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

1. Whole blood transcriptome profiles of trypanotolerant and trypanosusceptible cattle highlight a differential modulation of metabolism and immune response during infection by Trypanosoma congolense

Author: Moana Peylhard, David Berthier, Guiguigbaza-Kossigan Dayo, Isabelle Chantal, Souleymane Sylla, Sabine Nidelet, Emeric Dubois, Guillaume Martin, Guilhem Sempéré, Laurence Flori, Sophie Thévenon, Interactions hôtes-vecteurs-parasites-environnement dans les maladies tropicales négligées dues aux trypanosomatides (UMR INTERTRYP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut de Recherche pour le Développement (IRD)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université de Bordeaux (UB), Département Systèmes Biologiques (Cirad-BIOS), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Centre international de recherche-développement sur l'élevage en zone sub-humide (CIRDES), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences de l'Evolution de Montpellier (UMR ISEM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université de Montpellier (UM)-Institut de recherche pour le développement [IRD] : UR226-Centre National de la Recherche Scientifique (CNRS), Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro Montpellier, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Université de Montpellier (UM), Systèmes d'élevage méditerranéens et tropicaux (UMR SELMET), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), ANR-11-JSV6-0001,AATTOL,Caractérisation des bases moléculaires de la tolérance à la Trypanosomose Animale Africaine: analyse conjointe des transcriptomes de l'hôte bovin et du parasite(2011), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), and ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010)
Subjects: Trypanosoma congolense, [SDV]Life Sciences [q-bio]
Abstract: Animal African trypanosomosis, caused by blood protozoan parasites transmitted mainly by tsetse flies, represents a major constraint for millions of cattle in sub-Saharan Africa. Exposed cattle include trypanosusceptible indicine breeds, severely affected by the disease, and West African taurine breeds called trypanotolerant owing to their ability to control parasite development, survive and grow in enzootic areas. Until now the genetic basis of trypanotolerance remains unclear. Here, to improve knowledge of the biological processes involved in trypanotolerance versus trypanosusceptibility, we identified bovine genes differentially expressed in five West African cattle breeds during an experimental infection byTrypanosoma congolenseand their biological functions. To this end, whole blood genome-wide transcriptome of three trypanotolerant taurine breeds (N’Dama, Lagune and Baoulé), one susceptible zebu (Zebu Fulani) and one African taurine x zebu admixed breed (Borgou) were profiled by RNA sequencing at four time points, one before and three during infection. As expected, infection had a major impact on cattle blood transcriptome regardless of the breed. The functional analysis of differentially expressed genes over time in each breed confirmed an early activation of the innate immune response, followed by an activation of the humoral response and an inhibition of T cell functions at the chronic stage of infection. More importantly, we highlighted overlooked features, such as a strong disturbance in host metabolism and cellular energy production that differentiates trypanotolerant and trypanosusceptible breeds. N’Dama breed showed the earliest regulation of immune response, associated with a strong activation of cellular energy production, also observed in Lagune, and to a lesser extent in Baoulé. Susceptible Zebu Fulani breed differed from other breeds by the strongest modification in lipid metabolism regulation. Overall, this study provides a better understanding of the biological mechanisms at work during infection, especially concerning the interplay between immunity and metabolism that seems differentially regulated depending on the cattle breeds.
Published: 2023

2. Single cell RNA sequencing reveals hemocyte heterogeneity in Biomphalaria glabrata: Plasticity over diversity

Author: Pichon, Rémi, Pinaud, Silvain, Vignal, Emmanuel, Chaparro, Cristian, Pratlong, Marine, Portet, Anaïs, Duval, David, Galinier, Richard, Gourbal, Benjamin, Apollo - University of Cambridge Repository, Interactions Hôtes-Pathogènes-Environnements (IHPE), Université de Perpignan Via Domitia (UPVD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Cancer Research UK Cambridge Institute [Cambridge, Royaume-Uni] (CRUK), University of Cambridge [UK] (CAM), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, and Modat, Anne
Subjects: glabrata, Biomphalaria, Sequence Analysis, RNA, Snails, Immunology, innate immune system, Schistosoma mansoni, mansoni, B. glabrata, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Schistosomiasis mansoni, [SDV.BA.ZI]Life Sciences [q-bio]/Animal biology/Invertebrate Zoology, hemocytes, [SDV.BA.ZI] Life Sciences [q-bio]/Animal biology/Invertebrate Zoology, Animals, Humans, Immunology and Allergy, S. mansoni, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, single cell RNA seq
Abstract: Peer reviewed: True, The freshwater snail Biomphalaria glabrata is an intermediate host of Schistosoma mansoni, the agent of human intestinal schistosomiasis. However, much is to be discovered about its innate immune system that appears as a complex black box, in which the immune cells (called hemocytes) play a major role in both cellular and humoral response towards pathogens. Until now, hemocyte classification has been based exclusively on cell morphology and ultrastructural description and depending on the authors considered from 2 to 5 hemocyte populations have been described. In this study, we proposed to evaluate the hemocyte heterogeneity at the transcriptomic level. To accomplish this objective, we used single cell RNA sequencing (scRNAseq) technology coupled to a droplet-based system to separate hemocytes and analyze their transcriptome at a unique cell level in naive Biomphalaria glabrata snails. We were able to demonstrate the presence of 7 hemocyte transcriptomic populations defined by the expression of specific marker genes. As a result, scRNAseq approach showed a high heterogeneity within hemocytes, but provides a detailed description of the different hemocyte transcriptomic populations in B. glabrata supported by distinct cellular functions and lineage trajectory. As a main result, scRNAseq revealed the 3 main population as a super-group of hemocyte diversity but, on the contrary, a great hemocytes plasticity with a probable capacity of hemocytes to engage to different activation pathways. This work opens a new field of research to understand the role of hemocytes particularly in response to pathogens, and towards S. mansoni parasites.
Published: 2022

3. SUMOylation controls the rapid transcriptional reprogramming induced by anthracyclines in Acute Myeloid Leukemias

Author: Mathias Boulanger, Chamseddine Kifagi, Marko Ristic, Ludovic Gabellier, Denis Tempé, Jon-Otti Sigurdsson, Tony Kaoma, Charlotte Andrieu-Soler, Thierry Forné, Eric Soler, Yosr Hicheri, Elise Gueret, Laurent Vallar, Jesper V Olsen, Guillaume Cartron, Marc Piechaczyk, Guillaume Bossis, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), University of Copenhagen = Københavns Universitet (UCPH), Luxembourg Institute of Health (LIH), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Département d'Hématologie [CHU de Montpellier], Centre Hospitalier Universitaire de Montpellier (CHU Montpellier ), and bossis, guillaume
Subjects: [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio]
Abstract: Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces broad transcriptional changes in AML cells before cell death induction. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, which limits both the positive and negative effects of DNR on transcription. Quantitative proteomics shows that proteins that are deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. Its induction is preceded by a SUMO-dependent reconfiguration of chromatin loops engaging its CTCF- and SUMO-bound promoter with distal cis-regulatory regions. Altogether, our work suggests that one of the earliest effects of DNR in AML cells is a SUMO-dependent transcriptional reprogramming.
Published: 2022

4. FishmiRNA: An Evolutionarily Supported MicroRNA Annotation and Expression Database for Ray-Finned Fishes

Author: Thomas Desvignes, Philippe Bardou, Jérôme Montfort, Jason Sydes, Cervin Guyomar, Simon George, John H Postlethwait, Julien Bobe, Girard, Agnès, APPEL À PROJETS GÉNÉRIQUE 2018 - Elucider les bases cellulaires de la fécondité chez le poisson : dynamique et régulation de l'ovogenèse chez le medaka - - DynaMO2018 - ANR-18-CE20-0004 - AAPG2018 - VALID, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, University of Oregon [Eugene], Système d'Information des GENomes des Animaux d'Elevage (SIGENAE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire de Physiologie et Génomique des Poissons (LPGP), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the National Institutes of Health (grant numbers NIH R24 OD011199, NIH 5R01 OD011116, and NIH R01 GM085318 to JHP), the National Science Foundation Office of Polar Program (NSF OPP-1543383 to JHP and TD), and the Agence Nationale de la Recherche (ANR-18-CE20-0004 to JB). This work benefited from access to the University of Oregon high performance computer, Talapas. MGX acknowledges financial support from France Génomique National infrastructure, funded as part of 'Investissement d'Avenir' program managed by Agence National pour la Recherche (ANR-10-INBS-09)., ANR-18-CE20-0004,DynaMO,Elucider les bases cellulaires de la fécondité chez le poisson : dynamique et régulation de l'ovogenèse chez le medaka(2018), and ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010)
Subjects: [INFO.INFO-DB]Computer Science [cs]/Databases [cs.DB], Teleost, [SDV]Life Sciences [q-bio], non-coding RNA, Fishes, bowfin Amia calva, Holostei, [SDV] Life Sciences [q-bio], Whole Genome Duplication, MicroRNAs, actinopterygian, Genetics, [INFO.INFO-DB] Computer Science [cs]/Databases [cs.DB], Animals, Molecular Biology, Ecology, Evolution, Behavior and Systematics
Abstract: MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression involved in countless biological processes and are widely studied across metazoans. Although miRNA research continues to grow, the large community of fish miRNA researchers lacks exhaustive resources consistent among species. To fill this gap, we developed FishmiRNA, an evolutionarily supported miRNA annotation and expression database for ray-finned fishes: www.fishmirna.org. The self-explanatory database contains detailed, manually curated miRNA annotations with orthology relationships rigorously established by sequence similarity and conserved syntenies, and expression data provided for each detected mature miRNA. In just few clicks, users can download the annotation and expression database in several convenient formats either in its entirety or a subset. Simple filters and Blast search options also permit the simultaneous exploration and visual comparison of expression data for up to any ten mature miRNAs across species and organs. FishmiRNA was specifically designed for ease of use to reach a wide audience.
Published: 2022

5. Transcriptional differences between the two host strains of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Author: Marion Orsucci, Yves Moné, Philippe Audiot, Sylvie Gimenez, Sandra Nhim, Rima Naït-Saïdi, Marie Frayssinet, Guillaume Dumont, Jean-Paul Boudon, Marin Vabre, Stéphanie Rialle, Rachid Koual, Gael J. Kergoat, Rodney N. Nagoshi, Robert L. Meagher, Emmanuelle d’Alençon, Nicolas Nègre, Centre de Biologie pour la Gestion des Populations (UMR CBGP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut de Recherche pour le Développement (IRD [France-Sud])-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro Montpellier, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Université de Montpellier (UM), Diversité, Génomes & Interactions Microorganismes - Insectes [Montpellier] (DGIMI), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Montpellier (UM), Plant Biology, Linnean Centre for Plant Biology, Uppsala BioCentre, Swedish University of Agricultural Sciences (SLU), Drexel University College of Medicine, DIASCOPE, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), USDA/ARS, and USDA-ARS : Agricultural Research Service
Subjects: [SDV]Life Sciences [q-bio]
Abstract: International audience; Spodoptera frugiperda, the fall armyworm (FAW), is an important agricultural pest in the Americas and an emerging pest in sub-Saharan Africa, India, East-Asia and Australia, causing damage to major crops such as corn, sorghum and soybean. While FAW larvae are considered polyphagous, differences in diet preference have been described between two genetic variants: the corn strain (sf-C) and the rice strain (sf-R). These two strains are sometimes considered as distinct species, raising the hypothesis that host plant specialization might have driven their divergence. To test this hypothesis, we first performed controlled reciprocal transplant (RT) experiments to address the impact of plant diet on several traits linked to the fitness of the sf-C and sf-R strains. The phenotypical data suggest that sf-C is specialized to corn. We then used RNA-Se to identify constitutive transcriptional differences between strains, regardless of diet, in laboratory as well as in natural populations. We found that variations in mitochondrial transcription levels are among the most substantial and consistent differences between the two strains. Since mitochondrial genotypes also vary between the strains, we believe the mitochondria may have a significant role in driving strain divergence.
Published: 2022

