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46 results on '"B3S"'

1. Point-of-care testing (POCT) and IT security concepts

Author

Johannis Wibke, Bietenbeck Andreas, Malchau Gebhart, and Streichert Thomas

Subjects
b3s, bsig, din en iso 15189, it, kritis, poct, poct symposium munich, 1st round table poct-it-security meeting, protection of (information) infrastructures, security, Medical technology, R855-855.5
Abstract

Point-of-care testing (POCT) has been an essential service in hospitals for many years with a main focus on reliability, classical laboratory quality criteria and easy handling. Hospital information technology (IT) security regulations, however, have not yet been adapted to the specificities of POCT. Following the POCT Symposium in Munich, the “1st Round Table POCT-IT-Security Meeting” held in October 2019 in Cologne addressed these issues and managed to establish first consensus results in the essential fields of user, data and update management, as well as network connections and user-friendliness. First practical steps include optimizing the user management by connection to a directory service and definition of access control (including emergency authorization). Patient data economy on analyzers in combination with data and data transmission encryption as well as technically secure communication protocols are relevant steps in the fields of data management and network connections. An update management needs to be contractually defined for remote services and generally includes testing in a protocol-based scenario. Providing an organizational structure for POCT-IT security is a necessary prerequisite, as are continuous training and awareness for this topic with a strong focus on usability.

Published
2020
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2. Photobiological systems studied by time-resolved infrared spectroscopy (2015-2018)

Author

Alberto Mezzetti, 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), Photocatalyse et Biohydrogène (LPB), 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), 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), Albini, A. and Protti, and S.

Subjects
Materials science, quantum cascade lasers, business.industry, Infrared, [SDV]Life Sciences [q-bio], Infrared spectroscopy, photosynthetic reaction centers, intracellular acidification enhancement, orange carotenoid protein, AUTH U, LPB, Photobiology, step-scan ftir, conformational-changes, photosystem-i, Optoelectronics, Sample preparation, Spectroscopy, business, difference spectroscopy, Time range, B3S, site-specific incorporation, Spectral simulation, proton-transfer reactions
Abstract

ISSN: 0556-3860 WOS:000550058500006; International audience; Time-resolved infrared (IR) spectroscopy is a well established technique for the study of photobiological systems. In this review attention is focused on technical improvements (instrumental, data analysis, spectral simulation, methodology, sample preparation, isotope-labelling strategies, vibrational probes) and on the scientific results obtained since 2015 for time-resolved IR spectroscopy in the (sub-) ns - second time range. A peculiar emphasis will be given to studies in the field of photosynthesis. Finally, after the general conclusions, the perspectives for the application of time-resolved IR spectroscopy in photobiology are described.

Published
2020

3. The Long and Winding Road Toward Efficient High-Performance Computing

Author

William Jalby, Michel Masella, David Kuck, Allen D. Malony, Mihail Popov, Abdelhafid Mazouz, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Laboratoire d'Informatique Parallélisme Réseaux Algorithmes Distribués (LI-PaRAD), Computer Science Department [Oregon], University of Oregon [Eugene], Laboratoire de Biologie Structurale et Radiobiologie (LBSR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), 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), 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), Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), 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 Bull atos technologies

Subjects
Computer science, [SDV]Life Sciences [q-bio], design, molecular-dynamics, performance evaluation tools, 02 engineering and technology, INTGEN, Software, Autotuning, 0202 electrical engineering, electronic engineering, information engineering, Code (cryptography), benchmarking, Electrical and Electronic Engineering, implementation, business.industry, Benchmarking, Supercomputer, molecular dynamics, 020202 computer hardware & architecture, hardware design, Computer architecture, code, Key (cryptography), Software design, 020201 artificial intelligence & image processing, Analysis tools, business, B3S
Abstract

WOS:000448616200009; The major challenge to Exaflop computing, and more generally, efficient high-end computing, is in finding the best "matches" between advanced hardware capabilities and the software used to program applications, so that top performance will be achieved. Several benchmarks show very disappointing performance progress over the last decade, clearly indicating a mismatch between hardware and software. To remedy this problem, it is important that key performance enablers at the software level-autotuning, performance analysis tools, full application optimization-are understood. For each area, we highlight major limitations and most promising approaches to reaching better performance and energy levels. Finally, we conclude by analyzing hardware and software design, trying to pave the way for more tightly integrated hardware and software codesign.

Published
2018

4. Electronic Structure and Triplet-Triplet Energy Transfer in Artificial Photosynthetic Antennas

Author

Julio L. Palma, Marely E. Tejeda-Ferrari, Vladimiro Mujica, Gabriela C. C. C. Coutinho, Chelsea L. Brown, Devens Gust, Ana L. Moore, Thomas A. Moore, Manuel J. Llansola-Portoles, Gerdenis Kodis, Ghabriel A. Gomes de Sá, Junming Ho, 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 Bioénergétique Membranaire et Stress (LBMS), 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), 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), and Arizona State University [Tempe] (ASU)

Subjects
Double bond, [SDV]Life Sciences [q-bio], carotenoid triplet, mechanism, Conjugated system, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, LBMS, chemistry.chemical_compound, 0103 physical sciences, Physical and Theoretical Chemistry, chemistry.chemical_classification, 010304 chemical physics, General Medicine, Chromophore, Tetrapyrrole, states, 0104 chemical sciences, photoprotection, Intersystem crossing, chemistry, Covalent bond, Phthalocyanine, Density functional theory, B3S
Abstract

International audience; Three Pd(II) phthalocyanine-carotenoid dyads featuring chromophores linked by amide bonds were prepared in order to investigate the rate of triplet-triplet (T-T) energy transfer from the tetrapyrrole to the covalently attached carotenoid as a function of the number of conjugated double bonds in the carotenoid. Carotenoids having 9, 10 and 11 conjugated double bonds were studied. Transient absorption measurements show that intersystem crossing in the Pd(II) phthalocyanine takes place in 10 ps in each case and that T-T energy transfer occurs in 126, 81 and 132 ps in the dyads bearing 9, 10 and 11 double bond carotenoids, respectively. To identify the origin of this variation in T-T energy transfer rates, density functional theory (DFT) was used to calculate the T-T electronic coupling in the three dyads. According to the calculations, the primary reason for the observed T-T energy transfer trend is larger T-T electronic coupling between the tetrapyrrole and the 10-double bond carotenoid. A methyl group adjacent to the amide linker that connects the Pd(II) phthalocyanine and the carotenoid in the 9 and 11-double bond carotenoids is absent in the 10-double bond carotenoid, and this difference alters its electronic structure to increase the coupling.

Published
2018

5. Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough

Author

Dominique Durand, Christian Malosse, Dorothée Raoux-Barbot, Véronique Hourdel, Mahmoud Ghomi, Bertrand Raynal, Darragh P. O'Brien, Daniel Ladant, Ana Cristina Sotomayor Pérez, Bruno Baron, Alexis Voegele, Patrick England, Véronique Yvette Ntsogo Enguéné, Orso Subrini, Alexandre Chenal, Sara E. Cannella, Patrice Vachette, Marilyne Davi, Johanna C. Karst, Belén Hernández, Sébastien Brier, Audrey Hessel, J. Iñaki Guijarro, Julia Chamot-Rooke, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Résonance Magnétique Nucléaire des Biomolécules, Biophysique Moléculaire (Plate-forme), Université Paris 13 (UP13), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), 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), 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), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), 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), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Protein Folding, Bordetella pertussis, FAAM, CyaA toxin, [SDV]Life Sciences [q-bio], chemistry.chemical_element, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Calcium, Toxicology, Models, Biological, 03 medical and health sciences, Protein Domains, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Secretion, Whooping cough, biology, Disorder-to-order transition, Chemistry, Folding, cyaA, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Protein Transport, Cytosol, Chaotropic agent, Eukaryotic Cells, 030104 developmental biology, Secretory protein, Adenylate Cyclase Toxin, Protein Translocation Systems, Biophysics, Protein secretion, B3S
Abstract

International audience; The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.

Published
2018

6. Glycolate Induces Redox Tuning Of Photosystem II in Vivo: Study of a Photorespiration Mutant

Author

Andrea Fantuzzi, Hermann Bauwe, A. William Rutherford, Wolfram Weckwerth, Anja Krieger-Liszkay, Marine Messant, Stefan Timm, Department of Life Sciences, Imperial College London, Molecular Systems Biology, universite de Vienne, Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), 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
0106 biological sciences, 0301 basic medicine, Photosystem II, Physiology, [SDV]Life Sciences [q-bio], Bicarbonate, Arabidopsis, Plant Science, Photosynthesis, Photosystem I, Thylakoids, 01 natural sciences, Fluorescence, Electron Transport, 03 medical and health sciences, chemistry.chemical_compound, Spinacia oleracea, Genetics, Chlorophyll fluorescence, Singlet Oxygen, Arabidopsis Proteins, Chemistry, Photosystem II Protein Complex, food and beverages, Articles, Electron transport chain, Glycolates, Plant Leaves, Bicarbonates, 030104 developmental biology, Thylakoid, Luminescent Measurements, Mutation, Biophysics, Photorespiration, MROP, B3S, 010606 plant biology & botany
Abstract

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor Q(A) to a more positive potential and lowers the yield of singlet oxygen ((1)O(2)) production. The presence of Q(A)(−) results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against (1)O(2) in vivo under photorespiratory conditions. Here, we compared the properties of Q(A) in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between Q(A) and Q(B) in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of Q(A) to a more positive value. The yield of (1)O(2) production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of Q(A)/Q(A)(−). In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO(2) conditions.

Published
2018

7. Apoprotein heterogeneity increases spectral disorder and a step-wise modification of the B850 fluorescence peak position

Author

Rienk van Grondelle, Oana Ilioaia, Bruno Robert, Andrew Gall, Tjaart P. J. Krüger, Cristian Ilioaia, Biophysics Photosynthesis/Energy, LaserLaB - Energy, Spectroscopies Électroniques (SE), Département Plateforme (PF I2BC), 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)-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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire Bioénergétique Membranaire et Stress (LBMS), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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), Department of Physics and Astronomy [Amsterdam], Vrije Universiteit Amsterdam [Amsterdam] (VU), Institut de Biologie et de Technologies de Saclay (IBITECS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay

Subjects
0301 basic medicine, Limiting factor, [SDV]Life Sciences [q-bio], Light-Harvesting Protein Complexes, Biophysics, Biochemistry, Purple bacteria, Fluorescence, LBMS, Light-harvesting complex, 03 medical and health sciences, Bacterial Proteins, SDG 7 - Affordable and Clean Energy, Photosynthesis, Absorption (electromagnetic radiation), Light harvesting complexes, biology, Spectrometry, Chemistry, Cell Biology, Single-molecule experiment, biology.organism_classification, Rhodopseudomonas, Spectrometry, Fluorescence, 030104 developmental biology, Chemical physics, Protein quaternary structure, Single molecule spectroscopy, Rhodopseudomonas palustris, Apoproteins, B3S
Abstract

It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric ‘ring’ of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 ‘rings’ are present in “low-light” adapted cells and that an unknown chaperon process creates multiple sub-types of ‘rings’ with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.

