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  • L. Becker, S. Bellow, V. Carré, G. Latouche, A. Poutaraud, D. Merdinoglu, S. C. Brown, Z. G. Cerovic, and P. Chaimbault, “Correlative Analysis of Fluorescent Phytoalexins by Mass Spectrometry Imaging and Fluorescence Microscopy in Grapevine Leaves”, Analytical Chemistry, 2017.
    Abstract: Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.
    Tags: IMAGIF, PF, PHOT.

  • C. Bou-Nader, D. Cornu, V. Guerineau, T. Fogeron, M. Fontecave, and D. Hamdane, “Enzyme Activation with a Synthetic Catalytic Co-enzyme Intermediate: Nucleotide Methylation by Flavoenzymes”, Angewandte Chemie (International Ed. in English), 2017.
    Abstract: To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.
    Tags: artificial enzymes, flavoenzyme mechanism, Methylation, Nucleotides, PF, reaction intermediates, SICAPS.

  • C. Bou-Nader, L. Pecqueur, D. Cornu, M. Lombard, M. Dezi, M. Nicaise, C. Velours, M. Fontecave, and D. Hamdane, “Power of protein/tRNA functional assembly against aberrant aggregation”, Physical chemistry chemical physics: PCCP, 2017.
    Abstract: Understanding the mechanisms of protein oligomerization and aggregation is a major concern for biotechnology and medical purposes. However, significant challenges remain in determining the mechanism of formation of these superstructures and the environmental factors that can precisely modulate them. Notably the role that a functional ligand plays in the process of protein aggregation is largely unexplored. We herein address these issues with an original flavin-dependent RNA methyltransferase (TrmFO) used as a protein model since this protein employs a complex set of cofactors and ligands for catalysis. Here, we show that TrmFO carries an unstable protein structure that can partially mis-unfold leading to either formation of irregular and nonfunctional soluble oligomers endowed with hyper-thermal stability or large amorphous aggregates in the presence of salts. Mutagenesis confirmed that this peculiarity is an intrinsic property of a polypeptide and it is independent of the flavin coenzyme. Structural characterization and kinetic studies identified several regions of the protein that enjoy conformational changes and more particularly pinpointed the N-terminal subdomain as being a key element in the mechanisms of oligomerization and aggregation. Only stabilization of this region via tRNA suppresses these aberrant protein states. Although protein chaperones emerged as major actors against aggregation, our study emphasizes that other powerful mechanisms exist such as the stabilizing effect of functional assemblies that provide an additional layer of protection against the instability of the proteome.
    Tags: PF, PIM, SICAPS.

  • S. C. Brown, M. Bourge, N. Maunoury, M. Wong, M. W. Bianchi, S. Lepers-Andrzejewski, P. Besse, S. Siljak-Yakovlev, M. Dron, and B. Satiat-Jeunemaître, “DNA remodelling by Strict Partial Endoreplication in orchids, an original process in the plant kingdom”, Genome Biology and Evolution, Apr. 2017.
    Abstract: DNA remodelling during endoreplication appears to be a strong developmental characteristic in orchids. In this study, we analysed DNA content and nuclei in 41 species of orchids to further map the genome evolution in this plant family. We demonstrate that the DNA remodelling observed in 36 out of 41 orchids studied corresponds to strict partial endoreplication. Such process is developmentally regulated in each wild species studied. Cytometry data analyses allowed us to propose a model where nuclear states 2C, 4E, 8E, etc. form a series comprising a fixed proportion, the euploid genome 2C, plus 2 to 32 additional copies of a complementary part of the genome. The fixed proportion ranged from 89% of the genome in Vanilla mexicana down to 19% in V. pompona, the lowest value for all 148 orchids reported. Insterspecific hybridisation did not suppress this phenomenon. Interestingly, this process was not observed in mass-produced epiphytes. Nucleolar volumes grow with the number of endocopies present, coherent with high transcription activity in endoreplicated nuclei. Our analyses suggest species-specific chromatin rearrangement. Towards understanding endoreplication, V. planifolia constitutes a tractable system for isolating the genomic sequences that confer an advantage via endoreplication from those that apparently suffice at diploid level.
    Tags: BIOCELL, CYTO, cytogenetics, cytometry, DYNBSJ, endoreplication, genome imbalance, Genome Size, IMAGIF, MINION, PF, PHOT, Vanilla.