6. Assessing the Impact of Persistent HIV Infection on Innate Lymphoid Cells Using In Vitro Models

Author: Aude Boulay, Sara Trabanelli, Stéphanie Boireau, Myriam Boyer-Clavel, Sébastien Nisole, Pedro Romero, Camilla Jandus, Anne-Sophie Beignon, Nathalie J. Arhel, Institut de Recherche en Infectiologie de Montpellier (IRIM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Université de Genève = University of Geneva (UNIGE), Ludwig Institute for Cancer Research, Montpellier Ressources Imagerie, Biocampus, CNRS, INSERM, Universite Montpellier, Montpellier, France, BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Université de Lausanne = University of Lausanne (UNIL), Immunologie des maladies virales, auto-immunes, hématologiques et bactériennes (IMVA-HB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Infectious Diseases Models for Innovative Therapies (IDMIT), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, This work was supported by the Montage de Reseaux Scientifiques Européens ou Internationaux grant (N.J.A.) from the Agence Nationale de la Recherche , France, Sidaction grant for the salary of A.B., and by the Institut des sciences biologiques du Centre national de la recherche scientifique, and The Swiss Cancer League (KFS-4404-02-2018)
Subjects: [SDV]Life Sciences [q-bio], [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Immunology, Immunology and Allergy, General Medicine, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity
Abstract: Pathogens that persist in their host induce immune dysfunctions even in the absence of detectable replication. To better understand the phenotypic and functional changes that persistent infections induce in sentinel innate immune cells, we developed human PBMC-based HIV models of persistent infection. Autologous nonactivated PBMCs were cocultured with chronically infected, acutely infected, or uninfected cells and were then analyzed by unsupervised high-dimensional flow cytometry. Using this approach, we identified prevalent patterns of innate immune dysfunctions associated with persistent HIV infections that at least in part mirror immune dysfunctions observed in patients. In one or more models of chronic infection, bystander CD16+ NK cells expressing markers of activation, such as CD94, CD45RO, CD62L, CD69, CD25, and immune checkpoints PD1, Tim3, TIGIT, NKG2A and Lag3, were significantly reduced. Conversely, helper ILC subsets expressing PDL1/PDL2 were significantly enriched in chronic infection compared with either uninfected or acute infection, suggesting that chronic HIV-1 infection was associated with an inhibitory environment for bystander ILC and NK subsets. The cell-based models of persistent infection that we describe here provide versatile tools to explore the molecular mechanisms of these immune dysfunctions and unveil the contribution of innate immunity in sustaining pathogen persistence.
Published: 2023

7. Multivariate Analysis of RNA Chemistry Marks Uncovers Epitranscriptomics-Based Biomarker Signature for Adult Diffuse Glioma Diagnostics

Author: S. Relier, A. Amalric, A. Attina, I.B. Koumare, V. Rigau, F. Burel Vandenbos, D. Fontaine, M. Baroncini, J.P. Hugnot, H. Duffau, L. Bauchet, C. Hirtz, E. Rivals, A. David, Gouat, Isabelle, Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Plateforme de Protéomique Clinique de Montpellier (PPC), BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), 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), Service de Neurochirurgie [Montpellier], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-CHU Gui de Chauliac [Montpellier], Hôpital Gabriel Touré [Mali], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Centre Hospitalier Universitaire de Nice (CHU Nice), Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Méthodes et Algorithmes pour la Bioinformatique (MAB), Laboratoire d'Informatique de Robotique et de Microélectronique de Montpellier (LIRMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)
Subjects: Proteomics, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multivariate Analysis, Humans, Metabolomics, RNA, Glioma, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Biomarkers, Analytical Chemistry
Abstract: International audience; One of the main challenges in cancer management relates to the discovery of reliable biomarkers, which could guide decision-making and predict treatment outcome. In particular, the rise and democratization of high-throughput molecular profiling technologies bolstered the discovery of "biomarker signatures" that could maximize the prediction performance. Such an approach was largely employed from diverse OMICs data (i.e., genomics, transcriptomics, proteomics, metabolomics) but not from epitranscriptomics, which encompasses more than 100 biochemical modifications driving the post-transcriptional fate of RNA: stability, splicing, storage, and translation. We and others have studied chemical marks in isolation and associated them with cancer evolution, adaptation, as well as the response to conventional therapy. In this study, we have designed a unique pipeline combining multiplex analysis of the epitranscriptomic landscape by high-performance liquid chromatography coupled to tandem mass spectrometry with statistical multivariate analysis and machine learning approaches in order to identify biomarker signatures that could guide precision medicine and improve disease diagnosis. We applied this approach to analyze a cohort of adult diffuse glioma patients and demonstrate the existence of an "epitranscriptomics-based signature" that permits glioma grades to be discriminated and predicted with unmet accuracy. This study demonstrates that epitranscriptomics (co)evolves along cancer progression and opens new prospects in the field of omics molecular profiling and personalized medicine.
Published: 2022

8. A simulated marine heatwave impacts European sea bass sperm quantity, but not quality

Author: Benjamin Geffroy, Leydy Sandoval‐Vargas, Myriam Boyer‐Clavel, Maritza Pérez‐Atehortúa, Stephane Lallement, Iván Valdebenito Isler, MARine Biodiversity Exploitation and Conservation (UMR MARBEC), Institut de Recherche pour le Développement (IRD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Montpellier Ressources Imagerie, Biocampus, CNRS, INSERM, Universite Montpellier, Montpellier, France, BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)
Subjects: [SDE.MCG]Environmental Sciences/Global Changes, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Aquatic Science, Ecology, Evolution, Behavior and Systematics
Abstract: International audience; Rapid environmental changes will be the major challenge that most biota will have to deal with in the near future. Extreme events, such as marine heatwaves, are becoming more frequent and could be spatially uniform at a regional scale for a relatively long period of time. To date, most research studies on heatwaves have focused on sessile organisms, but these extreme events can also impact mobile species. Here, a 3-week marine heatwave was simulated to investigate its effects on the male reproductive performance of a Mediterranean Sea emblematic species, the European sea bass Dicentrarchus labrax. Males from the control condition (c. 13°C) produced significantly more sperm than those exposed to a relatively warm thermal treatment (c. 16°C). Nonetheless, neither the percentage of motile spermatozoa nor most of the other sperm motility parameters were significantly affected by the rearing temperature over the whole period. Overall, the results of this study suggest only moderated effects of a potential winter heatwave on the reproductive performance of male European sea bass.
Published: 2023

9. The autism-associated Meis2 gene is necessary for cardiac baroreflex regulation in mice

Author: J Roussel, R Larcher, P Sicard, P Bideaux, S Richard, F Marmigère, J Thireau, MORNET, Dominique, Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Montpellier Ressources Imagerie, Biocampus, CNRS, INSERM, Universite Montpellier, Montpellier, France, BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut des Neurosciences de Montpellier (INM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
Subjects: Mice, Multidisciplinary, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, Sensory Receptor Cells, Autism Spectrum Disorder, Heart Rate, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Animals, Autistic Disorder, Baroreflex, [SDV.MHEP.CSC] Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system
Abstract: Recent understanding of Autism Spectrum Disorder (ASD) showed that peripheral primary mechanosensitive neurons involved in touch sensation and central neurons affected in ASD share transcriptional regulators. Mutant mice for ASD-associated transcription factors exhibit impaired primary tactile perception, and restoring those genes specifically in primary sensory neurons rescue some of the anxiety-like behavior and social interaction defects.Interestingly, peripheral mechanosensitive sensory neurons also project to internal organs including the cardio-vascular system, and an imbalance of the cardio-vascular sympatho-vagal regulation is evidenced in ASD and intellectual disability. ASD patients have decreased vagal tone, suggesting dysfunction of sensory neurons involved in cardio-vascular sensing.In light of our previous finding that the ASD-associated Meis2 gene is necessary for normal touch neurons development and function, we investigated here if its inactivation in mouse peripheral sensory neurons also affects cardio-vascular sympatho-vagal regulation and baroreflex. Combining echocardiography, pharmacological challenge, blood pressure monitoring and heart rate variability analysis, we found that Meis2 mutant mice exhibited a blunted vagal response independently of any apparent cardiac malformation. These results suggest that defects in primary sensory neurons with mechanosensitive identity could participate in the imbalanced cardio-vascular sympatho-vagal tone found in ASD patients, reinforcing current hypotheses on the role of primary sensory neurons in the etiology of ASD.
Published: 2022

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

Author: Alexandre Martinière, Yvon Jaillais, Matthieu Pierre Platre, Marcelo Nollmann, Claire Burny, Vincent Bayle, Jean-Bernard Fiche, Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut für Populationsgenetik [Vienna], Veterinärmedizinische Universität Wien, Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-18-CE13-0025,caLIPSO,Mécanismes du pattern lipidique du réseau trans-Golgien (trans-Golgi Network) et rôles dans le tri des protéines, la polarité cellulaire et le développement des plantes(2018), and ANR-19-CE20-0008,CellOsmo,Nanodomain membranaire: un role dans la signalisation osmotique(2019)
Subjects: Fluorescence-lifetime imaging microscopy, Computer science, [SDV]Life Sciences [q-bio], single particle tracking, Arabidopsis, Total internal reflection microscopy, Fluorescence correlation spectroscopy, total internal reflection fluorescence microscopy, General Biochemistry, Genetics and Molecular Biology, Diffusion, molecular diffusion, 03 medical and health sciences, 0302 clinical medicine, Plant Cells, super-resolution microscopy, Microscopy, Photoactivated localization microscopy, 030304 developmental biology, 0303 health sciences, plants, Super-resolution microscopy, sptPALM, Optical Imaging, photo-activated localization microscopy, Rho GTPase, Membrane Proteins, Fluorescence recovery after photobleaching, root, Single Molecule Imaging, Autofluorescence, Spectrometry, Fluorescence, Microscopy, Fluorescence, single molecule localization microscopy, auxin, Biological system, 030217 neurology & neurosurgery, Fluorescence Recovery After Photobleaching
Abstract: Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3–4 h of imaging followed by 3–4 d of image processing and data analysis. This protocol describes how to perform single-particle tracking photoactivated localization microscopy (sptPALM) of membrane proteins in living plant tissues. The procedure covers all stages, from sample preparation to data analysis.
Published: 2021

11. TAT-RasGAP 317-326 kills cells by targeting inner-leaflet–enriched phospholipids

Author: Christian Widmann, Filip Stojceski, Pierre-Emmanuel Milhiet, Gabriel Ichim, Gianvito Grasso, Robyn Roth, Cédric Godefroy, Kushal Kumar Das, Andrea Danani, Marc Serulla, Tim Schober, Ana J. García-Sáez, Sergii Afonin, Mathieu Heulot, Milhiet, Pierre-Emmanuel, Université de Lausanne = University of Lausanne (UNIL), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Istituto Dalle Molle di Studi sull'Intelligenza Artificiale (IDSIA), Università della Svizzera italiana = University of Italian Switzerland (USI)-Scuola universitaria professionale della Svizzera italiana = University of Applied Sciences and Arts of Southern Switzerland [Manno] (SUPSI), Karlsruhe Institute of Technology (KIT), Karlsruher Institut für Technologie (KIT), Washington University School of Medicine [Saint Louis, MO], Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Eberhard Karls Universität Tübingen = Eberhard Karls University of Tuebingen, University of Lausanne (UNIL), Università della Svizzera italiana = University of Italian Switzerland (USI)-Scuola universitaria professionale della Svizzera italiana [Manno] (SUPSI), Washington University School of Medecine [Saint Louis, MO], Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Interfaculty Institute of Biochemistry (IFIB), University of Tübingen, 72076, Tübingen, Germany
Subjects: Phosphatidylinositol 4,5-Diphosphate, 0301 basic medicine, Programmed cell death, Necrosis, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Peptide, CHO Cells, Phosphatidylserines, Molecular Dynamics Simulation, 03 medical and health sciences, chemistry.chemical_compound, Cricetulus, 0302 clinical medicine, Neoplasms, medicine, Animals, Humans, Nuclear Magnetic Resonance, Biomolecular, chemistry.chemical_classification, Multidisciplinary, Cell Death, Chemistry, Cell Membrane, GTPase-Activating Proteins, Phosphatidylserine, Biological Sciences, Peptide Fragments, Cell biology, Point mutant, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Microscopy, Electron, Cytosol, 030104 developmental biology, Membrane, 030220 oncology & carcinogenesis, Liposomes, Cancer cell, lipids (amino acids, peptides, and proteins), medicine.symptom, HeLa Cells
Abstract: International audience; TAT-RasGAP 317–326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP 317–326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP 2 ) and phosphatidylserine (PS). Decreasing the amounts of PIP 2 in cells renders them more resistant to TAT-RasGAP 317–326 , while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP 317–326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP 2 - and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP 317–326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.
Published: 2020