Published
2018

8. Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts

Author

Naïma Nhiri, Michel Desmadril, Sonia Fieulaine, Julien Nusbaum, Thierry Meinnel, Carmela Giglione, Renata Grzela, Alain Van Dorsselaer, Francesco Lavecchia, Sarah Cianférani, Eric Jacquet, Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Interactions et mécanismes d’assemblage des protéines et des peptides (IMAPP), Département Biochimie, Biophysique et Biologie Structurale (B3S), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Maturation des proteines, destinée cellulaire et thérapeutique (PROMTI), Département Biologie des Génomes (DBG), Modélisation et Ingénierie des Protéines (MIP), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), 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)-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), 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)-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)-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), Mesures d'interactions protéine-protéine (PIM), Département Plateforme (PF I2BC), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-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 Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-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 Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
0301 basic medicine, Enzyme mechanism, DBG, [SDV]Life Sciences [q-bio], viruses, Biophysics, Peptide, Biochemistry, Genome, Amidohydrolases, Analytical Chemistry, Bacteriophage, Viral Proteins, 03 medical and health sciences, Peptide deformylase, chemistry.chemical_compound, Catalytic Domain, Enzyme Stability, Hydrolase, N-terminal methionine excision, Bacteriophages, Actinonin, PROMTI, Molecular Biology, Gene, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, IMAPP, AMIG, biology, fungi, Structure, biology.organism_classification, MIP, PIM, Virus, 030104 developmental biology, chemistry, Specific activity, Vibrio parahaemolyticus, PF, B3S
Abstract

International audience; Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 specific activity to values similar to those of bacterial PDFs. We next show that Vp16PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.

Published
2018

9. Influence of Force-Field Parameters on the Atomistic Simulations of Metallic Surfaces and Nanoparticles

Author

Massimo Marchi, Stéphane Abel, Johannes Richardi, Takieddine Djebaili, De la Molécule aux Nanos-objets : Réactivité, Interactions et Spectroscopies (MONARIS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Laboratoire Bioénergétique Membranaire et Stress (LBMS), 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), 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), and Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, [SDV]Life Sciences [q-bio], Nanoparticle, Interaction model, 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, Force field (chemistry), 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, LBMS, Metal, Crystallography, Molecular dynamics, General Energy, Adsorption, Nanocrystal, Chemical physics, visual_art, visual_art.visual_art_medium, Physical and Theoretical Chemistry, 0210 nano-technology, B3S
Abstract

The influence of the interaction model on the adsorption of butanethiolate on gold surfaces and nanocrystals has been studied with molecular dynamics simulations. The results obtained for three different head group sizes are compared to experiments. The use of the largest head group induces new organizations of the ligands in the case of nanocrystals and Au(100) surfaces, while no such difference is observed for Au(111). As a consequence, this model does not reproduce the higher surface coverage experimentally observed for nanocrystals. Our results show that the evaluation of the quality of force fields cannot be restricted to the study of specific surfaces. Some properties such as the occupation frequencies of adsorption sites markedly depend on the nanocrystal size.

Published
2017

10. αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design

Author

Jean-Pierre Mahy, Christophe Velours, Agathe Urvoas, Wadih Ghattas, Thibault Di Meo, Christian Herrero, Philippe Minard, Rémy Ricoux, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay, Département Plateforme (PF I2BC), 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), Mesures d'interactions protéine-protéine (PIM), 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), Centre de Recherches Historiques (CRH) _ Unité Mixte de Recherches (UMR 8558 CNRS / EHESS) (CRH (UMR 8558 CNRS / EHESS)), École des hautes études en sciences sociales (EHESS)-Centre National de la Recherche Scientifique (CNRS), Institutions et Dynamiques Historiques de l'Économie et de la Société (IDHES), Université Paris 1 Panthéon-Sorbonne (UP1)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Département Biochimie, Biophysique et Biologie Structurale (B3S), Modélisation et Ingénierie des Protéines (MIP), 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, Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), 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-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, 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-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-Institut de Biologie Intégrative de la Cellule (I2BC), Centre de recherches historiques (CRH), École normale supérieure - Cachan (ENS Cachan)-Université Panthéon-Sorbonne (UP1)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre de Recherches Historiques (CRH), and Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Nanterre (UPN)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris 1 Panthéon-Sorbonne (UP1)

Subjects
Protein Folding, Circular dichroism, Stereochemistry, [SDV]Life Sciences [q-bio], Dimer, Phenanthroline, 010402 general chemistry, 01 natural sciences, Mass Spectrometry, Catalysis, chemistry.chemical_compound, Residue (chemistry), Metalloproteins, Amino Acid Sequence, Diels-Alder reaction, Cycloaddition Reaction, 010405 organic chemistry, Circular Dichroism, Organic Chemistry, Electron Spin Resonance Spectroscopy, Enantioselective Catalysis, Stereoisomerism, General Chemistry, Protein engineering, artificial repeat proteins, MIP, PIM, 0104 chemical sciences, chemistry, Covalent bond, Protein folding, PF, Dimerization, Sequence Alignment, B3S, Copper, Phenanthrolines, Cysteine
Abstract

International audience; αRep is a new family of artificial proteins based on a thermostable alpha-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to receive metal complexes and thus appears as suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen and changed independently into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine of each protein variant and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was bound specifically by the two biohybrids with two different binding modes and, in addition, the holo biohybrid A3F119NPH was found to be able to catalyze enantioselectively the Diels-Alder (D-A) cycloaddition with up to 62% ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a new promising route for the design and production of new enantioselective biohybrids based on entirely artificial proteins issued from a highly diverse library.

Published
2017

11. Spectral and 3D model studies of the interaction of orphan human cytochrome P450 2U1 with substrates and ligands

Author

Lionel Ducassou, François André, Maryse Jaouen, Nicolas Pietrancosta, Julien Dairou, Jean-Luc Boucher, Laura Dhers, Booma Ramassamy, Daniel Mansuy, 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), Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences du Vivant Frédéric JOLIOT (JOLIOT), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Département Biochimie, Biophysique et Biologie Structurale (B3S), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Stress Oxydants et Détoxication (LSOD), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Collège de France (CdF (institution))-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), 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), 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), Systèmes glutamatergiques normaux et pathologiques = Normal and Pathologic Glutamatergic Neurons (NPS), Neuroscience Paris Seine (NPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Analyse, Interactions Moléculaires et Cellulaires (LBM-E2), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Chimie Moléculaire de Paris Centre (FR 2769), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, 0301 basic medicine, Serotonin, Pyridines, Stereochemistry, [SDV]Life Sciences [q-bio], Biophysics, Debrisoquin, Arachidonic Acids, Ligands, Biochemistry, Molecular Docking Simulation, Substrate Specificity, LSOD, 03 medical and health sciences, chemistry.chemical_compound, Escherichia coli, Humans, Imidazole, Homology modeling, Cytochrome P450 Family 2, Molecular Biology, Arachidonic Acid, 030102 biochemistry & molecular biology, biology, Electron Spin Resonance Spectroscopy, Imidazoles, Hexacoordinate, Lauric Acids, Active site, Cytochrome P450, Recombinant Proteins, 030104 developmental biology, chemistry, Docking (molecular), Biocatalysis, biology.protein, Spectrophotometry, Ultraviolet, Oxidation-Reduction, B3S, Protein Binding
Abstract

Background Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an “orphan” protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N -arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine. Methods We have expressed CYP2U1 in E. coli and performed UV–vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS. Results The UV–vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate Fe III state. Imidazole (Im) derivatives, such as miconazole, acted as Fe III ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to “reverse type I” difference UV–vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model. Major conclusion and general significance Our study describes for the first time the mode of interaction of several Fe III -heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP.

Published
2017

12. Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly

Author

Klemens Rottner, Frieda Kage, Marie-France Carlier, Georgi Dimchev, Anika Steffen, Julien Pernier, Vanessa Dimchev, Institute of Genetics, Rheinische Friedrich-Wilhelms-Universität Bonn, Dynamique du cytosquelette et motilité (ACTIN), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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)-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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and Helmholtz Centre for Infection Research (HZI)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], macromolecular substances, Actin-Related Protein 2-3 Complex, ACTIN, 03 medical and health sciences, Cytosol, Cell Movement, Animals, Humans, Pseudopodia, Cytoskeleton, Molecular Biology, Actin, biology, fungi, Microfilament Proteins, Cell Biology, Articles, Actin cytoskeleton, Phosphoproteins, Actins, Cell Motility, Actin Cytoskeleton, 030104 developmental biology, Formins, biology.protein, Biophysics, Lamellipodium, Filopodia, B3S, Cell Adhesion Molecules
Abstract

Lamellipodia protrusion requires actin network formation driven by the Arp2/3 complex and its upstream regulators WAVE complex and Rac. Actin assembly factors of the formin and Ena/VASP families can influence protrusion, in particular by maintaining a balance between lamellipodial and cytosolic actin filament assembly., Cell migration and cell–cell communication involve the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through the Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamellipodial actin networks, but the regulation of nucleation or elongation of Arp2/3-independent filament populations within the network by, for example, formins or Ena/VASP family members and its influence on the effectiveness of protrusion have been unclear. Here we analyzed the effects of a set of distinct formin fragments and VASP on site-specific, lamellipodial versus cytosolic actin assembly and resulting consequences on protrusion. Surprisingly, expression of formin variants but not VASP reduced lamellipodial protrusion in B16-F1 cells, albeit to variable extents. The rates of actin network polymerization followed a similar trend. Unexpectedly, the degree of inhibition of both parameters depended on the extent of cytosolic but not lamellipodial actin assembly. Indeed, excess cytosolic actin assembly prevented actin monomer from rapid translocation to and efficient incorporation into lamellipodia. Thus, as opposed to sole regulation by actin polymerases operating at their tips, the protrusion efficiency of lamellipodia is determined by a finely tuned balance between lamellipodial and cytosolic actin assembly.

Published
2017

13. Tuning antenna function through hydrogen bonds to chlorophyll a

Author

Llansola-Portoles, Manuel J., Pascal, Andrew A., Llansola-Portoles, Manuel, Li, Fei, Xu, Pengqi, Streckaite, Simona, Ilioaia, Cristian, Yang, Chunhong, Gall, Andrew, Croce, Roberta, Robert, Bruno, Pascal, Andrew, 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 Bioénergétique Membranaire et Stress (LBMS), 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), 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), Biophysics Photosynthesis/Energy, and LaserLaB - Energy

Subjects
Light-harvesting, energy regulation, 0301 basic medicine, Photosystem II, [SDV]Life Sciences [q-bio], Light-Harvesting Protein Complexes, Biophysics, macromolecular substances, Conjugated system, Spectrum Analysis, Raman, 010402 general chemistry, Photosynthesis, Photochemistry, 7. Clean energy, 01 natural sciences, Biochemistry, Hydrogen bonds, LBMS, 03 medical and health sciences, symbols.namesake, Oxygenic photosynthesis, Molecule, SDG 7 - Affordable and Clean Energy, Absorption (electromagnetic radiation), Hydrogen bond, Chemistry, Chlorophyll A, Energy regulation, Hydrogen Bonding, Cell Biology, Resonance (chemistry), 0104 chemical sciences, 030104 developmental biology, hydrogen bonds, symbols, Raman spectroscopy, B3S, Chl-a
Abstract

We describe a molecular mechanism tuning the functional properties of chlorophyll a (Chl-a) molecules in photosynthetic antenna proteins. Light-harvesting complexes from photosystem II in higher plants – specifically LHCII purified with α- or β-dodecyl-maltoside, along with CP29 – were probed by low-temperature absorption and resonance Raman spectroscopies. We show that hydrogen bonding to the conjugated keto carbonyl group of protein-bound Chl-a tunes the energy of its Soret and Qy absorption transitions, inducing red-shifts that are proportional to the strength of the hydrogen bond involved. Chls-a with non-H-bonded keto C131 groups exhibit the blue-most absorption bands, while both transitions are progressively red-shifted with increasing hydrogen-bonding strength – by up 382 & 605 cm−1 in the Qy and Soret band, respectively. These hydrogen bonds thus tune the site energy of Chl-a in light-harvesting proteins, determining (at least in part) the cascade of energy transfer events in these complexes.