  • E. Dambroise, M. Simion, T. Bourquard, S. Bouffard, B. Rizzi, Y. Jaszczyszyn, M. Bourge, P. Affaticati, A. Heuzé, J. Jouralet, J. Edouard, S. Brown, C. Thermes, A. Poupon, E. Reiter, F. Sohm, F. Bourrat, and J. - S. Joly, “Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile: Features of Neuroepithelial Cells in Fish”, STEM CELLS, 2017.
    Tags: BMgif, CYTO, IMAGIF, NGS, PF, PHOT.

  • T. Di Meo, W. Ghattas, C. Herrero, C. Velours, P. Minard, J. - P. Mahy, R. Ricoux, and A. Urvoas, “αRep A3: A versatile artificial scaffold for metalloenzyme design”, Chemistry (Weinheim an Der Bergstrasse, Germany), 2017.
    Abstract: 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.
    Tags: artificial repeat proteins, B3S, Diels-Alder reaction, Enantioselective Catalysis, MIP, PF, PIM.

  • R. El Helou, G. Pinna, O. Cabaud, J. Wicinski, R. Bhajun, L. Guyon, C. Rioualen, P. Finetti, A. Gros, B. Mari, P. Barbry, F. Bertucci, G. Bidaut, A. Harel-Bellan, D. Birnbaum, E. Charafe-Jauffret, and C. Ginestier, “miR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling”, Cell Reports, vol. 18, no. 9, p. 2256-2268, 2017.

  • C. Esnault, D. Leiber, C. Toffano-Nioche, Z. Tanfin, and M. - J. Virolle, “Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity”, Applied Microbiology and Biotechnology, vol. 101, no. 1, p. 139-145, 2017.
    Abstract: Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [(3)H]phosphatidic acid (PA) released from [(3)H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.
    Tags: Amino Acid Motifs, Cell Membrane, Choline, DBG, eBio, Hydrolysis, Lipid droplets, MESMIC, MICROBIO, PF, Phosphatidic Acids, Phosphatidylcholines, Phospholipase D, Phosphotransferases (Phosphate Group Acceptor), Polyphosphate kinase, Promiscuous enzyme, Protein Conformation, SSFA, Streptomyces lividans.

  • R. Grzela, J. Nusbaum, S. Fieulaine, F. Lavecchia, M. Desmadril, N. Nhiri, A. Van Dorsselaer, S. Cianferani, E. Jacquet, T. Meinnel, and C. Giglione, “Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts”, Biochimica Et Biophysica Acta, Oct. 2017.
    Abstract: 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.
    Tags: B3S, DBG, Enzyme mechanism, IMAPP, N-terminal methionine excision, Peptide deformylase, PF, PIM, PROMTI, structure, Virus.

  • J. Lang, A. Vigouroux, A. El Sahili, A. Kwasiborski, M. Aumont-Nicaise, Y. Dessaux, J. A. Shykoff, S. Moréra, and D. Faure, “Fitness costs restrict niche expansion by generalist niche-constructing pathogens”, The ISME Journal, vol. 11, no. 2, p. 374-385, 2017.

  • T. N. Le Lam, C. Morvan, W. Liu, C. Bohn, Y. Jaszczyszyn, and P. Bouloc, “Finding sRNA-associated phenotypes by competition assays: An example with Staphylococcus aureus”, Methods, vol. 117, p. 21-27, 2017.

  • L. Loiseau, C. Fyfe, L. Aussel, M. Hajj Chehade, S. B. Hernández, B. Faivre, D. Hamdane, C. Mellot-Draznieks, B. Rascalou, L. Pelosi, C. Velours, D. Cornu, M. Lombard, J. Casadesús, F. Pierrel, M. Fontecave, and F. Barras, “The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ”, The Journal of Biological Chemistry, 2017.
    Abstract: Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, since both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.
    Tags: bioenergetics, coenzyme Q10 (CoQ10), Electron transfer, Escherichia coli (E. coli), Microbiology, PF, PIM, SICAPS, Ubiquinone.