12. Relaxation time asymmetry in stator dynamics of the bacterial flagellar motor

Author: Jerome Dorignac, Jean-Charles Walter, Andrea Parmeggiani, María-José Franco-Oñate, Nils-Ole Walliser, Ashley L. Nord, Fred Geniet, John Palmeri, Ruben Perez-Carrasco, Department of Life Sciences, Imperial College London, Laboratoire Charles Coulomb (L2C), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Centre de Biologie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and Nord, Ashley
Subjects: Stator, media_common.quotation_subject, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Flagellum, 010402 general chemistry, 01 natural sciences, Asymmetry, law.invention, 03 medical and health sciences, Bacterial Proteins, law, Molecular motor, Torque, Ion channel, 030304 developmental biology, media_common, Physics, 0303 health sciences, Multidisciplinary, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Bacteria, Rotor (electric), Molecular Motor Proteins, Dynamics (mechanics), 0104 chemical sciences, Flagella, Biophysics
Abstract: The bacterial flagellar motor (BFM) is the membrane-embedded rotary molecular motor which turns the flagellum that provides thrust to many bacterial species. This large multimeric complex, composed of a few dozen constituent proteins, has emerged as a hallmark of dynamic subunit exchange. The stator units are inner-membrane ion channels which dynamically bind and unbind to the peptidoglycan at the rotor periphery, consuming the ion motive force (IMF) and applying torque to the rotor when bound. The dynamic exchange is known to be a function of the viscous load on the flagellum, allowing the bacterium to dynamically adapt to its local viscous environment, but the molecular mechanisms of exchange and mechanosensitivity remain to be revealed. Here, by actively perturbing the steady-state stator stoichiometry of individual motors, we reveal a stoichiometry-dependent asymmetry in stator remodeling kinetics. We interrogate the potential effect of next-neighbor interactions and local stator unit depletion and find that neither can explain the observed asymmetry. We then simulate and fit two mechanistically diverse models which recapitulate the asymmetry, finding stator assembly dynamics to be particularly well described by a two-state catch-bond mechanism.
Published: 2022

13. Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury

Author: Muriel Gelin, Fred Allemand, Cong Trung Nguyen, Mathieu Panel, Abdelhakim Ahmed-Belkacem, Bijan Ghaleh, Rozenn Brillet, Isaac Ruiz, Julien Calderaro, Fouad Lafdil, Jean-François Guichou, Fatima Teixeira-Clerc, Didier Morin, Jean-Michel Pawlotsky, 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), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), morin, didier, and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Male, 0301 basic medicine, Isomerase activity, [SDV.MHEP.CHI] Life Sciences [q-bio]/Human health and pathology/Surgery, Ischemia, Mitochondria, Liver, [SDV.MHEP.CHI]Life Sciences [q-bio]/Human health and pathology/Surgery, Pharmacology, Mitochondrion, Mitochondrial Membrane Transport Proteins, Mouse model, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, medicine, Animals, Humans, Calcium Signaling, Enzyme Inhibitors, Inner mitochondrial membrane, Cells, Cultured, Mice, Knockout, Hepatology, Mitochondrial Permeability Transition Pore, Chemistry, Liver Diseases, MPTP, Gastroenterology, PPIF, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, medicine.disease, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, 3. Good health, PPIase activity, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, Liver, Mitochondrial permeability transition pore, Cytoprotection, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Reperfusion Injury, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, 030211 gastroenterology & hepatology, Drug, Mitochondrial Swelling, Reperfusion injury, Cyclophilin D, Signal Transduction
Abstract: International audience; Background & Aims: Hepatic ischemia-reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injuryMethods: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif–/– (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis.Results: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2–16.2 μmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4–132 μmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif–/– mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle.Conclusions:Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.
Published: 2019

14. Preparing sequencing grade RNAs from a small number of FACS-sorted larvae macrophages isolated from enzyme free dissociated zebrafish larvae

Author: Christina Begon-Pescia, Stéphanie Boireau, Myriam Boyer-Clavel, Georges Lutfalla, Mai Nguyen-Chi, LPHI - Laboratory of Pathogen Host Interactions (LPHI), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Montpellier Ressources Imagerie, Biocampus, CNRS, INSERM, Universite Montpellier, Montpellier, France, BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and Nguyen Chi, Mai
Subjects: FACS-sorted, Medical Laboratory Technology, Zebrafish larva, [SDV.IMM] Life Sciences [q-bio]/Immunology, RNA quality, Clinical Biochemistry, [SDV.IMM]Life Sciences [q-bio]/Immunology, Macrophages dissociation
Abstract: International audience; Macrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages.•An optimized protocol in 5 steps to extract high quality RNAs from zebrafish macrophages.•A cell dissociation method using an enzyme-free and osmotically controlled buffer to prevent the alteration of macrophage activation status and limit cell mortality.•Production of high integrity RNAs from a small number of isolated macrophages.
Published: 2021

15. A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers

Author: Joan Font, Robert B. Quast, Patrick Chames, Jordi Haubrich, Anne Goupil-Lamy, Damien Nevoltris, Jean-Philippe Pin, Pauline Scholler, Philippe Rondard, Francine Acher, Laurent Prézeau, Institut de Génomique Fonctionnelle (IGF), 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), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Dassault Systèmes, Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Université de Paris (UP), ANR-18-CE11-0004,DynaMuR2,Dynamique structurale d'un RCPG dimérique en molécule unique(2018), ANR-10-LABX-0053,MAbImprove,Optimization of therapeutic monoclonal antibodies development Better antibodies, better developed AND better used(2010), ANR-15-CE18-0020,nanoGluAct,Modulation de la synapse glutamatergique à l'aide de nanobodies(2015), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), Université Paris Cité - UFR Sciences du Vivant [Sciences] (UPCité - UFR SDV), Université Paris Cité (UPCité), 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), Guerineau, Nathalie C., Laboratoires d'excellence - Optimization of therapeutic monoclonal antibodies development Better antibodies, better developed AND better used - - MAbImprove2010 - ANR-10-LABX-0053 - LABX - VALID, Modulation de la synapse glutamatergique à l'aide de nanobodies - - nanoGluAct2015 - ANR-15-CE18-0020 - AAPG2015 - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Dynamique structurale d'un RCPG dimérique en molécule unique - - DynaMuR22018 - ANR-18-CE11-0004 - AAPG2018 - VALID, Université de Paris - UFR Sciences du Vivant [Sciences] (UP - UFR SDV), and UFR Sciences du Vivant [Sciences] - Université Paris Cité (UFR SDV UPCité)
Subjects: Allosteric modulator, Protein Conformation, [SDV]Life Sciences [q-bio], Allosteric regulation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Receptors, Metabotropic Glutamate, Models, Biological, 03 medical and health sciences, 0302 clinical medicine, Humans, Receptor, agonist, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, single-domain antibody, 030304 developmental biology, G protein-coupled receptor, 0303 health sciences, Multidisciplinary, Chemistry, Metabotropic glutamate receptor 4, Glutamate receptor, Glutamate binding, Single-Domain Antibodies, Biological Sciences, activation mechanism, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Metabotropic receptor, Gene Expression Regulation, G protein–coupled receptor, Mutation, 030217 neurology & neurosurgery, Protein Binding
Abstract: International audience; There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.
Published: 2021

16. Melanotransferrin is efficiently sorted on the surface of exosomes secreted by melanoma cells

Author: Gaëtan Bellot, Nesrine Aissaoui, Laurent Henry, Marie Morille, Michel Vidal, Anne Bonhoure, Pierre-Emmanuel Stoebner, Dynamique des interactions membranaires normales et pathologiques (DIMNP), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université Montpellier 1 (UM1), 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), Institut Charles Gerhardt Montpellier - Institut de Chimie Moléculaire et des Matériaux de Montpellier (ICGM ICMMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut de Chimie du CNRS (INC), Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)
Subjects: Cancer Research, Skin Neoplasms, Dermatology, Exosomes, Mice, Melanoma Biomarker, medicine, Animals, Humans, [CHIM]Chemical Sciences, Melanoma, neoplasms, ComputingMilieux_MISCELLANEOUS, Membrane Glycoproteins, CD63, biology, Chemistry, medicine.disease, Microvesicles, 3. Good health, Oncology, Cutaneous melanoma, Cancer cell, biology.protein, Cancer research, Melanotransferrin, Antibody
Abstract: Cutaneous melanoma is the most lethal type of skin cancer. Early detection is crucial to improve the outcome of melanoma patients. The identification of noninvasive prognostic biomarkers for the follow-up of melanoma patients is still in demand for clinical use. We show here that exosomal melanotransferrin fulfills the biomarker characteristics required to meet this demand. Melanotransferrin is typically overexpressed in melanoma cells compared to other cell types - including cancer cells - and is efficiently sorted and secreted with nanovesicles, or so-called exosomes, due to its membrane-anchoring by a glycosylphosphatidylinositol. Melanotransferrin is exposed on the surface of exosomes and is accessible for antibody recognition. An ELISA was set up to quantify melanotransferrin after immobilization of nanovesicles through the exosomal constituent tetraspanins CD63. Melanotransferrin was detected using a low number of exosomes purified from melanoma cell line cultures, and melanotransferrin detection was abolished by phosphatidylinositol-specific phospholipase C treatment. This exosomal melanotransferrin ELISA was able to discriminate an equal number of assayed exosomes purified from two different melanoma cell lines (A-375 vs. SK-MEL-28). Moreover, plasma samples from patients with melanoma and noncancer disease were assayed using this ELISA and elevated levels of exosomal melanotransferrin were seen in the plasma of patients with melanoma. We propose that exosomal melanotransferrin should be assessed as a potential melanoma biomarker.
Published: 2021

17. Mammalian Mechanoelectrical Transduction: Structure and Function of Force-Gated Ion Channels

Author: Dominique Douguet, Eric Honoré, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (... - 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), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and Honoré, Eric
Subjects: 0303 health sciences, [SDV]Life Sciences [q-bio], PIEZO1, Mechanoelectrical transduction, Depolarization, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Gating, Biology, General Biochemistry, Genetics and Molecular Biology, Autonomic regulation, Structure and function, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 0302 clinical medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Mechanosensitive channels, Neuroscience, ComputingMilieux_MISCELLANEOUS, 030217 neurology & neurosurgery, Ion channel, 030304 developmental biology
Abstract: The conversion of force into an electrical cellular signal is mediated by the opening of different types of mechanosensitive ion channels (MSCs), including TREK/TRAAK K2P channels, Piezo1/2, TMEM63/OSCA, and TMC1/2. Mechanoelectrical transduction plays a key role in hearing, balance, touch, and proprioception and is also implicated in the autonomic regulation of blood pressure and breathing. Thus, dysfunction of MSCs is associated with a variety of inherited and acquired disease states. Significant progress has recently been made in identifying these channels, solving their structure, and understanding the gating of both hyperpolarizing and depolarizing MSCs. Besides prototypical activation by membrane tension, additional gating mechanisms involving channel curvature and/or tethered elements are at play.
Published: 2019

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

Author: Erwan Thouennon, Vanessa Delfosse, Rémy Bailly, Pauline Blanc, Abdelhay Boulahtouf, Marina Grimaldi, Alessandro Barducci, William Bourguet, Patrick Balaguer, Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Transcriptional Activation, Estrogen-related receptor γ, Molecular Dynamics Simulation, Crystallography, X-Ray, Ligands, 03 medical and health sciences, Cellular and Molecular Neuroscience, 0302 clinical medicine, Phenols, Cell Line, Tumor, Humans, Benzhydryl Compounds, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Endocrine disruptors, Molecular Biology, 030304 developmental biology, Pharmacology, 0303 health sciences, Binding Sites, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Xenoestrogens, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Recombinant Proteins, Protein Structure, Tertiary, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Receptors, Estrogen, Thermodynamics, Molecular Medicine, Protein–ligand interaction, 030217 neurology & neurosurgery, Protein Binding
Abstract: International audience; The estrogen-related receptor γ (ERRγ, NR3B3) is a constitutively active nuclear receptor which has been proposed to act as a mediator of the low-dose effects of a number of environmental endocrine-disrupting chemicals (EDCs) such as the xenoestrogen bisphenol-A (BPA). To better characterize the ability of exogenous compounds to bind and activate ERRγ, we used a combination of cell-based, biochemical, structural and computational approaches. A purposely created stable cell line allowed for the determination of the EC50s for over 30 environmental ERRγ ligands, including previously unknown ones. Interestingly, affinity constants (Kds) of the most potent compounds measured by isothermal titration calorimetry were in the 50–500 nM range, in agreement with their receptor activation potencies. Crystallographic analysis of the interaction between the ERRγ ligand-binding domain (LBD) and compounds of the bisphenol, alkylphenol and naphthol families revealed a partially shared binding mode and minimal alterations of the receptor conformation upon ligand binding. Further biophysical characterizations coupled to molecular dynamics simulations suggested a mechanism through which ERRγ ligands would exhibit their agonistic properties by preserving the transcriptionally active form of the receptor while rigidifying some loop regions with associated functions. This unique mechanism contrasts with the classical one involving a ligand-induced repositioning and stabilization of the C-terminal activation helix H12.
Published: 2019

19. Osmotic Stress Activates Two Reactive Oxygen Species Pathways with Distinct Effects on Protein Nanodomains and Diffusion