Published
2019
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14. Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer

Author

Anja Krieger-Liszkay, Pierre Sétif, Alain Boussac, Mécanismes régulateurs chez les organismes photosynthétiques (MROP), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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)-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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Photosystème II (PS2), and ANR-16-CE05-0026,ReCyFuel,Régulations de la photosynthèse et production de biofuels par les cyanobactéries(2016)

Subjects
0106 biological sciences, 0301 basic medicine, PS2, Cytochrome, [SDV]Life Sciences [q-bio], Analytical chemistry, P700, Plant Science, Photosystem I, Cytochrome c 6, 01 natural sciences, Biochemistry, Redox, Electron Transport, 03 medical and health sciences, Electron transfer, Cytochromes c6, Bacterial Proteins, Spinacia oleracea, Photosystem I terminal acceptor, Tobacco, Plastocyanin, Ferredoxin, Plant Proteins, chemistry.chemical_classification, Spectroscopy, Near-Infrared, Photosystem I Protein Complex, biology, Synechocystis, Cell Biology, General Medicine, Electron acceptor, Infrared spectral deconvolution, 030104 developmental biology, chemistry, biology.protein, Ferredoxins, Spectrophotometry, Ultraviolet, MROP, Oxidation-Reduction, B3S, 010606 plant biology & botany
Abstract

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, – 0.033 ± 0.006, – 0.036 ± 0.008, and – 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.

Published
2019

15. Importing Manganese into the Chloroplast: Many Membranes to Cross

Author

Sébastien Thomine, Anja Krieger-Liszkay, Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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)-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), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), 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), Approches intégratives du Transport Ionique (MINION), Département Biologie Cellulaire (BioCell), and ANR-16-CE20-0019,ISISTOR,Amélioration du contenu en fer de la graine(2016)

Subjects
0106 biological sciences, 0301 basic medicine, Chloroplasts, Photosystem II, [SDV]Life Sciences [q-bio], Golgi Apparatus, chemistry.chemical_element, Plant Science, Manganese, Photosynthesis, Thylakoids, 01 natural sciences, 03 medical and health sciences, Arabidopsis, homeostasis, BIOCELL, MINION, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Plant Proteins, photosynthesis, biology, Photosystem II Protein Complex, Biological Transport, Intracellular Membranes, biology.organism_classification, Chloroplast, arabidopsis, 030104 developmental biology, Membrane, Biochemistry, chemistry, photosystem-ii, Minion, Vacuoles, cells, MROP, protein, B3S, 010606 plant biology & botany
Abstract

International audience

Published
2018

16. Visible-Light-Driven Reduction of CO$_2$ to CO and Its Subsequent Valorization in Carbonylation Chemistry and $^{13}$C Isotope Labeling

Author

Antonio Del Vecchio, Ally Aukauloo, Annamaria Quaranta, Winfried Leibl, Philipp Gotico, Davide Audisio, Zakaria Halime, 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), Service de Chimie Bio-Organique et de Marquage (SCBM), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), IRAMAT - Laboratoire Métallurgies et Cultures (IRAMAT - LMC), Institut de Recherches sur les Archéomatériaux (IRAMAT), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)-Université Bordeaux Montaigne-Université de Technologie de Belfort-Montbeliard (UTBM)-Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)-Université Bordeaux Montaigne-Université de Technologie de Belfort-Montbeliard (UTBM), Laboratoire de Marquage au Carbone 14 (LMC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), ANR-11-LABX-0039,CHARMMMAT,CHimie des ARchitectures MoléculairesMultifonctionnelles et des MATériaux(2011), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), European Project: 675071,H2020,H2020-MSCA-ITN-2015,ISOTOPICS(2016), Université de Technologie de Belfort-Montbeliard (UTBM)-Université d'Orléans (UO)-Université Bordeaux Montaigne (UBM)-Centre National de la Recherche Scientifique (CNRS)-Université de Technologie de Belfort-Montbeliard (UTBM)-Université d'Orléans (UO)-Université Bordeaux Montaigne (UBM)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des Mécanismes fondamentaux de la Bioénergétique (LMB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Photocatalyse et Biohydrogène (LPB), 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), 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), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay, and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
photoredox catalysis, [SDV]Life Sciences [q-bio], homogeneous catalysts, chemistry.chemical_element, carbonylation, 010402 general chemistry, Photochemistry, carbon-dioxide, 01 natural sciences, Analytical Chemistry, Artificial photosynthesis, Catalysis, Isotopic labeling, chemistry.chemical_compound, Physical and Theoretical Chemistry, isotopic labeling, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, continuous-flow, Organic Chemistry, Photoredox catalysis, photocatalytic reduction, carbon dioxide, [CHIM.CATA]Chemical Sciences/Catalysis, photochemical reduction, 0104 chemical sciences, Ruthenium, formic-acid, LPB, chemistry, highly efficient, 13. Climate action, artificial photosynthesis, Photocatalysis, rhenium(i) complex, Carbonylation, B3S, photocatalysis, Carbon monoxide, metal-complexes
Abstract

WOS:000441548500003; International audience; A convenient and safe approach in valorizing carbon monoxide (CO) produced from the photocatalytic reduction of carbon dioxide (CO2) has been investigated. Visible light was used to drive an optimized photocatalytic reduction using a ruthenium trisbipyridine complex as a sensitizer and a rhenium bipyridyl carbonyl complex as a catalyst to perform an efficient reduction of CO2 to CO, which was then simultaneously utilized in a palladium-catalyzed aminocarbonylation reaction at room temperature. This approach provides safe handling of the produced CO which also opens the way for a more efficient application of C-13-isotope and C-14-radioisotope-labeled CO2 in pharmaceutically relevant drug labeling.

Published
2018

17. Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation

Author

Ma, Emilie, Dupaigne, Pauline, Maloisel, Laurent, Guerois, Raphaël, Le Cam, Eric, Coïc, Eric, Institut de Biologie et Technologies de Saclay, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Laboratoire d'Etudes de la Réparation de l'ADN (LERA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), Assemblage moléculaire et intégrité du génome (AMIG), 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
chromosomes, Saccharomyces cerevisiae Proteins, QH301-705.5, Science, [SDV]Life Sciences [q-bio], genetic processes, Gene Conversion, S. cerevisiae, homologous recombination, chemical biology, Saccharomyces cerevisiae, Models, Biological, Protein Domains, Biochemistry and Chemical Biology, biochemistry, DNA Breaks, Double-Stranded, Amino Acid Sequence, Biology (General), DNA, Fungal, Alleles, Srs2, ComputingMilieux_MISCELLANEOUS, AMIG, fungi, DNA Helicases, Sumoylation, Chromosomes and Gene Expression, Rad52 DNA Repair and Recombination Protein, enzymes and coenzymes (carbohydrates), Mutation, Rad52, health occupations, gene expression, Rad51, Medicine, Mutant Proteins, Rad51 Recombinase, biological phenomena, cell phenomena, and immunity, B3S, genome stability, Research Article, Protein Binding
Abstract

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.

Published
2018

18. Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein

Author

Hutinet, Geoffrey, Besle, Arthur, Son, Olivier, McGovern, Stephen, Guérois, Raphaël, Petit, Marie-Agnès, Ochsenbein, Françoise, Lecointe, François, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Université Paris-Saclay, 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), Agence Nationale de la Recherche (Dynamophage contract), 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, Assemblage moléculaire et intégrité du génome (AMIG), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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-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-Institut de Biologie Intégrative de la Cellule (I2BC), and 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-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

Subjects
Microbiology (medical), RecA, AMIG, [SDV]Life Sciences [q-bio], lcsh:QR1-502, annealase, Microbiology, lcsh:Microbiology, Rad51 paralog, recombineering, bacteriophage, strand exchange protein, Sak4, B3S, SSB, Original Research
Abstract

International audience; Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.

Published
2018

19. The structure of the TsaB/TsaD/TsaE complex reveals an unexpected mechanism for the bacterial t6A tRNA-modification

Author

Missoury, Sophia, Plancqueel, Stéphane, Sierra-Gallay, Inès, Zhang, Wenhua, Liger, Dominique, Durand, Dominique, Dammak, Raoudha, Collinet, Bruno, Tilbeurgh, Herman, 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), 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), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), 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), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), UPMC - UFR Sciences de la vie (UFR 927 ), and Université Pierre et Marie Curie - Paris 6 (UPMC)

Subjects
Models, Molecular, 0303 health sciences, Adenosine, Binding Sites, FAAM, Protein Conformation, Archaeal Proteins, [SDV]Life Sciences [q-bio], 030302 biochemistry & molecular biology, Enzymes, 03 medical and health sciences, Adenosine Triphosphate, Bacterial Proteins, RNA, Transfer, Structural Biology, Catalytic Domain, Genetics, Thermotoga maritima, Corrigendum, Protein Structure, Quaternary, B3S, 030304 developmental biology
Abstract

International audience; The universal N6-threonylcarbamoyladenosine (t6A) modification at position A37 of ANN-decoding tRNAs is essential for translational fidelity. In bacteria the TsaC enzyme first synthesizes an l-threonylcarbamoyladenylate (TC-AMP) intermediate. In cooperation with TsaB and TsaE, TsaD then transfers the l-threonylcarbamoyl-moiety from TC-AMP onto tRNA. We determined the crystal structure of the TsaB-TsaE-TsaD (TsaBDE) complex of Thermotoga maritima in presence of a non-hydrolysable AMPCPP. TsaE is positioned at the entrance of the active site pocket of TsaD, contacting both the TsaB and TsaD subunits and prohibiting simultaneous tRNA binding. AMPCPP occupies the ATP binding site of TsaE and is sandwiched between TsaE and TsaD. Unexpectedly, the binding of TsaE partially denatures the active site of TsaD causing loss of its essential metal binding sites. TsaE interferes in a pre- or post-catalytic step and its binding to TsaBD is regulated by ATP hydrolysis. This novel binding mode and activation mechanism of TsaE offers good opportunities for antimicrobial drug development.

