinkinase–independent mechanism through changes in allostericeffectors of phosphofructokinase-1 and fructose bisphospha-tase-1, including AMP, Pi, and glycerol 3-phosphate
Published: 2020

11. High Sensitivity of Circulating Tumor Cells Derived from a Colorectal Cancer Patient for Dual Inhibition with AKT and mTOR Inhibitors

Author: Marie-Therese Haider, Laure Cayrefourcq, Nico Hinz, Klaus Pantel, Catherine Alix-Panabières, Daniel J Smit, Manfred Jücker, Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (UKE), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Aide à la Décision pour une Médecine Personnalisé - Laboratoire de Biostatistique, Epidémiologie et Recherche Clinique - EA 2415 (AIDMP), Université Montpellier 1 (UM1)-Université de Montpellier (UM), Laboratoire Cellules Circulantes Rares Humaines [CHRU Montpellier] (LCCRH), and Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-CHU Saint-Eloi
Subjects: 0301 basic medicine, MAPK/ERK pathway, MESH: Signal Transduction, AKT1, Apoptosis, AKT2, Phosphatidylinositol 3-Kinases, 0302 clinical medicine, Circulating tumor cell, MESH: Protein Kinase Inhibitors, MK2206, Phosphorylation, lcsh:QH301-705.5, MESH: Inhibitory Concentration 50, Chemistry, TOR Serine-Threonine Kinases, drug sensitivity assays, General Medicine, personalized medicine, MESH: Gene Expression Regulation, Neoplastic, Neoplastic Cells, Circulating, targeted therapy, 3. Good health, Gene Expression Regulation, Neoplastic, 030220 oncology & carcinogenesis, MESH: HEK293 Cells, PI3K/AKT/mTOR pathway, Signal transduction, CTCs, Colorectal Neoplasms, Heterocyclic Compounds, 3-Ring, Signal Transduction, MESH: Cell Line, Tumor, Antineoplastic Agents, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, circulating tumor cells, MESH: Everolimus, MESH: Neoplastic Cells, Circulating, Article, MESH: Cell Adhesion, Inhibitory Concentration 50, 03 medical and health sciences, colorectal carcinoma, Cell Line, Tumor, MESH: Cell Proliferation, Cell Adhesion, Humans, Everolimus, Protein Kinase Inhibitors, Protein kinase B, RAD001, MESH: TOR Serine-Threonine Kinases, Cell Proliferation, MESH: Heterocyclic Compounds, 3-Ring, MESH: Humans, MESH: Phosphorylation, Cell growth, MESH: Proto-Oncogene Proteins c-akt, AKT, dual targeting, MESH: Apoptosis, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, HEK293 Cells, 030104 developmental biology, lcsh:Biology (General), MESH: Phosphatidylinositol 3-Kinases, Cancer research, MESH: Antineoplastic Agents, Proto-Oncogene Proteins c-akt, MESH: Colorectal Neoplasms
Abstract: International audience; Circulating tumor cells (CTCs) are cells shed from the primary tumor into the bloodstream. While many studies on solid tumor cells exist, data on CTCs are scarce. The mortality of cancer is mostly associated with metastasis and recent research identified CTCs as initiators of metastasis. The PI3K/AKT/mTOR signaling pathway is an intracellular pathway that regulates essential functions including protein biosynthesis, cell growth, cell cycle control, survival and migration. Importantly, activating oncogenic mutations and amplifications in this pathway are frequently observed in a wide variety of cancer entities, underlining the significance of this signaling pathway. In this study, we analyzed the functional role of the PI3K/AKT/mTOR signaling pathway in the CTC-MCC-41 line, derived from a patient with metastatic colorectal cancer. One striking finding in our study was the strong sensitivity of this CTC line against AKT inhibition using MK2206 and mTOR inhibition using RAD001 within the nanomolar range. This suggests that therapies targeting AKT and mTOR could have been beneficial for the patient from which the CTC line was isolated. Additionally, a dual targeting approach of AKT/mTOR inside the PI3K/AKT/mTOR signaling pathway in the colorectal CTCs showed synergistic effects in vitro. Depending on the phenotypical behavior of CTC-MCC-41 in cell culture (adherent vs. suspension), we identified altered phosphorylation levels inside the PI3K/AKT/mTOR pathway. We observed a downregulation of the PI3K/AKT/mTOR signaling pathway, but not of the RAS/RAF/MAPK pathway, in CTCs growing in suspension in comparison to adherent CTCs. Our results highlight distinct functions of AKT isoforms in CTC-MCC-41 cells with respect to cell proliferation. Knockdown of AKT1 and AKT2 leads to significantly impaired proliferation of CTC-MCC-41 cells in vitro. Therefore, our data demonstrate that the PI3K/AKT/mTOR signaling pathway plays a key role in the proliferation of CTC-MCC-41.
Published: 2020

12. TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function

Author: Jose Ursic-Bedoya, Isabelle Doignon, Hayat Simerabet, Dominique Rainteau, Doris Cassio, Nicolas Kahale, Anne Davit-Spraul, Thierry Tordjmann, Grégory Merlen, Lydie Humbert, Catherine Guettier, Julien Gautherot, Valeska Bidault-Jourdainne, Christoph Ullmer, Isabelle Garcin, Noémie Péan, Klaus Ebnet, Zahra Tanfin, Intéractions cellulaires et physiopathologie hépathique (Orsay, Essonne) UMRS 1174 (ICPH ), Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris-Sud - Paris 11 - Faculté de médecine (UP11 UFR Médecine), Université Paris-Sud - Paris 11 (UP11), Service de Biochimie [AP-HP Hôpital Bicêtre], AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Service d’Anatomopathologie [Le Kremlin-Bicêtre], ER_7, Université Pierre et Marie Curie - Paris 6 (UPMC), Microorganismes, Molécules Bioactives et Physiopathologie Intestinale (LBM-E4), Institut National de la Santé et de la Recherche Médicale (INSERM)-Laboratoire des biomolécules (LBM UMR 7203), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-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)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Microorganismes et physiopathologie intestinale (ERL INSERM U1157 - CNRS UMR 7203), Laboratoire des biomolécules (LBM UMR 7203), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC), Zentrum für Molekularbiologie der Entzündung - Center for Molecular Biology of Inflammation [Münster, Germany] (ZMBE), Westfälische Wilhelms-Universität Münster (WWU), F. Hoffmann-La Roche [Basel], Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Chimie Moléculaire de Paris Centre (FR 2769), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), and Salvy-Córdoba, Nathalie
Subjects: Biliary epithelium, MESH: Signal Transduction, Stimulation, MESH: Receptors, G-Protein-Coupled, MESH: Mice, Knockout, Epithelium, Receptors, G-Protein-Coupled, MESH: Isonipecotic Acids, 0302 clinical medicine, MESH: Biliary Tract, Oximes, Electric Impedance, Bile, MESH: Animals, Phosphorylation, Biliary Tract, Cells, Cultured, Barrier function, MESH: Receptors, Cell Surface, Mice, Knockout, Liver injury, 0303 health sciences, Bile acid, Tight junction, Chemistry, Gastroenterology, MESH: Oximes, G protein-coupled bile acid receptor, Paracellular transport, MESH: Permeability, Cholestaticliver diseases, MESH: Cell Adhesion Molecules, 030211 gastroenterology & hepatology, Signal Transduction, MESH: Cells, Cultured, medicine.drug_class, MESH: Tight Junction Proteins, Receptors, Cell Surface, Cholestasis, Intrahepatic, MESH: Bile Acids and Salts, Permeability, Bile Acids and Salts, 03 medical and health sciences, Isonipecotic Acids, In vivo, MESH: Mice, Inbred C57BL, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Electric Impedance, MESH: Bile, 030304 developmental biology, Tight Junction Proteins, MESH: Phosphorylation, MESH: Cholestasis, Intrahepatic, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, medicine.disease, Molecular biology, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Mice, Inbred C57BL, MESH: Epithelium, Cell Adhesion Molecules
Abstract: ObjectiveWe explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability.DesignExperiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied.ResultsIn vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice.ConclusionThe BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.
Published: 2019

13. Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation

Author: Marie Octave, Laurence Pirotton, Audrey Ginion, Valentine Robaux, Sophie Lepropre, Jérôme Ambroise, Caroline Bouzin, Bruno Guigas, Martin Giera, Marc Foretz, Luc Bertrand, Christophe Beauloye, Sandrine Horman, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), FORETZ, Marc, UCL - SSS/IREC/CARD - Pôle de recherche cardiovasculaire, UCL - SSS/IREC/CTMA - Centre de technologies moléculaires appliquées (plate-forme technologique), and UCL - (SLuc) Service de cardiologie
Subjects: rac1 GTP-Binding Protein, actin cytoskeleton, Platelet Aggregation, Rac1 signaling, MESH: Tubulin, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Microtubules, acetyl-CoA carboxylase, platelet, platelet aggregation, acetylation, tubulin, MESH: Thrombin, Biology (General), Enzyme Inhibitors, Phosphorylation, Extracellular Signal-Regulated MAP Kinases, MESH: Extracellular Signal-Regulated MAP Kinases, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Blood Platelets, Spectroscopy, MESH: Platelet Aggregation, MESH: Lipid Metabolism, [SDV.MHEP.EM] Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, MESH: Microtubules, Thrombin, MESH: Reactive Oxygen Species, Acetylation, General Medicine, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Mitochondria, Computer Science Applications, Chemistry, MESH: Enzyme Inhibitors, MESH: Acetylation, Blood Platelets, MESH: p21-Activated Kinases, QH301-705.5, MESH: Mitochondria, macromolecular substances, Article, Catalysis, Inorganic Chemistry, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Physical and Theoretical Chemistry, QD1-999, Molecular Biology, MESH: Humans, MESH: Phosphorylation, MESH: rac1 GTP-Binding Protein, Organic Chemistry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Lipid Metabolism, p21-Activated Kinases, MESH: Acetyl-CoA Carboxylase, MESH: Actin Cytoskeleton, Reactive Oxygen Species
Abstract: International audience; Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.
Published: 2021

14. The Juxtamembrane Domain of Butyrophilin BTN3A1 Controls Phosphoantigen-Mediated Activation of Human Vγ9Vδ2 T Cells

Author: Peigné, Cassie-Marie, Léger, Alexandra, Gesnel, Marie-Claude, Konczak, Fabienne, Olive, Daniel, Bonneville, Marc, Breathnach, Richard, Scotet, Emmanuel, Leger, Alexandra, Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Recherche sur les effecteurs lymphocytaires T, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Recherches en cancérologie, Université de Nantes (UN)-IFR26-Institut National de la Santé et de la Recherche Médicale (INSERM), MITOYAN, Louciné, Aix Marseille Université (AMU)-Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, [SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], T cell, Immunology, Cell, Mutant Chimeric Proteins, MESH: T-Lymphocyte Subsets, Biology, Lymphocyte Activation, 03 medical and health sciences, MESH: Butyrophilins, Butyrophilin, Antigen, Antigens, CD, T-Lymphocyte Subsets, MESH: Receptors, Antigen, T-Cell, gamma-delta, medicine, Humans, Immunology and Allergy, Antigens, Phosphorylation, MESH: Lymphocyte Activation, Receptor, MESH: Antigens, CD, MESH: Humans, Butyrophilins, MESH: Phosphorylation, MESH: Mutant Chimeric Proteins, HEK 293 cells, Receptors, Antigen, T-Cell, gamma-delta, Cell biology, [SDV] Life Sciences [q-bio], Transmembrane domain, HEK293 Cells, 030104 developmental biology, medicine.anatomical_structure, MESH: HEK293 Cells, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Antigens, Intracellular
Abstract: Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1’s juxtamembrane domain for correct γδ T cell–related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.
Published: 2017

15. The kinase domain of CK1δ can be phosphorylated by Chk1

Author: Joachim Bischof, Thomas Böhm, Najma Rachidi, Zhigang Meng, Doris Henne-Bruns, Uwe Knippschild, Philipp Haas, Universitätsklinikum Ulm - University Hospital of Ulm, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung under grant number 108489 awarded to Uwe Knippschild and by the Else Kröner-Fresenius-Stiftung under grant number 2017_A142 awarded to Joachim Bischof., and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, MESH: Signal Transduction, Casein kinase 1, [SDV.CAN]Life Sciences [q-bio]/Cancer, Applied Microbiology and Biotechnology, Biochemistry, MESH: Checkpoint Kinase 1, Analytical Chemistry, 03 medical and health sciences, enzyme kinetics, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, CHEK1, Phosphorylation, Kinase activity, Molecular Biology, Gene, site-specific phosphorylation, MESH: Humans, 030102 biochemistry & molecular biology, MESH: Phosphorylation, Kinase, Chemistry, Organic Chemistry, phosphopeptide analysis, General Medicine, Cell biology, 030104 developmental biology, Protein kinase domain, Checkpoint kinase 1, Signal transduction, Signal Transduction, Biotechnology
Abstract: Members of the casein kinase 1 (CK1) family are key regulators in numerous cellular signal transduction pathways and in order to prevent the development of certain diseases, CK1 kinase activity needs to be tightly regulated. Modulation of kinase activity by site-specific phosphorylation within the C-terminal regulatory domain of CK1δ has already been shown for several cellular kinases. By using biochemical methods, we now identified residues T161, T174, T176, and S181 within the kinase domain of CK1δ as target sites for checkpoint kinase 1 (Chk1). At least residues T176 and S181 show full conservation among CK1δ orthologues from different eukaryotic species. Enzyme kinetic analysis furthermore led to the hypothesis that site-specific phosphorylation within the kinase domain finally contributes to fine-tuning of CK1δ kinase activity. These data provide a basis for the extension of our knowledge about the role of site-specific phosphorylation for regulation of CK1δ and associated signal transduction pathways.
Published: 2019