  • M. Ma, I. Li de La Sierra Gallay, N. Lazar, O. Pellegrini, J. Lepault, C. Condon, D. Durand, and H. van Tilbeurgh, “Trz1, the long form RNase Z from yeast, forms a stable heterohexamer with endonuclease Nuc1 and mutarotase”, The Biochemical Journal, 2017.
    Abstract: Proteomic studies haves established that Trz1, Nuc1 and mutarotase form a complex in yeast. Trz1 is a b-lactamase type RNase composed of two b-lactamase type domains connected by a long linker that is responsible for the endonucleolytic cleavage at the 3'-end of tRNAs during the maturation process (RNase Z activity); Nuc1 is a dimeric mitochondrial nuclease involved in apoptosis, while mutarotase (encoded by YMR099C) catalyzes the conversion between the a- and b-configuration of glucose-6-phosphate. Using gel-filtration, SAXS and electron microscopy we demonstrated that Trz1, Nuc1 and mutarotase form a very stable heterohexamer, composed of two copies of each of the three subunits. A Nuc1 homodimer is at the centre of the complex, creating a two-fold symmetry and interacting with both Trz1 and mutarotase. Enzymatic characterization of the ternary complex revealed that the activities of Trz1 and mutarotase are not affected by complex formation, but that the Nuc1 activity is completely inhibited by mutarotase and partially by Trz1. This suggests that mutarotase and Trz1 might be regulators of the Nuc1 apoptotic nuclease activity.
    Tags: B3S, complex, CRYOEM, endoglucanase, FAAM, mutarotase, PF, RNASeZ, structure.

  • J. Marion, R. Le Bars, B. Satiat-Jeunemaitre, and C. Boulogne, “Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots”, Journal of Structural Biology, 2017.
    Tags: Arabidopsis, BIOCELL, Correlative microscopy, DYNBSJ, GFP, GMA resin, IMAGIF, MET, PF, PHOT, Tokuyasu, Transmission electron microscopy.

  • T. Q. Nguyen, M. Chenon, F. Vilela, C. Velours, M. Aumont-Nicaise, J. Andreani, P. F. Varela, P. Llinas, and J. Ménétrey, “Structural plasticity of the N-terminal capping helix of the TPR domain of kinesin light chain”, PloS One, vol. 12, no. 10, p. e0186354, 2017.
    Abstract: 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.
    Tags: AMIG, Amino Acid Motifs, Amino Acid Sequence, Animals, B3S, Conserved Sequence, Humans, Ligands, Mice, Microtubule-Associated Proteins, MIKICA, Models, Molecular, PF, PIM, Protein Conformation, alpha-Helical, Protein Domains.

  • J. Nikolic, R. Le Bars, Z. Lama, N. Scrima, C. Lagaudrière-Gesbert, Y. Gaudin, and D. Blondel, “Negri bodies are viral factories with properties of liquid organelles”, Nature Communications, vol. 8, no. 1, p. 58, 2017.
    Abstract: Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.

  • J. - A. Pedroza-García, C. Mazubert, I. del Olmo, M. Bourge, S. Domenichini, R. Bounon, Z. Tariq, E. Delannoy, M. Piñeiro, J. A. Jarillo, C. Bergounioux, M. Benhamed, and C. Raynaud, “Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis”, Plant Physiology, vol. 173, no. 3, p. 1735-1749, 2017.

  • P. Pétriacq, L. de Bont, L. Genestout, J. Hao, C. Laureau, I. Florez-Sarasa, T. Rzigui, G. Queval, F. Gilard, C. Mauve, F. Guérard, M. Lamothe-Sibold, J. Marion, C. Fresneau, S. Brown, A. Danon, A. Krieger-Liszkay, R. Berthomé, M. Ribas-Carbo, G. Tcherkez, G. Cornic, B. Pineau, B. Gakière, and R. De Paepe, “Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants”, Plant Physiology, vol. 173, no. 1, p. 434-455, 2017.

  • J. Pirrello, C. Deluche, N. Frangne, F. Gévaudant, E. Maza, A. Djari, M. Bourge, J. - P. Renaudin, S. Brown, C. Bowler, M. Zouine, C. Chevalier, and N. Gonzalez, “Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation”, The Plant Journal: For Cell and Molecular Biology, 2017.
    Abstract: As part of normal development most eukaryotic organisms ranging from insects to mammals and plants display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains opened. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA-Seq approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to study RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location. This article is protected by copyright. All rights reserved.
    Tags: CYTO, data interpretation, endoreduplication, fruit development, PF, RNA-Seq profiling, Solanum lycopersicum, sorted nuclei, tomato.

