17 results on '"Thibault Tubiana"'
Search Results
2. Dissecting peripheral protein-membrane interfaces.
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Thibault Tubiana, Ian Sillitoe, Christine Orengo, and Nathalie Reuter
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Biology (General) ,QH301-705.5 - Abstract
Peripheral membrane proteins (PMPs) include a wide variety of proteins that have in common to bind transiently to the chemically complex interfacial region of membranes through their interfacial binding site (IBS). In contrast to protein-protein or protein-DNA/RNA interfaces, peripheral protein-membrane interfaces are poorly characterized. We collected a dataset of PMP domains representative of the variety of PMP functions: membrane-targeting domains (Annexin, C1, C2, discoidin C2, PH, PX), enzymes (PLA, PLC/D) and lipid-transfer proteins (START). The dataset contains 1328 experimental structures and 1194 AphaFold models. We mapped the amino acid composition and structural patterns of the IBS of each protein in this dataset, and evaluated which were more likely to be found at the IBS compared to the rest of the domains' accessible surface. In agreement with earlier work we find that about two thirds of the PMPs in the dataset have protruding hydrophobes (Leu, Ile, Phe, Tyr, Trp and Met) at their IBS. The three aromatic amino acids Trp, Tyr and Phe are a hallmark of PMPs IBS regardless of whether they protrude on loops or not. This is also the case for lysines but not arginines suggesting that, unlike for Arg-rich membrane-active peptides, the less membrane-disruptive lysine is preferred in PMPs. Another striking observation was the over-representation of glycines at the IBS of PMPs compared to the rest of their surface, possibly procuring IBS loops a much-needed flexibility to insert in-between membrane lipids. The analysis of the 9 superfamilies revealed amino acid distribution patterns in agreement with their known functions and membrane-binding mechanisms. Besides revealing novel amino acids patterns at protein-membrane interfaces, our work contributes a new PMP dataset and an analysis pipeline that can be further built upon for future studies of PMPs properties, or for developing PMPs prediction tools using for example, machine learning approaches.
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- 2022
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3. Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity
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Mohyeddine Omrane, Amanda Souza Camara, Cyntia Taveneau, Nassima Benzoubir, Thibault Tubiana, Jinchao Yu, Raphaël Guérois, Didier Samuel, Bruno Goud, Christian Poüs, Stéphane Bressanelli, Richard Charles Garratt, Abdou Rachid Thiam, and Ama Gassama-Diagne
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Science - Abstract
Summary: Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology. : Membrane Architecture; Molecular Interaction; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Membrane Architecture, Molecular Interaction, Cell Biology, Functional Aspects of Cell Biology
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- 2019
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4. Membrane models for molecular simulations of peripheral membrane proteins
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Mahmoud Moqadam, Thibault Tubiana, Emmanuel E. Moutoussamy, and Nathalie Reuter
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Physics ,QC1-999 - Abstract
Peripheral membrane proteins (PMPs) bind temporarily to the surface of biological membranes. They also exist in a soluble form and their tertiary structure is often known. Yet, their membrane-bound form and their interfacial-binding site with membrane lipids remain difficult to observe directly. Their binding and unbinding mechanism, the conformational changes of the PMPs and their influence on the membrane structure are notoriously challenging to study experimentally. Molecular dynamics simulations are particularly useful to fill some knowledge-gaps and provide hypothesis that can be experimentally challenged to further our understanding of PMP-membrane recognition. Because of the time-scales of PMP-membrane binding events and the computational costs associated with molecular dynamics simulations, membrane models at different levels of resolution are used and often combined in multiscale simulation strategies. We here review membrane models belonging to three classes: atomistic, coarse-grained and implicit. Differences between models are rooted in the underlying theories and the reference data they are parameterized against. The choice of membrane model should therefore not only be guided by its computational efficiency. The range of applications of each model is discussed and illustrated using examples from the literature.