16. ProNGF increases breast tumor aggressiveness through functional association of TrkA with EphA2

Author: Léo Aubert, Xuefen Le Bourhis, Nicolas Magné, Matthieu Guilbert, Eric Adriaenssens, Robert-Alain Toillon, Valérie Chopin, Cyril Corbet, Romain Lévêque, Daniel Birnbaum, Jérémy Duval, Chann Lagadec, François Bertucci, Pascal Finetti, Plasticité Cellulaire et Cancer - U908 (CPAC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Cancérologie Lucien Neuwirth, Centre Hospitalier Universitaire de Saint-Etienne [CHU Saint-Etienne] (CHU ST-E), Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Saint-Etienne, CCSD, Accord Elsevier, and UCL - SSS/IREC/FATH - Pôle de Pharmacologie et thérapeutique
Subjects: 0301 basic medicine, MESH: Signal Transduction, Cancer Research, MESH: Protein Precursors, MESH: Tumor Burden, [SDV]Life Sciences [q-bio], Mice, SCID, Proximity ligation assay, 0302 clinical medicine, Breast cancer, Nerve Growth Factor, MESH: Protein Kinase Inhibitors, proNGF, MESH: Animals, Phosphorylation, MESH: Mice, SCID, MESH: Nerve Growth Factor, Brain Neoplasms, Chemistry, TrkA, Receptor, EphA2, MESH: Receptor, trkA, Ephrin-A2, MESH: RNAi Therapeutics, Primary tumor, Tumor Burden, [SDV] Life Sciences [q-bio], MESH: MCF-7 Cells, src-Family Kinases, Oncology, 030220 oncology & carcinogenesis, MESH: Brain Neoplasms, MCF-7 Cells, Female, Tyrosine kinase, Protein Binding, Signal Transduction, Proto-oncogene tyrosine-protein kinase Src, MESH: Xenograft Model Antitumor Assays, animal structures, Antineoplastic Agents, Breast Neoplasms, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, EphA2, MESH: Ephrin-A2, 03 medical and health sciences, breast cancer, MESH: Cell Proliferation, medicine, Animals, Humans, MESH: Protein Binding, Protein Precursors, Receptor, trkA, Clonogenic assay, Autocrine signalling, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Protein Kinase Inhibitors, Protein kinase B, Cell Proliferation, MESH: Humans, MESH: Phosphorylation, MESH: Adaptor Proteins, Vesicular Transport, medicine.disease, Xenograft Model Antitumor Assays, Adaptor Proteins, Vesicular Transport, RNAi Therapeutics, 030104 developmental biology, nervous system, MESH: src-Family Kinases, Cancer research, MESH: Antineoplastic Agents, MESH: Female, MESH: Breast Neoplasms
Abstract: International audience; ProNGF expression has been linked to several types of cancers including breast cancer, and we have previously shown that proNGF stimulates breast cancer invasion in an autocrine manner through membrane receptors sortilin and TrkA. However, little is known regarding TrkA-associated protein partners upon proNGF stimulation. By proteomic analysis and proximity ligation assays, we found that proNGF binding to sortilin induced sequential formation of the functional sortilin/TrkA/EphA2 complex, leading to TrkA-phosphorylation dependent Akt activation and EphA2-dependent Src activation. EphA2 inhibition using siRNA approach abolished proNGF-stimulated clonogenic growth of breast cancer cell lines. Combinatorial targeting of TrkA and EphA2 dramatically reduced colony formation in vitro, primary tumor growth and metastatic dissemination towards the brain in vivo. Finally, proximity ligation assay in breast tumor samples revealed that increased TrkA/EphA2 proximity ligation assay signals were correlated with a decrease of overall survival in patients. All together, these data point out the importance of TrkA/EphA2 functional association in proNGF-induced tumor promoting effects, and provide a rationale to target proNGF/TrkA/EphA2 axis by alternative methods other than the simple use of tyrosine kinase inhibitors in breast cancer.
Published: 2019

17. New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach

Author: Roland Brosch, María Noel Alvarez, Mariana Piuri, Alessandro Cascioferro, Analía Lima, Rosario Durán, Paulo C. Carvalho, Estefanía Urdániz, María-Natalia Lisa, Ana Denicola, Marco Bellinzoni, Otto Pritsch, Federico Carrion, Magdalena Gil, Annemarie Wehenkel, Pedro M. Alzari, Carlos Batthyány, Bernardina Rivera, Jessica Rossello, Instituto de Investigaciones Biológicas Clemente Estable [Montevideo] (IIBCE), Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Dynamique des Interactions Hôte-Pathogène, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Universidad de Buenos Aires [Buenos Aires] (UBA), Pathogénomique mycobactérienne intégrée - Integrated Mycobacterial Pathogenomics, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Universidad de la República [Montevideo] (UCUR), Fiocruz Paraná - Instituto Carlos Chagas = Carlos Chagas Institute [Curitiba, Brésil] (ICC), Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), 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], This work was funded by grants from the Agencia Nacional de Investigación e Innovación, Uruguay (ANII) [FCE_3_2013_1_100358 and FCE_1_2014_1_104045] and FOCEM (MERCOSUR Structural Convergence Fund, COF 03/11). MG, JR and BR were supported by a fellowship from ANII [POS_NAC_2012_1_8824, POS_NAC_2015_1_109755, POS_FCE_2015_1_1005186] and AC was supported by Agence Nationale pour la Recherche (France) [grant 09 BLAN 0400 01]., ANR-05-PADD-0009,Eco-Serre,Viabilité des systèmes de cultures protégées dans un contexte d'agriculture durable(2005), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Facultad de Ciencias Exactas y Naturales [Buenos Aires] (FCEyN), Microbiologie structurale, University of the Republic of Uruguay, Fiocruz Paraná - Instituto Carlos Chagas / Carlos Chagas Institute [Curitiba, Brésil] (ICC), ANR-09-BLAN-0400,MuKit(2009), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ), Institut Pasteur [Paris], Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Instituto Carlos Chagas/Fundação Oswaldo Cruz [Curitiba]
Subjects: 0301 basic medicine, Affinity purification-mass spectrometry, MESH: Protein-Serine-Threonine Kinases / genetics, Serine threonine protein kinase, MESH: Antigens, Bacterial / metabolism, Biochemistry, Interactome, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Substrate Specificity, Serine, MESH: Protein-Serine-Threonine Kinases / metabolism, [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], Phosphorylation, MESH: Bacterial Proteins / genetics, Kinase, Chemistry, MESH: Antigens, Bacterial / chemistry, MESH: Ribosomal Proteins / genetics, MESH: Mycobacterium tuberculosis / enzymology, glutamine synthetase, MESH: Ribosomal Proteins / chemistry, 3. Good health, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, MESH: Mycobacterium tuberculosis / genetics, Proteome, MESH: Protein Domains, Ribosomal Proteins, MESH: Mutation, Biophysics, PknG, Protein Serine-Threonine Kinases, 03 medical and health sciences, Bacterial Proteins, Protein Domains, Ribosomal protein, FhaA, Serine/Threonine protein kinase, Protein kinase A, MESH: Bacterial Proteins / metabolism, MESH: Protein-Serine-Threonine Kinases / chemistry, Antigens, Bacterial, 030102 biochemistry & molecular biology, MESH: Phosphorylation, MESH: Antigens, Bacterial / genetics, Mycobacterium tuberculosis, MESH: Ribosomal Proteins / metabolism, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Mutation, MESH: Substrate Specificity, MESH: Bacterial Proteins / chemistry
Abstract: International audience; PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.
Published: 2019

18. Regulatory control of DNA end resection by Sae2 phosphorylation

Author: Cannavo, Elda, Johnson, Dominic, Andres, Sara, Kissling, Vera, Reinert, Julia, Garcia, Valerie, Erie, Dorothy, Hess, Daniel, Thomä, Nicolas, Enchev, Radoslav, Peter, Matthias, Williams, R Scott, Neale, Matt, Cejka, Petr, Williams, R. Scott, Friedrich Miescher Institute for Biomedical Research (FMI), Novartis Research Foundation, Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Institute of Biochemistry, Universität Zürich [Zürich] = University of Zurich (UZH), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: DNA End-Joining Repair, Saccharomyces cerevisiae Proteins, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, MESH: Exodeoxyribonucleases, MESH: Cell Cycle, Saccharomyces cerevisiae, Article, MESH: Saccharomyces cerevisiae Proteins, MESH: Endonucleases, MESH: Protein Binding, DNA Breaks, Double-Stranded, MESH: Endodeoxyribonucleases, Phosphorylation, DNA, Fungal, Endodeoxyribonucleases, MESH: Phosphorylation, MESH: Protein Multimerization, Cell Cycle, Recombinational DNA Repair, MESH: DNA End-Joining Repair, MESH: Multiprotein Complexes, Endonucleases, MESH: Saccharomyces cerevisiae, DNA-Binding Proteins, MESH: Recombinational DNA Repair, MESH: DNA, Fungal, Meiosis, enzymes and coenzymes (carbohydrates), MESH: Meiosis, Exodeoxyribonucleases, Multiprotein Complexes, Protein Multimerization, MESH: DNA-Binding Proteins, Protein Binding
Abstract: DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms., It has previously been established that DNA end resection in yeast and in humans is under CDK control. Here the authors explain how phosphorylation regulates the capacity of Sae2 — the yeast orthologue of human CtIP — to promote DNA end resection.
Published: 2018

19. Exercise-induced molecular mechanisms promoting glycogen supercompensation in human skeletal muscle

Author: David E. James, Benjamin L. Parker, Jesper B. Birk, Lea Bruhn, Marie F. Rosschou, Nils J. Færgeman, Kei Sakamoto, Joachim Fentz, Mads B. Hansen, Erik A. Richter, Bente Kiens, Marc Foretz, Jørgen F. P. Wojtaszewski, Janne R. Hingst, Jesper F. Havelund, Benoit Viollet, Jørgen Arendt Jensen, University of Copenhagen = Københavns Universitet (KU), Protocols and applications for the Internet (PLANETE), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Inria Sophia Antipolis - Méditerranée (CRISAM), Institut National de Recherche en Informatique et en Automatique (Inria), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Dundee, University of Southern Denmark (SDU), The University of Sydney, and Section of Molecular Physiology
Subjects: 0301 basic medicine, Male, Proteomics, MESH: Signal Transduction, MESH: Dietary Carbohydrates, Glucose uptake, medicine.medical_treatment, MESH: Carbohydrate Metabolism, MESH: Glycogen, MESH: Physical Conditioning, Animal, chemistry.chemical_compound, Mice, Insulin, MESH: Animals, Phosphorylation, MESH: Muscle, Skeletal, Glycogen, biology, MESH: Proteomics, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Glucose, medicine.anatomical_structure, Glycogen Synthase, Carbohydrate Metabolism, Signal Transduction, MESH: Carbohydrates, Adult, Glycogen synthase (GS), medicine.medical_specialty, lcsh:Internal medicine, MESH: Mice, Transgenic, Carbohydrates, Mice, Transgenic, MESH: Insulin, Carbohydrate metabolism, 03 medical and health sciences, Internal medicine, Physical Conditioning, Animal, AMP-activated protein kinase (AMPK), medicine, Dietary Carbohydrates, Animals, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Glycogen synthase, lcsh:RC31-1245, Muscle, Skeletal, TBC1 domain family member 4 (TBC1D4), Molecular Biology, Exercise, MESH: Mice, Insulin action, MESH: Glycogen Synthase, MESH: Humans, MESH: Phosphorylation, AMPK, Skeletal muscle, MESH: Adult, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, MESH: Male, 030104 developmental biology, Endocrinology, Glucose, chemistry, MESH: Exercise, biology.protein, Supercompensation
Abstract: Objective: A single bout of exercise followed by intake of carbohydrates leads to glycogen supercompensation in prior exercised muscle. Our objective was to illuminate molecular mechanisms underlying this phenomenon in skeletal muscle of man. Methods: We studied the temporal regulation of glycogen supercompensation in human skeletal muscle during a 5 day recovery period following a single bout of exercise. Nine healthy men depleted (day 1), normalized (day 2) and supercompensated (day 5) muscle glycogen in one leg while the contralateral leg served as a resting control. Euglycemic hyperinsulinemic clamps in combination with leg balance technique allowed for investigating insulin-stimulated leg glucose uptake under these 3 experimental conditions. Cellular signaling in muscle biopsies was investigated by global proteomic analyses and immunoblotting. We strengthened the validity of proposed molecular effectors by follow-up studies in muscle of transgenic mice. Results: Sustained activation of glycogen synthase (GS) and AMPK in combination with elevated expression of proteins determining glucose uptake capacity were evident in the prior exercised muscle. We hypothesize that these alterations offset the otherwise tight feedback inhibition of glycogen synthesis and glucose uptake by glycogen. In line with key roles of AMPK and GS seen in the human experiments we observed abrogated ability for glycogen supercompensation in muscle with inducible AMPK deletion and in muscle carrying a G6P-insensitive form of GS in muscle. Conclusion: Our study demonstrates that both AMPK and GS are key regulators of glycogen supercompensation following a single bout of glycogen-depleting exercise in skeletal muscle of both man and mouse. Keywords: AMP-activated protein kinase (AMPK), TBC1 domain family member 4 (TBC1D4), Glycogen synthase (GS), Glucose uptake, Exercise, Insulin action
Published: 2018

20. Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

Author: Stéphanie Miserey-Lenkei, Patricia Bassereau, Feng-Ching Tsai, Laura Picas, Yosuke Senju, Aurélie Bertin, Emmanuel Lemichez, Pekka Lappalainen, John Manzi, Evelyne Coudrier, Hugo Bousquet, Meng Chen Tsai, Laboratoire Physico-Chimie Curie [Institut Curie] (PCC), Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Helsingin yliopisto = Helsingfors universitet = University of Helsinki, Toxines bactériennes - Bacterial Toxins, Institut Pasteur [Paris] (IP), Institut de pharmacologie moléculaire et cellulaire (IPMC), 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)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Université Paris Descartes - Paris 5 (UPD5), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institut Curie, Centre National de la Recherche Scientifique (CNRS), the Agence Nationale pour la Recherche (grant ANR-15-CE18-0016 to F-CT, M-CT, EL and PB), the Investments for the Future’ LABEX SIGNALIFE (grant ANR-11-LABX-0028–01 for M-CT and EL), the European Research Council (EC and PB partners of the advanced grant, project 339847) and the Human Frontier Science Program Organization (RGP0005/2016 to YS, PL, F-CT, EC and PB). EC and PB groups belong to the CNRS consortium CellTiss, to the Labex CelTisPhyBio (ANR-11-LABX0038) and to Paris Sciences et Lettres (ANR-10-IDEX-0001–02). F-C Tsai was funded by the EMBO Long-Term fellowship (ALTF 1527–2014) and Marie Curie actions (H2020-MSCA-IF-2014, project membrane-ezrin-actin)., ANR-15-CE18-0016,TransEndotheliaTunnel,Vers la prévention de dysfonctions vasculaires et la délivrance de médicaments au travers des endothelia(2015), ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), European Project: 339847,EC:FP7:ERC,ERC-2013-ADG,MYODYN(2014), European Project: 656442,H2020,H2020-MSCA-IF-2014,membrane-ezrin-actin(2016), Institute of Biotechnology, Doctoral Programme in Integrative Life Science, Pekka Lappalainen / Principal Investigator, University Management, University of Helsinki, Institut Pasteur [Paris], Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Picas, Laura, Vers la prévention de dysfonctions vasculaires et la délivrance de médicaments au travers des endothelia - - TransEndotheliaTunnel2015 - ANR-15-CE18-0016 - AAPG2015 - VALID, Centres d'excellences - Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie - - SIGNALIFE2011 - ANR-11-LABX-0028 - LABX - VALID, Initiative d'excellence - Paris Sciences et Lettres - - PSL2010 - ANR-10-IDEX-0001 - IDEX - VALID, Myosins and the dynamics of intracellular membranes - MYODYN - - EC:FP7:ERC2014-02-01 - 2019-01-31 - 339847 - VALID, and Membrane-ezrin-actin interactions mediated by ezrin binding proteins in reconstituted systems - membrane-ezrin-actin - - H20202016-01-01 - 2018-01-01 - 656442 - VALID
Subjects: 0301 basic medicine, Protein Conformation, Lipid Bilayers, Mutant, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Ezrin, MESH: Protein Conformation, physics of living systems, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], ezrin-membrane interaction, cellular protrusions, Biology (General), phosphorylation, General Neuroscience, CYTOSKELETAL LINKER, EPITHELIAL-CELLS, General Medicine, QUANTITATIVE-ANALYSIS, I-BAR domain proteins, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Membrane, Membrane curvature, C. elegans, Phosphorylation, Medicine, MESH: Actins/chemistrys, MESH: Lipid Bilayers/chemistry, Protein Binding, Research Article, MESH: Cell Membrane/genetics, GIANT UNILAMELLAR VESICLES, MESH: Mutant Proteins/chemistry, QH301-705.5, MESH: Cytoskeletal Proteins/chemistry, Science, PHYSIOLOGICAL CONDITIONS, MESH: Cytoskeletal Proteins/metabolism, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, NATIVE MEMBRANES, macromolecular substances, PARIETAL-CELLS, Curvature, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Protein Domains, MESH: Cell Membrane/chemistry, MESH: Mutant Proteins/metabolism, Actin, MESH: Actins/geneticss, General Immunology and Microbiology, MESH: Phosphorylation, APICAL MEMBRANE, MESH: Mutant Proteins/genetics, Cell Membrane, MESH: Protein Binding/genetics, Apical membrane, Actins, F-ACTIN BINDING, In vitro, Protein enrichment, ezrin, Cytoskeletal Proteins, 030104 developmental biology, MESH: Lipid Bilayers/metabolisms, MESH: Protein Domains/genetics, membrane curvature, Biophysics, 1182 Biochemistry, cell and molecular biology, Mutant Proteins, ERM PROTEINS
Abstract: One challenge in current cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat or with positive or negative curvatures. Using in vitro and cell biology approaches, we assess mechanisms of ezrin’s enrichment on curved membranes. We evidence that ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD’s specific conformation reduces intermolecular ezrin interactions, allows binding to actin filaments, and promotes ezrin binding to positively curved membranes. While neither ezrinTD nor ezrinWT senses negative membrane curvature alone, we demonstrate that interacting with curvature sensors I-BAR-domain proteins facilitates ezrin enrichment in negatively curved membrane protrusions. Overall, our work reveals new mechanisms, specific conformation or binding to a curvature sensor partner, for targeting curvature insensitive proteins to curved membranes.
Published: 2018