  • S. Robinson, J. Nevalainen, G. Pinna, A. Campalans, J. P. Radicella, and L. Guyon, “Incorporating interaction networks into the determination of functionally related hit genes in genomic experiments with Markov random fields”, Bioinformatics (Oxford, England), vol. 33, no. 14, p. i170-i179, 2017.
    Abstract: Motivation: Incorporating gene interaction data into the identification of 'hit' genes in genomic experiments is a well-established approach leveraging the 'guilt by association' assumption to obtain a network based hit list of functionally related genes. We aim to develop a method to allow for multivariate gene scores and multiple hit labels in order to extend the analysis of genomic screening data within such an approach. Results: We propose a Markov random field-based method to achieve our aim and show that the particular advantages of our method compared with those currently used lead to new insights in previously analysed data as well as for our own motivating data. Our method additionally achieves the best performance in an independent simulation experiment. The real data applications we consider comprise of a survival analysis and differential expression experiment and a cell-based RNA interference functional screen. Availability and implementation: We provide all of the data and code related to the results in the paper. Contact: or Supplementary information: Supplementary data are available at Bioinformatics online.
    Tags: PARI, PF.

  • C. Samson, F. Celli, K. Hendriks, M. Zinke, N. Essawy, I. Herrada, A. - A. Arteni, F. - X. Theillet, B. Alpha-Bazin, J. Armengaud, C. Coirault, A. Lange, and S. Zinn-Justin, “Emerin self-assembly mechanism: role of the LEM domain”, The FEBS Journal, vol. 284, no. 2, p. 338-352, 2017.

  • P. V. Sauer, J. Timm, D. Liu, D. Sitbon, E. Boeri-Erba, C. Velours, N. Mücke, J. Langowski, F. Ochsenbein, G. Almouzni, and D. Panne, “Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1”, eLife, vol. 6, Mar. 2017.

  • A. K. Sinha, A. Durand, J. - M. Desfontaines, I. Iurchenko, H. Auger, D. R. F. Leach, F. - X. Barre, and B. Michel, “Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme”, PLoS genetics, vol. 13, no. 10, p. e1006895, 2017.
    Abstract: Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.
    Tags: DBG, EMC2, NGS, PF, STABAC.

  • Z. M. Song, L. Bouchab, E. Hudik, R. Le Bars, O. Nüsse, and S. Dupré-Crochet, “Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome”, Journal of Leukocyte Biology, p. jlb.1A0716-305R, Jan. 2017.

  • A. Talagas, L. Fontaine, L. Ledesma-García, J. Mignolet, I. Li de la Sierra-Gallay, N. Lazar, M. Aumont-Nicaise, M. J. Federle, G. Prehna, P. Hols, and S. Nessler, “Correction: Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS”, PLOS Pathogens, vol. 13, no. 2, p. e1006208, Feb. 2017.

  • P. P. Weil, Y. Jaszczyszyn, A. Baroin-Tourancheau, J. Postberg, and L. Amar, “Holistic and Affordable Analyses of MicroRNA Expression Profiles Using Tagged cDNA Libraries and a Multiplex Sequencing Strategy”, Methods in Molecular Biology (Clifton, N.J.), vol. 1654, p. 179-196, 2017.
    Abstract: Small and long noncoding RNAs (ncRNAs) are key regulators of gene expression. Variations in ncRNA expression patterns can consequently affect the control of many cellular processes. Not just since 2006, when Andrew Z Fire and Craig C Mello were jointly awarded The Nobel Prize in Physiology or Medicine for their discovery of RNA interference, great efforts were undertaken to unleash the biomedical applicability of small noncoding RNAs, in particular microRNAs. With the technological evolution of massive parallel sequencing technologies over the last years, which now are available for an increasing number of scientists, there is a demand for comprehensible and efficient workflows reliable even for unique and valuable clinical specimens. Here we describe a highly reproducible low-cost protocol for analyses of miRNA expression patterns using tagged cDNA libraries and a multiplex sequencing strategy following an Illumina-like protocol. This protocol easily allows the identification of expression differences from samples of tissues of 1-2 mm(3) and fluids of 50-200 μL. We further provide entry points into useful computational biology applications, whose target groups explicitly involve non-bioinformaticians.
    Tags: microRNA, miRNome, Multiplex sequencing, NGS, PF.