Author: Yvon Jaillais, Stéphane Mari, Jean-Bernard Fiche, Xavier Dumont, Marija Smokvarska, Alexandre Martinière, Christophe Maurel, Carine Alcon, Marcelo Nollmann, Kian Hématy, Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut Jean-Pierre Bourgin (IJPB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Baracco, Chantal, Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0106 biological sciences, Osmotic shock, Physiology, [SDV]Life Sciences [q-bio], Arabidopsis, Aquaporin, Plant Science, Aquaporins, Endocytosis, 01 natural sciences, Protein Domains, Osmotic Pressure, Genetics, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Arabidopsis thaliana, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, chemistry.chemical_classification, Reactive oxygen species, biology, Arabidopsis Proteins, Chemistry, Cell Biology, biology.organism_classification, Cell biology, [SDV] Life Sciences [q-bio], Second messenger system, Signal transduction, Reactive Oxygen Species, Signal Transduction, 010606 plant biology & botany
Abstract: Physiological acclimation of plants to an everchanging environment is governed by complex combinatorial signaling networks that perceive and transduce various abiotic and biotic stimuli. Reactive oxygen species (ROS) serve as one of the second messengers in plant responses to hyperosmotic stress. The molecular bases of ROS production and the primary cellular processes that they target were investigated in the Arabidopsis (Arabidopsis thaliana) root. Combined pharmacological and genetic approaches showed that the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) pathway and an additional pathway involving apoplastic ascorbate and iron can account for ROS production upon hyperosmotic stimulation. The two pathways determine synergistically the rate of membrane internalization, within minutes after activation. Live superresolution microscopy revealed at single-molecule scale how ROS control specific diffusion and nano-organization of membrane cargo proteins. In particular, ROS generated by RBOHs initiated clustering of the PLASMA MEMBRANE INTRINSIC PROTEIN2;1 aquaporin and its removal from the plasma membrane. This process is contributed to by clathrin-mediated endocytosis, with a positive role of RBOH-dependent ROS, specifically under hyperosmotic stress.
Published: 2019

20. Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM

Author: Laurent Fernandez, Antoine Le Gall, Eric Rubinstein, Célia Chamontin, Marylène Mougel, Pierre-Emmanuel Milhiet, Selma Dahmane, Patrice Dosset, Florent Murcy, Marcelo Nollmann, Desiree Salas, Christine Doucet, Mougel, Marylene, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Modèles de Cellules Souches Malignes et Thérapeutiques, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Viral protein, [SDV]Life Sciences [q-bio], viruses, Cell, 02 engineering and technology, Microscopy, Atomic Force, 010402 general chemistry, medicine.disease_cause, gag Gene Products, Human Immunodeficiency Virus, 01 natural sciences, Tetraspanin 29, Tetraspanin 28, Flow cytometry, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, General Materials Science, Budding, medicine.diagnostic_test, Chemistry, Super-resolution microscopy, Virus Assembly, Cell Membrane, Flow Cytometry, 021001 nanoscience & nanotechnology, 3. Good health, 0104 chemical sciences, Cell biology, [SDV] Life Sciences [q-bio], Membrane, medicine.anatomical_structure, Viral replication, embryonic structures, HIV-1, 0210 nano-technology, HeLa Cells, CD81
Abstract: International audience; Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell. Specifically assembly and release of HIV-1 particles require specific cellular components, which are recruited to the egress site by the viral protein Gag. We previously demonstrated that HIV-1 assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81, which are key players in many infectious processes, forming enriched areas where the virus buds. In this study we correlated super resolution microscopy mapping of tetraspanins with membrane topography delineated by atomic force microscopy (AFM) in Gag-expressing cells. We revealed that CD9 is specifically trapped within the nascent viral particles, especially at buds tips, suggesting that Gag mediates CD9 and CD81 depletion from the plasma membrane. In addition, we showed that CD9 is organized as small membrane assemblies of few tens of nanometers that can coalesce upon Gag expression.
Published: 2019

21. Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1

Author: Cécile Dubois, Philippe Barthe, Vicente J. Planelles-Herrero, Camille Tillatte-Tripodi, Elena M. Sirkia, Léa Mammri, Virginie Ropars, Christian Roumestand, Stephane Delbecq, Planelles-Herrero, Vicente J [0000-0002-1665-184X], Mammri, Léa [0000-0001-5064-3619], Roumestand, Christian [0000-0002-4082-3293], Barthe, Philippe [0000-0003-4282-6604], Apollo - University of Cambridge Repository, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Gestionnaire, Hal Sorbonne Université, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, Protein Denaturation, Magnetic Resonance Spectroscopy, Globular protein, Protein Conformation, [SDV]Life Sciences [q-bio], Hydrostatic pressure, Protein domain, Antiparallel (biochemistry), Article, Catalysis, lcsh:Chemistry, Inorganic Chemistry, 03 medical and health sciences, α-helical bundle, Protein Domains, protein folding, Humans, Denaturation (biochemistry), Physical and Theoretical Chemistry, lcsh:QH301-705.5, Molecular Biology, Spectroscopy, Adaptor Proteins, Signal Transducing, Protein Unfolding, chemistry.chemical_classification, 030102 biochemistry & molecular biology, Chemistry, high hydrostatic pressure, Organic Chemistry, Temperature, Energy landscape, General Medicine, Nuclear magnetic resonance spectroscopy, NMR, Computer Science Applications, [SDV] Life Sciences [q-bio], Kinetics, 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, Biophysics, thermodynamic stability, Thermodynamics, Protein folding
Abstract: International audience; When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.
Published: 2021
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22. Biology across scales: from atomic processes to bacterial communities through the lens of the microscope

Author: Marcelo Nollmann, Tâm Mignot, Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and LEROI, Delphine
Subjects: 0303 health sciences, Microscopy, Microscope, Bacteria, 030306 microbiology, [SDV]Life Sciences [q-bio], Nanotechnology, Biology, Microbiology, Through-the-lens metering, law.invention, [SDV] Life Sciences [q-bio], 03 medical and health sciences, Infectious Diseases, law, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: International audience
Published: 2021

23. Structure of the antidiuretic hormone vasopressin receptor signalling complex

Author: Julien Bous, Aurelie Ancelin, Nicolas Floquet, Hélène Orcel, Joséphine Lai-Kee-Him, Stefano Trapani, Julie Saint-Paul, Hélène Déméné, Sébastien Granier, Patrick Bron, Maxime Louet, Remy Sounier, Gérald Gaibelet, Bernard Mouillac, Cedric Leyrat, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Génomique Fonctionnelle (IGF), 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), 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), Déméné, Hélène, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Gs alpha subunit, Chemistry, [SDV]Life Sciences [q-bio], Protein subunit, Aquaporin, Cell biology, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 0302 clinical medicine, Arginine vasopressin receptor 2, Receptor, Ternary complex, 030217 neurology & neurosurgery, Function (biology), 030304 developmental biology, G protein-coupled receptor
Abstract: Arginine-vasopressin (AVP) is a neurohypophysial peptide known as the antidiuretic hormone. It forms an active signalling complex with the V2 receptor (V2R) and the Gs protein, promoting a cAMP/PKA-dependent aquaporin insertion in apical membranes of principal cells of the renal collecting ducts and ultimately, water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signalling system are still unknown. To fill this gap of knowledge, we report here the structure of the AVP-V2R-Gs complex using cryo-electron microscopy (cryo-EM). Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and NMR spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes and could thus represent distinct complex conformations along the signalling activation pathway. Importantly, as compared to those of other class A GPCR-Gs complexes, the structures revealed an original receptor-Gs interface in which the Gsα subunit penetrates deeper into the active V2R, notably forming an ionic bond between its free C-terminal carboxylic function and the side chain of R137 in the V2R. Interestingly, the structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases with opposite clinical outcomes, cNDI or NSIAD respectively. Our study thus provides important structural insights into the function of this clinically relevant GPCR signalling complex.
Published: 2020

24. A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

Author: Marie-Hélène Balesdent, Karine Blondeau, E. Zelie, Thierry Rouxel, Jérôme Gracy, Noureddine Lazar, Françoise Blaise, Alexander Idnurm, I. Li de la Sierra-Gallay, H. van Tilbeurgh, Carl H. Mesarich, Isabelle Fudal, N. Talbi, Bénédicte Ollivier, Y. Petit-Houdenot, 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), Laboratory of Molecular Plant Pathology, School of Agriculture and Environment, Massey University, Palmerston North, New Zealand, BIOlogie et GEstion des Risques en agriculture (BIOGER), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), The University of Melbourne, Melbourne, Australia., Grains R&D Corp UM00050, ANR-17-EURE-0007,SPS-GSR,Ecole Universitaire de Recherche de Sciences des Plantes de Paris-Saclay(2017), ANR-14-CE19-0019,StructuraLEP,Caractérisation structurale et fonctionnelle d'effecteurs de L. maculans et de leurs interactants(2014), ANR-13-BS07-0007,Ln23,Complexes de lanthanide comme nouveaux chevaux de Troie pour la détermination structurale de protéines(2013), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Massey University, Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Montpellier Ressources Imagerie, Biocampus, CNRS, INSERM, Universite Montpellier, Montpellier, France, BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), University of Melbourne, Bourse « Contrat Jeune Scientifique » de l’INRAE, and Université ParisSaclay
Subjects: 0106 biological sciences, structural family, Immunology, [SDV.SA.AGRO]Life Sciences [q-bio]/Agricultural sciences/Agronomy, Virulence, Leptosphaeria, medicine.disease_cause, avirulence, Microbiology, 01 natural sciences, Fungal Proteins, resistance, 03 medical and health sciences, Leptosphaeria maculans, Virology, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, medicine, Molecular Biology, Gene, Plant Diseases, Plant Proteins, 030304 developmental biology, 0303 health sciences, Mutation, biology, Effector, Brassica napus, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, [SDV.BV.PEP]Life Sciences [q-bio]/Vegetal Biology/Phytopathology and phytopharmacy, effector, plant pathogenic fungi, Epistasis, Parasitology, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Function (biology), 010606 plant biology & botany
Abstract: Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9- mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.Author summaryAn efficient strategy to control fungal diseases in the field is genetic control using resistant crop cultivars. Crop resistance mainly relies on gene-for-gene relationships between plant resistance (R) genes and pathogen avirulence (AVR) genes, as defined by Flor in the 1940s. However, such gene-for-gene relationships can increase in complexity over the course of plant-pathogen co-evolution. Resistance against the plant-pathogenic fungus Leptosphaeria maculans by Brassica napus and other Brassica species relies on the recognition of effector (AVR) proteins by R proteins; however, L. maculans produces an effector that suppresses a subset of these specific resistances. Using a protein structure approach, we revealed structural analogy between several of the resistance-triggering effectors, the resistance-suppressing effector, and effectors from other plant-pathogenic species in the Dothideomycetes and Sordariomycetes classes, defining a new family of effectors called LARS. Notably, cross-species expression of one LARS effector from Fulvia fulva, a pathogen of tomato, in L. maculans resulted in recognition by several resistant cultivars of oilseed rape. These results highlight the need to integrate knowledge on effector structures to improve resistance management and to develop broad-spectrum resistances for multi-pathogen control of diseases.
Published: 2020

25. MiSiC, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities

Author: Jean-Bernard Fiche, Tâm Mignot, Eugénie Martineau, Dorothée Murat, Marcelo Nollmann, Kelly Goldlust, Antoine Le Gall, Sara Rombouts, Swapnesh Panigrahi, Leon Espinosa, Centre National de la Recherche Scientifique (CNRS), Institut des Fonctions Optiques pour les Technologies de l'informatiON (Institut FOTON), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-École Nationale Supérieure des Sciences Appliquées et de Technologie (ENSSAT)-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Nollmann, Marcelo, Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), École Nationale Supérieure des Sciences Appliquées et de Technologie (ENSSAT)-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)
Subjects: Computer science, [SDV]Life Sciences [q-bio], 02 engineering and technology, computer.software_genre, 0302 clinical medicine, B. subtilis, Segmentation, Biology (General), Throughput (business), Microbiology and Infectious Disease, Microscopy, 0303 health sciences, General Neuroscience, myxococcus xanthus, Microbiota, General Medicine, Tools and Resources, [SDV] Life Sciences [q-bio], Identification (information), Medicine, Identification (biology), Computational and Systems Biology, QH301-705.5, Ecology (disciplines), Systems biology, Science, 0206 medical engineering, Cell segmentation, Bacterial Physiological Phenomena, Machine learning, Models, Biological, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Species Specificity, image analysis, 030304 developmental biology, Modality (human–computer interaction), General Immunology and Microbiology, business.industry, Deep learning, E. coli, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, High-Throughput Screening Assays, Biofilms, Other, Artificial intelligence, business, computer, 030217 neurology & neurosurgery, 020602 bioinformatics
Abstract: International audience; Studies of bacterial communities, biofilms and microbiomes, are multiplying due to their impact on health and ecology. Live imaging of microbial communities requires new tools for the robust identification of bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based 2D segmentation method that automatically segments single bacteria in complex images of interacting bacterial communities with very little parameter adjustment, independent of the microscopy settings and imaging modality. Using a bacterial predator-prey interaction model, we demonstrate that MiSiC enables the analysis of interspecies interactions, resolving processes at subcellular scales and discriminating between species in millimeter size datasets. The simple implementation of MiSiC and the relatively low need in computing power make its use broadly accessible to fields interested in bacterial interactions and cell biology.
Published: 2020