21. Staphylococcus aureus Adenosine Inhibits sPLA2-IIA–Mediated Host Killing in the Airways

Author: Jérémy Brunet, Erwan Pernet, Lhousseine Touqui, Laurent Guillemot, Michel Chignard, Yongzheng Wu, Défense innée et inflammation, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Génétique Fonctionnelle de la Souris, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by Domaine d’Intérêts Majeur-Maladies Infectieuses from the Région Ile de France (a Ph.D. fellowship to E.P.) and Vaincre la Mucoviscidose Grants RF20120600672 and RF20130500830 (to Y.W.)., We thank Dr. D. Missiakas (University of Chicago, Chicago, IL) for providing the S. aureus Newman WT, ∆adsA mutant, and AdsA complemented strains, and Dr. Tarek Msadek (Institut Pasteur, Paris, France) for the different WT strains of S. aureus., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Wu, Yongzheng
Subjects: Male, Adenosine, MESH: Macrophages, Alveolar, Pyridines, Mutant, Nod2 Signaling Adaptor Protein, MESH: NF-kappa B, MESH: Cricetinae, medicine.disease_cause, p38 Mitogen-Activated Protein Kinases, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, 0302 clinical medicine, MESH: Cricetulus, Cricetinae, MESH: Staphylococcus aureus, MESH: Nod2 Signaling Adaptor Protein, Immunology and Allergy, MESH: Animals, Phosphorylation, MESH: Phagocytosis, 0303 health sciences, Imidazoles, NF-kappa B, Staphylococcal Infections, MESH: Group II Phospholipases A2, 3. Good health, Staphylococcus aureus, Host-Pathogen Interactions, MESH: Receptor, Adenosine A2A, MESH: Receptor, Adenosine A2B, Bronchoalveolar Lavage Fluid, MESH: Imidazoles, medicine.drug, Receptor, Adenosine A2A, Phagocytosis, p38 mitogen-activated protein kinases, Guinea Pigs, Immunology, MESH: Staphylococcal Infections, CHO Cells, MESH: Cyclic AMP-Dependent Protein Kinases, Biology, Receptor, Adenosine A2B, Group II Phospholipases A2, Cell Line, MESH: Guinea Pigs, Microbiology, 03 medical and health sciences, Cricetulus, MESH: CHO Cells, Macrophages, Alveolar, medicine, Animals, Protein kinase A, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, 030304 developmental biology, MESH: Phosphorylation, MESH: Bronchoalveolar Lavage Fluid, MESH: Host-Pathogen Interactions, MESH: Pyridines, MESH: Adenosine, Cyclic AMP-Dependent Protein Kinases, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Adenosine receptor, MESH: Male, MESH: Cell Line, MESH: p38 Mitogen-Activated Protein Kinases, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030215 immunology
Abstract: Staphylococcus aureus is a common cause of bacterial infections in respiratory diseases. It secretes molecules to dampen host immunity, and the recently identified adenosine is one of these molecules. The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host protein endowed with antibacterial properties, especially against Gram-positive bacteria such as S. aureus. However, the role of adenosine in sPLA2-IIA–mediated S. aureus killing by host is still unknown. The present studies showed that the S. aureus mutant lacking adenosine production (∆adsA strain) increased sPLA2-IIA expression in guinea pig airways and was cleared more efficiently, compared with the wild-type strain. S. aureus ∆adsA strain induced sPLA2-IIA expression by alveolar macrophages after phagocytic process via NOD2–NF-κB–dependent mechanism. However, S. aureus adenosine (wild-type and adsA-complemented strains) and exogenous adenosine downregulated S. aureus phagocytosis by alveolar macrophages, leading to inhibition of sPLA2-IIA expression. This occurred through inhibition of p38 phosphorylation via adenosine receptors A2a-, A2b-, and protein kinase A–dependent pathways. Taken together, our studies suggest that, in the airway, S. aureus escapes sPLA2-IIA–mediated killing through adenosine-mediated inhibition of phagocytosis and sPLA2-IIA expression.
Published: 2015

22. Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

Author: Anna Noatynska, Nicolas Tavernier, Lionel Pintard, Monica Gotta, Lisa Martino, Françoise Schwager, Thibaut Léger, Costanza Panbianco, Lucie Van Hove, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Department of Cellular Physiology and Metabolism, Faculty of Medicine, University of Geneva, Swiss National Centre for Competence in Research Programme Chemical Biology (NCCR-Chemical Biology), University of Geneva [Switzerland], Fondation Association pour la Recherche sur le Cancer PhD fellowship, French Government, French National Research Agency under grant No. ANR-11-IDEX-0005-01, Swiss National Science Foundation, University of Geneva, French National Research Agency under grant No. ANR-2012-BSV2-0001-01, Foundation for Medical Research 'Equipe Fondation pour la Recherche Médicale' DEQ20140329538, and ANR-11-IDEX-0005-02/11-LABX-0071,WHO AM I,Determinants de l’Identité : de la molécule à l’individu(2011)
Subjects: Embryo, Nonmammalian, Cell division, Cell Cycle Proteins, MESH: Amino Acid Sequence, environment and public health, 0302 clinical medicine, Sf9 Cells, MESH: Sf9 Cells, MESH: Animals, Phosphorylation, Research Articles, Caenorhabditis elegans, Aurora Kinase A, 0303 health sciences, MESH: Spodoptera, Protein-Serine-Threonine Kinases/metabolism, MESH: Caenorhabditis elegans Proteins, MESH: CDC2 Protein Kinase, Cell biology, MESH: Aurora Kinase A, Larva, 030220 oncology & carcinogenesis, CDC2 Protein Kinase/physiology, biological phenomena, cell phenomena, and immunity, inorganic chemicals, MESH: Enzyme Activation, Molecular Sequence Data, Mitosis, Aurora Kinase A/metabolism, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Protein Serine-Threonine Kinases, Spodoptera, Biology, PLK1, MESH: Protein-Serine-Threonine Kinases, 03 medical and health sciences, Enzyme activator, MESH: Cell Cycle Proteins, Report, MESH: Caenorhabditis elegans, CDC2 Protein Kinase, Animals, Humans, Amino Acid Sequence, ddc:612, Caenorhabditis elegans Proteins, 030304 developmental biology, Cyclin-dependent kinase 1, MESH: Humans, MESH: Molecular Sequence Data, Cell Cycle Proteins/metabolism, MESH: Phosphorylation, Larva/cytology/enzymology, MESH: Embryo, Nonmammalian, Cell Biology, MESH: Mitosis, biology.organism_classification, Enzyme Activation, enzymes and coenzymes (carbohydrates), Caenorhabditis elegans/cytology/enzymology, MESH: Protein Processing, Post-Translational, Caenorhabditis elegans Proteins/metabolism, Protein Processing, Post-Translational, MESH: Larva, Embryo, Nonmammalian/cytology/metabolism
Abstract: Phosphorylation of SPAT-1/Bora by Cdk1 enhances Plk1 phosphorylation by Aurora A and promotes entry into mitosis in C. elegans., The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho–SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing.
Published: 2015
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23. Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

Author: David C. Schriemer, Cortt G. Piett, Michael Weinfeld, J. N. Mark Glover, Shujuan Fang, Ruiqiong Ye, John A. Tainer, Ross A. Edwards, Zahra Havali-Shahriari, R. Daniel Aceytuno, Fatima Javed, Rajam S. Mani, Martial Rey, Michal Hammel, Susan P. Lees-Miller, Department of Biochemistry [Edmonton, AB, Canada] (Faculty of Medicine and Dentistry), University of Alberta, Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, Cross Cancer Institute and Department of Oncology, Molecular Biophysics and Integrated Bioimaging Division [Berkeley, CA, USA], and Lawrence Berkeley National Laboratory [Berkeley] (LBNL)
Subjects: Models, Molecular, 0301 basic medicine, DNA End-Joining Repair, MESH: Deuterium, Protein Conformation, Developmental Disabilities, MESH: Catalytic Domain, medicine.disease_cause, Mass Spectrometry, MESH: Recombinant Proteins, DNA Ligase ATP, chemistry.chemical_compound, Protein structure, MESH: Protein Conformation, X-Ray Diffraction, Structural Biology, Catalytic Domain, Protein Interaction Mapping, MESH: Syndrome, Phosphorylation, chemistry.chemical_classification, Mutation, MESH: X-Ray Diffraction, Syndrome, DNA repair protein XRCC4, MESH: Seizures, Recombinant Proteins, Cell biology, DNA-Binding Proteins, Phosphotransferases (Alcohol Group Acceptor), [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: DNA Repair Enzymes, Biochemistry, Microcephaly, MESH: Models, Molecular, Protein Binding, DNA damage, Mutation, Missense, Biology, MESH: Microcephaly, 03 medical and health sciences, MESH: DNA Ligase ATP, Seizures, DNA repair complex, Scattering, Small Angle, Genetics, medicine, Humans, Point Mutation, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Scattering, Small Angle, MESH: Point Mutation, MESH: DNA Damage, MESH: Mass Spectrometry, DNA ligase, MESH: Mutation, Missense, MESH: Humans, 030102 biochemistry & molecular biology, MESH: Phosphorylation, Point mutation, MESH: Protein Interaction Mapping, MESH: DNA End-Joining Repair, MESH: Multiprotein Complexes, Deuterium, MESH: Phosphotransferases (Alcohol Group Acceptor), DNA Repair Enzymes, 030104 developmental biology, chemistry, MESH: Developmental Disabilities, Multiprotein Complexes, MESH: Protein Processing, Post-Translational, Protein Processing, Post-Translational, DNA, MESH: DNA-Binding Proteins, DNA Damage
Abstract: International audience; Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.
Published: 2017

24. High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions

Author: Philippe Champeil, Pierre Le Maréchal, Joseph A. Lyons, Poul Nissen, José Luis Vázquez-Ibar, Cédric Montigny, Aurore Jacquot, Thibaud Dieudonné, Jakob Ulstrup, Miriam-Rose Ash, Guillaume Lenoir, Hassina Azouaoui, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin (DANDRITE), Centre for Membrane Pumps in Cells and Disease (PUMPKIN), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut des Neurosciences de Paris-Saclay ( Neuro-PSI ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Nordic-EMBL Partnership for Molecular Medicine, and PUMPkin ( DANDRITE ), and Centre for Membrane Pumps in Cells and Disease ( PUMPKIN )
Subjects: 0301 basic medicine, Phosphatidylinositol 4-phosphate, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, MESH : Saccharomyces cerevisiae, Plasma protein binding, Biochemistry, MESH: Thrombin, chemistry.chemical_compound, MESH : Phosphorylation, [ SDV.NEU.SC ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, 0302 clinical medicine, Adenosine Triphosphate, MESH: Saccharomyces cerevisiae Proteins, Phosphatidylinositol Phosphates, ATP hydrolysis, MESH : Arginine, lipid-protein interaction, MESH: Adenosine Triphosphate, MESH : Adenosine Triphosphate, Phospholipid Transfer Proteins, Phosphorylation, Phospholipids, MESH : Phospholipid Transfer Proteins, medicine.diagnostic_test, [SDV.NEU.PC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, Hydrolysis, Autophosphorylation, MESH: Phospholipid Transfer Proteins, Thrombin, MESH: Arginine, [SDV.NEU.SC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, MESH : Protein Binding, Phosphatidylserine, MESH: Phosphatidylinositol Phosphates, MESH: Saccharomyces cerevisiae, phosphoinositide, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, inhibition mechanism, MESH: Protein Domains, autophosphorylation, MESH : Mutation, MESH: Hydrolysis, Protein Binding, CDC50 protein, phosphatidylserine, Saccharomyces cerevisiae Proteins, MESH : Proteolysis, MESH: Mutation, Proteolysis, MESH : Calcium-Transporting ATPases, MESH: Proteolysis, Calcium-Transporting ATPases, Saccharomyces cerevisiae, Biology, Arginine, MESH : Phospholipids, MESH: Calcium-Transporting ATPases, MESH : Saccharomyces cerevisiae Proteins, [ SDV.NEU.PC ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, 03 medical and health sciences, Protein Domains, MESH : Hydrolysis, Membrane Biology, medicine, Journal Article, MESH : Phosphatidylinositol Phosphates, MESH: Protein Binding, Phosphatidylinositol, Molecular Biology, MESH: Phospholipids, MESH: Phosphorylation, MESH : Protein Domains, MESH : Thrombin, Cell Biology, Flippase, flippase, 030104 developmental biology, chemistry, Mutation, 030217 neurology & neurosurgery, limited proteolysis
Abstract: International audience; P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104 and after Arg 1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.
Published: 2017

25. Type I interferon-enhanced IL-10 expression in human CD4 T cells is regulated by STAT3, STAT2, and BATF transcription factors

Author: Umeshree Govender, Frédérique Michel, Yasmine Bourdache, Sandra Pellegrini, Béatrice Corre, Signalisation des Cytokines, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by funding from the Aide à la Recherche sur la Sclérose En Plaques Foundation (ARSEP), Institut Pasteur, and Centre National de la Recherche Scientifique (CNRS). U.G. was supported by the Pasteur‐Paris University (PPU) International Ph.D. Program and by short funding from the Immunology Department of the Institut Pasteur and ARSEP., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, CD4-Positive T-Lymphocytes, MESH: Signal Transduction, MESH: Interleukin-10, MESH: CD3 Complex, CD3 Complex, MESH: Protein Isoforms, 0302 clinical medicine, Interferon, BATF, MESH: RNA, Small Interfering, MESH: STAT2 Transcription Factor, Immunology and Allergy, Protein Isoforms, STAT1, Cycloheximide, Phosphorylation, RNA, Small Interfering, MESH: Cycloheximide, Regulation of gene expression, MESH: CD28 Antigens, biology, Tr1 cells, MESH: STAT3 Transcription Factor, MESH: CD4-Positive T-Lymphocytes, MESH: Gene Expression Regulation, Cell biology, Interleukin-10, Interleukin 10, Basic-Leucine Zipper Transcription Factors, Enhancer Elements, Genetic, STAT1 Transcription Factor, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Interferon-alpha, signaling, medicine.drug, Protein Binding, Signal Transduction, STAT3 Transcription Factor, Immunology, Primary Cell Culture, MESH: Basic-Leucine Zipper Transcription Factors, Antibodies, MESH: Primary Cell Culture, 03 medical and health sciences, CD28 Antigens, medicine, Humans, MESH: Protein Binding, Enhancer, Transcription factor, Binding Sites, MESH: Humans, MESH: Phosphorylation, MESH: Antibodies, T-cell receptor, Interferon-alpha, STAT2 Transcription Factor, 030206 dentistry, Cell Biology, 030104 developmental biology, Gene Expression Regulation, MESH: Binding Sites, biology.protein, MESH: STAT1 Transcription Factor, MESH: Enhancer Elements, Genetic, gene regulation
Abstract: Type I IFN can exert pro- and anti-inflammatory activities in the immune system. Here, we have investigated the mechanism by which IFN-α enhances early expression of the anti-inflammatory cytokine IL-10 in human CD45RA+CD4+ T cells. With the use of transcriptomic and biochemical approaches, we found distinct and combined contributions of the IFN and the TCR signaling pathways to the induction of STAT1/2/3 and the basic leucine zipper activating transcription factor-like (BATF) family members. Moreover, IFN-induced STAT3 phosphorylation was prolonged by the TCR response, whereas IFN-induced STAT2 phosphorylation was of long duration. With the use of RNA interference (RNAi), we identified STAT3 as the major actor and STAT2 as a contributor of the IFN action on IL-10. Upon TCR/IFN costimulation, STAT3 directly bound at the IL-10 conserved noncoding sequence (CNS)- 9, an enhancer element known to recruit BATF in CD4 T cells. The cosilencing of the 3 BATFs resulted in an overall reduction of IL-10 expression, but the promoting activity of IFN-α was retained. These results support the notion that the IFN action is indexed on BATF function and provide evidence for a cooperation between BATFs and STAT3, the latter being activated via early IFN and delayed TCR effects.
Published: 2017