  • Y. Wu, V. Pons, A. Goudet, L. Panigai, A. Fischer, J. - A. Herweg, S. Kali, R. A. Davey, J. Laporte, C. Bouclier, R. Yousfi, C. Aubenque, G. Merer, E. Gobbo, R. Lopez, C. Gillet, S. Cojean, M. R. Popoff, P. Clayette, R. Le Grand, C. Boulogne, N. Tordo, E. Lemichez, P. M. Loiseau, T. Rudel, D. Sauvaire, J. - C. Cintrat, D. Gillet, and J. Barbier, “ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments”, Scientific Reports, vol. 7, no. 1, p. 15567, 2017.
    Abstract: Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
    Tags: MET, PF.


  • S. Ahmad, L. Pecqueur, B. Dreier, D. Hamdane, M. Aumont-Nicaise, A. Plückthun, M. Knossow, and B. Gigant, “Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin”, Scientific Reports, vol. 6, p. 28922, Jul. 2016.

  • C. Aillaud, C. Bosc, Y. Saoudi, E. Denarier, L. Peris, L. Sago, N. Taulet, A. Cieren, O. Tort, M. M. Magiera, C. Janke, V. Redeker, A. Andrieux, and M. - J. Moutin, “Evidence for new C-terminally truncated variants of α- and β-tubulins”, Molecular Biology of the Cell, vol. 27, no. 4, p. 640-653, 2016.
    Abstract: Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.
    Tags: Amino Acid Sequence, Animals, Brain, Carboxypeptidases, cell cycle, Gene Knockdown Techniques, HEK293 Cells, HeLa Cells, Humans, Mass Spectrometry, Mice, Microtubules, Molecular Sequence Data, Neurogenesis, Neurons, Peptide Synthases, PF, Protein Processing, Post-Translational, SICAPS, Tubulin, Tyrosine.

  • M. Benincasa, Q. Barrière, G. Runti, O. Pierre, M. Bourge, M. Scocchi, and P. Mergaert, “Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria”, BIO-PROTOCOL, vol. 6, no. 23, 2016.

  • C. Chaintreuil, D. Gully, C. Hervouet, P. Tittabutr, H. Randriambanona, S. C. Brown, G. P. Lewis, M. Bourge, F. Cartieaux, M. Boursot, H. Ramanankierana, A. D'Hont, N. Teaumroong, E. Giraud, and J. - F. Arrighi, “The evolutionary dynamics of ancient and recent polyploidy in the African semiaquatic species of the legume genus Aeschynomene”, The New Phytologist, vol. 211, no. 3, p. 1077-1091, 2016.
    Abstract: The legume genus Aeschynomene is notable in the ability of certain semiaquatic species to develop nitrogen-fixing stem nodules. These species are distributed in two clades. In the first clade, all the species are characterized by the use of a unique Nod-independent symbiotic process. In the second clade, the species use a Nod-dependent symbiotic process and some of them display a profuse stem nodulation as exemplified in the African Aeschynomene afraspera. To facilitate the molecular analysis of the symbiotic characteristics of such legumes, we took an integrated molecular and cytogenetic approach to track occurrences of polyploidy events and to analyze their impact on the evolution of the African species of Aeschynomene. Our results revealed two rounds of polyploidy: a paleopolyploid event predating the African group and two neopolyploid speciations, along with significant chromosomal variations. Hence, we found that A. afraspera (8x) has inherited the contrasted genomic properties and the stem-nodulation habit of its parental lineages (4x). This study reveals a comprehensive picture of African Aeschynomene diversification. It notably evidences a history that is distinct from the diploid Nod-independent clade, providing clues for the identification of the specific determinants of the Nod-dependent and Nod-independent symbiotic processes, and for comparative analysis of stem nodulation.
    Tags: Aeschynomene, CYTO, dysploidy, genome downsizing, IMAGIF, PF, PHOT, Polyploidy, stem nodulation, Symbiosis.

  • A. de Saint Germain, G. Clavé, M. - A. Badet-Denisot, J. - P. Pillot, D. Cornu, J. - P. Le Caer, M. Burger, F. Pelissier, P. Retailleau, C. Turnbull, S. Bonhomme, J. Chory, C. Rameau, and F. - D. Boyer, “An histidine covalent receptor and butenolide complex mediates strigolactone perception”, Nature Chemical Biology, vol. 12, no. 10, p. 787-794, 2016.
    Abstract: Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.
    Tags: PF, SICAPS.

  • E. Deforzh, T. Vargas, J. Kropp, M. Vandamme, G. Pinna, and A. Polesskaya, “IMP-3 protects the mRNAs of cyclins D1 and D3 from GW182/AGO2-dependent translational repression”, International Journal of Oncology, Oct. 2016.