Published
2018

20. Anti-osteoclastic effects of C-glucosidic ellagitannins mediated by actin perturbation

Author

Christophe Le Clainche, Denis Deffieux, Daniela Melanie Delannoy López, Elisabeth Génot, Thierry Dakhli, Damien Le Nihouannen, Stéphane Quideau, Isabelle Fremaux, Pierre Jurdic, Dan Georgess, Pirjo Spuul, Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure de Lyon (ENS de 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), Dynamique du cytosquelette et motilité (ACTIN), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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)-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), 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), Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), 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), Université de Bordeaux (UB), Centre de recherche Cardio-Thoracique de Bordeaux [Bordeaux] (CRCTB), Université Bordeaux Segalen - Bordeaux 2-CHU Bordeaux [Bordeaux]-Institut National de la Santé et de la Recherche Médicale (INSERM), Departement Sciences du Vivant et de la Sante [Bordeaux], Institut des Sciences Moléculaires (ISM), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1 (UB)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ANR-17-CE11-0033,POLYPHENOLPROT,Sondes Protéomiques pour l'Elucidation des Interactions Polyphénol-Protéines – Vers le Développement d'Agents Thérapeutiques à Base de Polyphénols(2017), 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)-École normale supérieure - Lyon (ENS Lyon), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), 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-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-Institut de Biologie Intégrative de la Cellule (I2BC), 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-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, 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, IGFL - Institut de Génomique Fonctionnelle de Lyon, and Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Histology, Podosome, Cell Survival, [SDV]Life Sciences [q-bio], Osteoclasts, macromolecular substances, Medicinal biology, Pathology and Forensic Medicine, Polymerization, ACTIN, 03 medical and health sciences, Glucosides, Actin dynamics, Cell Adhesion, Animals, Bone Resorption, Cytoskeleton, Actin, Chemistry, Cell Differentiation, Cell Biology, General Medicine, Actin cytoskeleton, In vitro, Actins, Hydrolyzable Tannins, Osteoclast biology, Cell biology, Extracellular Matrix, Mice, Inbred C57BL, Polyphenol sciences, Actin Cytoskeleton, 030104 developmental biology, Podosomes, Ultrastructure, B3S
Abstract

Actin subunits assemble into actin filaments whose dynamics and three-dimensional architectures are further regulated by a variety of cellular factors to establish the functional actin cytoskeleton. The C-glucosidic ellagitannin vescalagin and its simpler analogue vescalin, affect both the dynamics and the ultrastructure of the actin cytoskeleton by directly binding to F-actin. Herein, we show that in vitro, the two compounds induce the formation of distinct F-actin networks characterized by different superstructures and dynamics. In living mature osteoclasts, highly specialized bone-degrading cells that constantly remodel their cytoskeleton, vescalagin and vescalin alter actin dynamics at podosomes and compromise the integrity of the podosome belt that forms the bone-degrading apparatus. Both compounds target the bone-resorbing activity at concentrations that preserve osteoclastic maturation and survival and with no detectable impact on the behaviour of bone-forming osteoblastic cells. This anti-osteoclastic activity of vescalagin and vescalin reveals the potential of targeting actin dynamics as a new therapeutic opportunity and, in this case, as a plausible approach for the local treatment of osteoporosis.

Published
2018

21. Screening of Detergents for Stabilization of Functional Membrane Proteins

Author

Cédric Montigny, Thibaud Dieudonné, Maylis Lejeune, Anaïs Lamy, José Luis Vázquez-Ibar, Guillaume Lenoir, Hycole Sarl, 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), 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), Laboratoire des Protéines et Systèmes Membranaires (LPSM), 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)-Institut de Biologie Intégrative de la Cellule (I2BC)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Context (language use), Drs2p/Cdc50p, Biochemistry, lipids, 03 medical and health sciences, LPSM, Structural Biology, Animals, Humans, membrane protein, SERCA1a, Micelles, Chemistry, Protein Stability, Membrane Proteins, stabilization, 030104 developmental biology, Membrane, Membrane protein, Solubilization, detergents, Critical micelle concentration, LMNG, Biophysics, B3S, Function (biology)
Abstract

Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent. This article provides a convenient protocol for estimating the CMC of detergents in given experimental conditions. Then, it presents two protocols aimed at monitoring the function of a membrane protein in the presence of detergent. Such experiments may help to test various detergents for their inactivating or stabilizing effects on long incubation times, ranging from few hours to some days. © 2018 by John Wiley & Sons, Inc.

Published
2018

22. The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum

Author

Céline Lavire, Magali Aumont-Nicaise, Gilles Comte, Solange Moréra, Thibault Meyer, Ludovic Vial, Armelle Vigouroux, Institut de Biologie Intégrative de la Cellule (I2BC), 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, Département Biochimie, Biophysique et Biologie Structurale (B3S), 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-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, Microbiologie et enzymologie structurale (MESB3S), 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-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-Institut de Biologie Intégrative de la Cellule (I2BC), Mesures d'interactions protéine-protéine (PIM), Département Plateforme (PF I2BC), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Lyon (ENVL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), 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)-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)-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), 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)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Ecole Nationale Vétérinaire de Lyon (ENVL), FRISBI ANR-10-INSB-05-01, and Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Protein Conformation, [SDV]Life Sciences [q-bio], ATP-binding cassette transporter, crystal structure, sugar transport, bacteria, ABC transporter, microbiology, agrobacterium, galactinol, periplasmic binding protein, RFOs, plant defense, Agrobacterium fabrum, Crystallography, X-Ray, Disaccharides, Biochemistry, chemistry.chemical_compound, Plant defense against herbivory, Raffinose, MESB3S, bactérie, Rhizosphere, food and beverages, Plants, Protein Structure and Folding, PF, rhizosphère, B3S, agent pathogène, Agrobacterium, 030106 microbiology, Biology, Stachyose, 03 medical and health sciences, Bacterial Proteins, oligosaccharide, Melibiose, Molecular Biology, fungi, Nutrients, Cell Biology, biology.organism_classification, PIM, chemistry, Agrobacterium tumefaciens, Galactose
Abstract

The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose–containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.

Published
2018

23. Triple resonance EPR spectroscopy determines the Mn2+ coordination to ATP

Author

Aleksei Litvinov, Daniella Goldfarb, Akiva Feintuch, Sun Un, Weizmann Institute of Science [Rehovot, Israël], 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), RPE Haut Champ des systèmes biologiques (BHFMR), 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), 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), and Departments of Chemical Physics

Subjects
inorganic chemicals, Nuclear and High Energy Physics, ATPase, [SDV]Life Sciences [q-bio], Biophysics, Context (language use), divalent metal, 010402 general chemistry, ELDOR detected NMR, 01 natural sciences, Biochemistry, BHFMR, law.invention, thycos, atp, law, ATP hydrolysis, molecular-orbital methods, Molecule, Electron paramagnetic resonance, High field EPR, biology, 010405 organic chemistry, Chemistry, endor, metal-ion-binding, Active site, crystal-structure, organic-molecules, Condensed Matter Physics, Resonance (chemistry), Mn2+-ATP, 0104 chemical sciences, ternary complexes, Crystallography, Mn2+ ion, nuclear-magnetic-resonance, biology.protein, gaussian-type basis, Two-dimensional nuclear magnetic resonance spectroscopy, B3S, adenosine 5-triphosphate, eldor-detected nmr
Abstract

WOS:000442065200016; Mn2+ often serves as a paramagnetic substitute to Mg2+, providing means for exploring the close environment of Mg2+ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg2+-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band P-31 electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) - detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring N-15-P-31 correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn2+ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for Mn-55-P-31 correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with N-15-P-31 correlations. (C) 2018 Elsevier Inc. All rights reserved.

Published
2018

24. Photoinduced electron transfer in a molecular dyad by nanosecond pump-pump-probe spectroscopy

Author

Christophe Lefumeux, Ally Aukauloo, Van-Thai Pham, Winfried Leibl, V. Maslova, Minh-Huong Ha-Thi, Annamaria Quaranta, Thomas Pino, Institut des Sciences Moléculaires d'Orsay (ISMO), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Photophysique Moléculaire, Université Paris-Sud - Paris 11 (UP11), Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), Photocatalyse et Biohydrogène (LPB), 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), 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), Centre de Photonique Biomédicale (CPBM), Centre Laser Université Paris Sud, Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre Laser de l'Université Paris Sud (CLUPS), Université Paris-Sud - Paris 11 (UP11)-Université Paris-Sud - Paris 11 (UP11), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, photosensitizer, [SDV]Life Sciences [q-bio], reduction, 010402 general chemistry, Photochemistry, 7. Clean energy, 01 natural sciences, Redox, Photoinduced electron transfer, photocatalysts, Electron transfer, catalytic water oxidation, solar-energy, Molecule, Physical and Theoretical Chemistry, chemistry.chemical_classification, 010405 organic chemistry, accumulative charge separation, Electron acceptor, Chromophore, Nanosecond, 0104 chemical sciences, LPB, Photoinduced charge separation, chemistry, artificial photosynthesis, photosystem-ii, co2, B3S, complex
Abstract

International audience; The design of robust and inexpensive molecular photocatalysts for the conversion of abundant stable molecules like H2O and CO2 into an energetic carrier is one of the major fundamental questions for scientists nowadays. The outstanding challenge is to couple single photoinduced charge separation events with the sequential accumulation of redox equivalents at the catalytic unit for performing multielectronic catalytic reactions. Herein, double excitation by nanosecond pump-pump-probe experiments was used to interrogate the photoinduced charge transfer and charge accumulation on a molecular dyad composed of a porphyrin chromophore and a ruthenium-based catalyst in the presence of a reversible electron acceptor. An accumulative charge transfer state is unattainable because of rapid reverse electron transfer to the photosensitizer upon the second excitation and the low driving force of the forward photodriven electron transfer reaction. Such a method allows the fundamental understanding of the relaxation mechanism after two sequential photon absorptions, deciphering the undesired electron transfer reactions that limit the charge accumulation efficiency. This study is a step toward the improvement of synthetic strategies of molecular photocatalysts for light-induced charge accumulation and more generally, for solar energy conversion.

Published
2018

25. Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

Author

Lauriane Lecoq, Thomas Wiegand, Anja Böckmann, Michael Nassal, Beat H. Meier, Shishan Wang, Stéphane Bressanelli, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratory of Physical Chemistry [ETH Zürich] (LPC), Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), 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), 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), University Hospital Freiburg, Physical Chemistry [ETH Zürich], Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich)-Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich), Institut de biologie et chimie des protéines [Lyon] (IBCP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon

Subjects
0301 basic medicine, Models, Molecular, Icosahedral symmetry, Protein Conformation, media_common.quotation_subject, asymmetric unit, [SDV]Life Sciences [q-bio], nucleocapsid, Dihedral angle, 010402 general chemistry, 01 natural sciences, Asymmetry, 03 medical and health sciences, Protein structure, Capsid, core protein, Molecule, Physical and Theoretical Chemistry, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, media_common, hepatitis B virus, solid-state NMR, IMAPP, Chemistry, Viral Core Proteins, Computational Biology, Atomic and Molecular Physics, and Optics, 3. Good health, 0104 chemical sciences, NMR spectra database, Crystallography, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Solid-state nuclear magnetic resonance, B3S
Abstract

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of "dynamic" domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.

Published
2018

26. Lycopene crystalloids exhibit singlet exciton fission in tomatoes

Author

Mikas Vengris, Manuel J. Llansola-Portoles, Bruno Robert, Andrew A. Pascal, Alison Telfer, Simona Streckaite, Kipras Redeckas, Cristian Ilioaia, Leonas Valkunas, 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), 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), Laboratoire Bioénergétique Membranaire et Stress (LBMS), 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), Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), IMSAR, and Vilnius University [Vilnius]

Subjects
0301 basic medicine, Fission, [SDV]Life Sciences [q-bio], Singlet exciton, General Physics and Astronomy, 010402 general chemistry, Photochemistry, 01 natural sciences, LBMS, 03 medical and health sciences, chemistry.chemical_compound, Lycopene, Solanum lycopersicum, Chromoplast, Ultrafast laser spectroscopy, Plastids, Physical and Theoretical Chemistry, Carotenoid, chemistry.chemical_classification, Crystalloid Solutions, Carotenoids, 3. Good health, 0104 chemical sciences, 030104 developmental biology, chemistry, Excited state, Singlet fission, Isotonic Solutions, B3S
Abstract

Transient absorption studies conducted on in vitro lycopene aggregates, as well as on lycopene crystalloids inside tomato chromoplasts, reveal the appearance of a long-lived excited state, which we unambiguously identified as lycopene triplet. These triplet states must be generated by singlet exciton fission, which occurs from the lycopene 2Ag state. This is the first time the singlet fission process has ever been shown to occur in a biological material. We propose that the formation of carotenoid assemblies in chromoplasts may constitute a photoprotective process during chromoplast maturation, in addition to their function in signaling processes.