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- 2021
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5. Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity
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Mohyeddine Omrane, Amanda Souza Camara, Cyntia Taveneau, Nassima Benzoubir, Thibault Tubiana, Jinchao Yu, Raphaël Guérois, Didier Samuel, Bruno Goud, Christian Poüs, Stéphane Bressanelli, Richard Charles Garratt, Abdou Rachid Thiam, and Ama Gassama-Diagne
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Science - Published
- 2020
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6. Membrane alterations induced by nonstructural proteins of human norovirus.
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Sylvie Y Doerflinger, Mirko Cortese, Inés Romero-Brey, Zach Menne, Thibault Tubiana, Christian Schenk, Peter A White, Ralf Bartenschlager, Stéphane Bressanelli, Grant S Hansman, and Volker Lohmann
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies.
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- 2017
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7. Dynamics and asymmetry in the dimer of the norovirus major capsid protein.
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Thibault Tubiana, Yves Boulard, and Stéphane Bressanelli
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Medicine ,Science - Abstract
Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single ca 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.
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- 2017
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8. A Perspective on the Prospective Use of AI in Protein Structure Prediction.
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Raphaelle Versini, Sujith Sritharan, Burcu Aykaç Fas, Thibault Tubiana, Sana Zineb Aimeur, Julien Henri, Marie Erard, Oliver Nüsse, Jessica Andreani, Marc Baaden, Patrick Fuchs, Tatiana Galochkina, Alexios Chatzigoulas, Zoe Cournia, Hubert Santuz, Sophie Sacquin-Mora, and Antoine Taly
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- 2024
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9. TTClust: A Versatile Molecular Simulation Trajectory Clustering Program with Graphical Summaries.
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Thibault Tubiana, Jean-Charles Carvaillo, Yves Boulard, and Stéphane Bressanelli
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- 2018
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10. Ensemble-based molecular docking and spectrofluorometric analysis of interaction between cytotoxin and tumor necrosis factor receptor 1
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Nurhamimah Misuan, Saharuddin Mohamad, Thibault Tubiana, and Michelle Khai Khun Yap
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Structural Biology ,General Medicine ,Molecular Biology - Abstract
Cytotoxin (CTX) is a three-finger toxin presents predominantly in cobra venom. The functional site of the toxin is located at its three hydrophobic loop tips. Its actual mechanism of cytotoxicity remains inconclusive as few conflicting hypotheses have been proposed in addition to direct cytolytic effects. The present work investigated the interaction between CTX and death receptor families via ensemble-based molecular docking and fluorescence titration analysis. Multiple sequence alignments of different CTX isoforms obtained a conserved CTX sequence. The three-dimensional structure of the conserved CTX was later determined using homology modelling, and its quality was validated. Ensemble-based molecular docking of CTX was performed with different death receptors, such as Fas-ligand and tumor necrosis factor receptor families. Our results showed that tumor necrosis factor receptor 1 (TNFR1) was the best receptor interacting with CTX attributed to the interaction of all three functional loops and evinced with low HADDOCK, Z-score and RMSD value. The interaction between CTX and TNFR1 was also supported by a concentration-dependent reduction of fluorescence intensity with increasing binding affinity. The possible intermolecular interactions between CTX and TNFR1 were Van der Waals forces and hydrogen bonding. Our findings suggest a possibility that CTX triggers apoptosis cell death through non-covalent interactions with TNFR1. Communicated by Ramaswamy H. Sarma
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- 2023
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11. De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation
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Sonia Fieulaine, Thibault Tubiana, Stéphane Bressanelli, 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), ANRS ECTZ105819, ANRS ECTZ188022, and ANRS ECTZ189696
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Replication polyprotein pORF1 ,Single-stranded positive-sense RNA virus ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Virology ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Hepatitis E virus ,AlphaFold2 ,In vitro translation - Abstract
International audience; Hepatitis E virus (HEV), a major cause of acute viral hepatitis, is a single-stranded, positive-sense RNA virus. As such, it encodes a 1700-residue replication polyprotein pORF1 that directs synthesis of new viral RNA in infected cells. Here we report extensive modeling with AlphaFold2 of the full-length pORF1, and its production by in vitro translation. From this, we give a detailed update on the breakdown into domains of HEV pORF1. We also provide evidence that pORF1’s N-terminal domain is likely to oligomerize to form a dodecameric pore, homologously to what has been described for Chikungunya virus. Beyond providing accurate folds for its five domains, our work highlights that there is no canonical protease encoded in pORF1 and that flexibility in several functionally important regions rather than proteolytic processing may serve to regulate HEV RNA synthesis.