26. MAGI1 inhibits the AMOTL2/p38 stress pathway and prevents luminal breast tumorigenesis

Author: Diala Kantar, Vera Slaninova, Charles Géminard, Charles Theillet, Jacques Colinge, Antonio Maraver, Florence Boissière-Michot, Emilie Bousquet Mur, Christine Benistant, Patrice Lassus, Elodie Forest, Luca Costa, Lisa Heron-Milhavet, Marion Larroque, Yezza Ben Salah, Alexandre Djiane, Béatrice Orsetti, Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut du Cancer de Montpellier (ICM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Lassus, Patrice
Subjects: Scaffold protein, Carcinogenesis, [SDV]Life Sciences [q-bio], [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], medicine.disease_cause, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell junction, p38 Mitogen-Activated Protein Kinases, 0302 clinical medicine, Breast cancer, Cell polarity, beta Catenin, Cancer, 0303 health sciences, rho-Associated Kinases, Effector, Chemistry, Cadherins, Cell biology, Phenotype, 030220 oncology & carcinogenesis, Medicine, Female, Protein Binding, Signal Transduction, Cell signalling, p38 mitogen-activated protein kinases, Science, [SDV.CAN]Life Sciences [q-bio]/Cancer, Breast Neoplasms, Stress activated protein kinase, Article, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, Stress, Physiological, Cell Line, Tumor, medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Humans, Actin, 030304 developmental biology, Adaptor Proteins, Signal Transducing, Cell Proliferation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Epithelial Cells, YAP-Signaling Proteins, Angiomotins, Cell Adhesion Molecules, Guanylate Kinases
Abstract: Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.
Published: 2020

27. FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Author: Lerner, Eitan, Barth, Anders, Hendrix, Jelle, Ambrose, Benjamin, Birkedal, Victoria, Blanchard, Scott C., Börner, Richard, Chung, Hoi Sung, Cordes, Thorben, Craggs, Timothy D., Deniz, Ashok A., Diao, Jiajia, Fei, Jingyi, Gonzalez, Ruben L., Gopich, Irina V., Ha, Taekjip, Hanke, Christian A., Haran, Gilad, Hatzakis, Nikos S., Hohng, Sungchul, Hong, Seok Cheol, Hugel, Thorsten, Ingargiola, Antonino, Joo, Chirlmin, Kapanidis, Achillefs N., Kim, Harold D., Laurence, Ted, Lee, Nam Ki, Lee, Tae Hee, Lemke, Edward A., Margeat, Emmanuel, Michaelis, Jens, Michalet, Xavier, Myong, Sua, Nettels, Daniel, Peulen, Thomas Otavio, Ploetz, Evelyn, Razvag, Yair, Robb, Nicole C., Schuler, Benjamin, Soleimaninejad, Hamid, Tang, Chun, Vafabakhsh, Reza, Lamb, Don C., Seidel, Claus A.M., Weiss, Shimon, Boudker, Olga, Institute of Chemistry, The Hebrew University of Jerusalem, The Hebrew University of Jerusalem (HUJ), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Hasselt University (UHasselt), University of Sheffield [Sheffield], Aarhus University [Aarhus], St Jude Children's Research Hospital, Hochschule Mittweida - University of Applied Sciences, National Institutes of Health [Bethesda] (NIH), Ludwig-Maximilians-Universität München (LMU), The Scripps Research Institute [La Jolla], University of California [San Diego] (UC San Diego), University of California-University of California, University of Cincinnati (UC), University of Chicago, Columbia University [New York], Howard Hughes Medical Institute (HHMI), Weizmann Institute of Science [Rehovot, Israël], University of Copenhagen = Københavns Universitet (KU), Seoul National University [Seoul] (SNU), Korea University [Seoul], University of Freiburg [Freiburg], University of California [Los Angeles] (UCLA), University of California, Delft University of Technology (TU Delft), University of Oxford [Oxford], Georgia Institute of Technology [Atlanta], Lawrence Livermore National Laboratory (LLNL), Pennsylvania State University (Penn State), Penn State System, University Medical Center of the Johannes Gutenberg-University Mainz, Institute of Molecular Biology (IMB), Johannes Gutenberg - Universität Mainz (JGU), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Ulm (UUlm), Johns Hopkins University (JHU), Université de Zurich [Switzerland], University of California [San Francisco] (UCSF), University of Warwick [Coventry], University of Melbourne, Peking University [Beijing], Northwestern University [Evanston], The Scripps Research Institute [La Jolla, San Diego], University of Copenhagen = Københavns Universitet (UCPH), University of California (UC), University of Oxford, Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), University of California [San Francisco] (UC San Francisco), and Margeat, Emmanuel
Subjects: 0301 basic medicine, conformation, Open science, Computer science, Structural Biology and Molecular Biophysics, AMINOACYL-TRANSFER-RNA, INTRAMOLECULAR DISTANCE DISTRIBUTIONS, Review Article, RESONANCE ENERGY-TRANSFER, 01 natural sciences, biomolecules, FREELY DIFFUSING MOLECULES, Documentation, Fluorescence Resonance Energy Transfer, Mainstream, structural biology, Biology (General), General Neuroscience, NANO-POSITIONING SYSTEM, General Medicine, dynamics, INTRINSICALLY DISORDERED PROTEINS, Single Molecule Imaging, FLUORESCENCE CORRELATION SPECTROSCOPY, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Medicine, community, single-molecule, QH301-705.5, Science, Appeal, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Bioengineering, chemical biology, 010402 general chemistry, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, ALTERNATING-LASER EXCITATION, Biochemistry and Chemical Biology, molecular biophysics, biochemistry, Molecular Biology, Structure (mathematical logic), General Immunology and Microbiology, SINGLE-MOLECULE FRET, TRANSITION PATH TIMES, Data science, 0104 chemical sciences, 030104 developmental biology, FRET, Position paper, Generic health relevance, Biochemistry and Cell Biology
Abstract: International audience; Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB- Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage‘open science’ practices.
Published: 2020

28. A plasma membrane nanoplatform ensures signal specificity during osmotic signaling in plants

Author: Yvon Jaillais, Xavier Dumont, Philippe Nacry, V. Bayle, Matthieu Pierre Platre, Jean-Bernard Fiche, C. Francis, Marija Smokvarska, Christophe Maurel, Carine Alcon, Marcelo Nollmann, Alexandre Martinière, Baracco, Chantal, Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Plateforme d'histocytologie et d'imagerie cellulaire végétale (PHIV), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro - Montpellier SupAgro, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro - Montpellier SupAgro, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
Subjects: 0106 biological sciences, chemistry.chemical_classification, 0303 health sciences, Reactive oxygen species, NADPH oxidase, Osmotic concentration, biology, [SDV]Life Sciences [q-bio], Cell, GTPase, Stimulus (physiology), 01 natural sciences, Cell biology, [SDV] Life Sciences [q-bio], 03 medical and health sciences, medicine.anatomical_structure, chemistry, Auxin, Second messenger system, medicine, biology.protein, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, 030304 developmental biology, 010606 plant biology & botany
Abstract: In the course of their growth and development plants have to constantly perceive and react to their environment. This is achieved in cells, by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependant nanodomains within the PM. These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS),that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, ROP6 activation triggers the nanoclustering of two NADPH oxidases that subsequently generates ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically-driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensure signal specificity downstream of independent stimuli.
Published: 2020

29. Compression, Rupture, and Puncture of Model Membranes at the Molecular Scale

Author: Luca Costa, Thales F. D. Fernandes, V Oscar Saavedra, Pierre-Emmanuel Milhiet, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Costa, Luca, and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Materials science, Scale (ratio), [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Modulus, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Quantitative Biology::Cell Behavior, Quantitative Biology::Subcellular Processes, [SPI.MECA.BIOM] Engineering Sciences [physics]/Mechanics [physics.med-ph]/Biomechanics [physics.med-ph], Electrochemistry, General Materials Science, Composite material, Spectroscopy, Physics::Biological Physics, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], technology, industry, and agriculture, [SPI.MECA.BIOM]Engineering Sciences [physics]/Mechanics [physics.med-ph]/Biomechanics [physics.med-ph], Biological membrane, Surfaces and Interfaces, musculoskeletal system, 021001 nanoscience & nanotechnology, Condensed Matter Physics, Compression (physics), 0104 chemical sciences, body regions, Membrane, Bending stiffness, 0210 nano-technology
Abstract: International audience; Elastic properties of biological membranes are involved in a large number of membrane functionalities and activities. Conventionally characterized in terms of Young’s modulus, bending stiffness and stretching modulus, membrane mechanics can be assessed at high lateral resolution by means of atomic force microscopy (AFM). Here we show that the mechanical response of biomimetic model systems such as supported lipid bilayers (SLBs) is highly affected by the size of the AFM tip employed as a membrane indenter. Our study is focused on phase-separated fluid-gel lipid membranes at room temperature. In a small tip radius regime (≈ 2 nm) and in the case of fluid phase membranes, we show that the tip can penetrate through the membrane minimizing molecular vertical compression and in absence of molecular membrane rupture. In this case, AFM indentation experiments cannot assess the vertical membrane Young’s modulus. In agreement with the data reported in the literature, in the case of larger indenters (>2 nm) SLBs can be compressed leading to an evaluation of Young’s modulus and membrane maximal withstanding force before rupture. We show that such force increases with the indenter in agreement with the existing theoretical frame. Finally, we demonstrate that the latter has no influence on the number of molecules involved in the rupture process that is observed to be constant and rather dependent on the indenter chemical composition.
Published: 2020

30. Structural basis of nanobody recognition of grapevine fanleaf virus and of virus resistance loss

Author: Olivier Lemaire, Christophe Ritzenthaler, Aurélie Marmonier, Sophie Gersch, Bruno P. Klaholz, Léa Ackerer, Corinne Schmitt-Keichinger, Vianney Poignavent, Kamal Hleibieh, Lorène Belval, Serge Muyldermans, Véronique Komar, Caroline Hemmer, Patrick Bron, Ahmed Ghannam, Claude Sauter, Igor Orlov, Pascale Schellenberger, Jean-Michel Hily, Gérard Demangeat, Bernard Lorber, Emmanuelle Vigne, Klaholz, Bruno, Appel à projets générique - Résistances antivirales combinées pour lutter contre la maladie du court-noué de la vigne - - COMBiNiNG2014 - ANR-14-CE19-0022 - Appel à projets générique - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Centre for Integrative Biology - CBI (Inserm U964 - CNRS UMR7104 - IGBMC), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie moléculaire des plantes (IBMP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Santé de la vigne et qualité du vin (SVQV), Université de Strasbourg (UNISTRA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut Français de la Vigne et du Vin [Le Grau du Roi], Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Vrije Universiteit Brussel (VUB), ANR-14-CE19-0022,COMBiNiNG,Résistances antivirales combinées pour lutter contre la maladie du court-noué de la vigne(2014), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0106 biological sciences, Models, Molecular, GFLV, Nematoda, Cryo-electron microscopy, Protein Conformation, [SDV]Life Sciences [q-bio], Nepovirus, Biology, Antibodies, Viral, 01 natural sciences, Virus, Epitope, Plant Viruses, 03 medical and health sciences, Epitopes, Capsid, Plant virus, structural biology, Animals, Vitis, Binding site, 030304 developmental biology, Plant Diseases, 0303 health sciences, Multidisciplinary, Cryoelectron Microscopy, Grapevine fanleaf virus, virus, Biological Sciences, biology.organism_classification, Virology, [SDV] Life Sciences [q-bio], Plant Leaves, nanobody, Structural biology, Capsid Proteins, 010606 plant biology & botany, Single-Chain Antibodies
Abstract: International audience; Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.
Published: 2020