Published
2018
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27. Sub-nanosecond tryptophan radical deprotonation mediated by a protein-bound water cluster in class II DNA photolyases

Author

Pavel Müller, Elisabeth Ignatz, Stephan Kiontke, Lars-Oliver Essen, Klaus Brettel, I2BC, Centre National de la Recherche Scientifique (CNRS), Département Plateforme (PF I2BC), 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)-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), Philipps University Marburg, Philipps Universität Marburg, Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Département Biochimie, Biophysique et Biologie Structurale (B3S), Photocatalyse et Biohydrogène (LPB), 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), Philipps Universität Marburg = Philipps University of Marburg, 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)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, DNA repair, Stereochemistry, [SDV]Life Sciences [q-bio], Pyrimidine dimer, 010402 general chemistry, 01 natural sciences, Cofactor, 03 medical and health sciences, chemistry.chemical_compound, Deprotonation, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Photolyase, ComputingMilieux_MISCELLANEOUS, Flavin adenine dinucleotide, biology, General Chemistry, DNA photolyase, B3S, LPB, 0104 chemical sciences, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, LPB, Chemistry, 030104 developmental biology, chemistry, biology.protein, B3S, DNA
Abstract

Light activation of class II DNA photolyases is enhanced by a unique cluster of protein-bound water molecules., Class II DNA photolyases are flavoenzymes occurring in both prokaryotes and eukaryotes including higher plants and animals. Despite considerable structural deviations from the well-studied class I DNA photolyases, they share the main biological function, namely light-driven repair of the most common UV-induced lesions in DNA, the cyclobutane pyrimidine dimers (CPDs). For DNA repair activity, photolyases require the fully reduced flavin adenine dinucleotide cofactor, FADH–, which can be obtained from oxidized or semi-reduced FAD by a process called photoactivation. Using transient absorption spectroscopy, we have examined the initial electron and proton transfer reactions leading to photoactivation of the class II DNA photolyase from Methanosarcina mazei. Upon photoexcitation, FAD is reduced via a distinct (class II-specific) chain of three tryptophans, giving rise to an FAD˙– TrpH˙+ radical pair. The distal Trp388H˙+ deprotonates to Trp388˙ in 350 ps, i.e., by three orders of magnitude faster than TrpH˙+ in aqueous solution or in any previously studied photolyase. We identified a class II-specific cluster of protein-bound water molecules ideally positioned to serve as the primary proton acceptor. The high rate of Trp388H˙+ deprotonation counters futile radical pair recombination and ensures efficient photoactivation.

Published
2018

28. Involvement of the FOXO6 transcriptional factor in breast carcinogenesis

Author

Sophie Vacher, Bernard S. Lopez, Karim Taouis, François Lallemand, Ivan Bièche, Leanne De Koning, Ambre Petitalot, Rosette Lidereau, Sophie Zinn-Justin, Nicole Dalla-Venezia, Sandrine M. Caputo, Walid Chemlali, Anne Schnitzler, Genetique Moleculaire des Cancers d'Origine Epitheliale, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de génétique et biologie des cancers (U830), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Département Microbiologie (Dpt Microbio), 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), Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), 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), Institut Curie [Paris], Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Génétique moléculaire, signalisation et cancer (GMSC), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Oncogénétique, Institut Curie [Paris]-Hôpital René HUGUENIN (Saint-Cloud), ANR-16-CE12-0011,2R-POL,ADN polymérase theta : lien entre réplication et réparation de l'ADN(2016), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris], Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Institut Curie, Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, and Hôpital René HUGUENIN (Saint-Cloud)-Institut Curie

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], endometrial adenocarcinoma, Context (language use), Biology, medicine.disease_cause, 03 medical and health sciences, INTGEN, 0302 clinical medicine, Breast cancer, medicine, skin and connective tissue diseases, uc.189, Transcription factor, Cancer, FOXO Family, Cell cycle, 16. Peace & justice, medicine.disease, 3. Good health, 030104 developmental biology, Oncology, Apoptosis, 030220 oncology & carcinogenesis, Cancer research, cervical squamous cell carcinoma, gynecological cancers, prognosis, Carcinogenesis, B3S, Research Paper
Abstract

// Francois Lallemand 1, * , Ambre Petitalot 1, 2, * , Sophie Vacher 1 , Leanne de Koning 3 , Karim Taouis 1 , Bernard S. Lopez 4 , Sophie Zinn-Justin 5 , Nicole Dalla-Venezia 6 , Walid Chemlali 1 , Anne Schnitzler 1 , Rosette Lidereau 1 , Ivan Bieche 1, 7, * and Sandrine M. Caputo 2, * 1 Service de genetique, unite de pharmacogenomique, Institut Curie, Paris, France 2 Service de genetique, unite de genetique constitutionnelle, Institut Curie, Paris, France 3 Departement de transfert, Institut Curie, Paris, France 4 CNRS UMR 8200, Gustave Roussy Cancer Institute, Universite Paris-Saclay, equipe labelisee par la Ligue contre le cancer, Villejuif, France 5 Laboratoire de biologie structurale et radiobiologie, IBITEC-S (CEA) and I2BC (UMR 9198, CEA, CNRS, Univ. Paris South), Gif-sur-Yvette, France 6 Centre de Recherche en Cancerologie de Lyon (CRCL)/INSERM U1052-CNRS UMR5286, Lyon, France 7 EA7331, Universite Paris Descartes, Paris, France * These authors contributed equally to this work Correspondence to: Sandrine M. Caputo, email: sandrine.caputo@curie.fr Keywords: gynecological cancers; cervical squamous cell carcinoma; endometrial adenocarcinoma; uc.189; prognosis Received: May 12, 2017 Accepted: December 22, 2017 Published: December 30, 2017 ABSTRACT In mammals, FOXO transcriptional factors form a family of four members (FOXO1, 3, 4, and 6) involved in the modulation proliferation, apoptosis, and carcinogenesis. The role of the FOXO family in breast cancer remains poorly elucidated. According to the cellular context and the stage of the disease, FOXOs can have opposite effects on carcinogenesis. To study the role of FOXOs in breast carcinogenesis in more detail, we examined their expression in normal tissues, breast cell lines, and a large series of breast tumours of human origin. We found a very low physiological level of FOXO6 expression in normal adult tissues and high levels of expression in foetal brain. FOXO gene expressions fluctuate specifically in breast cancer cells compared to normal cells, suggesting that these genes may have different roles in breast carcinogenesis. For the first time, we have shown that, among the various FOXO genes, only FOXO6 was frequently highly overexpressed in breast cell lines and tumours. We also found that inhibition of the endogenous expression of FOXO6 by a specific siRNA inhibited the growth of the human breast cell lines MDA-MB-468 and HCC-38. FACS and Western blot analysis showed that inhibition of endogenous expression of FOXO6 induced accumulation of cells in G0/G1 phase of the cell cycle, but not apoptosis. These results tend to demonstrate that the overexpression of the human FOXO6 gene that we highlighted in the breast tumors stimulates breast carcinogenesis by activating breast cancer cell proliferation.

Published
2018

29. Switching an Individual Phycobilisome Off and On

Author

Adjélé Wilson, Diana Kirilovsky, Tjaart P. J. Krüger, Michal Gwizdala, Joshua L. Botha, Rienk van Grondelle, Institute for Lasers, Life and Biophotonics, Faculty of Sciences, VU University Amsterdam, Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, 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), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), 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), 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), Department of Physics and Astronomy [Amsterdam], Vrije Universiteit Amsterdam [Amsterdam] (VU), LaserLaB - Energy, and Biophysics Photosynthesis/Energy

Subjects
0106 biological sciences, 0301 basic medicine, Brightness, Orange carotenoid protein, Chemistry, [SDV]Life Sciences [q-bio], Dissipation, Photosynthesis, 01 natural sciences, 03 medical and health sciences, Light intensity, 030104 developmental biology, Biophysics, Intermediate state, General Materials Science, Phycobilisome, MROP, SDG 7 - Affordable and Clean Energy, Physical and Theoretical Chemistry, B3S, 010606 plant biology & botany
Abstract

Photosynthetic organisms have found various smart ways to cope with unexpected changes in light conditions. In many cyanobacteria, the lethal effects of a sudden increase in light intensity are mitigated mainly by the interaction between phycobilisomes (PBs) and the orange carotenoid protein (OCP). The latter senses high light intensities by means of photoactivation and triggers thermal energy dissipation from the PBs. Due to the brightness of their emission, PBs can be characterized at the level of individual complexes. Here, energy dissipation from individual PBs was reversibly switched on and off using only light and OCP. We reveal the presence of quasistable intermediate states during the binding and unbinding of OCP to PB, with a spectroscopic signature indicative of transient decoupling of some of the PB rods during docking of OCP. Real-time control of emission from individual PBs has the potential to contribute to the development of new super-resolution imaging techniques.

Published
2018

30. Protein-Protein Docking Using Evolutionary Information

Author

Raphael Guerois, Aravindan Arun Nadaradjane, Jessica Andreani, 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), Assemblage moléculaire et intégrité du génome (AMIG), 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), 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), and Marsh, Joseph A.

Subjects
0301 basic medicine, AMIG, Computer science, [SDV]Life Sciences [q-bio], Protein protein, Computational biology, Proteomics, Protein–protein interaction, 03 medical and health sciences, 030104 developmental biology, Protein structure, Docking (molecular), Macromolecular docking, B3S, ComputingMilieux_MISCELLANEOUS
Abstract

The structural modeling of protein complexes by docking simulations has been attracting increasing interest with the rise of proteomics and of the number of experimentally identified binary interactions. Structures of unbound partners, either modeled or experimentally determined, can be used as input to sample as extensively as possible all putative binding modes and single out the most plausible ones. At the scoring step, evolutionary information contained in the joint multiple sequence alignments of both partners can provide key insights to recognize correct interfaces. Here, we describe a computational protocol based on the InterEvDock web server to exploit coevolution constraints in protein-protein docking methods. We provide methodology guidelines to prepare the input protein structures and generate improved alignments. We also explain how to extract and use the information returned by the server through the analysis of two representative examples.