26. A second Wpl1 anti‐cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP 4

Author: Birot, Adrien, Eguienta, Karen, Vazquez, Stéphanie, Claverol, Stéphane, Bonneu, Marc, Ekwall, Karl, Javerzat, Jean-Paul, Vaur, Sabine, Javerzat, Jean‐Paul, Institut de biochimie et génétique cellulaires (IBGC), and Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Chromosomal Proteins, Non-Histone, [SDV]Life Sciences [q-bio], Mutant, cohesin, Cell Cycle Proteins, Chromosome segregation, Phosphoprotein Phosphatases, Protein Phosphatase 4, Phosphorylation, Genetics, General Neuroscience, Nuclear Proteins, Articles, fission yeast, Establishment of sister chromatid cohesion, MESH: Schizosaccharomyces, WAPL, Separase, biological phenomena, cell phenomena, and immunity, MESH: Mutation, Protein subunit, MESH: Carrier Proteins, Biology, MESH: Phosphoproteins, General Biochemistry, Genetics and Molecular Biology, Dephosphorylation, 03 medical and health sciences, MESH: Cell Cycle Proteins, MESH: Chromosomal Proteins, Non-Histone, Schizosaccharomyces, MESH: Phosphoprotein Phosphatases, Immunoprecipitation, Molecular Biology, General Immunology and Microbiology, Cohesin, MESH: Phosphorylation, MESH: Immunoprecipitation, MESH: Schizosaccharomyces pombe Proteins, Phosphoproteins, 030104 developmental biology, MESH: Gene Deletion, MESH: Protein Processing, Post-Translational, Mutation, Schizosaccharomyces pombe Proteins, Carrier Proteins, Protein Processing, Post-Translational, MESH: Nuclear Proteins, Gene Deletion
Abstract: International audience; Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.
Published: 2017

27. ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription

Author: Richard Treisman, Stuart Horswell, Aengus Stewart, Gavin Kelly, Phil East, Francesco Gualdrini, Nik Matthews, Cyril Esnault, Lincoln's Inn Fields Laboratories, and Cancer Research UK
Subjects: 0301 basic medicine, MESH: Signal Transduction, Serum Response Factor, Transcription, Genetic, [SDV]Life Sciences [q-bio], Transcription coregulator, MESH: Mice, Knockout, Histones, Mice, Elk-1, Histone methylation, MESH: Early Growth Response Protein 1, Histone code, histone modification, MESH: Animals, Phosphorylation, Extracellular Signal-Regulated MAP Kinases, MESH: Extracellular Signal-Regulated MAP Kinases, Mice, Knockout, MESH: Histones, General transcription factor, biology, Activating transcription factor 2, ternary complex factor, 3. Good health, ERK, MESH: Serum Response Factor, H3 phosphorylation, embryonic structures, Tetradecanoylphorbol Acetate, RNA Interference, SRF, MESH: Transcription Initiation Site, Transcription Initiation Site, TCF Transcription Factors, transcription, Signal Transduction, MESH: Enzyme Activation, MESH: Mutation, MESH: RNA Interference, Transfection, immediate-early genes, Article, Cell Line, MESH: Chromatin, 03 medical and health sciences, Sp3 transcription factor, Animals, MESH: ets-Domain Protein Elk-1, Transcription factor, Molecular Biology, MESH: Mice, MESH: Tetradecanoylphorbol Acetate, Early Growth Response Protein 1, ets-Domain Protein Elk-1, MESH: Phosphorylation, MESH: Transcription, Genetic, MESH: Transfection, Pioneer factor, MESH: Chromatin Assembly and Disassembly, Cell Biology, Chromatin Assembly and Disassembly, Molecular biology, MESH: Cell Line, Enzyme Activation, 030104 developmental biology, Mutation, biology.protein, chromatin, MESH: TCF Transcription Factors
Abstract: Summary We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription., Graphical Abstract, Highlights • TPA-driven ERK activation induces a common pattern of histone modifications at TSSs • Most ERK-induced histone modifications are dependent on TCF family of SRF cofactors • At these TSSs, induced histone modifications require ERK-dependent TCF phosphorylation • TCF-dependent histone modifications require TCF to recruit the transcription machinery, In MEFs, TPA-induced ERK activation induces a common pattern of histone modifications at more than 2,000 TSSs. Modifications at most TSSs are dependent on the TCF family of ERK-regulated SRF cofactors. At these TSSs, induced modifications are dependent both on phosphorylation of TCF and on its ability to recruit the transcriptional machinery.
Published: 2017

28. A naturally occurring variant of HPV-16 E7 exerts increased transforming activity through acquisition of an additional phospho-acceptor site

Author: Amira Zine El Abidine, Ikram Guizani, Samir Boubaker, Paola Massimi, Vjekoslav Tomaić, Lawrence Banks, Emna Ennaifer, Rahima Bel Haj Rhouma, Laboratoire d'Epidémiologie Moléculaire et de Pathologie Expérimentale Appliquée aux Maladies Infectieuses (LR11IPT04), Université de Tunis El Manar (UTM)-Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Rudjer Boskovic Institute [Zagreb], Tumour Virology Laboratory [Trieste], International Centre for Genetic Engineering and Biotechnology (ICGEB), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP), This work was supported in part by a research grant to LB from the Associazione Italiana per la Ricerca sul Cancro. Amira Zine El Abidine is a recipient of an ICGEB Arturo Falaschi Pre-doctoral Fellowship and was supported by the Ministry of Higher Education and Scientific Research of Tunisia (LR111PT04)., and We are grateful to Miranda Thomas for valuable comments on the manuscript.
Subjects: 0301 basic medicine, Papillomavirus E7 Proteins, Amino Acid Motifs, MESH: Papillomavirus E7 Proteins/metabolism, Retinoblastoma Protein, MESH: Human papillomavirus 16/physiology, MESH: Amino Acid Motifs, Phosphorylation, TBP, E7, Human papillomavirus 16, MESH: Papillomavirus E7 Proteins/chemistry, HPV, CKII phosphorylation, pRb, MESH: Human papillomavirus 16/genetics, 3. Good health, Cell biology, MESH: TATA-Box Binding Protein/genetics, Biochemistry, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Amino acid change, Cancer development, Protein Binding, MESH: Retinoblastoma Protein/metabolism, MESH: TATA-Box Binding Protein/metabolism, MESH: Papillomavirus Infections/metabolism, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, MESH: Retinoblastoma Protein/genetics, MESH: Cell Transformation, Viral, 03 medical and health sciences, Virology, Humans, MESH: Protein Binding, MESH: Human papillomavirus 16/chemistry, Human papillomavirus, Binding Sites, MESH: Humans, MESH: Phosphorylation, TATA-Box Binding Protein, Papillomavirus Infections, MESH: Papillomavirus Infections/virology, Cell Transformation, Viral, 030104 developmental biology, MESH: Binding Sites, MESH: Papillomavirus Infections/genetics, MESH: Papillomavirus E7 Proteins/genetics, Function (biology), [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Human Papillomavirus E6 and E7 play critical roles in cancer development, although not all isolates of the viral oncoproteins are identical. A common E7 variant encodes an amino acid change at N29S. We show that this change increases the levels of phosphorylation by CKII by creating an additional phospho-acceptor site at S29. This confers increased phospho-dependent interaction with a number of cellular targets, including TATA Box Binding Protein (TBP) and pRb. A further consequence is an increased ability to target pRb and p130 for degradation. Biologically, these biochemical differences are reflected in an increased ability of the N29S variant to transform primary rodent cells. This is the first study to demonstrate an important biochemical change in E7 function caused by a naturally occurring variation, and we suggest that the N29S variant merits further assessment to determine whether it has an increased association with the development of HPV-associated malignancies.
Published: 2017

29. Site-Specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and Phospho-Protein Affinity SDS-PAGE

Author: Sébastien Huault, Krittalak Chakrabandhu, Anne-Odile Hueber, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)
Subjects: inorganic chemicals, 0301 basic medicine, Phos-tag™, medicine.drug_class, [SDV]Life Sciences [q-bio], Monoclonal antibody, Immunoblot, MESH: Phosphoproteins, environment and public health, 03 medical and health sciences, 0302 clinical medicine, Protein purification, medicine, Mobility shift, Phosphorylation, Tyrosine, Receptor, Polyacrylamide gel electrophoresis, ComputingMilieux_MISCELLANEOUS, Death domain, MESH: Humans, MESH: Immunoblotting, MESH: Phosphorylation, Chemistry, MESH: fas Receptor, MESH: Chromatography, Affinity, Fas receptor, MESH: Cell Line, 3. Good health, enzymes and coenzymes (carbohydrates), 030104 developmental biology, Biochemistry, 030220 oncology & carcinogenesis, MESH: Electrophoretic Mobility Shift Assay, CD95, bacteria, SDS-PAGE, MESH: Electrophoresis, Polyacrylamide Gel, MESH: Phosphotyrosine
Abstract: International audience; Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
Published: 2017

30. Annonacin, a natural lipophilic mitochondrial complex I inhibitor, increases phosphorylation of tau in the brain of FTDP-17 transgenic mice

Author: Wolfgang H. Oertel, Bertrand Friguet, Gesine Respondek, Günter U. Höglinger, Matthias Höllerhage, Agathe Tarze, Stefanie Müssner, Mohamed Salama, Christel Depienne, Agnès Rastetter, Anderson de Andrade, Pierre Champy, Elizabeth Sumi Yamada, Guellaen, Georges, Experimental Neurology, Philipps University, Experimental Neuropathology Laboratory, University Hospital João de Barros Barreto-Federal University of Para - Universidade Federal do Pará - UFPA [Belém, Brazil] (UFPA), German Research Center for Neurodegenerative Diseases - Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Department of Neurology, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Neurologie et thérapeutique expérimentale, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR70-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Laboratoire de Biologie Cellulaire du vieillissement, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR83, Laboratoire de Pharmacognosie (BioCIS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Deutsche Forschungsgemeinschaft (HO2402/6-1, HO2402/8-1), the German Ministry of Education and Research (BMBF EF 10-54), the Centre National de la Recherche Scientifique (CNRS), a Marie Curie Incoming International Fellowship (21996 to E.S.Y.) and the German Academic Exchange Service (DAAD to A.C.F.d.A.)., University Hospital João de Barros Barreto-Federal University of Para - Universidade Federal do Para [Belem - Brésil], Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR70-Université Pierre et Marie Curie - Paris 6 (UPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Intégrative (IFR-BI), Université Pierre et Marie Curie - Paris 6 (UPMC)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Neurons, pharmacology [Enzyme Inhibitors], drug effects [Gene Expression Regulation], genetics [Gene Expression Regulation], drug effects [Microglia], MESH: Dose-Response Relationship, Drug, Mice, Lactones, chemistry.chemical_compound, 0302 clinical medicine, MESH: Animals, Phosphorylation, pharmacology [Furans], Enzyme Inhibitors, genetics [Arginine], Neurons, 0303 health sciences, Kinase, Neurodegeneration, neurodegeneration, MESH: Arginine, Tryptophan, Brain, MESH: Gene Expression Regulation, Mitochondria, MESH: tau Proteins, Cell biology, MESH: Microglia, Neurology, Biochemistry, MESH: Enzyme Inhibitors, drug effects [Brain], Microglia, genetics [Tryptophan], Tauopathy, Microtubule-Associated Proteins, MESH: Lactones, Genetically modified mouse, MESH: Mutation, MESH: Mice, Transgenic, MESH: Mitochondria, Annonacin, Mice, Transgenic, genetics [Mutation], MAPT protein, human, tau Proteins, Biology, Arginine, MESH: Brain, 03 medical and health sciences, drug effects [Phosphorylation], Developmental Neuroscience, MESH: Mice, Inbred C57BL, mental disorders, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Animals, Humans, drug effects [Neurons], [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ddc:610, Mortality, genetics [Phosphorylation], Furans, MESH: Mice, MESH: Tryptophan, 030304 developmental biology, MESH: Humans, MESH: Mortality, MESH: Phosphorylation, Dose-Response Relationship, Drug, Cyclin-dependent kinase 5, tauopathy, MESH: Furans, metabolism [Microtubule-Associated Proteins], annonacin, metabolism [Mitochondria], medicine.disease, Mice, Inbred C57BL, MESH: Microtubule-Associated Proteins, genetics [tau Proteins], Somatodendritic compartment, Gene Expression Regulation, chemistry, environmental neurotoxin, metabolism [Brain], Mutation, pharmacology [Lactones], genetics [Mitochondria], microtubule-associated protein tau, 030217 neurology & neurosurgery
Abstract: International audience; Both genetic and environmental factors likely contribute to the neuropathology of tauopathies, but it remains unclear how specific genetic backgrounds affect the susceptibility towards environmental toxins. Mutations in the tau gene have been associated with familial tauopathies, while annonacin, a plant-derived mitochondrial inhibitor, has been implicated in an environmental form of tauopathy. We therefore determined whether there was a pathogenic synergy between annonacin exposure and the expression of the R406W-tau mutation in transgenic mice. We found that annonacin exposure caused an increase in the number of neurons with phosphorylated tau in the somatodendritic compartment in several brain areas in R406W(+/+) mice as opposed to mice that had only the endogenous mouse tau (R406W(-/-)). Western blot analysis demonstrated a concomitant increase in total tau protein without increase in tau mRNA, but reduced proteasomal proteolytic activity in R406W(+/+), but not R406W(-/-) mice, upon annonacin-treatment. Phosphorylated tau levels exceeded the increase in total tau protein, along with increased levels of different tau kinases, foremost a striking increase in the p25/p35 ratio, known to activate the tau kinase Cdk5. In summary, we observed a synergistic interaction between annonacin exposure and the presence of the R406W-tau mutation, which resulted in reduced degradation, increased phosphorylation and redistribution of neuronal tau.
Published: 2014