31. ATP-driven separation of liquid phase condensates in bacteria

Author: Jean-Charles Walter, Baptiste Guilhas, A. Le Gall, Jean-Yves Bouet, Andrea Parmeggiani, Marcelo Nollmann, Céline Mathieu-Demazière, Nils-Ole Walliser, Jérôme Rech, John Palmeri, Gabriel David, Le Gall, Antoine, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Charles Coulomb (L2C), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: DNA, Bacterial, Quantitative Biology - Subcellular Processes, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Liquid phase, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], DNA Primase, Bacterial Physiological Phenomena, Phase Transition, 03 medical and health sciences, Adenosine Triphosphate, 0302 clinical medicine, Phase (matter), 0502 economics and business, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Nucleoid, 050207 economics, Molecular Biology, Subcellular Processes (q-bio.SC), 030304 developmental biology, Microscopy, 0303 health sciences, 050208 finance, biology, Chemistry, Escherichia coli Proteins, 05 social sciences, Cell Biology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Single Molecule Imaging, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Membrane, Single-particle tracking, FOS: Biological sciences, Biophysics, Nanoparticles, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030217 neurology & neurosurgery, Bacteria
Abstract: Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.
Published: 2020
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32. TADs or no TADS: Lessons From Single-cell Imaging of Chromosome Architecture

Author: Andrés M. Cardozo Gizzi, Marcelo Nollmann, Diego I. Cattoni, Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Universidad Nacional de Córdoba [Argentina], Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Macromolecular Substances, Population, Molecular Conformation, Computational biology, Biology, Genome, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Chromosome architecture, Hierarchical organization, education, Molecular Biology, 030304 developmental biology, Genomic organization, optical microscopy, Microscopy, genome architecture, 0303 health sciences, education.field_of_study, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Eukaryota, Chromatin, Single Molecule Imaging, Regulatory sequence, transcription, 030217 neurology & neurosurgery, Genome architecture
Abstract: International audience; Eukaryotic genomes are folded in a hierarchical organization that reflects and possibly regulates their function. Genomewide studies revealed a new level of organization at the kilobase-to-megabase scale termed “topological associating domains” (TADs). TADs are characterized as stable units of chromosome organization that restrict the action of regulatory sequences within one “functional unit.” Consequently, TADs are expected to appear as physical entities in most cells. Very recent single-cell studies have shown a notable variability in genome architecture at this scale, raising concerns about this model. Furthermore, the direct and simultaneous observation of genome architecture and transcriptional output showed the lack of stable interactions between regulatory sequences in transcribing cells. These findings are consistent with a large body of evidence suggesting that genome organization is highly heterogeneous at different scales. In this review, we discuss the main strategies employed to image chromatin organization, present the latest state-of-the-art developments, and propose an interpretation reconciling population-based findings with direct single-cell chromatin organization observations. All in all, we propose that TADs are made of multiple, low-frequency, low-affinity interactions that increase the probability, but are not deterministic, of regulatory interactions.
Published: 2020
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33. Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3

Author: Jean-Philippe Pin, Ann Van Eeckhaut, Pauline Scholler, Ilse Julia Smolders, Emmanuel Margeat, Linnea Olofsson, Alexandre Cabayé, Philippe Rondard, Fanny Malhaire, Amélie S. Tora, Xavier Rovira, Cyril Goudet, Anne-Marinette Cao, Francine Acher, Hayeon Baik, Institut de Génomique Fonctionnelle (IGF), 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), Universitat de Vic, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Vrije Universiteit [Brussels] (VUB), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Vrije Universiteit Brussel (VUB), Pharmaceutical and Pharmacological Sciences, Experimental Pharmacology, and Alliance for Modulation in Epilepsy
Subjects: 0301 basic medicine, Agonist, medicine.drug_class, Allosteric regulation, Glutamic Acid, Chloride, Radioligand Assay, 03 medical and health sciences, Cellular and Molecular Neuroscience, GPCR, Allosteric Regulation, Chlorides, Constitutive activity, Inosine Monophosphate, medicine, Humans, Receptors, AMPA, Allostery, Receptor, Cells, Cultured, G protein-coupled receptor, Pharmacology, Binding Sites, Chemistry, Glutamate receptor, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 030104 developmental biology, Metabotropic glutamate receptor, Luminescent Measurements, Mutation, Excitatory postsynaptic potential, Biophysics, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Metabotropic glutamate receptor 3, Glutamate
Abstract: International audience; Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl− ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive “chloride-lock” network. Indeed, Cl− ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors’ large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl−mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl− ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.
Published: 2018

34. Emerging fields in chaperone proteins: A French workshop

Author: Reynald Gillet, Elisabetta Mileo, Patricia Bordes, Chantal Iobbi-Nivol, Marie-Thérèse Giudici-Orticoni, Alessandro Barducci, Marie-Pierre Castanié-Cornet, Cyrille Garnier, Brigitte Gontero, Pierre Goloubinoff, Pierre Genevaux, Françoise Ochsenbein, Olivier Genest, Marianne Ilbert, Gilbert Richarme, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Mécanismes moléculaires dans les démences neurodégénératives (MMDN), Université de Montpellier (UM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES), Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Université de Lausanne (UNIL), 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), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), BIP laboratory, Agence Nationale de la Recherche [ANR-16-CE11-0002-01], Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Université de Rennes (UR), 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 ), Université de Lausanne = University of Lausanne (UNIL), ANR-16-CE11-0002,ChaperomEnvBact,Exploration du chaperome des bactéries environnementales : découverte de nouveaux chaperons(2016), Laboratoire de Bioénergétique et Ingénierie des Protéines, Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Montpellier 2 - Sciences et Techniques (UM2)-École pratique des hautes études (EPHE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, and Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, [SDV.GEN]Life Sciences [q-bio]/Genetics, Engineering, Plasticity, biology, business.industry, Folding/misfolding, education, Library science, General Medicine, Biochemistry, GroEL, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Aggregation, 03 medical and health sciences, 030104 developmental biology, Chaperone proteins, Chaperone (protein), Hsp33, Multifunctionality, biology.protein, Structural dynamics, Protein folding, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], business
Abstract: International audience; The "Bioenergetique et Ingenierie des Proteines (BIP)" laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and post-translational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field.
Published: 2018

35. Lipid tempering simulation of model biological membranes on parallel platforms

Author: Chiara Cardelli, Alessandro Barducci, Piero Procacci, Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, 030103 biophysics, Materials science, Lipid Bilayers, Biophysics, Degrees of freedom (statistics), Molecular Dynamics Simulation, Dihedral angle, 01 natural sciences, Biochemistry, Quantitative Biology::Subcellular Processes, 03 medical and health sciences, Molecular dynamics, 0103 physical sciences, Diffusion (business), Lipid bilayer, ComputingMilieux_MISCELLANEOUS, Alkyl, chemistry.chemical_classification, Physics::Biological Physics, 010304 chemical physics, Biological membrane, Cell Biology, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Condensed Matter::Soft Condensed Matter, Membrane, chemistry, Gauche/trans ratio, Hamiltonian Replica Exchange Simulation (HREM), POPC, Slipids, Chemical physics, Thermodynamics, lipids (amino acids, peptides, and proteins)
Abstract: In this report we have tested a parallel implementation for the simulation of lipid bilayers at the atomistic level, based on a generalized ensemble protocol where only the torsional degrees of freedom of the alkyl chains of the lipids are heated. The results in terms of configurational sampling enhancement have been compared with a conventional simulation produced with a widespread molecular dynamics code. Results show that the proposed thermodynamic-based multiple trajectories parallel protocol for membrane simulations allows for an efficient use of CPU resources with respect to the conventional single trajectory, providing accurate results for area and volume per lipid, membrane thickness, undulation spectra and boosting significantly diffusion and mixing in lipid bilayers due to the sampling enhancement of gauche/trans ratios of the alkyl chain dihedral angles.
Published: 2018

36. Multivalent Metallosupramolecular Assemblies as Effective DNA Binding Agents

Author: Sébastien Ulrich, Xiao-Yu Cao, Yannick Bessin, Wojciech Drożdż, Jean-Marie Lehn, Virginie Gervais, Artur R. Stefankiewicz, Anna Walczak, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut de Science et d'ingénierie supramoléculaires (ISIS), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Adam Mickiewicz University in Poznań (UAM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), 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), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)
Subjects: chemistry.chemical_element, Antineoplastic Agents, Zinc, Calorimetry, Crystallography, X-Ray, 010402 general chemistry, 01 natural sciences, Catalysis, Coordination complex, chemistry.chemical_compound, Cations, [CHIM]Chemical Sciences, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, Gel electrophoresis, 010405 organic chemistry, Organic Chemistry, Cationic polymerization, Isothermal titration calorimetry, DNA, General Chemistry, Nuclear magnetic resonance spectroscopy, Combinatorial chemistry, 0104 chemical sciences, 3. Good health, chemistry, Self-assembly
Abstract: We report the implementation of coordination chemistry onto the generation of new types of metallosupramolecular complexes with laterally appended cationic moieties for DNA binding in buffered aqueous media. Utilization of an N,N,O-type coordination pocket along with an octahedral zinc(II) metal ion allowed us to obtain mono- and tetranuclear complexes in both solution and solid state, as confirmed by NMR spectroscopy and single-crystal X-ray diffraction, respectively. By using isothermal titration calorimetry and gel electrophoresis, multiply charged cationic assemblies were observed to effectively bind to DNA through multivalent electrostatic interactions. Furthermore, we observed a correlation between the multivalency of the compounds employed and the effectiveness of DNA binding.
Published: 2018

37. Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin

Author: Lionel Chiron, Alice M. Lynch, Marc-André Delsuc, Federico Floris, Peter B. O’Connor, Mark P. Barrow, Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Proteomics, Calmodulin, 010402 general chemistry, Top-down proteomics, Mass spectrometry, 01 natural sciences, Fourier transform ion cyclotron resonance, Fragmentation (mass spectrometry), Sequence Analysis, Protein, Tandem Mass Spectrometry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Structural Biology, Animals, [CHIM]Chemical Sciences, QD, Infrared multiphoton dissociation, ComputingMilieux_MISCELLANEOUS, Spectroscopy, Chromatography, biology, Tandem, Chemistry, 010401 analytical chemistry, FT-ICR MS, 2DMS, QP, 0104 chemical sciences, biology.protein, Cattle, Research Article
Abstract: Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Graphical AbstractTwo-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin. Electronic supplementary material The online version of this article (10.1007/s13361-018-1978-y) contains supplementary material, which is available to authorized users.
Published: 2018

38. A Tumor-Imaging Method Targeting Cancer-Associated Fibroblasts

Author: Clemens Kratochwil, Jürgen Debus, Dirk Jäger, Annette Altmann, Christian Roumestand, Frederik L. Giesel, Uwe Haberkorn, Thomas Lindner, Anastasia Loktev, Philippe Barthe, Walter Mier, Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Biodistribution, Stromal cell, Chemistry, [SDV]Life Sciences [q-bio], Cancer, medicine.disease, 3. Good health, 030218 nuclear medicine & medical imaging, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Fibroblast activation protein, alpha, In vivo, 030220 oncology & carcinogenesis, medicine, Cancer research, Adenocarcinoma, Cancer-Associated Fibroblasts, DOTA, Radiology, Nuclear Medicine and imaging, ComputingMilieux_MISCELLANEOUS
Abstract: The tumor stroma, which accounts for a large part of the tumor mass, represents an attractive target for the delivery of diagnostic and therapeutic compounds. Here, the focus is notably on a subpopulation of stromal cells, known as cancer-associated fibroblasts, which are present in more than 90% of epithelial carcinomas, including pancreatic, colon, and breast cancer. Cancer-associated fibroblasts feature high expression of fibroblast activation protein (FAP), which is not detectable in adult normal tissue but is associated with a poor prognosis in cancer patients. Methods: We developed an iodinated and a DOTA-coupled radiotracer based on a FAP-specific enzyme inhibitor (FAPI) and evaluated them in vitro using uptake, competition, and efflux studies as well as confocal microscopy of a fluorescence-labeled variant. Furthermore, we performed imaging and biodistribution studies on tumor-bearing animals. Finally, proof of concept was realized by imaging patients with 68Ga-labeled FAPI. Results: Both FAPIs showed high specificity, affinity, and rapid internalization into FAP-expressing cells in vitro and in vivo. Biodistribution studies on tumor-bearing mice and on the first cancer patients demonstrated high intratumoral uptake of the tracer and fast body clearance, resulting in high-contrast images and negligible exposure of healthy tissue to radiation. A comparison with the commonly used radiotracer 18F-FDG in a patient with locally advanced lung adenocarcinoma revealed that the new FAP ligand was clearly superior. Conclusion: Radiolabeled FAPIs allow fast imaging with very high contrast in tumors having a high stromal content and may therefore serve as pantumor agents. Coupling of these molecules to DOTA or other chelators allows labeling not only with 68Ga but also with therapeutic isotopes such as 177Lu or 90Y.
Published: 2018