Published
2018

31. Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1)

Author

T Quyen, Nguyen, Magali, Aumont-Nicaise, Jessica, Andreani, Christophe, Velours, Mélanie, Chenon, Fernando, Vilela, Clémentine, Geneste, Paloma F, Varela, Paola, Llinas, Julie, Ménétrey, 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), Mesures d'interactions protéine-protéine (PIM), Département Plateforme (PF I2BC), 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), 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), Assemblage moléculaire et intégrité du génome (AMIG), Département Biochimie, Biophysique et Biologie Structurale (B3S), Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), and Biochimie Structurale des Microtubules, des Kinésines et de leurs Cargos (MIKICA)

Subjects
TPR domain, axonal-transport, MIKICA, [SDV]Life Sciences [q-bio], Kinesins, Calorimetry, autoinhibitory LFP-acidic motif, Binding, Competitive, kinesin, protein-protein interaction, motor protein, Animals, Humans, cargo, isothermal titration calorimetry (ITC), Adaptor Proteins, Signal Transducing, MST, Binding Sites, AMIG, SH2D6, Y-acidic motif, protein engineering, sequence, Alcadein, PIM, Protein Transport, TorsinA, PF, JNK-interacting protein 1, site-directed mutagenesis, Microtubule-Associated Proteins, B3S, Molecular Biophysics, performance, kinesin light chain, Protein Binding
Abstract

JIP1 was first identified as scaffold protein for the MAP kinase JNK and is a cargo protein for the kinesin1 molecular motor. JIP1 plays significant and broad roles in neurons, mainly as a regulator of kinesin1-dependent transport, and is associated with human pathologies such as cancer and Alzheimer disease. JIP1 is specifically recruited by the kinesin-light chain 1 (KLC1) of kinesin1, but the details of this interaction are not yet fully elucidated. Here, using calorimetry, we extensively biochemically characterized the interaction between KLC1 and JIP1. Using various truncated fragments of the tetratricopeptide repeat (TPR) domain of KLC1, we narrowed down its JIP1-binding region and identified seven KLC1 residues critical for JIP1 binding. These isothermal titration calorimetry (ITC)-based binding data enabled us to footprint the JIP1-binding site on KLC1-TPR. This footprint was used to uncover the structural basis for the marginal inhibition of JIP1 binding by the autoinhibitory LFP-acidic motif of KLC1, as well as for the competition between JIP1 and another cargo protein of kinesin1, the W-acidic motif–containing alcadein-α. Also, we examined the role of each of these critical residues of KLC1 for JIP1 binding in light of the previously reported crystal structure of the KLC1-TPR:JIP1 complex. Finally, sequence search in eukaryotic genomes identified several proteins, among which is SH2D6, that exhibit a motif similar to the KLC1-binding motif of JIP1. Overall, our extensive biochemical characterization of the KLC:JIP1 interaction, as well as identification of potential KLC1-binding partners, improves the understanding of how this growing family of cargos is recruited to kinesin1 by KLC1.

Published
2018

32. Regulation of hydroxycinnamic acid degradation drives Agrobacterium fabrum lifestyles

Author

Meyer, Thibault, Renoud, Sébastien, Vigouroux, Armelle, Miomandre, Aurélie, Gaillard, Vincent, Kerzaon, Isabelle, Prigent-Combaret, Claire, Comte, Gilles, Moréra, Solange, Vial, Ludovic, Lavire, Céline, Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), 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), 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), Microbiologie et enzymologie structurale (MESB3S), 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), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, CNRS (Mission pour l'interdisciplinarite, Agromics 2014-2016), EcoGenome project of the French Agence Nationale de la Recherche ANR-BLAN-08-0090, French national program EC2CO-Biohefect/Ecodyn//Dril/MicrobiEn (IBAD), French Ministere de l'Education Nationale, de l'Enseignement Superieur et de la Recherche, FRISBI ANR-10-INSB-05-01, FR BioEnviS Research Federation, Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Ecole Nationale Vétérinaire de Lyon (ENVL), Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Lyon (ENVL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), 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, 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-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, 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-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-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
phenolic-compounds, Coumaric Acids, Molecular Structure, catabolic genes, rhodopseudomonas-palustris, molecular-mechanism, virh2 protein, [SDV]Life Sciences [q-bio], Agrobacterium, family transcription factors, DNA, Gene Expression Regulation, Bacterial, Extinction, Biological, dna-binding, Bacterial Proteins, coenzyme-a, ti plasmid, pseudomonas-fluorescens, B3S, Gene Deletion, ComputingMilieux_MISCELLANEOUS, Protein Binding, MESB3S
Abstract

WOS:000439338300005; International audience; Regulatory factors are key components for the transition between different lifestyles to ensure rapid and appropriate gene expression upon perceiving environmental cues. Agrobacterium fabrum C58 (formerly called A. tumefaciens C58) has two contrasting lifestyles: it can interact with plants as either a rhizo-sphere inhabitant (rhizospheric lifestyle) or a pathogen that creates its own ecological niche in a plant tumor via its tumor-inducing plasmid (pathogenic lifestyle). Hydroxycinnamic acids are known to play an important role in the pathogenic lifestyle of Agrobacterium spp. but can be degraded in A. fabrum species. We investigated the molecular and ecological mechanisms involved in the regulation of A. fabrum species-specific genes responsible for hydroxycinnamic acid degradation. We characterized the effectors (feruloyl-CoA and p-coumaroyl-CoA) and the DNA targets of the MarR transcriptional repressor, which we named HcaR, which regulates hydroxycinnamic acid degradation. Using an hcaR-deleted strain, we further revealed that hydroxycinnamic acid degradation interfere with virulence gene expression. The HcaR deletion mutant shows a contrasting competitive colonization ability, being less abundant than the wild-type strain in tumors but more abundant in the rhizosphere. This supports the view that A. fabrum C58 HcaR regulation through ferulic and p-coumaric acid perception is important for the transition between lifestyles.

Published
2018

33. Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

Author

Claudio Romano, Paola De Angelis, Jie Pan, Marc Bras, Anne M. Griffiths, Karoline Fiedler, Paola Francalanci, Marianna Parlato, Nadine Cerf-Bensussan, Mohammed Zarhrate, Frank M. Ruemmele, Eileen Crowley, Aleixo M. Muise, Rémi Duclaux-Loras, Bernadette Bègue, Fabienne Charbit-Henrion, Nicolas Guegan, Julie Bruneau, Sabine Rakotobe, Nathalie Kapel, Marie‐Helene Le Du, Neil Warner, SERRE, Marie-Claude, Laboratory of Intestinal Immunity (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), GENIUS Group, Service de Gastroentérologie, d'hépatologie et nutrition pédiatrique [CHU Necker], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), The Hospital for sick children [Toronto] (SickKids), University of Messina, Department of Pathology, University of Alabama at Birmingham [ Birmingham] (UAB), 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), Children's Hospital Bambino Gesù IRCCS [Rome], Université Paris Descartes - Paris 5 (UPD5), Service de pathologie [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Plateforme de bioinformatique (UNIV Paris Descartes), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), University of Toronto, and Université Sorbonne Paris Cité (USPC)

Subjects
0301 basic medicine, Agonist, Medicine (General), medicine.drug_class, [SDV]Life Sciences [q-bio], Biology, QH426-470, GPI-Linked Proteins, Inflammatory bowel disease, inflammatory bowel diseases, intestinal phosphatase alkaline, Lipid A, 03 medical and health sciences, INTGEN, R5-920, medicine, Genetics, Homeostasis, Humans, Receptor, Loss function, Research Articles, HEK 293 cells, medicine.disease, Alkaline Phosphatase, 3. Good health, Intestines, [SDV] Life Sciences [q-bio], 030104 developmental biology, HEK293 Cells, Metabolism, inflammatory bowel diseases, intestinal phosphatase alkaline, monogenic disease, Molecular Medicine, Immunology, Mutation, Alkaline phosphatase, monogenic disease, Molecular Medicine, Heterologous expression, Genetics, Gene Therapy & Genetic Disease, Digestive System, B3S, Signal Transduction, Research Article
Abstract

International audience; Herein, we report the first identification of biallelic-inherited mutations inALPIas a Mendelian cause of inflammatory bowel disease in two unrelated patients.ALPIencodes for intestinal phosphatase alkaline, a brush border metalloenzyme that hydrolyses phosphate from the lipid A moiety of lipopolysaccharides and thereby drastically reduces Toll-like receptor 4 agonist activity. Prediction tools and structural modelling indicate that all mutations affect critical residues or inter-subunit interactions, and heterologous expression in HEK293T cells demonstrated that allALPImutations were loss of function.ALPImutations impaired either stability or catalytic activity of ALPI and rendered it unable to detoxify lipopolysaccharide-dependent signalling. Furthermore, ALPI expression was reduced in patients' biopsies, and ALPI activity was undetectable in ALPI-deficient patient's stool. Our findings highlight the crucial role of ALPI in regulating host-microbiota interactions and restraining host inflammatory responses. These results indicate thatALPImutations should be included in screening for monogenic causes of inflammatory bowel diseases and lay the groundwork for ALPI-based treatments in intestinal inflammatory disorders.

Published
2018

34. Cosolubilization of phenanthrene and pyrene in surfactant micelles: Experimental and atomistic simulations studies

Author

Yanfu Wei, Shasha Liu, Xujun Liang, Xiaoyun Yi, Stéphane Abel, Chuling Guo, Zhi Dang, School of Information and Electronics Engineering, Shandong Institute of Business and Technology, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Laboratoire Bioénergétique Membranaire et Stress (LBMS), 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
Hydrogen, [SDV]Life Sciences [q-bio], Inorganic chemistry, chemistry.chemical_element, 02 engineering and technology, 010501 environmental sciences, 01 natural sciences, Micelle, LBMS, Molecular dynamics, chemistry.chemical_compound, Pulmonary surfactant, Phenanthrene, Surfactant, Materials Chemistry, Physical and Theoretical Chemistry, Sodium dodecyl sulfate, Spectroscopy, Cosolubilization, 0105 earth and related environmental sciences, Chemistry, Molecular dynamics simulations, Pyrene, 021001 nanoscience & nanotechnology, Condensed Matter Physics, Atomic and Molecular Physics, and Optics, Electronic, Optical and Magnetic Materials, Solubilization, 0210 nano-technology, B3S
Abstract

Solubilization of mixed phenanthrene (PHE) and pyrene (PYR) in Triton X-100 (TX), sodium dodecyl sulfate (SDS) and mixed TX-SDS surfactant solutions were done to observe their cosolubilization effect. Moreover, molecular dynamics (MD) simulations were performed to reveal how polycyclic aromatic hydrocarbons (PAHs) coexist in the micelle. Cosolubilization of PHE and PYR exhibited synergism along with decreasing synergistic extent with increasing SDS in mixed micelle. MD simulations verified the distribution of PHE and PYR in the shell and core regions of pure SDS and mixed SDS-TX micelles (with molar ratio of 1: 1), which were chosen as the representative systems for simulation study. The movement of PHE and PYR inside the micelle along with their different probability to contact with SDS non‑hydrogen atoms in pure SDS and mixed SDS-TX solubilization systems suggests the different solubilization sites of the two PAHs inside the micelle leading to their coexistence in the micelle. This study implies the significance of considering cosolubilization effects between PAH mixtures in determining surfactant concentration for environmental remediation.

Published
2018

35. Membrane alterations induced by nonstructural proteins of human norovirus

Author

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]

Subjects
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.

Published
2017

36. Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity

Author

Celma, L., Corbinais, C., Vercruyssen, J., Veaute, X., de La Sierra-Gallay, I.L., Guérois, R., Busso, D., Mathieu, A., Marsin, S., Quevillon-Cheruel, S., Radicella, J.P., 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), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), 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), 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), Laboratoire de Recherche sur l'Instabilité Génétique (LRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire d'Etudes de la Réparation de l'ADN (LERA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Assemblage moléculaire et intégrité du génome (AMIG), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Stabilité génétique, Cellules Souches et Radiations (SCSR (U_967)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Commissariat a l'Energie Atomique, Centre National de la Recherche Scientifique, Institut National pour la Santé et la Recherche Médicale [UMR967], University Paris-Sud 11 [UMR8619], French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-01], Indo-French Centre for Promotion of Advanced research (CEFIPRA) [5203-5], French Ministry of Education, DIM-Malinf, Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), and SERRE, Marie-Claude

Subjects
FAAM, Protein Conformation, Hydrolases, [SDV]Life Sciences [q-bio], Oligonucleotides, lcsh:Medicine, Crystallography, X-Ray, Pathology and Laboratory Medicine, Biochemistry, Substrate Specificity, Nucleic Acids, Helicobacter, Medicine and Health Sciences, Amino Acids, lcsh:Science, Crystallography, AMIG, Nucleotides, Organic Compounds, Physics, Condensed Matter Physics, Enzymes, Bacterial Pathogens, [SDV] Life Sciences [q-bio], Physical sciences, Chemistry, Medical Microbiology, Crystal Structure, Pathogens, Basic Amino Acids, B3S, Research Article, Nucleases, Chemical physics, Microbiology, DNA-binding proteins, Solid State Physics, Histidine, Amino Acid Sequence, Microbial Pathogens, Helicobacter pylori, Sequence Homology, Amino Acid, Biology and life sciences, Bacteria, lcsh:R, Organic Chemistry, Organisms, Chemical Compounds, Proteins, Dimers (Chemical physics), Endonucleases, Purines, Enzymology, lcsh:Q
Abstract

International audience; The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 angstrom resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site.