31. Structural Coupling between Autokinase and Phosphotransferase Reactions in a Bacterial Histidine Kinase

Author: Silvia Soto Diaz, Ariel E. Mechaly, Alejandro Buschiazzo, Nathalie Sassoon, Pedro M. Alzari, Jean-Michel Betton, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), 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), A.E.M. was the recipient of postdoctoral fellowships from the Basque Country Government and ANII (Agencia Nacional de Investigación e Innovación, Uruguay). This work has been partly supported by grants from the Institut Pasteur and the CNRS (France)., and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Models, Molecular, phosphoryl transfer, Histidine Kinase, Stereochemistry, Protein Conformation, Allosteric regulation, MESH: Escherichia coli Proteins, Protomer, response regulator, Crystallography, X-Ray, Phosphotransferase, 03 medical and health sciences, allosteric switch, MESH: Protein Conformation, Bacterial Proteins, Structural Biology, [CHIM.CRIS]Chemical Sciences/Cristallography, Transferase, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Phosphorylation, Molecular Biology, MESH: Bacterial Proteins, MESH: Protein Kinases, 030102 biochemistry & molecular biology, MESH: Phosphorylation, Chemistry, MESH: Protein Multimerization, Escherichia coli Proteins, Histidine kinase, Autophosphorylation, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Response regulator, 030104 developmental biology, two-component systems, MESH: Histidine Kinase, autophosphorylation, Signal transduction, Protein Multimerization, Protein Kinases, signal transduction, MESH: Models, Molecular
Abstract: International audience; Bacterial two-component systems consist of a sensor histidine kinase (HK) and a response regulator (RR). HKs are homodimers that catalyze the autophosphorylation of a histidine residue and the subsequent phosphoryl transfer to its RR partner, triggering an adaptive response. How the HK autokinase and phosphotransferase activities are coordinated remains unclear. Here, we report X-ray structures of the prototypical HK CpxA trapped as a hemi-phosphorylated dimer, and of the receiver domain from the RR partner, CpxR. Our results reveal that the two catalytic reactions can occur simultaneously, one in each protomer of the asymmetric CpxA dimer. Furthermore, the increase of autokinase activity in the presence of phosphotransfer-impaired CpxR put forward the idea of an allosteric switching mechanism, according to which CpxR binding to one CpxA protomer triggers autophosphorylation in the second protomer. The ensuing dynamical model provides a mechanistic explanation of how HKs can efficiently orchestrate two catalytic reactions involving large-scale protein motions.
Published: 2016

32. Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action

Author: Marcelo A. Martí, Ariel E. Mechaly, Gonzalo Obal, J.A. Imelio, Nicole Larrieux, Alejandro Buschiazzo, Felipe Trajtenberg, Matías R. Machado, 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), Biomolecular Simulations / Simulaciones Biomoleculares [Montevideo], Universidad de Buenos Aires [Buenos Aires] (UBA), Protein Biophysics [Montevideo] (UBP), Integrative Microbiology of Zoonotic Agents [Paris and Montevideo] (IMiZA), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris], Agencia Nacional de Investigación e Innovación (FCE2009_1_2679)Agence Nationale de la Recherche (PCV06_138918)FOCEM (MERCOSUR Structural Convergence Fund) (COF 03/11)Centro de Biologia Estructural del MercosurAgencia Nacional de Investigación e Innovación (FCE2007_219), and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, Models, Molecular, MESH: Signal Transduction, Histidine Kinase, Protein Conformation, Regulator, Crystallography, X-Ray, Phosphotransferase, MESH: Protein Conformation, biophysics, B. subtilis, Transferase, structural biology, Phosphorylation, Biology (General), Microbiology and Infectious Disease, Kinase, General Neuroscience, General Medicine, MESH: Transcription Factors, Biophysics and Structural Biology, Cell biology, Biochemistry, MESH: Histidine Kinase, Medicine, phosphoryl-transfer mechanism, MESH: Models, Molecular, Research Article, Bacillus subtilis, Signal Transduction, Cell signaling, QH301-705.5, infectious disease, Science, Phosphatase, Biology, General Biochemistry, Genetics and Molecular Biology, Dephosphorylation, 03 medical and health sciences, [CHIM.CRIS]Chemical Sciences/Cristallography, cell signaling, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030102 biochemistry & molecular biology, General Immunology and Microbiology, MESH: Phosphorylation, microbiology, E. coli, MESH: Bacillus subtilis, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Structural biology, MESH: Protein Processing, Post-Translational, two component systems, Protein Processing, Post-Translational, Transcription Factors, allosteric control of protein function
Abstract: Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring. DOI: http://dx.doi.org/10.7554/eLife.21422.001
Published: 2016

33. Body fat distribution in Parkinson's disease: An MRI-based body fat quantification study

Author: Jan Kassubek, Albert C. Ludolph, Hans-Peter Müller, Denise Bernhardt, Luc Dupuis, University of Ulm (UUlm), Heidelberg Institute of Radiation Oncology [Heidelberg, Germany] (HIRO), Heidelberg University Hospital [Heidelberg], Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Dieterle, Stéphane
Subjects: Leptin, Male, Parkinson's disease, [SDV]Life Sciences [q-bio], Statistics as Topic, Adipose tissue, Gastroenterology, Weight loss, Body Mass Index, MESH: Magnetic Resonance Imaging, [SCCO]Cognitive science, 0302 clinical medicine, MESH: Sequestosome-1 Protein, Image Processing, Computer-Assisted, Medicine, Mass index, 030212 general & internal medicine, MESH: Statistics, Nonparametric, medicine.diagnostic_test, MESH: Peptides, Parkinson Disease, MESH: Follow-Up Studies, MESH: Case-Control Studies, MESH: Image Processing, Computer-Assisted, [SDV] Life Sciences [q-bio], Adipose Tissue, Neurology, Female, medicine.symptom, MESH: Adipose Tissue, medicine.medical_specialty, Body fat distribution, MESH: Zebrafish Proteins, Statistics, Nonparametric, MESH: Protein-Serine-Threonine Kinases, MESH: Body Mass Index, 03 medical and health sciences, Magnetic resonance imaging, Internal medicine, Humans, MESH: Body Fat Distribution, MESH: Zebrafish, MESH: Statistics as Topic, MESH: Humans, MESH: Phosphorylation, business.industry, Body Weight, Case-control study, [SCCO] Cognitive science, MESH: Leptin, medicine.disease, MESH: Male, MESH: Body Weight, Endocrinology, Metabolism, Case-Control Studies, Neurology (clinical), Geriatrics and Gerontology, business, Body mass index, MESH: Female, 030217 neurology & neurosurgery, MESH: Parkinson Disease, Follow-Up Studies
Abstract: International audience; Introduction: There is some evidence that Parkinson's Disease (PD) patients have lower body weight and lower fat mass when compared to healthy subjects and that lower body weight and fat mass influence disease risk and progression. It remains unclear, however, if weight loss of fat mass loss occurs only in a subgroup of patients and whether fat distribution is altered during PD. The aim of this study was to prospectively investigate adipose tissue content and distribution in PD patients.Methods: The body fat composition of PD patients (N = 54) was compared with age matched healthy controls (N = 55) using a magnetic resonance imaging (MRI)-based method. A longitudinal MRI scan was acquired in 25 PD patients after a mean follow up period of 12 months.Results: The volume of total body fat as well as of visceral fat showed no difference between PD patients and healthy controls at baseline or at follow up. However, PD patients displayed decreased subcutaneous fat tissue (p = 0.01) and a higher visceral to subcutaneous fat ratio as compared to controls (p = 0.004). After follow up, 16 PD patients did not lose weight, while 9 PD patients lost between 0.5 and 10 kg.Conclusion: Fat distribution is altered in PD patients, with an increased ratio of visceral to subcutaneous fat.
Published: 2016

34. PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development

Author: Drelon, Coralie, Berthon, Annabel, Sahut-Barnola, Isabelle, Mathieu, Mickael, Dumontet, Typhanie, Rodriguez, Stéphanie, Batisse-Lignier, Marie, Tabbal, Houda, Tauveron, Igor, Martinez, Anne-Marie, Pointud, Jean-Christophe, Gomez-Sanchez, Celso, Vainio, Seppo, Shan, Jingdong, Val, Sonia, Val, Andreas, Stratakis, Constantine, Martinez, Antoine, VAL, Pierre, Lefrançois-Martinez, Marie, Sacco, Sonia, Schedl, Andreas, Génétique, Reproduction et Développement - Clermont Auvergne (GReD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne (UCA)-Centre National de la Recherche Scientifique (CNRS), Génétique, Reproduction et Développement - Clermont Auvergne (GReD ), Interactions génétiques et cellulaires au cours de la différenciation, Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Clermont-Ferrand, University of Mississippi Medical Center (UMMC), Laboratory of Developmental Biology, Department of Medical Biochemistry and Molecular Biology, University of Oulu and Biocenter Oulu, Section on Endocrinology and Genetics, National Institutes of Health (NIH)-National Institute of Child Health and Human Development, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Génétique, Reproduction et Développement (GReD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), Génétique, Reproduction et Développement (GReD ), 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), Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020]), 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), Centre de Recherche Universitaire Lorrain d'Histoire (CRULH), Université de Lorraine (UL), 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), Génétique du développement normal et pathologique, and COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Carcinogenesis, Cellular differentiation, MESH: beta Catenin, Adrenal Gland Neoplasms, General Physics and Astronomy, MESH: Zona Fasciculata, Mice, chemistry.chemical_compound, WNT4, MESH: Animals, Phosphorylation, Wnt Signaling Pathway, beta Catenin, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, Aldosterone, biology, Adrenal cortex, Wnt signaling pathway, MESH: Wnt Signaling Pathway, Cell Differentiation, MESH: Carcinogenesis, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, 3. Good health, Cell biology, medicine.anatomical_structure, Female, Zona Glomerulosa, MESH: Cell Differentiation, endocrine system, medicine.medical_specialty, Beta-catenin, MESH: Cell Line, Tumor, MESH: Zona Glomerulosa, Science, [SDV.CAN]Life Sciences [q-bio]/Cancer, MESH: Cyclic AMP-Dependent Protein Kinases, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Zona fasciculata, Cell Line, Tumor, Internal medicine, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Animals, Humans, MESH: Mice, MESH: Humans, MESH: Phosphorylation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, Cyclic AMP-Dependent Protein Kinases, MESH: Adrenal Gland Neoplasms, [SDV.GEN.GA]Life Sciences [q-bio]/Genetics/Animal genetics, 030104 developmental biology, Endocrinology, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, chemistry, Zona glomerulosa, biology.protein, Zona Fasciculata, MESH: Female
Abstract: Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates β-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling., The adrenal cortex undergoes functional zonation to generate an outer zona glomerulosa (ZG) and inner zona fasciculata (ZF), but how this is regulated at a molecular level is unclear. Here, the authors show that ZG differentiation is stimulated by WNT signalling and that PKA blocks WNT signalling to allow ZF differentiation and also prevents WNT-induced cancer development.
Published: 2016

35. The octadecaneuropeptide ODN prevents 6-hydroxydopamine-induced apoptosis of cerebellar granule neurons through a PKC-MAPK-dependent pathway

Author: Laurence Desrues, Hubert Vaudry, Jérôme Leprince, Mohamed Amri, David Vaudry, Olfa Masmoudi-Kouki, Hadhemi Kaddour, Yosra Hamdi, Hélène Castel, Marie-Christine Tonon, M. Basille, Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Biological Sciences, Université de Tunis El Manar (UTM)-Laboratory of Functional Neurophysiology and Pathology, Research Unit UR/11ES09, Faculty of Science of Tunis-Faculty of Science of Tunis, Différenciation et communication neuronale et neuroendocrine (DC2N), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Neuroendocrinologie cellulaire et moléculaire, Télécom SudParis (TSP), and Université de Tunis El Manar (UTM)
Subjects: MAPK/ERK pathway, MESH: Cyclin D1, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, MESH: Neurons, Apoptosis, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, MESH: Neuropeptides, medicine.disease_cause, Biochemistry, 0302 clinical medicine, Cerebellum, MESH: Caspase 3, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Cyclin D1, MESH: Animals, Phosphorylation, MESH: Peptide Fragments, Protein Kinase C, Diazepam Binding Inhibitor, Neurons, MESH: Glutathione, 0303 health sciences, MESH: Oxidative Stress, Caspase 3, Chemistry, MESH: Reactive Oxygen Species, hemic and immune systems, MESH: Neuroprotective Agents, respiratory system, Glutathione, 3. Good health, Cell biology, Neuroprotective Agents, Programmed cell death, MESH: Rats, MAP Kinase Signaling System, Neuroprotection, 03 medical and health sciences, Cellular and Molecular Neuroscience, medicine, Animals, Rats, Wistar, Oxidopamine, Protein kinase C, 030304 developmental biology, Hydroxydopamine, MESH: Phosphorylation, MESH: MAP Kinase Signaling System, MESH: Apoptosis, Neuropeptides, MESH: Sympatholytics, MESH: Rats, Wistar, MESH: Protein Kinase C, Peptide Fragments, MESH: Cerebellum, Rats, MESH: Oxidopamine, Oxidative Stress, Metabotropic receptor, nervous system, Sympatholytics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH: Diazepam Binding Inhibitor, Reactive Oxygen Species, 030217 neurology & neurosurgery, Oxidative stress
Abstract: International audience; Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6-hydroxydopamine (6-OHDA)-induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central-type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10⁻¹⁸ to 10⁻¹² M) inhibited 6-OHDA-evoked cell death in a concentration-dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo₁₋₈ [DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6-OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6-OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro-apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase-3 activity. Exposure of 6-OHDA-treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6-OHDA-induced oxidative stress and apoptotic cell death.
Published: 2013

36. JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

Author: Marie-Paule Wautier, Wassim El Nemer, Marie Cambot, Bruno Cassinat, Maria De Grandis, Jean-Luc Wautier, Christine Chomienne, Yves Colin, Caroline Le Van Kim, Protéines de la membrane érythrocytaire et homologues non-érythroides, Université des Antilles et de la Guyane (UAG)-Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM), GR-Ex, Laboratoire d'Excellence, Institut National de la Transfusion Sanguine [Paris] (INTS), Unite de Biologie Cellulaire, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Hématologie -Immunologie -Cibles thérapeutiques, Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), and The work was funded by the Institut National de la Santé et de la Recherche Médicale (Inserm), the Institut National de la Transfusion Sanguine (INTS), and a grant from Région Île-de-France (SESAME 2007 no. F-08-1104/R). The PhD student, Maria De Grandis, was funded by the Ministère de l'Enseignement Supérieur et de la Recherche at the Ecole Doctorale B3MI.
Subjects: Male, MESH: Signal Transduction, Erythrocytes, MESH: rap1 GTP-Binding Proteins, MESH: Polycythemia Vera, Biochemistry, MESH: Janus Kinase 2, Mice, MESH: Aged, 80 and over, 0302 clinical medicine, Receptors, Erythropoietin, MESH: Animals, Phosphorylation, Polycythemia Vera, Aged, 80 and over, MESH: Aged, 0303 health sciences, MESH: Middle Aged, Janus kinase 2, biology, Cell adhesion molecule, MESH: Erythrocytes, rap1 GTP-Binding Proteins, MESH: Laminin, Hematology, Middle Aged, Lutheran Blood-Group System, MESH: Lutheran Blood-Group System, 030220 oncology & carcinogenesis, MESH: Cell Adhesion Molecules, Female, Signal Transduction, Immunology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Cell Line, MESH: Cell Adhesion, 03 medical and health sciences, BCAM, Cell Adhesion, Animals, Humans, MESH: Receptors, Erythropoietin, Cell adhesion, MESH: Mice, Protein kinase B, PI3K/AKT/mTOR pathway, Aged, 030304 developmental biology, MESH: Humans, MESH: Phosphorylation, MESH: Proto-Oncogene Proteins c-akt, Akt/PKB signaling pathway, Cell Biology, Janus Kinase 2, Molecular biology, MESH: Male, MESH: Cell Line, Erythropoietin receptor, biology.protein, Laminin, Cell Adhesion Molecules, Proto-Oncogene Proteins c-akt, MESH: Female
Abstract: Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.
Published: 2013

37. Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance

Author: Paul D. Carr, Faisal Younus, Jian-Wei Liu, Robyn J. Russell, Chris M. Coppin, Tamara Meirelles, John G. Oakeshott, David L. Ollis, Mathilde Lethier, Martin Weik, Gunjan Pandey, Colin J. Jackson, Research School of Chemistry, Australian National University (ANU), Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), CSIRO - Land & Water National Research Flagship, 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), Commonwealth Scientific and Industrial Research Organisation [Canberra] (CSIRO), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-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), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0106 biological sciences, Insecticides, Drug Resistance, MESH: Protein Structure, Secondary, MESH: Catalytic Domain, Genes, Insect, MESH: Genes, Insect, Crystallography, X-Ray, 01 natural sciences, Protein Structure, Secondary, Carboxylesterase, Substrate Specificity, chemistry.chemical_compound, Catalytic Domain, MESH: Animals, MESH: Carboxylesterase, Phosphorylation, directed evolution, MESH: Diptera, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Sheep Diseases, Organophosphate, Biological Sciences, Directed evolution, Acetylcholinesterase, Biochemistry, Lucilia cuprina, MESH: Drug Resistance, ali-esterase, MESH: Australia, Sheep Diseases, MESH: Sheep, 03 medical and health sciences, Animals, 030304 developmental biology, Enzyme substrate complex, Sheep, MESH: Phosphorylation, Diptera, Australia, Active site, protein engineering, MESH: Acetylcholinesterase, biology.organism_classification, MESH: Crystallography, X-Ray, MESH: Insecticides, 010602 entomology, Enzyme, chemistry, biology.protein, MESH: Substrate Specificity
Abstract: Insect carboxylesterases from the α Esterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina ( Lc αE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of Lc αE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of Lc αE7 crystals in OPs led to the capture of a crystallographic snapshot of Lc αE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of Lc αE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10 6 M −1 s −1 ) indicative of a natural substrate.
Published: 2013

38. Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast

Author: Etienne Almayrac, Emmanuelle Fabre, Sébastien Herbert, Fabiola García Fernández, Yasmine Khalil, Jyotsana J. Parmar, Eleonore Birgy, Adeline Veillet, Jean-Michel Arbona, Christophe Zimmer, Mickaël Lelek, Alice Brion, Benoît Lelandais, Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), Biologie et Dynamique des Chromosomes [Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal] (Equipe), Pathologie cellulaire : aspects moléculaires et viraux / Pathologie et Virologie Moléculaire, Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)-Institut Universitaire d'Hématologie (IUH), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)-Université Sorbonne Paris Cité (USPC), A.B, A.V, F.G‐F, E.A, Y.K, and E.F. acknowledge support from Agence Nationale de la Recherche (ANR‐13‐BSV8‐0013‐01), IDEX SLI (DXCAIUHSLI‐EF14), Labex Who am I (ANR‐11‐LABX‐0071, IDEX ANR‐11‐IDEX‐0005‐02), Cancéropôle Ile de France (ORFOCRISE PME‐2015). Y.K and EF acknowledge support from Fondation pour la Recherche Médicale (ING20160435205). S.H., J‐M.A., M.L., B.L., J.P., and C.Z. acknowledge funding by Institut Pasteur (including a Roux fellowship for J‐M.A.), Agence Nationale de la Recherche (ANR‐11‐MONU‐020–02), Institut National du Cancer (INCa 2015‐135), and Fondation pour la Recherche Médicale (Equipe FRM DEQ20150331762)., We thank K. N'Thiombane for initial experiments, R. Henriques and J‐Y. Tinevez for help with image analysis, D. Durocher and S. Marcand for cep3S575A and H2AS129A mutants, respectively, M. Kupiec for suggestions on an early version of the manuscript, J. Miné‐Hattab, X. Darzacq, C. Muchardt and J. Haber for helpful discussions. C.Z. acknowledges support from the Siebel Stem Cell Foundation during a visit to UC Berkeley, where an early version of the manuscript was completed., ANR-13-BSV8-0013,SPAREDAM,Organisation tridimensionnelle des dommages à l'ADN(2013), ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), ANR-11-MONU-0020,ProbAlg,Algorithmes efficients pour modèles réalistes à grand échelle : développements fondamentaux et applications(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Lemesle, Marie, Blanc 2013 - Organisation tridimensionnelle des dommages à l'ADN - - SPAREDAM2013 - ANR-13-BSV8-0013 - Blanc 2013 - VALID, Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID, Modèles Numériques - Algorithmes efficients pour modèles réalistes à grand échelle : développements fondamentaux et applications - - ProbAlg2011 - ANR-11-MONU-0020 - MN - VALID, Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Université Sorbonne Paris Cité (USPC), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Universitaire d'Hématologie (IUH), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Sorbonne Paris Cité (USPC), Agence Nationale de la Recherche (ANR‐13‐BSV8‐0013‐01), IDEX SLI (DXCAIUHSLI‐EF14), Labex Who am I (ANR‐11‐LABX‐0071, IDEX ANR‐11‐IDEX‐0005‐02), Cancéropôle Ile de France (ORFOCRISE PME‐2015). Y.K and EF acknowledge support from Fondation pour la Recherche Médicale (ING20160435205). S.H., J‐M.A., M.L., B.L., J.P., and C.Z. acknowledge funding by Institut Pasteur (including a Roux fellowship for J‐M.A.), Agence Nationale de la Recherche (ANR‐11‐MONU‐020–02), Institut National du Cancer (INCa 2015‐135), and Fondation pour la Recherche Médicale (Equipe FRM DEQ20150331762)., and ANR-11-IDEX-0005-02/11-LABX-0071,WHO AM I,Determinants de l’Identité : de la molécule à l’individu(2011)
Subjects: 0301 basic medicine, Histone-modifying enzymes, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, yeast, Histones, Systems & Computational Biology, Histone code, DNA Breaks, Double-Stranded, Phosphorylation, DNA, Fungal, ComputingMilieux_MISCELLANEOUS, Chromatin Fiber, [PHYS]Physics [physics], chromatin structure, MESH: Histones, General Neuroscience, Articles, MESH: Bleomycin, Chromatin, Cell biology, chromatin dynamics, Epigenetics, DNA Replication, Biology, General Biochemistry, Genetics and Molecular Biology, Chromatin remodeling, MESH: Chromatin, 03 medical and health sciences, Bleomycin, Histone H1, MESH: Mutagens, Histone H2A, yeast Subject Categories Chromatin, Scaffold/matrix attachment region, MESH: Saccharomycetales, Molecular Biology, polymers, General Immunology and Microbiology, MESH: Phosphorylation, MESH: Chromatin Assembly and Disassembly, Chromatin Assembly and Disassembly, Molecular biology, MESH: DNA, Fungal, Genomics & Functional Genomics, 030104 developmental biology, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, Saccharomycetales, DNA damage, Repair & Recombination, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Mutagens
Abstract: International audience; DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.
Published: 2016

39. CD99 isoforms regulate CD1a expression in human monocyte-derived DCs through ATF-2/CREB-1 phosphorylation

Author: Frédéric Larbret, Karim Mahiddine, Alain Bernard, Hanen Bziouech, Ghislaine Bernard, Aude Mallavialle, 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), Régulations des réactions immunitaires et inflammatoires, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre méditerranéen de médecine moléculaire (C3M), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Laboratoire Tolérance Immunitaire (TIM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), and Centre Hospitalier Universitaire de Nice (CHU Nice)
Subjects: 0301 basic medicine, MESH: Antigens, CD1, Transcription, Genetic, Nonclassical MHC, MESH: Protein Isoforms, MESH: Monocytes, Dendritic cells, Monocytes, alternative splicing-ATF2-CREB1, Small hairpin RNA, Antigens, CD1, 0302 clinical medicine, Transcription (biology), hemic and lymphatic diseases, Immunology and Allergy, Protein Isoforms, Phosphorylation, Cyclic AMP Response Element-Binding Protein, Cells, Cultured, biology, integumentary system, MESH: Dendritic Cells, Cell Differentiation, hemic and immune systems, MESH: Gene Expression Regulation, Cell biology, CD99, MESH: Cells, Cultured, MESH: Cyclic AMP Response Element-Binding Protein, Gene isoform, MESH: Cell Differentiation, Immunology, 12E7 Antigen, CREB, Cell Line, 03 medical and health sciences, MESH: Activating Transcription Factor 2, Humans, Transcription factor, MESH: Humans, Activating Transcription Factor 2, MESH: Phosphorylation, MESH: Transcription, Genetic, Alternative splicing, MESH: 12E7 Antigen, In vitro, MESH: Cell Line, 030104 developmental biology, Gene Expression Regulation, biology.protein, Cancer research, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 030215 immunology
Abstract: International audience; CD1a expression is considered one of the major characteristics qualifying in vitro human dendritic cells (DCs) during their generation process. Here, we report that CD1A transcription is regulated by a mechanism involving the long and short isoforms of CD99. Using a lentiviral construct encoding for a CD99 short hairpin RNA, we were able to inhibit CD99 expression in human primary DCs. In such cells, CD1a membrane expression increased and CD1A transcripts were much higher in abundance compared to cells expressing CD99 long form (CD99LF). We also show that CD1A transcription is accompanied by a switch in expression from CD99LF to expression at comparable levels of both CD99 isoforms during immature DCs generation in vitro. We demonstrate that CD99LF maintains a lower level of CD1A transcription by up-regulating the phosphorylated form of the ATF-2 transcription factor and that CD99 short form (SF) is required to counteract this regulatory mechanism. Elucidation of the molecular mechanisms related to CD99 alternative splicing will be very helpful to better understand the transcriptional regulatory mechanism of CD1a molecules during DCs differentiation and its involvement in the immune response.
Published: 2016

40. Naringin inhibits the invasion and migration of human glioblastoma cell via downregulation of MMP-2 and MMP-9 expression and inactivation of p38 signaling pathway

Author: Hamadi Fetoui, Yassine Chtourou, Amel Laajimi, Abderraouf Kenani, Sonia Aroui, Annie-Claire Meunier, Feten Najlaoui, Laboratory of Biochemistry, Molecular Mechanisms and Diseases Research Unit, UR12ES08. Faculty of Medicine, Université de Monastir - University of Monastir (UM), Université de Poitiers, Laboratory of Toxicology-Microbiology and Environmental Health (UR11ES70), Faculté des Sciences de Sfax, Université de Sfax - University of Sfax-Université de Sfax - University of Sfax, Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), and Animal Physiology Laboratory, Life Sciences Department
Subjects: 0301 basic medicine, Signaling pathways, Pyridines, [SDV]Life Sciences [q-bio], Matrix metalloproteinase, Bioinformatics, p38 Mitogen-Activated Protein Kinases, MESH: Down-Regulation, MESH: Drug Synergism, MESH: Dose-Response Relationship, Drug, chemistry.chemical_compound, 0302 clinical medicine, Invasion, Cell Movement, Enzyme Inhibitors, Phosphorylation, MESH: Cell Movement, Anisomycin, Migration, MESH: Immunoblotting, MESH: Glioblastoma, Imidazoles, Drug Synergism, General Medicine, 3. Good health, MESH: Flavanones, Matrix Metalloproteinase 9, MESH: Cell Survival, MESH: Enzyme Inhibitors, 030220 oncology & carcinogenesis, Flavanones, Matrix Metalloproteinase 2, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Signal transduction, MESH: Imidazoles, MESH: Cell Line, Tumor, Cell Survival, MAP Kinase Signaling System, p38 mitogen-activated protein kinases, Immunoblotting, Down-Regulation, 03 medical and health sciences, Downregulation and upregulation, Glioma, Cell Line, Tumor, medicine, Humans, Neoplasm Invasiveness, Naringin, Tissue Inhibitor of Metalloproteinase-2, Tissue Inhibitor of Metalloproteinase-1, MESH: Humans, Dose-Response Relationship, Drug, MESH: Phosphorylation, Activator (genetics), business.industry, MESH: MAP Kinase Signaling System, MESH: Pyridines, MESH: Matrix Metalloproteinase 9, MESH: Neoplasm Invasiveness, medicine.disease, MESH: Matrix Metalloproteinase 2, MESH: p38 Mitogen-Activated Protein Kinases, 030104 developmental biology, Matrix metalloproteinases, MESH: Tissue Inhibitor of Metalloproteinase-2, chemistry, MESH: Tissue Inhibitor of Metalloproteinase-1, Cancer research, business, Glioblastoma, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Gliomas are the most common and malignant primary brain tumors. They are associated with a poor prognosis despite the availability of multiple therapeutic options. Naringin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong anti-proliferative and anti-cancer properties. However, there are no reports describing its effects on the invasion and migration of glioblastoma cell lines. Our results showed that the treatment of U251 glioma cell lines with different concentrations of naringin inhibited the invasion and migration of these cells. In addition, we revealed a decrease in the levels of matrix metalloproteinases (MMP-2) and (MMP-9) expression as well as proteinase activity in U251 glioma cells. In contrast, the expression of tissue inhibitor of metalloproteinases (TIMP-1) and (TIMP-2) was increased. Furthermore, naringin treatment decreased significantly the phosphorylated level of p38. Combined treatment with a p38 inhibitor (SB203580) resulted in the synergistic reduction of MMP-2 and MMP-9 expressions correlated with an increase of TIMP-1 and TIMP-2 expressions and the anti-invasive properties. However, p38 chemical activator (anisomycin) could block these effects produced by naringin, suggesting a direct downregulation of the p38 signaling pathway. These data suggest that naringin may have therapeutic potential for controlling invasiveness of malignant gliomas by inhibiting of p38 signal transduction pathways.
Published: 2016

41. The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila

Author: Schell, Ursula, Simon, Sylvia, Sahr, Tobias, Hager, Dominik, Albers, Michael F., Kessler, Aline, Fahrnbauer, Felix, Trauner, Dirk, Hedberg, Christian, Buchrieser, Carmen, Hilbi, Hubert, Max Von Pettenkofer Institute (MVP), Ludwig-Maximilians-Universität München (LMU), Institute of Medical Microbiology [Zurich], Universität Zürich [Zürich] = University of Zurich (UZH), Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, LMU University of Munich, University of Munich, Chemical Biology Center (KBC), Umeå University, Work in the group of H.H. was supported by the German Research Foundation (DFG, HI 1511/2-1, HI 1511/5-1) and the Swiss National Science Foundation (SNF, 31003A_153200). Work in the C.B. laboratory was ﬁnanced by the Institut Pasteur, the Institut Carnot-Pasteur MI, the French Region Ile de France (DIM Malinf),the grant N°ANR-10-LABX-62-IBEID, the Fondation pour la Recherche Médicale (FRM) grant N° DEQ20120323697 and the Pasteur-Weizmann consortium ‘The roles of non-codingRNAs in regulation of microbial life styles and virulence’. C.H. and M.F.A thank the Knut and Alice Wallenberg foundation (Sweden) for generous support, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), University of Zurich, Hilbi, Hubert, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Signal Transduction, MESH: Gene Expression, Movement, viruses, Gene Expression, 610 Medicine & health, MESH: Ketones, MESH: Movement, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Quorum Sensing, Legionella pneumophila, MESH: Gene Expression Profiling, Cell Movement, Alkanes, 1312 Molecular Biology, Escherichia coli, Phosphorylation, Vibrio cholerae, MESH: Cell Movement, MESH: Gene Expression Regulation, Bacterial, MESH: Phosphorylation, 10179 Institute of Medical Microbiology, MESH: Escherichia coli, Gene Expression Profiling, 2404 Microbiology, MESH: Host-Pathogen Interactions, Quorum Sensing, Gene Expression Regulation, Bacterial, MESH: Transcription Factors, Ketones, MESH: Legionella pneumophila, MESH: Alkanes, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Host-Pathogen Interactions, 570 Life sciences, biology, MESH: Vibrio cholerae, Signal Transduction, Transcription Factors
Abstract: International audience; The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [γ-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.
Published: 2016