39. Quantifying the Precision of Single-Molecule Torque and Twist Measurements Using Allan Variance

Author: Mina Lee, Jan Lipfert, Tessa Jager, Maarten M. van Oene, Nynke H. Dekker, Seungkyu Ha, Francesco Pedaci, Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Physics, Millisecond, Magnetic moment, Magnetic Phenomena, Statistics as Topic, Biophysics, Torque wrench, Rotation, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 03 medical and health sciences, 030104 developmental biology, Torque, Control theory, Position (vector), Tweezers, Molecular Machines, Motors, and Nanoscale Biophysics, Astrophysics::Earth and Planetary Astrophysics, Allan variance, ComputingMilieux_MISCELLANEOUS
Abstract: Single-molecule manipulation techniques have provided unprecedented insights into the structure, function, interactions, and mechanical properties of biological macromolecules. Recently, the single-molecule toolbox has been expanded by techniques that enable measurements of rotation and torque, such as the optical torque wrench (OTW) and several different implementations of magnetic (torque) tweezers. Although systematic analyses of the position and force precision of single-molecule techniques have attracted considerable attention, their angle and torque precision have been treated in much less detail. Here, we propose Allan deviation as a tool to systematically quantitate angle and torque precision in single-molecule measurements. We apply the Allan variance method to experimental data from our implementations of (electro)magnetic torque tweezers and an OTW and find that both approaches can achieve a torque precision better than 1 pN · nm. The OTW, capable of measuring torque on (sub)millisecond timescales, provides the best torque precision for measurement times [Formula: see text] 10 s, after which drift becomes a limiting factor. For longer measurement times, magnetic torque tweezers with their superior stability provide the best torque precision. Use of the Allan deviation enables critical assessments of the torque precision as a function of measurement time across different measurement modalities and provides a tool to optimize measurement protocols for a given instrument and application.
Published: 2018

40. Pulling lipid tubes from supported bilayers unveils the underlying substrate contribution to the membrane mechanics

Author: Lidia Ferrer-Tasies, Imma Ratera, Marina I. Giannotti, Fausto Sanz, Nora Ventosa, Luca Costa, Berta Gumí-Audenis, Universitat de Barcelona, Costa, Luca, Institute for Bioengineering of Catalonia [Barcelona] (IBEC), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Centro de Investigación Biomédica en Red Salud Mental [Madrid] (CIBER-SAM), Institut de Ciència de Materials de Barcelona (ICMAB), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Materials science, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Lipid Bilayers, 02 engineering and technology, Microscopy, Atomic Force, 010402 general chemistry, Endocytosis, 01 natural sciences, Indentation, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, General Materials Science, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Lipid bilayer, Cytoskeleton, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Phospholipids, Mechanical Phenomena, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Nanociència, Vesicle, Cell Membrane, Lipid membranes, 021001 nanoscience & nanotechnology, Membranes lipídiques, [PHYS.COND.CM-SCM] Physics [physics]/Condensed Matter [cond-mat]/Soft Condensed Matter [cond-mat.soft], 0104 chemical sciences, Cell membranes, Nanoscience, Membrane, Models, Chemical, Optical tweezers, Biophysics, 0210 nano-technology, [PHYS.COND.CM-SCM]Physics [physics]/Condensed Matter [cond-mat]/Soft Condensed Matter [cond-mat.soft], Membranes cel·lulars, Nanomechanics
Abstract: International audience; Cell processes like endocytosis, membrane resealing, signaling and transcription involve conformational changes which depend on the chemical composition and the physicochemical properties of the lipid membrane. The better understanding of the mechanical role of lipids in cell membrane force-triggered and sensing mechanisms has recently become the focus of attention. Different membrane models and experimental methodologies are commonly explored. While general approaches involve controlled vesicle deformation using micropipettes or optical tweezers, due to the local and dynamic nature of the membrane, high spatial resolution atomic force microscopy (AFM) has been widely used to study the mechanical compression and indentation of supported lipid bilayers (SLBs). However, the substrate contribution remains unkown. Here, we demonstrate how pulling lipid tubes with an AFM out of model SLBs can be used to assess the nanomechanics of SLBs through the evaluation of the tube growing force (F tube), allowing for very local evaluation with high spatial and force resolution of the lipid membrane tension. We first validate this approach to determine the contribution of different phospholipids, by varying the membrane composition, in both one-component and phase-segregated membranes. Finally, we successfully assess the contribution of the underlying substrate to the membrane mechanics, demonstrating that SLB models may represent an intermediate scenario between a free membrane (blebs) and a cytoskeleton supported membrane. † Electronic supplementary information (ESI) available: Breakthrough force (F b) values for different membrane chemical compositions. F tube histograms for DOPC and DSPC SLBs at different tip retracting velocities. DOPC on PEG-graftedmica preliminary studies. See
Published: 2018

41. Exploring the conformational space of a receptor for drug design: An ERα case study

Author: Bourguet, William, Schneider, Melanie, Pons, Jean-Luc, Labesse, Gilles, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Behavioural Ecology and Evolutionary Genetics, Max Planck Institute for Ornithology, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, LABESSE, Gilles, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV.SP.MED] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Protein Conformation, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Conformation, Crystal structure, Computational biology, Molecular Dynamics Simulation, Ligands, 01 natural sciences, 03 medical and health sciences, Molecular dynamics, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, 0103 physical sciences, Materials Chemistry, Physical and Theoretical Chemistry, Spectroscopy, [INFO.INFO-BI] Computer Science [cs]/Bioinformatics [q-bio.QM], 030304 developmental biology, Physics, Flexibility (engineering), 0303 health sciences, Virtual screening, Binding Sites, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010304 chemical physics, Estrogen Receptor alpha, Active site, Computer Graphics and Computer-Aided Design, Pharmaceutical Preparations, Nuclear receptor, Docking (molecular), Drug Design, Helix, biology.protein, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], BindingDB, Estrogen receptor alpha, Protein Binding
Abstract: MotivationProtein flexibility is challenging for both experimentalists and modellers, especially in the field of drug design. Estrogen Receptor alpha (ERα) is an extensively studied Nuclear Receptor (NR) and a well-known therapeutic target with an important role in development and physiology. It is also a frequent off-target in standard toxicity tests for endocrine disruption. Here, we aim to evaluate the degree to which the conformational space and macromolecular flexibility of this well-characterized drug target can be described. Our approach exploits hundreds of crystallographic structures by means of molecular dynamics simulations and of virtual screening.ResultsThe analysis of hundreds of crystal structures confirms the presence of two main conformational states, known as ‘agonist’ and ‘antagonist’, that mainly differ by the orientation of the C-terminal helix H12 which serves to close the binding pocket. ERα also shows some loop flexibility, as well as variable side-chain orientations in its active site. We scrutinized the extent to which standard molecular dynamics simulations or crystallographic refinement as ensemble recapitulate most of the variability features seen by the array of available crystal structure. In parallel, we investigated on the kind and extent of flexibility that is required to achieve convincing docking for all high-affinity ERα ligands present in BindingDB. Using either only one conformation with a few side-chains set flexible, or static structure ensembles in parallel during docking led to good quality and similar pose predictions. These results suggest that the several hundreds of crystal structures already known can properly describe the whole conformational universe of ERα’s ligand binding domain. This opens the road for better drug design and affinity computation.Contactlabesse@cbs.cnrs.fr
Published: 2021

42. Molecular insights into the biased signaling mechanism of the μ-opioid receptor

Author: Damien Maurel, Xiaojing Cong, Joanna Hagelberger, Fanny Peysson, Sébastien Granier, Hélène Déméné, Jérôme Golebiowski, Remy Sounier, Julie Saint-Paul, Ieva Vasiliauskaité-Brooks, Institut de Génomique Fonctionnelle (IGF), 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), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Chimie de Nice (ICN), 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 de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Daegu Gyeongbuk Institute of Science and Technology (DGIST), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)-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)
Subjects: G protein, medicine.drug_class, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Allosteric regulation, Biology, 03 medical and health sciences, GPCR, 0302 clinical medicine, Opioid receptor, Functional selectivity, medicine, Receptor, Molecular Biology, 030304 developmental biology, G protein-coupled receptor, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Mechanism (biology), Cell Biology, NMR, molecular dynamics, 3. Good health, Cell biology, biased agonism, opioid, 030217 neurology & neurosurgery, Intracellular
Abstract: International audience; GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the β-arrestin pathways through the μ-opioid receptor (μOR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific μOR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists trigger μOR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair β-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.
Published: 2021

43. Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images

Author: Patrick Bron, Alessandro Valeri, Jean-Bernard Fiche, Gaëtan Bellot, Yonggang Ke, Antoine Le Gall, Desiree Salas, Marcelo Nollmann, KUHN-BERAUD, Sandrine, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique Fonctionnelle (IGF), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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, Diffraction, [SDV]Life Sciences [q-bio], 02 engineering and technology, law.invention, superresolution microscopy, 03 medical and health sciences, Optics, Optical microscope, law, Microscopy, structural biology, DNA origami, 3D reconstruction, Projection (set theory), multivariate statistical analysis, Physics, Multidisciplinary, business.industry, Resolution (electron density), 021001 nanoscience & nanotechnology, [SDV] Life Sciences [q-bio], 030104 developmental biology, Physical Sciences, Electron microscope, 0210 nano-technology, business
Abstract: International audience; Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions.
Published: 2017

44. Aromatic biobased polymer latex from cardanol

Author: Renaud Jeannin, Patrick Bron, Vincent Ladmiral, Joséphine Lai-Kee-Him, Sylvain Caillol, Patrick Lacroix-Desmazes, Kepa Fernandes Lizarazu, Institut Charles Gerhardt Montpellier - Institut de Chimie Moléculaire et des Matériaux de Montpellier (ICGM ICMMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut de Chimie du CNRS (INC), Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Materials science, Polymers and Plastics, Radical polymerization, General Physics and Astronomy, Emulsion polymerization, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Styrene, biobased polymer, chemistry.chemical_compound, Polymer chemistry, Materials Chemistry, cardanol, Methyl methacrylate, ComputingMilieux_MISCELLANEOUS, Cardanol, latex, Organic Chemistry, emulsion polymer, 021001 nanoscience & nanotechnology, 0104 chemical sciences, [CHIM.POLY]Chemical Sciences/Polymers, Monomer, chemistry, Polymerization, Radical initiator, 0210 nano-technology
Abstract: We present for the first time the aqueous emulsion radical polymerization of cardanol derived methacrylic monomer (CAMA) in order to pave the way for the replacement of styrene. This monomer was synthesized in two-step routes by epoxidation of cardanol prior to methacrylation. Polymerization of CAMA was studied both in toluene solution and in aqueous emulsion. Radical aqueous emulsion homo- and co-polymerization of CAMA with methyl methacrylate (MMA) were performed with sodium dodecyl sulfate as surfactant. Stable latexes were obtained with particle size between 25 and 75 nm. CAMA and MMA conversions were monitored versus time. Influence of radical initiator was studied on gel content, showing transfer to monomer in the case of persulfate as initiator. Glass transition temperature of homoPoly(CAMA) was determined and is suitable for coating application. Photo-crosslinking was performed by thiol-ene chemistry and allowed yielding cross-linked biobased aromatic coatings.
Published: 2017

45. COLIN trial: Value of colchicine in the treatment of patients with acute myocardial infarction and inflammatory response