Published
2017

37. Structural plasticity of the N-terminal capping helix of the TPR domain of kinesin light chain

Author

Nguyen, The Quyen, Chenon, Mélanie, Vilela, Fernando, Velours, Christophe, Aumont-Nicaise, Magali, Andreani, Jessica, Fernandez Varela, Paloma, Llinas, Paola, Menetrey, Julie, 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), Mesures d'interactions protéine-protéine (PIM), Département Plateforme (PF I2BC), 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), 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), Biochimie Structurale des Microtubules, des Kinésines et de leurs Cargos (MIKICA), and Département Biochimie, Biophysique et Biologie Structurale (B3S)

Subjects
Models, Molecular, Protein Conformation, alpha-Helical, Protein Conformation, MIKICA, [SDV]Life Sciences [q-bio], Amino Acid Motifs, lcsh:Medicine, Kinesins, Ligands, Mice, Protein Domains, Models, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, lcsh:Science, Conserved Sequence, AMIG, alpha-Helical, lcsh:R, Molecular, Correction, PIM, lcsh:Q, PF, B3S, Microtubule-Associated Proteins
Abstract

International audience; Kinesin1 plays a major role in neuronal transport by recruiting many different cargos through its kinesin light chain (KLC). Various structurally unrelated cargos interact with the conserved tetratricopeptide repeat (TPR) domain of KLC. The N-terminal capping helix of the TPR domain exhibits an atypical sequence and structural features that may contribute to the versatility of the TPR domain to bind different cargos. We determined crystal structures of the TPR domain of both KLC1 and KLC2 encompassing the N-terminal capping helix and show that this helix exhibits two distinct and defined orientations relative to the rest of the TPR domain. Such a difference in orientation gives rise, at the N-terminal part of the groove, to the formation of one hydrophobic pocket, as well as to electrostatic variations at the groove surface. We present a comprehensive structural analysis of available KLC1/2-TPR domain structures that highlights that ligand binding into the groove can be specific of one or the other N-terminal capping helix orientations. Further, structural analysis reveals that the N-terminal capping helix is always involved in crystal packing contacts, especially in a TPR1:TPR1' contact which highlights its propensity to be a protein-protein interaction site. Together, these results underline that the structural plasticity of the N-terminal capping helix might represent a structural determinant for TPR domain structural versatility in cargo binding.

Published
2017

38. Mutations in KEOPS-complex genes cause nephritic syndrome with primary microcephaly

Author

Sebastian A. Leidel, Sarah Vergult, Olivier Gribouval, Olivia Boyer, Annapurna Poduri, Fatih Ozaltin, Heon Yung Gee, Oraly Sanchez-Ferras, Ankana Daga, David A. Sweetser, Chyong Hsin Hsu, Carolin E. Sadowski, Nithiwat Vatanavicharn, Shirlee Shril, Bruno Collinet, Verena Matejas, Jeremy F.P. Ullmann, Jennifer A. Lawson, Weizhen Tan, David Chitayat, Peter Kannu, Emmanuelle Lemyre, Megan T. Cho, Gaëlle H. Martin, Amber Begtrup, Jui Hsing Chang, Matthias T.F. Wolf, Agnieszka Prytuła, Jennifer Hu, Peter C. Dedon, Sik Nin Wong, Gessica Truglio, Maxime Bouchard, Sandra D. Kienast, Tobias Hermle, Merlin Airik, Manish D. Sinha, Rebecca O. Littlejohn, Takashi Shiihara, Daniella Magen, Yu Yuan Ke, Kenza Soulami, Denny Schanze, Chitra Prasad, Dominique Liger, Svjetlana Lovric, Kazuyuki Nakamura, Jameela A. Kari, Wai Ming Lai, Wen Hui Tsai, Jeng Daw Tsai, Eugen Widmeier, Neveen A. Soliman, Tilman Jobst-Schwan, Shazia Ashraf, Amira Masri, Jia Rao, Jillian K. Warejko, Tamara Basta, Martin Zenker, Brendan Beeson, Corinne Antignac, Malcolm Bruce, Patrick E. Gipson, Mónica Furlano, Géraldine Mollet, Johanna Magdalena Schmidt, Jessica L. Waxler, Daniela A. Braun, Karin Scharmann, David Schapiro, Shrikant Mane, Shuan-Pei Lin, Marleen Praet, Patrick M. Gaffney, Werner L. Pabst, Charlotte A. Hoogstraten, Björn Menten, Nina De Rocker, Richard P. Lifton, Anne Claire Boschat, Klaas J. Wierenga, Chao Huei Chen, Cathy Kiraly-Borri, Nathalie Boddaert, Marie Claire Daugeron, Bert Callewaert, Gaik Siew Ch’ng, Sylvia Sanquer, Won-Il Choi, Udo Vester, Herman van Tilbeurgh, Rezan Topaloglu, David Viskochil, Elizabeth Roeder, Friedhelm Hildebrandt, I. Chiara Guerrera, Rhonda E. Schnur, Patrick Rump, Babak Behnam, Patrick Revy, Mastaneh Moghtaderi, Université Paris Descartes - Paris 5 (UPD5), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Sorbonne Paris Cité (USPC), Laboratoire des Maladies Rénales Héréditaires, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), 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), Département Microbiologie (Dpt Microbio), 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), Biologie Cellulaire des Archées (ARCHEE), 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), Service de néphrologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Division of Nephrology, Boston Children's Hospital, Institute of Human Genetics (University Hospital Magdeburg), University Hospital of the Otto von Guericke University of Magdeburg, Service de Radiologie et imagerie médicale [CHU Necker], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), UPMC - UFR Sciences de la vie (UFR 927 ), Université Pierre et Marie Curie - Paris 6 (UPMC), Département Biochimie, Biophysique et Biologie Structurale (B3S), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Plateforme Protéomique Necker [SFR Necker] (PPN - 3P5), Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service de biochimie métabolique [CHU Necker], Toxicité environnementale, cibles thérapeutiques, signalisation cellulaire (T3S - UMR_S 1124), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ghent University Hospital, Institut de génétique et microbiologie [Orsay] (IGM), Institute of Human Genetics [Erlangen, Allemagne], Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, GeneDx [Gaithersburg, MD, USA], Université de Montréal (UdeM), CHU Sainte Justine [Montréal], University of Amman, Cabinet de Néphrologie pédiatrique [Casablanca, Maroc], Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Singapore-MIT Alliance for Research and Technology (SMART), Massachusetts Institute of Technology (MIT), Yale University [New Haven], Yale University School of Medicine, University of Toronto, Center for Medical Genetics [Ghent], Institute of Human Genetics, University Hospital Magdeburg, Service de Génétique Médicale [CHU Necker], Howard Hughes Medical Institute [Chevy Chase] (HHMI), Howard Hughes Medical Institute (HHMI), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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, 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-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, 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-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-Institut de Biologie Intégrative de la Cellule (I2BC), CHU Necker - Enfants Malades [AP-HP]-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University Hospital Erlangen, Université de Montréal [Montréal], Sapienza University [Rome], Singapore-MIT Alliance for Research and Technology (SMART) Centre, CREATE Tower, Singapore 138602, Singapore (SMART), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Howard Hugues Medical Institute, Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), Yale School of Medicine [New Haven, Connecticut] (YSM), and Çocuk Sağlığı ve Hastalıkları

Subjects
Microcephaly, Nephrotic Syndrome, MICROBIO, FAAM, DNA Repair, cell migration, congenital nephrotic syndrome, Apoptosis, DNA damage response, Pediatrics, Galloway Mowat syndrome, Mice, gene silencing, Models, Cell Movement, GALLOWAY-MOWAT SYNDROME, molecular pathology, newborn, caspase 3, KEOPS complex, ARCHEE, microcephaly, Cytoskeleton, Genetics & Heredity, Mutation, Gene knockdown, clinical article, UNFOLDED PROTEIN RESPONSE, Intracellular Signaling Peptides and Proteins, Endoplasmic Reticulum Stress, transfer RNA, 3. Good health, Cell biology, TRANSFER-RNA MODIFICATION, Nephrosis, TPRKB protein, actin filament, phenotype, embryo, Article, loss of function mutation, in vivo study, 03 medical and health sciences, OSGEP protein, protein serine threonine kinase, Genetics, Humans, YEAST, human, mouse, autosomal recessive disorder, animal model, Molecular, MASS-SPECTROMETRY, DNA, zebrafish protein, medicine.disease, LAGE3 protein, Transfer, carrier protein, 030104 developmental biology, proteasome, cell proliferation, Multiprotein Complexes, Unfolded protein response, CRISPR-Cas Systems, Carrier Proteins, Models, Molecular, 0301 basic medicine, SECKEL-SYNDROME, Hernia, Protein Conformation, [SDV]Life Sciences [q-bio], Medizin, Post-Transcriptional, medicine.disease_cause, lethality, Gene Knockout Techniques, TP53RK protein, RNA, Transfer, multiprotein complex, gene mutation, RNA Processing, Post-Transcriptional, Zebrafish, Hiatal, child, biology, Podocytes, LAGE3 protein, human, apoptosis, Metalloendopeptidases, Protein-Serine-Threonine Kinases, unclassified drug, epidermal growth factor, female, O-sialoglycoprotein endopeptidase, endoplasmic reticulum stress, metalloproteinase, B3S, WDR73, RNA Processing, DNA repair, CRISPR-CAS9 system, animal experiment, Protein Serine-Threonine Kinases, GENOME MAINTENANCE, male, medicine, KINASE, Animals, signal peptide, controlled study, TPRKB protein, human, TP53RK protein, human, nonhuman, gene deletion, Telomere Homeostasis, Zebrafish Proteins, Actin cytoskeleton, biology.organism_classification, Molecular biology, actin related protein 2-3 complex, infant, Hernia, Hiatal, adolescent, RNA, homozygosity
Abstract

Galloway-Mowat syndrome (GAMOS) is a severe autosomal-recessive disease characterized by the combination of early-onset steroid-resistant nephrotic syndrome (SRNS) and microcephaly with brain anomalies. To date, mutations of WDR73 are the only known monogenic cause of GAMOS and in most affected individuals the molecular diagnosis remains elusive. We here identify recessive mutations of OSGEP, TP53RK, TPRKB, or LAGE3, encoding the 4 subunits of the KEOPS complex in 33 individuals of 30 families with GAMOS. CRISPR/Cas9 knockout in zebrafish and mice recapitulates the human phenotype of microcephaly and results in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibits cell proliferation, which human mutations fail to rescue, and knockdown of either gene activates DNA damage response signaling and induces apoptosis. OSGEP and TP53RK molecularly interact and co-localize with the actin-regulating ARP2/3 complex. Furthermore, knockdown of OSGEP and TP53RK induces defects of the actin cytoskeleton and reduces migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identify 4 novel monogenic causes of GAMOS, describe the first link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.