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- 2023
12. TTClust: A Versatile Molecular Simulation Trajectory Clustering Program with Graphical Summaries
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Jean-Charles Carvaillo, Yves Boulard, Stéphane Bressanelli, Thibault Tubiana, 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), Interactions et mécanismes d’assemblage des protéines et des peptides (IMAPP), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)
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0301 basic medicine ,Protein Conformation ,Computer science ,[SDV]Life Sciences [q-bio] ,General Chemical Engineering ,Molecular Conformation ,Molecular simulation ,Hepacivirus ,Molecular Dynamics Simulation ,Viral Nonstructural Proteins ,Library and Information Sciences ,computer.software_genre ,01 natural sciences ,03 medical and health sciences ,0103 physical sciences ,Cluster Analysis ,Cluster analysis ,computer.programming_language ,010304 chemical physics ,dynamics ,General Chemistry ,Extremely Helpful ,Python (programming language) ,Computer Science Applications ,030104 developmental biology ,Trajectory clustering ,User control ,Data mining ,computer ,Algorithms ,Software - Abstract
WOS:000451650400002; International audience; It is extremely helpful to be able to partition the thousands of frames produced in molecular dynamics simulations into a limited number of most dissimilar conformations. While robust clustering algorithms are already available to do so, there is a distinct need for an easy-to-use clustering program with complete user control, taking as input a trajectory from any molecular dynamics (MD) package and outputting an intuitive display of results with plots allowing at-a-glance analysis. We present TTClust (for Trusty Trajectory Clustering), a python program that uses the MDTraj package to fill this need.
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- 2018
13. Septin 9 has two polybasic domains critical to septin filament assembly and golgi integrity
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Stéphane Bressanelli, Richard Charles Garratt, Raphael Guerois, Christian Poüs, Ama Gassama-Diagne, Jinchao Yu, Bruno Goud, Abdou Rachid Thiam, Amanda Souza Camara, Didier Samuel, Mohyeddine Omrane, Nassima Benzoubir, Cyntia Taveneau, Thibault Tubiana, Physiopathologie et traitement des maladies du foie, Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Laboratoire de Physique Théorique de l'ENS [École Normale Supérieure] (LPTENS), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Universidade de São Paulo = University of São Paulo (USP), 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), Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique Théorique de l'ENS (LPTENS), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Universidade de São Paulo (USP), and CCSD, Accord Elsevier
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0301 basic medicine ,[SDV]Life Sciences [q-bio] ,Membrane Architecture ,02 engineering and technology ,macromolecular substances ,Septin ,01 natural sciences ,Article ,Protein filament ,03 medical and health sciences ,symbols.namesake ,Organelle ,0103 physical sciences ,lcsh:Science ,POLIMERIZAÇÃO ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Functional Aspects of Cell Biology ,010304 chemical physics ,Molecular Interaction ,Chemistry ,fungi ,virus diseases ,Cell Biology ,Golgi apparatus ,biochemical phenomena, metabolism, and nutrition ,021001 nanoscience & nanotechnology ,Protein enrichment ,Cell biology ,[SDV] Life Sciences [q-bio] ,030104 developmental biology ,Membrane ,Membrane remodeling ,symbols ,lcsh:Q ,biological phenomena, cell phenomena, and immunity ,0210 nano-technology - Abstract
Summary Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology., Graphical Abstract, Highlights • Two polybasic domains mediate septin 9 interactions with PIs • Human septins have amphipathic helices suitable for membrane binding • Septin 9 polybasic domains mediate the formation of septin higher-order structures • The mutation or depletion of septin polybasic domains induces Golgi fragmentation, Membrane Architecture; Molecular Interaction; Cell Biology; Functional Aspects of Cell Biology