42. Molecular mechanisms of gravity perception and signal transduction in plants

Author: Igor D. Volotovsky, Elizabeth Kordyum, S. V. Kretynin, Yaroslav S. Kolesnikov, V. S. Kravets, Eric Ruelland, Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine (NASU), Institute of Biophysics and Cell Engineering, NASB, Minsk, National Academy of Sciences of Belarus (NASB), Institut d'écologie et des sciences de l'environnement de Paris (iEES Paris), Institut National de la Recherche Agronomique (INRA)-Sorbonne Université (SU)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Centre National de la Recherche Scientifique (CNRS), and Institut d'écologie et des sciences de l'environnement de Paris (IEES (UMR_7618 / UMR_D_242 / UMR_A_1392 / UM_113) )
Subjects: 0106 biological sciences, 0301 basic medicine, MESH: Signal Transduction, MESH: Gravity Sensing, Gravity, MESH: Plants, Plant Development, Plant Science, Signalling, Stimulus (physiology), Biology, 01 natural sciences, 03 medical and health sciences, MESH: Cytoskeleton, Amyloplast, MESH: Plant Development, Phospholipase, Gravity Sensing, Phosphorylation, Cytoskeleton, Plant Proteins, MESH: Phosphorylation, MESH: Plant Proteins, Cell Biology, General Medicine, Membrane transport, Plants, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, Cell biology, 030104 developmental biology, MESH: Protein Processing, Post-Translational, Proteome, Calcium, Signal transduction, Protein Processing, Post-Translational, Biogenesis, 010606 plant biology & botany, Signal Transduction
Abstract: International audience; Gravity is one of the environmental cues that direct plant growth and development. Recent investigations of different gravity signalling pathways have added complexity to how we think gravity is perceived. Particular cells within specific organs or tissues perceive gravity stimulus. Many downstream signalling events transmit the perceived information into subcellular, biochemical, and genomic responses. They are rapid, non-genomic, regulatory, and cell-specific. The chain of events may pass by signalling lipids, the cytoskeleton, intracellular calcium levels, protein phosphorylation-dependent pathways, proteome changes, membrane transport, vacuolar biogenesis mechanisms, or nuclear events. These events culminate in changes in gene expression and auxin lateral redistribution in gravity response sites. The possible integration of these signalling events with amyloplast movements or with other perception mechanisms is discussed. Further investigation is needed to understand how plants coordinate mechanisms and signals to sense this important physical factor.
Published: 2016

43. Interaction Between CaMKII and GluN2B Controls ERK-Dependent Plasticity

Author: Olivier Moustié, Paul De Koninck, Farida El Gaamouch, Bruno Bontempi, Simon Labrecque, Olivier Nicole, Alain Buisson, Mado Lemieux, Centre-Imagerie, Neurosciences, et Application aux Pathologies (CI-NAPS - UMR 6232), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Equipe 12 : NEuRopathologies et Dysfonctions Synaptiques, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-[GIN] Grenoble Institut des Neurosciences, Institut universitaire en santé mentale de Québec, Institut des Maladies Neurodégénératives [Bordeaux] (IMN), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from the French Ministry of Research, Alzheimer Foundation, the Agence National de la Recherche (ANR MALZ 2010-001-02 CoRehAlz to B.B.), and Regional Council of Basse-Normandie France (F.E.G., O.N., A.B.), and the Canadian Institutes of Health Research (CIHR) (P.D.K.). M.L. has been supported by studentships from the Natural Science and Engineering Research Council of Canada (NSERC), CIHR, and the Fond de la Recherche en Santé du Québec. S.L. was supported by a CIHR Neurophysics training grant. P.D.K. was a career awardee of CIHR., French Ministry of ResearchAlzheimer FoundationAgence National de la Recherche (ANR MALZ 2010-001-02 CoRehAlz to B.B.)Regional Council of Basse-NormandieCanadian Institutes of Health Research (CIHR)Natural Science and Engineering Research Council of Canada (NSERC),Fond de la Recherche en Santé, NEuRopathologies et Dysfonctions Synaptiques, [GIN] Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Centre de recherche de l’Institut universitaire en santé mentale de Québec [Canada] (CERVO), Département de réadaptation (Faculté de médecine de l'Université Laval) [Canada], Faculté de médecine de l'Université Laval [Québec] (ULaval), Université Laval [Québec] (ULaval)-Université Laval [Québec] (ULaval)-Faculté de médecine de l'Université Laval [Québec] (ULaval), Université Laval [Québec] (ULaval)-Université Laval [Québec] (ULaval), Faculté de médecine de l'Université Laval [Québec] (ULaval)-Faculté de médecine de l'Université Laval [Québec] (ULaval), and NICOLE, Olivier
Subjects: Cortical neurons, Male, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, MESH: Neurons, Nonsynaptic plasticity, Mice, 0302 clinical medicine, MESH: Neuronal Plasticity, 4-Aminopyridine, Enzyme Inhibitors, Phosphorylation, RNA, Small Interfering, Neuronal memory allocation, Cells, Cultured, Cerebral Cortex, Neurons, 0303 health sciences, Neuronal Plasticity, Photobleaching, Synaptic scaling, General Neuroscience, Articles, MESH: Calcium-Calmodulin-Dependent Protein Kinase Type 2, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Disks Large Homolog 4 Protein, Microtubule-Associated Proteins, MESH: Photobleaching, MAP Kinase Signaling System, Dendritic Spines, AMPA receptor, In Vitro Techniques, Biology, Bicuculline, Transfection, NMDA receptors, Receptors, N-Methyl-D-Aspartate, Synaptic plasticity, MESH: Dendritic Spines, 03 medical and health sciences, Synaptic augmentation, Metaplasticity, Potassium Channel Blockers, Animals, Immunoprecipitation, GABA-A Receptor Antagonists, MESH: Guanylate Kinase, 030304 developmental biology, Analysis of Variance, MESH: Receptors, N-Methyl-D-Aspartate, MESH: Phosphorylation, MESH: MAP Kinase Signaling System, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Membrane Proteins, MESH: Cerebral Cortex, Rats, Mice, Inbred C57BL, Luminescent Proteins, Synaptic fatigue, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Excitatory Amino Acid Antagonists, Guanylate Kinases, Neuroscience, 030217 neurology & neurosurgery
Abstract: International audience; Understanding how brief synaptic events can lead to sustained changes in synaptic structure and strength is a necessary step in solving the rules governing learning and memory. Activation of ERK1/2 (extracellular signal regulated protein kinase 1/2) plays a key role in the control of functional and structural synaptic plasticity. One of the triggering events that activates ERK1/2 cascade is an NMDA receptor (NMDAR)-dependent rise in free intracellular Ca(2+) concentration. However the mechanism by which a short-lasting rise in Ca(2+) concentration is transduced into long-lasting ERK1/2-dependent plasticity remains unknown. Here we demonstrate that although synaptic activation in mouse cultured cortical neurons induces intracellular Ca(2+) elevation via both GluN2A and GluN2B-containing NMDARs, only GluN2B-containing NMDAR activation leads to a long-lasting ERK1/2 phosphorylation. We show that αCaMKII, but not βCaMKII, is critically involved in this GluN2B-dependent activation of ERK1/2 signaling, through a direct interaction between GluN2B and αCaMKII. We then show that interfering with GluN2B/αCaMKII interaction prevents synaptic activity from inducing ERK-dependent increases in synaptic AMPA receptors and spine volume. Thus, in a developing circuit model, the brief activity of synaptic GluN2B-containing receptors and the interaction between GluN2B and αCaMKII have a role in long-term plasticity via the control of ERK1/2 signaling. Our findings suggest that the roles that these major molecular elements have in learning and memory may operate through a common pathway.
Published: 2012

44. Trypanosomal histone γH2A and the DNA damage response

Author: Lucy Glover, David Horn, London School of Hygiene and Tropical Medicine (LSHTM), and We thank The Wellcome Trust (Project grant: 089172) for funding.
Subjects: DNA Repair, MESH: Sequence Homology, Amino Acid, [SDV]Life Sciences [q-bio], Amino Acid Motifs, Protozoan Proteins, MESH: Amino Acid Sequence, I-SceI, MESH: Cell Cycle Checkpoints, Histones, MESH: Amino Acid Motifs, MESH: Protein Structure, Tertiary, MESH: Rad51 Recombinase, Cancer epigenetics, γH2AX, Phosphorylation, MESH: Protozoan Proteins, MESH: DNA Repair, MESH: Histones, 0303 health sciences, biology, Checkpoint, 030302 biochemistry & molecular biology, Chromatin, MMS, 3. Good health, Protein Transport, Histone, DNA mismatch repair, MESH: Protein Transport, DNA repair, DNA damage, Molecular Sequence Data, Trypanosoma brucei brucei, Article, 03 medical and health sciences, Histone H2A, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, MESH: DNA Damage, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, MESH: Phosphorylation, MESH: Trypanosoma brucei brucei, Cell Cycle Checkpoints, G2-M DNA damage checkpoint, Molecular biology, Protein Structure, Tertiary, Proliferating cell nuclear antigen, MESH: Protein Processing, Post-Translational, biology.protein, Parasitology, Rad51 Recombinase, Protein Processing, Post-Translational, Repair, DNA Damage
Abstract: Graphical abstract Highlights ► Histone H2A phosphorylated at Thr130 is identified as trypanosomal γH2A. ► γH2A nuclear foci mark the putative sites of replication fork stalling. ► γH2A nuclear foci form in response to meganuclease or chemical-induced DNA damage. ► Trypanosomal γH2A and RAD51 positive damage and repair foci are most often seen in S-phase and G2. ► The findings implicate γH2A in mitosis-entry checkpoint signaling., DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is γH2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of γH2A in Trypanosoma brucei. We identified Thr130 within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal γH2A increased in vivo in response to DNA damage. Nuclear γH2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced γH2A and RAD51 double-positive repair foci are typically found in S-phase or G2 nuclei. The results link trypanosomal γH2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling.
Published: 2012

45. Epidermal Growth Factor Receptor Protein Detection in Head and Neck Cancer Patients: A Many-Faceted Picture

Author: Ellen Van Obberghen-Schilling, Jean-Louis Formento, Dominique Grall, Anne Cayre, Dominique de Raucourt, Philippe Giraud, Olivier Dassonville, Sylvain Morinière, Frédérique Penault-Llorca, Marie-Christine Etienne-Grimaldi, René-Jean Bensadoun, Séverine Racadot, Mireille Francoual, Gérard Milano, Juliette Thariat, Laurence Veracini, Lionel Geoffrois, 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)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Centre Jean Perrin [Clermont-Ferrand] (UNICANCER/CJP), UNICANCER, Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), Dept of Oncopharmacology, Centre de Lutte contre le Cancer Antoine Lacassagne [Nice] (UNICANCER/CAL), UNICANCER-Université Côte d'Azur (UCA)-UNICANCER-Université Côte d'Azur (UCA), Service d'ORL, Centre Alexis Vautrin (CAV), and Département de pharmacogénomique
Subjects: Cancer Research, Pathology, medicine.medical_specialty, MESH: Receptor, Epidermal Growth Factor, 03 medical and health sciences, 0302 clinical medicine, MESH: Polymorphism, Genetic, medicine, Carcinoma, Humans, Epidermal growth factor receptor, Phosphorylation, [SDV.BDD]Life Sciences [q-bio]/Development Biology, 030304 developmental biology, 0303 health sciences, MESH: Humans, Polymorphism, Genetic, Tissue microarray, MESH: Phosphorylation, biology, business.industry, Head and neck cancer, Cancer, MESH: Carcinoma, Squamous Cell, MESH: Gene Expression Regulation, Neoplastic, medicine.disease, Head and neck squamous-cell carcinoma, MESH: Head and Neck Neoplasms, ErbB Receptors, Gene Expression Regulation, Neoplastic, Oncology, Head and Neck Neoplasms, 030220 oncology & carcinogenesis, Carcinoma, Squamous Cell, Cancer research, biology.protein, Biomarker (medicine), Immunohistochemistry, business
Abstract: Purpose: Epidermal growth factor receptor (EGFR) overexpression is associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC). Despite intensive biomarker studies, a consensual method for assessing EGFR protein expression is still lacking. Here we set out to compare three EGFR detection methods in tumor specimens from HNSCC patients. Experimental Design: Tumors were prospectively excised from a series of 79 high-risk HNSCC patients enrolled in a GORTEC-sponsored clinical trial. EGFR expression was determined using a ligand-binding assay on membranes, Western blotting (WB) on membranes and total homogenates, and immunohistochemistry (IHC) on tissue microarrays. In addition, phosphorylated EGFR (pEGFR) was measured by WB on membranes. Results: Distributions and ranges of tumor EGFR expression were method dependent. Moderate positive correlations (Spearman coefficient r ≈ 0.50) were observed between EGFR expression measured by the binding assay and WB or IHC. pEGFR levels positively and significantly correlated with total EGFR expression measured by WB or ligand binding, but not by IHC. The highest correlation (r = 0.85) was observed between EGFR and pEGFR levels, both measured by WB on membranes. Interestingly, the fraction of phosphorylated receptor (pEGFR/EGFR both measured by WB on membranes) significantly declined with increasing tumor EGFR expression, by all assessment methods used. Conclusion: This study shows significant correlations between EGFR detection methods. The observed relationships between EGFR and pEGFR indicate that high-throughput pEGFR/EGFR analyses merit further investigations and consideration for routine use in patient samples. Clin Cancer Res; 18(5); 1313–22. ©2012 AACR.
Published: 2012

46. β-arrestin-1 participates in thrombosis and regulates integrin aIIbβ3 signalling without affecting P2Y receptors desensitisation and function.: Role of β-arrestins in platelet function

Author: Pierre Mangin, Catherine Bourdon, Mathieu Schaff, François Lanza, Philippe Ohlmann, Christian Gachet, Nicolas Receveur, UMR_S949, Biologie et pharmacologie des plaquettes sanguines: hémostase, thrombose, transfusion, Université de Strasbourg (UNISTRA)-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Strasbourg (UNISTRA)-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Strasbourg (UNISTRA)-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), and This work was supported by ARMESA (Association de Recherche et Développement en Médecine et Santé Publique). Mathieu Schaff was supported by a ″Bourse Grenelle″ from the French government.
Subjects: MESH: Signal Transduction, 0301 basic medicine, P2Y receptor, Arrestins, MESH: Mesenteric Arteries, 030204 cardiovascular system & hematology, MESH: Receptors, Purinergic P2Y, Mice, 0302 clinical medicine, MESH: Animals, Platelet, Phosphorylation, Receptor, beta-Arrestins, MESH: Blood Platelets, β-arrestins, MESH: Enzyme-Linked Immunosorbent Assay, αIIbβ3, Hematology, beta-Arrestin 2, MESH: Platelet Glycoprotein GPIIb-IIIa Complex, Mesenteric Arteries, P-Selectin, Carotid Arteries, beta-Arrestin 1, Receptors, Purinergic P2Y, MESH: Calcium, MESH: P-Selectin, MESH: Arrestins, Signal transduction, MESH: Hemorrhage, Signal Transduction, Blood Platelets, Platelets, medicine.medical_specialty, MESH: Microscopy, Electron, Scanning, MESH: Mice, Transgenic, Integrin, Enzyme-Linked Immunosorbent Assay, Hemorrhage, Mice, Transgenic, Platelet Glycoprotein GPIIb-IIIa Complex, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, MESH: Cell Adhesion, 03 medical and health sciences, Internal medicine, Cell Adhesion, medicine, Animals, MESH: Thrombosis, Platelet activation, MESH: Mice, thrombosis, MESH: Carotid Arteries, MESH: Phosphorylation, Beta-Arrestins, AKT, Fibrinogen, Fibrinogen binding, 030104 developmental biology, Endocrinology, MESH: Fibrinogen, Microscopy, Electron, Scanning, biology.protein, Calcium
Abstract: Summaryβ-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1−/− and β-arr2−/− platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1−/− but not β-arr2−/− mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1−/− and β-arr2−/− mice, suggesting no defect in haemostasis. β-arr1−/− platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p
Published: 2012

47. Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells

Author: Zeineb Es-Salah-Lamoureux, Laurence Murillo, Alain Hamon, Bruno Lapied, Sophie Quinchard, Valerie Itier, LIttoral ENvironnement et Sociétés - UMR 7266 (LIENSs), Université de La Rochelle (ULR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de mécanique des sols, structures et matériaux (MSSMat), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec, 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), Stratégies S.A. [Rungis], Récepteurs Canaux Ioniques Membranaires (RCIM), Université d'Angers (UA)-Institut National de la Recherche Agronomique (INRA), LIttoral ENvironnement et Sociétés - UMRi 7266 (LIENSs), CentraleSupélec-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 de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Récepteurs et Canaux Ioniques Membranaires (RCIM), Zeineb Es-Salah-Lamoureux was supported by a doctoral fellowship of the Region Pays de la Loire., Laboratoire Récepteurs et Canaux Ioniques Membranaires (RCIM), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Laboratoire de Planétologie et Géodynamique UMR6112 (LPG), Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Nantes - Faculté des Sciences et des Techniques, and Université de Nantes (UN)-Université de Nantes (UN)-Université d'Angers (UA)
Subjects: Male, Insecticides, Patch-Clamp Techniques, Time Factors, [SDV]Life Sciences [q-bio], MESH: Calcium Channel Blockers, Pharmacology, Toxicology, MESH: Dose-Response Relationship, Drug, Membrane Potentials, KN-62, chemistry.chemical_compound, 0302 clinical medicine, GABA receptor, MESH: Protein Kinase Inhibitors, Periplaneta, MESH: Animals, Phosphorylation, Receptor, Protein Kinase C, ComputingMilieux_MISCELLANEOUS, Fipronil, gaba, 0303 health sciences, GABAA receptor, Kinase, [SDV.BA]Life Sciences [q-bio]/Animal biology, General Neuroscience, Calcium Channel Blockers, 3. Good health, MESH: Receptors, GABA, Biochemistry, MESH: Calcium-Calmodulin-Dependent Protein Kinase Type 2, [SDE]Environmental Sciences, pkc, dum neurons, Enzyme Activators, camkinase II, Biology, pharmacology and pharmacy, 03 medical and health sciences, fibronil, Receptors, GABA, MESH: Patch-Clamp Techniques, MESH: Membrane Potentials, Animals, MESH: Enzyme Activators, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, GABA Modulators, Protein Kinase Inhibitors, Protein kinase C, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Phosphorylation, Dose-Response Relationship, Drug, MESH: Time Factors, insecticide, MESH: Protein Kinase C, Neurosecretory Systems, MESH: Male, MESH: Insecticides, chemistry, MESH: Neurosecretory Systems, MESH: Periplaneta, Pyrazoles, neurosciences and neurology, Calcium-Calmodulin-Dependent Protein Kinase Type 2, MESH: GABA Modulators, MESH: Pyrazoles, 030217 neurology & neurosurgery
Abstract: International audience; Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil.
Published: 2011

48. p16INK4a deficiency promotes IL-4-induced polarization and inhibits proinflammatory signaling in macrophages.: p16INK4a function in macrophages

Author: Réjane Paumelle, Sébastien Fleury, Emmanuel Bouchaert, Jonathan Vanhoutte, Céline Cudejko, Giulia Chinetti-Gbaguidi, Bart Staels, Anne Tailleux, Sarah Anissa Hannou, Patrick Rémy, Lucía Fuentes, Kadiombo Bantubungi, Kristiaan Wouters, David Dombrowicz, Charlotte Paquet, Récepteurs nucléaires, maladies cardiovasculaires et diabète - U 1011 (RNMCD), 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), Service de Production des Antigènes, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), and This work was supported by the Fondation pour la Recherche Médicale (DCV20070409276 to B.S.), the EFSD/GSK Program 2009, the Cost Action (BM0602) and the Conseil régional Nord Pas-de-Calais and FEDER. C.Cudejko was supported by a doctoral fellowship from the Nouvelle Société Française d'Athérosclérose/ Schering-Plough/MSD. K.Wouters was supported by a European FP7 Marie Curie grant (PIEF-GA-2009-235221) and a European Atherosclerosis Society grant.
Subjects: MESH: Signal Transduction, MESH: Genes, p16, MESH: Inflammation, Lipopolysaccharides, Lipopolysaccharide, p16, Inbred C57BL, Biochemistry, MESH: Janus Kinase 2, chemistry.chemical_compound, Mice, 0302 clinical medicine, Schistosomiasis, MESH: Animals, STAT1, Phosphorylation, MESH: Bone Marrow Transplantation, Non-U.S. Gov't, Bone Marrow Transplantation, 0303 health sciences, MESH: Cytokines, Research Support, Non-U.S. Gov't, MESH: Macrophage Activation, Hematology, I-kappa B Kinase, STAT1 Transcription Factor, Liver, MESH: STAT6 Transcription Factor, 030220 oncology & carcinogenesis, MESH: Cyclin-Dependent Kinase Inhibitor p16, Radiation Chimera, MESH: Schistosomiasis, Cytokines, medicine.symptom, Signal transduction, Signal Transduction, MESH: Radiation Chimera, MESH: Interferon-gamma, Immunology, Macrophage polarization, Inflammation, Biology, Research Support, Article, Proinflammatory cytokine, 03 medical and health sciences, Interferon-gamma, MESH: Mice, Inbred C57BL, medicine, Journal Article, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: I-kappa B Kinase, neoplasms, MESH: Mice, Interleukin 4, Cyclin-Dependent Kinase Inhibitor p16, Protein Processing, 030304 developmental biology, MESH: Phosphorylation, Genes, p16, Macrophages, Post-Translational, MESH: Macrophages, Cell Biology, MESH: Interleukin-4, Janus Kinase 2, Macrophage Activation, Molecular biology, Mice, Inbred C57BL, chemistry, Genes, MESH: Protein Processing, Post-Translational, biology.protein, MESH: STAT1 Transcription Factor, Interleukin-4, MESH: Lipopolysaccharides, STAT6 Transcription Factor, Protein Processing, Post-Translational, MESH: Liver
Abstract: The CDKN2A locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize toward classically (CAMφ) or alternatively (AAMφ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. Here, we show that p16INK4a deficiency (p16−/−) modulates the macrophage phenotype. Transcriptome analysis revealed that p16−/− BM-derived macrophages (BMDMs) exhibit a phenotype resembling IL-4–induced macrophage polarization. In line with this observation, p16−/− BMDMs displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16−/− BM displayed higher hepatic AAMφ marker expression levels on Schistosoma mansoni infection, an in vivo model of AAMφ phenotype skewing. Surprisingly, p16−/− BMDMs did not display increased IL-4–induced STAT6 signaling, but decreased IFNγ-induced STAT1 and lipopolysaccharide (LPS)–induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16INK4a as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.
Published: 2011

49. Excess Type I Interferon Signaling in the Mouse Seminiferous Tubules Leads to Germ Cell Loss and Sterility

Author: Zhiguo He, Séverine Mazaud-Guittot, Anne-Pascale Satie, Isabelle Seif, Guilhem Jouve, Bernard Jégou, Dominique Mahé, Nathalie Dejucq-Rainsford, Groupe d'Etude de la Reproduction Chez l'Homme et les Mammiferes (GERHM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Sérotonine et neuropharmacologie, Université Paris-Sud - Paris 11 (UP11)-IFR141, and Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Anti-Mullerian Hormone, Male, MESH: Signal Transduction, Time Factors, Mouse, MESH: Pachytene Stage, Somatic cell, Apoptosis, Receptor, Interferon alpha-beta, Biochemistry, Transgenic, Mice, 0302 clinical medicine, Interferon, Testis, MESH: Animals, Phosphorylation, 0303 health sciences, 030219 obstetrics & reproductive medicine, MESH: Spermatozoa, Seminiferous Tubules, Sertoli cell, Spermatozoa, Innate Immunity, Cell biology, STAT1 Transcription Factor, medicine.anatomical_structure, Female, MESH: Spermatogenesis, Germ cell, Signal Transduction, medicine.drug, Cytokine Action, endocrine system, medicine.medical_specialty, Cell type, MESH: Mice, Transgenic, Transgene, Immunology, MESH: Seminiferous Tubules, Mice, Transgenic, MESH: Infertility, Male, Biology, 03 medical and health sciences, MESH: Sertoli Cells, Internal medicine, medicine, Animals, MESH: Receptor, Interferon alpha-beta, Inhibins, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Spermatogenesis, MESH: Mice, Molecular Biology, Infertility, Male, 030304 developmental biology, Sertoli Cells, MESH: Phosphorylation, MESH: Interferon-beta, Spermatid, MESH: Apoptosis, MESH: Time Factors, Interferon-beta, Idiopathic Infertilities, Cell Biology, MESH: Inhibins, MESH: Male, Endocrinology, MESH: Anti-Mullerian Hormone, Pachytene Stage, MESH: STAT1 Transcription Factor, MESH: Female
Abstract: International audience; Type I (α and β) interferons (IFNs) elicit antiproliferative and antiviral activities via the surface receptor IFNAR. Serendipitous observations in transgenic mice in 1988 strongly suggested that IFNα/β overexpression in the testis disrupts spermatogenesis. Here, we compare a new mouse strain transgenic for IFNβ (Tg10) and a sister strain lacking the IFNAR1 subunit of IFNAR (Tg10-Ifnar1(-/-)), both strains expressing the transgene in the testis. The main source of IFNβ RNA was the spermatid population. Importantly, the Tg10 mice, but not the double mutant Tg10-Ifnar1(-/-), showed altered spermatogenesis. The first IFNAR-dependent histological alteration was a higher apoptosis index in all germ cell categories apart from non-dividing spermatogonia. This occurred 3 weeks after the onset of IFNβ production at postnatal day 20 and in the absence of somatic cell defects in terms of cell number, expression of specific cell markers, and hormonal activities. Several known interferon-stimulated genes were up-regulated in Tg10 Sertoli cells and prepachytene germ cells but not in pachytene spermatocytes and spermatids. In concordance with this, pachytene spermatocytes and spermatids isolated from wild-type testes did not display measurable amounts of IFNAR1 and phosphorylated STAT1 upon IFNβ challenge in vitro, suggesting hyporesponsiveness of these cell types to IFN. At day 60, Tg10 males were sterile, and Sertoli cells showed increased amounts of anti-Mullerian hormone and decreased production of inhibin B, both probably attributable to the massive germ cell loss. Type I interferon signaling may lead to idiopathic infertilities by affecting the interplay between germ cells and Sertoli cells.
Published: 2011

50. CD5 Promotes IL-10 Production in Chronic Lymphocytic Leukemia B Cells through STAT3 and NFAT2 Activation

Author: Sébastien Lemoine, Soizic Garaud, Sophie Hillion, Christian Berthou, Anne Bordron, Jacques-Olivier Pers, Ahsen Morva, Yves Renaudineau, Rizgar A. Mageed, Pierre Youinou, Immunologie et Pathologie (EA2216), Université de Brest (UBO)-IFR148, CHRU Brest - Service d'Hématologie (CHU-Brest-Hemato), Centre Hospitalier Régional Universitaire de Brest (CHRU Brest), William Harvey Research Institute, Barts and the London Medical School, and Laboratoire d'Immunologie et Immunothérapie
Subjects: Male, MESH: Interleukin-13, MESH: Interleukin-10, viruses, Chronic lymphocytic leukemia, MESH: Antigens, CD5, Gene Expression, MESH: Aged, 80 and over, 0302 clinical medicine, immune system diseases, MESH: Reverse Transcriptase Polymerase Chain Reaction, MESH: Child, hemic and lymphatic diseases, Gene expression, Serine, Immunology and Allergy, Phosphorylation, Child, MESH: Leukemia, Lymphocytic, Chronic, B-Cell, Cells, Cultured, Aged, 80 and over, MESH: Aged, B-Lymphocytes, 0303 health sciences, Interleukin-13, MESH: Middle Aged, Reverse Transcriptase Polymerase Chain Reaction, MESH: Infant, Newborn, MESH: STAT3 Transcription Factor, hemic and immune systems, Hep G2 Cells, Transfection, Middle Aged, Interleukin-10, medicine.anatomical_structure, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, RNA Interference, MESH: Cells, Cultured, STAT3 Transcription Factor, MESH: Cell Line, Tumor, MESH: Gene Expression, Blotting, Western, MESH: RNA Interference, Immunology, MESH: NFATC Transcription Factors, MESH: Hep G2 Cells, chemical and pharmacologic phenomena, Biology, CD5 Antigens, 03 medical and health sciences, Cell Line, Tumor, MESH: B-Lymphocytes, medicine, Humans, MESH: Blotting, Western, MESH: Serine, Enhancer, Transcription factor, B cell, Aged, 030304 developmental biology, MESH: Humans, NFATC Transcription Factors, MESH: Phosphorylation, Infant, Newborn, MESH: Interleukin-5, medicine.disease, Leukemia, Lymphocytic, Chronic, B-Cell, Molecular biology, MESH: Male, Interleukin-5, CD5, MESH: Female, Chromatin immunoprecipitation, 030215 immunology
Abstract: B lymphocytes from chronic lymphocytic leukemia (CLL) display some CD5 transcripts for CD5 containing the known exon 1 (E1A) and other CD5 transcripts containing the new exon 1 (E1B). These malignant B cells, as well as B cell lines transfected with cDNA for E1A-cd5 or with cDNA for E1B-cd5 produce IL-10, raising the possibility that CD5 participates in the secretion of IL-10. We identified transcription factors involved in this production in CD5+ B lymphocytes from CLL patients and in E1A-cd5–transfected or E1B-cd5–transfected Jok cells. STAT3 is activated via phosphorylation of serine 727 but also NFAT2 through its translocation into the nucleus. Chromatin immunoprecipitation experiments confirmed the role of STAT3 and allowed the discovery of a role for NFAT2 in IL-10 production. Both transcription factors bind not only to the enhancer of the Il-10 gene but also to the promoter of the Il-5 and Il-13 genes. Furthermore, transfection of B cell lines with E1A-cd5 or E1B-cd5 established that activation of STAT3 and NFAT2 is regulated by CD5. The same holds true for the production of IL-10, IL-5, and IL-13 and the expression of the receptors for these cytokines. This interpretation was confirmed by two experiments. In the first, downregulation of CD5 by small interfering RNAs lowered the production of IL-10. In the second experiment, transfection of the GFP-NFAT2 gene into B lymphocytes induced nuclear translocation of NFAT2 in CD5+ but not in CD5− B cells. Thus, CD5 expression is associated with NFAT2 activity (and mildly STAT3 activity), indicating that CD5 controls IL-10 secretion.
Published: 2011

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