Published
2017

39. The paralogs to the C-terminal domain of the cyanobacterial OCP are carotenoid donors to HCPs

Author

Muzzopappa, Fernando, Wilson, Adjélé, Yogarajah, Vinosa, Cot, Sandrine, Perreau, François, Montigny, Cédric, Bourcier de Carbon, Céline, Kirilovsky, Diana, 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), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), 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), 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), Laboratoire des Protéines et Systèmes Membranaires (LPSM), Agence Nationale de la Recherche (ANR projects CYANOPROTECT [ANR-11-BSV8-0003] and RECYFUEL [ANR-16-CE05-0026]) and by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 675006 (SE2B). F.M.’s salary is financed by SE2B. Labex Saclay Plant Sciences-SPS (ANR- 10-LABX-0040-SPS). French Infrastructure for Integrated Structural Biology (ANR-10-INBS-05), and ANR-16-CE05-0026,ReCyFuel,Régulations de la photosynthèse et production de biofuels par les cyanobactéries(2016)

Subjects
LPSM, organic chemicals, [SDV]Life Sciences [q-bio], polycyclic compounds, food and beverages, MROP, macromolecular substances, B3S, biological factors
Abstract

The photoactive Orange Carotenoid Protein photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain (NTD) is the active part of the protein and the C-terminal domain (CTD) regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins or HCPs), paralogs of NTD-OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized and proved to be very good singlet oxygen quenchers. When synthesized in E. coli or Synechocystis PCC 6803, CTDHs form dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable. HCPs need the presence of CTDH to become holo-proteins. We propose that in cyanobacteria the CTDHs are carotenoid donors to HCPs.

Published
2017

40. The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation

Author

Grzela, Renata, Nusbaum, Julien, Fieulaine, Sonia, Lavecchia, Francesco, Bienvenut, Willy V., Dian, Cyril, Meinnel, Thierry, Giglione, Carmela, Interactions et mécanismes d’assemblage des protéines et des peptides (IMAPP), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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)-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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Maturation des proteines, destinée cellulaire et thérapeutique (PROMTI), and Département Biologie des Génomes (DBG)

Subjects
Models, Molecular, IMAPP, Protein Conformation, DBG, [SDV]Life Sciences [q-bio], lcsh:R, fungi, lcsh:Medicine, Crystallography, X-Ray, Article, Amidohydrolases, lcsh:Q, Bacteriophages, Vibrio parahaemolyticus, lcsh:Science, B3S, PROMTI, Phylogeny
Abstract

International audience; Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

Published
2017

41. Structures of biomolecular complexes by combination of NMR and cryoEM methods

Author

Sophie Zinn-Justin, Elena V. Orlova, Paulo Tavares, Philippe Cuniasse, 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), 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), Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), 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), Bactériophages des bactéries gram positive (PHAG+ ), Département Virologie (Dpt Viro), Institute of Structural & Molecular Biology, Birkbeck College, and University College of London [London] (UCL)

Subjects
0301 basic medicine, Amyloid, VIRO, Macromolecular Substances, Functional dynamics, [SDV]Life Sciences [q-bio], Supramolecular chemistry, Biology, bcs, 03 medical and health sciences, INTGEN, PHAG+, Structural Biology, Humans, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, Biological sciences, Cryoelectron Microscopy, Virion, Hybrid approach, Amyloid fibril, Solution structure, Cellular machines, Crystallography, 030104 developmental biology, Structural biology, Biophysics, B3S
Abstract

CryoEM is presently providing structures of biocomplexes considered intractable to analysis\ud by other structural techniques. NMR is playing an important role in delivering structural information\ud on dynamics events and conformational heterogeneity. Impressive results were obtained by\ud combining cryoEM and either liquid- or solid-state NMR, revealing the structures of cellular\ud machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were\ud fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher\ud their assembly mechanisms and describe their functional dynamics. Modeling based on solid-state\ud NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches\ud validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new\ud structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR\ud became an established hybrid approach in structural biology that significantly contributes to our\ud understanding of functional mechanisms in supramolecular assemblies.

Published
2017

42. Polyvinylpyrrolidone Behavior in Water/Ethanol Mixed Solvents: Comparison of Modeling Predictions with Experimental Results

Author

Moez Guettari, Stéphane Abel, Tahar Tajouri, Amal Belaidi, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Laboratoire Bioénergétique Membranaire et Stress (LBMS), 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), 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 Préparatoire aux Etudes d'Ingénieur de Tunis (IPEIT), and Université de Tunis

Subjects
Hydrodynamic radius, [SDV]Life Sciences [q-bio], Biophysics, Thermodynamics, 02 engineering and technology, 010402 general chemistry, Mole fraction, 01 natural sciences, Biochemistry, LBMS, Dynamic light scattering, medicine, Organic chemistry, Physical and Theoretical Chemistry, Molecular Biology, chemistry.chemical_classification, Polyvinylpyrrolidone, Polymer, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Solvent, chemistry, Virial coefficient, 0210 nano-technology, Ternary operation, B3S, medicine.drug
Abstract

The objective of this work is to study the behavior of a neutral polymer, polyvinylpyrrolidone (PVP), in a mixture of water and ethanol. A comparison of the experimental results with a theoretical model of effective solvent interaction with polymer (ESIP) was made. To do so, dynamic light scattering experiments were used to measure the hydrodynamic radius of PVP (M w = 3.6 × 105 g·mol−1) as a function of the ethanol fraction, x A, in the medium at 25 °C. We show that the polymer adopts an ideal chain–globule–coil conformation transition as the ethanol molar fraction varies. This transition is attributed to the change of the solvent quality which results from water and ethanol complex formation. On the other hand, the ternary PVP/water/ethanol system was described by the ESIP model. From the polymer–effective solvent interaction, the second virial coefficient of the polymer/effective solvent and the preferential adsorption parameter were calculated. The obtained results are in agreement with the reported experiments.

Published
2017

43. Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter

Author

Ekaitz Errasti-Murugarren, Arturo Rodríguez-Banqueri, José Luis Vázquez-Ibar, 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), 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), Laboratoire des Protéines et Systèmes Membranaires (LPSM), 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 Burgess-Brown, Nicola A.

Subjects
0301 basic medicine, 030102 biochemistry & molecular biology, biology, Membrane transport protein, [SDV]Life Sciences [q-bio], Mutagenesis (molecular biology technique), Protein engineering, Molecular biology, Green fluorescent protein, 03 medical and health sciences, Transmembrane domain, 030104 developmental biology, LPSM, Biochemistry, Membrane protein, biology.protein, Amino acid transporter, Heterologous expression, B3S, ComputingMilieux_MISCELLANEOUS
Abstract

Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.

Published
2017

44. Editorial

Author

Anja Krieger-Liszkay, Pierre Cardol, Génétique et Physiologie des Microalgues, Université de Liège, 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), 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), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), 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 Université de Liège

Subjects
0106 biological sciences, 0301 basic medicine, Cyanobacteria, Light, Physiology, [SDV]Life Sciences [q-bio], Plant Science, Biology, Photosynthesis, 01 natural sciences, Electron Transport, 03 medical and health sciences, Algae, Chlorophyta, Genetics, Microalgae, Ecology, Evolution of photosynthesis, Cell Biology, General Medicine, Carbon Dioxide, Plants, biology.organism_classification, Electron transport chain, Oxygen reduction, Oxygen, 030104 developmental biology, Electron flow, MROP, Excess energy, B3S, 010606 plant biology & botany
Abstract

Understanding of the molecular mechanisms of photosynthetic electron and proton transports and their regulation in plants and algae in response to changes in environmental conditions is an important issue for fundamental research on photosynthesis, and may extend even to practical applications by identifying important sites for improvement of photosynthesis. The significance and often centrality of regulatory mechanisms of photosynthetic electron transport is well established for processes in plant acclimation. In recent years, significant advancements have been achieved in understanding of regulatory processes such as dissipation of excess energy in the antenna systems, state transitions, cyclic electron flow, oxygen reduction by flavodiiron enzymes and many others.

Published
2017

45. ComB proteins expression levels determine Helicobacter pylori competence capacity

Author

Corbinais, Christopher, Mathieu, Aurélie, Damke, Prashant P., Kortulewski, Thierry, Busso, Didier, Prado-Acosta, Mariano, Radicella, J. Pablo, Marsin, Stéphanie, Laboratoire de Recherche sur l'Instabilité Génétique (LRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Stabilité génétique, Cellules Souches et Radiations (SCSR (U_967)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, 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), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), and SERRE, Marie-Claude

Subjects
DNA, Bacterial, FAAM, DNA Repair, Helicobacter pylori, [SDV]Life Sciences [q-bio], Gene Expression Regulation, Bacterial, Dna, Article, Helicobacter Pylori, Transformation, [SDV] Life Sciences [q-bio], Ciencias Biológicas, purl.org/becyt/ford/1 [https], Transformation, Genetic, Bacterial Proteins, Biología Celular, Microbiología, Competence, Mutation, Operon, purl.org/becyt/ford/1.6 [https], B3S, CIENCIAS NATURALES Y EXACTAS, DNA Damage
Abstract

Helicobacter pylori chronically colonises half of the world's human population and is the main cause of ulcers and gastric cancers. Its prevalence and the increase in antibiotic resistance observed recently reflect the high genetic adaptability of this pathogen. Together with high mutation rates and an efficient DNA recombination system, horizontal gene transfer through natural competence makes of H. pylori one of the most genetically diverse bacteria. We show here that transformation capacity is enhanced in strains defective for recN, extending previous work with other homologous recombination genes. However, inactivation of either mutY or polA has no effect on DNA transformation, suggesting that natural competence can be boosted in H. pylori by the persistence of DNA breaks but not by enhanced mutagenesis. The transformation efficiency of the different DNA repair impaired strains correlates with the number of transforming DNA foci formed on the cell surface and with the expression of comB8 and comB10 competence genes. Overexpression of the comB6-B10 operon is sufficient to increase the transformation capacity of a wild type strain, indicating that the ComB complex, present in the bacterial wall and essential for DNA uptake, can be a limiting factor for transformation efficiency. Fil: Corbinais, Christopher. Inserm; Francia. Institute of Molecular and Cellular Radiobiology; Francia. Université Paris Sud; Francia Fil: Mathieu, Aurélie. Institute of Molecular and Cellular Radiobiology; Francia Fil: Damke, Prashant P.. Institute of Molecular and Cellular Radiobiology; Francia. Inserm; Francia. Université Paris Sud; Francia Fil: Kortulewski, Thierry. Institute of Molecular and Cellular Radiobiology; Francia. Inserm; Francia. Université Paris Sud; Francia Fil: Busso, Didier. Institute of Molecular and Cellular Radiobiology; Francia. Inserm; Francia. Université Paris Sud; Francia Fil: Prado Acosta, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Institute of Molecular and Cellular Radiobiology; Francia Fil: Radicella, J. Pablo. Institute of Molecular and Cellular Radiobiology; Francia. Inserm; Francia. Université Paris Sud; Francia Fil: Marsin, Stéphanie. Institute of Molecular and Cellular Radiobiology; Francia. Inserm; Francia. Université Paris Sud; Francia

Published
2017

46. Ultrafast Dynamics at the Nanoscale: Biomolecules and Supramolecular Assemblies

Author

Burghardt, Irene, Haacke, Stefan, Institut für Physikalische und Theoretische Chemie [Frankfurt am Main] (PTC), Goethe-Universität Frankfurt am Main, Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), Université de Strasbourg (UNISTRA)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-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)-Institut de Chimie du CNRS (INC)-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)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-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), Burghardt, Irene and Haacke, and Stefan

Subjects
LPB, [SDV]Life Sciences [q-bio], B3S, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2017

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