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- 2019
14. Mapping Amino Acids at Protein-Membrane Interfaces to Update the Current Membrane Binding Model
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Thibault Tubiana and Nathalie Reuter
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chemistry.chemical_classification ,Membrane ,chemistry ,Biophysics ,Membrane binding ,Current (fluid) ,Amino acid - Published
- 2021
15. Identification of a Second Conserved Polybasic Domain in Septin 9 Involved in MicrotubuleeDependent Golgi Assembly
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Thibault Tubiana, Juan Peng, Stéphane Bressanelli, Cyntia Taveneau, Ama Gassama-Diagne, Bruno Goud, Christian Poüs, Mohyeddine Omrane, Didier Samuel, Jinchao Yua, and Raphaël Guérois
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Chemistry ,viruses ,Regulator ,virus diseases ,macromolecular substances ,biochemical phenomena, metabolism, and nutrition ,Golgi apparatus ,Septin ,Homology (biology) ,Cell biology ,symbols.namesake ,medicine.anatomical_structure ,Secretory protein ,Microtubule ,Organelle ,symbols ,medicine ,biological phenomena, cell phenomena, and immunity ,Nucleus - Abstract
Septins belong to a family of GTP‐binding proteins that bind to phosphoinositides (PIs) by a polybasic domain (PB1). We reported that the deletion of PB1 in septin 9 markedly reduced, its binding to PIs and its assembly. Here, using homology modelling, we reveal the presence of another polybasic domain (PB2) conserved among the different human septins. PB1 and PB2 from two distinct molecules form an extended basic cluster in the NC interface of the filament. Importantly binding of septin 9 to PIs through PB1 and PB2 is required for recruitment of microtubules to Golgi stacks and their assembly. Knockdown of septin 9 recapitulates these effects and affects nucleus/centrosomal positioning and protein secretion. Thus septin 9 could be considered as a crucial regulator of events require for membrane trafficking and organelle morphology.
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- 2018
16. Membrane alterations induced by nonstructural proteins of human norovirus
- Author
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Peter A. White, Christian Schenk, Ralf Bartenschlager, Inés Romero-Brey, Zach Menne, Sylvie Y. Doerflinger, Mirko Cortese, Thibault Tubiana, Grant S. Hansman, Stéphane Bressanelli, Volker Lohmann, Doerflinger, S. Y., Cortese, M., Romero-Brey, I., Menne, Z., Tubiana, T., Schenk, C., White, P. A., Bartenschlager, R., Bressanelli, S., Hansman, G. S., Lohmann, V., Department of Infectious Diseases, Molecular Virology, Heidelberg University, Universität Heidelberg [Heidelberg] = Heidelberg University, 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), Department of Infectious Diseases [Heidelberg, Germany], Heidelberg University Hospital [Heidelberg], Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Interactions et mécanismes d’assemblage des protéines et des peptides (IMAPP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Universität Heidelberg [Heidelberg]
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RNA viruses ,0301 basic medicine ,[SDV]Life Sciences [q-bio] ,viruses ,ved/biology.organism_classification_rank.species ,Viral Nonstructural Proteins ,Pathology and Laboratory Medicine ,Endoplasmic Reticulum ,Virus Replication ,Biochemistry ,Lipid droplet ,Medicine and Health Sciences ,Macromolecular Structure Analysis ,Membrane Technology ,lcsh:QH301-705.5 ,Vesicle ,virus diseases ,Built Structures ,3. Good health ,Cell biology ,Membrane ,Medical Microbiology ,Viral Pathogens ,Viruses ,Engineering and Technology ,RNA, Viral ,Cellular Structures and Organelles ,Pathogens ,B3S ,Research Article ,lcsh:Immunologic diseases. Allergy ,Protein Structure ,Structural Engineering ,Immunology ,Viral Structure ,Biology ,Transfection ,Research and Analysis Methods ,Microbiology ,Caliciviruses ,Membrane Structures ,Cell Line ,03 medical and health sciences ,Virology ,Organelle ,Genetics ,Animals ,Humans ,Vesicles ,Molecular Biology Techniques ,Microbial Pathogens ,Molecular Biology ,NS3 ,IMAPP ,ved/biology ,Endoplasmic reticulum ,Norovirus ,Organisms ,Biology and Life Sciences ,Proteins ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,Viral Replication ,030104 developmental biology ,Viral replication ,lcsh:Biology (General) ,Parasitology ,lcsh:RC581-607 ,Murine norovirus - Abstract
Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies., Author summary Positive-strand RNA viruses induce membrane alterations harboring the viral replication complexes. In the case of human noroviruses (huNoV), the major cause of acute viral gastroenteritis, these are induced by the ORF1 polyprotein, which is post-translationally processed into the functional nonstructural (NS) proteins. Partly due to the lack of efficient cell culture models, little is known so far about membrane alterations induced by huNoV belonging to the most clinically relevant genogroup II, genotype 4 (GII.4), nor about the function of individual NS proteins in their formation. We therefore expressed ORF1 proteins of GII.4 and individual NS proteins in cells to study their contribution to viral replication complex formation. Expression of ORF1 proteins of GII.4 induced vesicular membrane alterations comparable to those found in infected cells and similar to picornaviruses and hepatitis C virus (HCV). GII.4 NS1-2, NS3 and NS4 are contributing to viral membrane alterations. Our work provides new insights into their function in huNoV induced replication complex formation while identifying NS4 as the most important single determinant. This knowledge might provide novel attractive targets for future therapies inhibiting the formation of the membranous viral replication complex, as exemplified by the efficacy of HCV NS5A inhibitors.
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- 2017
17. Dynamics and asymmetry in the dimer of the norovirus major capsid protein
- Author
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Yves Boulard, Stéphane Bressanelli, Thibault Tubiana, 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), Interactions et mécanismes d’assemblage des protéines et des peptides (IMAPP), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Secondary ,Molecular model ,Hepacivirus ,Pathology and Laboratory Medicine ,Biochemistry ,01 natural sciences ,Biochemical Simulations ,Electron Microscopy ,Simulated Annealing ,lcsh:Science ,education.field_of_study ,Crystallography ,Physics ,virus diseases ,fluorescence spectroscopy ,Physical sciences ,Solutions ,Medical Microbiology ,Viral Pathogens ,Protons ,Protein Structure ,Chemical physics ,Molecular modeling ,Microbiology ,03 medical and health sciences ,Protein Domains ,Genetic ,Amino Acid Sequence ,Homology modeling ,education ,Microbial Pathogens ,Cryoelectron Microscopy ,lcsh:R ,Organisms ,Biology and Life Sciences ,Computational Biology ,Dimers (Chemical physics) ,MD simulation ,biochemical phenomena, metabolism, and nutrition ,Surface Plasmon Resonance ,Virology ,0104 chemical sciences ,030104 developmental biology ,X-Ray ,Capsid Proteins ,lcsh:Q ,Protein Multimerization ,Mathematics ,RNA viruses ,0301 basic medicine ,Protein Conformation ,Dimer ,viruses ,[SDV]Life Sciences [q-bio] ,lcsh:Medicine ,Crystallography, X-Ray ,Virus Replication ,Viral Packaging ,chemistry.chemical_compound ,Protein structure ,Medicine and Health Sciences ,Cluster Analysis ,Viral ,Conserved Sequence ,Microscopy ,Multidisciplinary ,LDAO surfactant ,Chemistry ,Simulation and Modeling ,Applied Mathematics ,Condensed Matter Physics ,RNA Replicase ,Capsid ,Viruses ,Crystal Structure ,Pathogens ,Transcription ,Algorithms ,Research Article ,Icosahedral symmetry ,Protein domain ,Population ,Molecular Dynamics Simulation ,Research and Analysis Methods ,010402 general chemistry ,Caliciviruses ,Scattering, Small Angle ,micelle ,Solid State Physics ,lipid kinase PI4KA ,Norovirus ,Electron Cryo-Microscopy ,Viral Replication ,Structural Homology, Protein ,RNA - Abstract
Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single ca 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.
- Published
- 2017
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