Author: Anne-Marie Dupuy, Nicolas Nagot, Mariama Akodad, Mathilde Buisson, Frédéric Cransac, Florence Leclercq, Jessica Labour, Jean-Paul Cristol, Thien-Tri Cung, Benoit Lattuca, Jean-Christophe Macia, Vera Georgescu, Richard Gervasoni, Stéphane Cade, François Roubille, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire [Montpellier] ( CHRU Montpellier ), Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] ( PhyMedExp ), and Centre National de la Recherche Scientifique ( CNRS ) -Université de Montpellier ( UM ) -Institut National de la Santé et de la Recherche Médicale ( INSERM )
Subjects: Male, Time Factors, medicine.medical_treatment, Anti-Inflammatory Agents, 030204 cardiovascular system & hematology, Pathologies cardiovasculaires, 0302 clinical medicine, Prospective Studies, 030212 general & internal medicine, Myocardial infarction, biology, General Medicine, Middle Aged, Cardiovascular disease, Magnetic Resonance Imaging, 3. Good health, C-Reactive Protein, Treatment Outcome, medicine.anatomical_structure, Echocardiography, Cardiology, Female, Acute coronary syndrome, France, Inflammation Mediators, Cardiology and Cardiovascular Medicine, TIMI, medicine.medical_specialty, Infarctus du myocarde, Heart failure, 03 medical and health sciences, Percutaneous Coronary Intervention, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, Internal medicine, medicine, Humans, Aged, Inflammation, [ SDV ] Life Sciences [q-bio], business.industry, C-reactive protein, Percutaneous coronary intervention, medicine.disease, Insuffisance cardiaque, Coronary arteries, Coronary Occlusion, Coronary occlusion, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, biology.protein, ST Elevation Myocardial Infarction, Colchicine, business, Syndrome coronarien aigu, Biomarkers
Abstract: International audience; Background: Inflammation is involved during acute myocardial infarction, and could be an interesting target to prevent ischaemia-reperfusion injuries. Colchicine, known for its pleiotropic anti-inflammatory effects, could decrease systemic inflammation in this context.Aims: To evaluate the impact of colchicine on inflammation in patients admitted for ST-segment elevation myocardial infarction (STEMI).Methods: All patients admitted for STEMI with one of the main coronary arteries occluded, and successfully treated with percutaneous coronary intervention, were included consecutively. Patients were randomized to receive either 1 mg colchicine once daily for 1 month plus optimal medical treatment or optimal medical treatment only. C-reactive protein (CRP) was assessed at admission and daily until hospital discharge. The primary endpoint was CRP peak value during the index hospitalization.Results: Forty-four patients were included: 23 were treated with colchicine; 21 received conventional treatment only. At baseline, both groups were well balanced regarding age, sex, risk factors, thrombolysis in myocardial infarction flow and reperfusion delay. The culprit artery was more often the left anterior descending artery in the colchicine group (P = 0.07), reflecting a more severe group. There was no significant difference in mean CRP peak value between the colchicine and control groups (29.03 mg/L vs 21.86 mg/L, respectively; P = 0.36), even after adjustment for type of culprit artery (26.99 vs 24.99 mg/L, respectively; P = 0.79).Conclusion: In our study, the effect of colchicine on inflammation in the context of STEMI could not be demonstrated. Further larger studies may clarify the impact of colchicine in acute myocardial infarction.; Contexte: L’inflammation, impliquée au cours de l’infarctus du myocarde, pourrait représenter une cible thérapeutique intéressante et limiter les lésions d’ischémie-reperfusion. La colchicine, aux effets anti-inflammatoires pléiotropes, pourrait diminuer l’inflammation systémique au cours de l’infarctus du myocarde.Objectif: Évaluer l’impact de la colchicine sur l’inflammation systémique, dans l’infarctus du myocarde avec sus-décalage du segment ST.Méthodes: Tous les patients admis pour infarctus du myocarde avec occlusion de l’une des trois artères principales traités avec succès par angioplastie primaire étaient inclus de manière consécutive. Ils étaient randomisés pour recevoir 1 mg de colchicine par jour pendant 1 mois, en sus du traitement médical optimal, ou le traitement médical optimal seul. La C-réactive protéine était dosée à l’admission et quotidiennement jusqu’à la sortie. Le critère de jugement principal était le pic de CRP au cours de l’hospitalisation.Résultats: Quarante-quatre patients ont été inclus, 23 ont reçu la colchicine et 21 le traitement conventionnel seul. Les caractéristiques de base étaient comparables entre les 2 groupes concernant l’âge, le sexe, les facteurs de risque cardiovasculaires, le flux TIMI et le délai de reperfusion. L’artère interventriculaire antérieure était le plus souvent l’artère coupable dans le groupe colchicine (p = 0,07) reflétant un groupe plus sévère. Il n’y avait aucune différence significative entre les 2 groupes concernant la valeur du pic de CRP (29,03 mg/L dans le groupe colchicine vs 21,86 mg/L dans le groupe témoin ; p = 0,36), même après ajustement sur le type d’artère coupable (26,99 vs 24,99 mg/L ; p = 0,79).Conclusion: Aucun effet de la colchicine sur l’inflammation systémique dans l’infarctus du myocarde n’a pu être démontré. Des études complémentaires de plus grande envergure apparaissent nécessaires pour clarifier l’impact de la colchicine dans l’infarctus du myocarde.
Published: 2017

46. 1H, 13C and 15N backbone chemical shift assignments of camelid single-domain antibodies against active state µ-opioid receptor

Author: Hélène Déméné, Yinshan Yang, Sébastien Granier, Remy Sounier, Joanna Hagelberger, KUHN-BERAUD, Sandrine, Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Stereochemistry, [SDV]Life Sciences [q-bio], [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biochemistry, Article, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Single domain, Receptor, G protein-coupled receptor, biology, Chemistry, Chemical shift, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, G protein Coupled Receptor, Combinatorial chemistry, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, Membrane protein, Camelid antibody, Nanobody, biology.protein, µ opioid receptor, Antibody, 030217 neurology & neurosurgery, Single-Chain Antibodies
Abstract: International audience; Nanobodies are single chain antibodies that have become a highly valuable and versatile tool for biomolecular and therapeutic research. One application field is the stabilization of active states of flexible proteins, among which G-protein coupled receptors (GPCRs) represent a very important class of membrane proteins. Here we present the backbone and side-chain assignment of the 1H, 13C and 15N resonances of Nb33 and Nb39, two nanobodies that recognize and stabilize the μ-opioid receptor (μOR) to opioids in its active agonist-bound conformation. In addition, we present a comparison of their secondary structures as derived from NMR chemical shifts.
Published: 2017

47. Competitive folding of RNA structures at a termination–antitermination site

Author: Caroline Clerté, Emmanuel Margeat, Nathalie Declerck, Soraya Aït-Bara, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Montpellier (UM), Département Microbiologie et Chaîne Alimentaire (MICA), Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, French National Research Agency [ANR 2010 BLAN 1525 01, ANR-10-INBS-04], 'Chercheur d'Avenir' grant from the Region Languedoc Roussillon, Universite de Montpellier, France-BioImaging infrastructure, GIS 'IBiSA: Infrastructures en Biologie Sante et Agronomie', Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and ProdInra, Archive Ouverte
Subjects: 0301 basic medicine, RNA Folding, Transcription, Genetic, [SDV]Life Sciences [q-bio], single molecule, Bacillus subtilis, Article, 03 medical and health sciences, Bacterial Proteins, Transcription (biology), Fluorescence Resonance Energy Transfer, Molecular Biology, Gene, biology, Oligonucleotide, RNA-Binding Proteins, RNA, biology.organism_classification, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, Förster resonance energy transfer, Transcription Termination, Genetic, Antitermination, Nucleic Acid Conformation, antitermination, fluorescence, Terminator (franchise), 5' Untranslated Regions, transcription, Transcription Factors
Abstract: Antitermination is a regulatory process based on the competitive folding of terminator–antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in β-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination–antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript.
Published: 2017

48. 8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode

Author: Julie Paoletti, Gilles Labesse, Olivier Dussurget, Marie Anne Nahori, Liliane Assairi, Sylvie Pochet, Muriel Gelin, Valérie Huteau, Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), U759 Unité Imagerie intégrative, Institut Curie, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM), Interactions Bactéries-Cellules (UIBC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Université Sorbonne Paris Cité (USPC), Agence Nationale de la Recherche [ANR-06-BLAN-0324, ANR-11-EMM-0019], French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-01], ANR [ANR-11-EMM-0019], ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-11-EMMA-0019,ANAKIN (Anti-NAd-Kinase),Nouveaux composés antibactériens ciblant la NAD kinase(2011), ANR-06-BLAN-0324,Gram(+) NADK,Rational design and in situ selection of drug targetting NAD kinases from Gram(+) bacterial pathogens(2006), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Adenosine, MESH: Adenosine/chemistry, Protein Conformation, Ribose, [SDV]Life Sciences [q-bio], MESH: Staphylococcus aureus/enzymology, MESH: Amino Acid Sequence, MESH: Ribose/chemistry, Nicotinamide adenine dinucleotide, 01 natural sciences, MESH: Adenosine/pharmacology, chemistry.chemical_compound, MESH: Protein Conformation, MESH: Structure-Activity Relationship, Drug Discovery, Transferase, Enzyme Inhibitors, MESH: Listeria monocytogenes/enzymology, biology, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Kinase, General Medicine, MESH: Phosphotransferases (Alcohol Group Acceptor)/metabolism, Molecular Docking Simulation, 010101 applied mathematics, Phosphotransferases (Alcohol Group Acceptor), Biochemistry, Phosphorylation, Nicotinamide adenine dinucleotide phosphate, Protein Binding, medicine.drug, Staphylococcus aureus, Inhibitor, MESH: Phosphotransferases (Alcohol Group Acceptor)/chemistry, Stereochemistry, Cofactor, Structure-Activity Relationship, 03 medical and health sciences, MESH: Adenosine/metabolism, Adenosine analogue, MESH: Molecular Docking Simulation, medicine, Humans, MESH: Protein Binding, Amino Acid Sequence, MESH: Enzyme Inhibitors/chemistry, 0101 mathematics, NAD kinase, X-ray crystallography, Pharmacology, MESH: Humans, MESH: Enzyme Inhibitors/pharmacology, Organic Chemistry, MESH: Enzyme Inhibitors/metabolism, Listeria monocytogenes, Antibacterial, 030104 developmental biology, chemistry, biology.protein, NAD+ kinase, MESH: Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors
Abstract: Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.
Published: 2016

49. Load-dependent adaptation near zero load in the bacterial flagellar motor

Author: Jasmine A. Nirody, Ashley L. Nord, Richard M. Berry, University of Oxford [Oxford], Rockefeller University [New York], Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Oxford, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Nord, Ashley
Subjects: Quantitative Biology - Subcellular Processes, Stator, Biomedical Engineering, Biophysics, FOS: Physical sciences, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Bioengineering, Catch bond, 01 natural sciences, Biochemistry, Models, Biological, law.invention, Biomaterials, 03 medical and health sciences, Bacterial Proteins, Control theory, law, 0103 physical sciences, Limit (music), Molecular motor, Physics - Biological Physics, 010306 general physics, Subcellular Processes (q-bio.SC), ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Physics, 0303 health sciences, Rotating magnetic field, Bacteria, Rotor (electric), Molecular Motor Proteins, Rotational speed, Life Sciences–Physics interface, Mechanism (engineering), [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Biological Physics (physics.bio-ph), Flagella, FOS: Biological sciences, Biotechnology
Abstract: The bacterial flagellar motor is an ion-powered transmembrane protein complex which drives swimming in many bacterial species. The motor consists of a cytoplasmic 'rotor' ring and a number of 'stator' units, which are bound to the cell wall of the bacterium. Recently, it has been shown that the number of functional torque-generating stator units in the motor depends on the external load, and suggested that mechanosensing in the flagellar motor is driven via a 'catch bond' mechanism in the motor's stator units. We present a method that allows us to measure -- on a single motor -- stator unit dynamics across a large range of external loads, including near the zero-torque limit. By attaching superparamagnetic beads to the flagellar hook, we can control the motor's speed via a rotating magnetic field. We manipulate the motor to four different speed levels in two different ion-motive force (IMF) conditions. This framework allows for a deeper exploration into the mechanism behind load-dependent remodelling by separating out motor properties, such as rotation speed and energy availability in the form of IMF, that affect the motor torque., 13 pages, 5 figures
Published: 2019

50. Automatised pharmacophoric deconvolution of plant extracts - application to Cinchona bark crude extract

Author: Mélanie Bourjot, Marc-André Delsuc, Laure Margueritte, Laura Duciel, Catherine Vonthron-Sénécheau, Laboratoire d'Innovation Thérapeutique (LIT), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC), Signalisation normale et pathologique de l'embryon aux thérapies innovantes des cancers, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Cinchona Bark, Internet, Computer science, Plant Extracts, 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, 0104 chemical sciences, Matrix (chemical analysis), Set (abstract data type), Machine Learning, Automation, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Plant Bark, [CHIM]Chemical Sciences, Statistical analysis, Cinchona, Deconvolution, Physical and Theoretical Chemistry, 0210 nano-technology, Biological system, ComputingMilieux_MISCELLANEOUS
Abstract: We present a development of the "Plasmodesma" dereplication method [Margueritte et al., Magn. Reson. Chem., 2018, 56, 469]. This method is based on the automatic acquisition of a standard set of NMR experiments from a medium sized set of samples differing by their bioactivity. From this raw data, an analysis pipeline is run and the data is analysed by leveraging machine learning approaches in order to extract the spectral fingerprints of the active compounds. The optimal conditions for the analysis are determined and tested on two different systems, a synthetic sample where a single active molecule is to be isolated and characterized, and a complex bioactive matrix with synergetic interactions between the components. The method allows the identification of the active compounds and performs a pharmacophoric deconvolution. The program is freely available on the Internet, with an interactive visualisation of the statistical analysis, at https://plasmodesma.igbmc.science.
Published: 2019

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