  • T. Eychenne, E. Novikova, M. - B. Barrault, O. Alibert, C. Boschiero, N. Peixeiro, D. Cornu, V. Redeker, L. Kuras, P. Nicolas, M. Werner, and J. Soutourina, “Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture”, Genes & Development, vol. 30, no. 18, p. 2119-2132, 2016.
    Abstract: Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts.
    Tags: DBG, GTR, Mediator, PEPS, PF, preinitiation complex, promoter architecture, RNA polymerase II transcription, Saccharomyces cerevisiae, SICAPS, TFIIB.

  • S. Fetics, A. Thureau, V. Campanacci, M. Aumont-Nicaise, I. Dang, A. Gautreau, J. Pérez, and J. Cherfils, “Hybrid Structural Analysis of the Arp2/3 Regulator Arpin Identifies Its Acidic Tail as a Primary Binding Epitope”, Structure (London, England: 1993), vol. 24, no. 2, p. 252-260, 2016.
    Abstract: Arpin is a newly discovered regulator of actin polymerization at the cell leading edge, which steers cell migration by exerting a negative control on the Arp2/3 complex. Arpin proteins have an acidic tail homologous to the acidic motif of the VCA domain of nucleation-promoting factors (NPFs). This tail is predicted to compete with the VCA of NPFs for binding to the Arp2/3 complex, thereby mitigating activation and/or tethering of the complex to sites of actin branching. Here, we investigated the structure of full-length Arpin using synchrotron small-angle X-ray scattering, and of its acidic tail in complex with an ankyrin repeats domain using X-ray crystallography. The data were combined in a hybrid model in which the acidic tail extends from the globular core as a linear peptide and forms a primary epitope that is readily accessible in unbound Arpin and suffices to tether Arpin to interacting proteins with high affinity.
    Tags: Actin-Related Protein 2-3 Complex, Animals, B3S, Binding Sites, Carrier Proteins, Crystallography, X-Ray, Epitopes, Fish Proteins, Fishes, Humans, MIKICA, Models, Molecular, PF, PIM, Protein Binding, Protein Conformation, Scattering, Small Angle, X-Ray Diffraction.

  • E. Galli, M. Poidevin, R. Le Bars, J. - M. Desfontaines, L. Muresan, E. Paly, Y. Yamaichi, and F. - X. Barre, “Cell division licensing in the multi-chromosomal Vibrio cholerae bacterium”, Nature Microbiology, vol. 1, no. 9, p. 16094, Jun. 2016.

  • J. Hai, N. Serradji, L. Mouton, V. Redeker, D. Cornu, J. - M. El Hage Chahine, P. Verbeke, and M. Hémadi, “Targeted Delivery of Amoxicillin to C. trachomatis by the Transferrin Iron Acquisition Pathway”, PloS One, vol. 11, no. 2, p. e0150031, 2016.
    Abstract: Weak intracellular penetration of antibiotics makes some infections difficult to treat. The Trojan horse strategy for targeted drug delivery is among the interesting routes being explored to overcome this therapeutic difficulty. Chlamydia trachomatis, as an obligate intracellular human pathogen, is responsible for both trachoma and sexually transmitted diseases. Chlamydia develops in a vacuole and is therefore protected by four membranes (plasma membrane, bacterial inclusion membrane, and bacterial membranes). In this work, the iron-transport protein, human serum-transferrin, was used as a Trojan horse for antibiotic delivery into the bacterial vacuole. Amoxicillin was grafted onto transferrin. The transferrin-amoxicillin construct was characterized by mass spectrometry and absorption spectroscopy. Its affinity for transferrin receptor 1, determined by fluorescence emission titration [KaffTf-amox = (1.3 ± 1.0) x 108], is very close to that of transferrin [4.3 x 108]. Transmission electron and confocal microscopies showed a co-localization of transferrin with the bacteria in the vacuole and were also used to evaluate the antibiotic capability of the construct. It is significantly more effective than amoxicillin alone. These promising results demonstrate targeted delivery of amoxicillin to suppress Chlamydia and are of interest for Chlamydiaceae and maybe other intracellular bacteria therapies.
    Tags: Amoxicillin, Anti-Bacterial Agents, Chlamydia Infections, Chlamydia trachomatis, Drug Delivery Systems, Humans, Iron, PF, SICAPS, Trachoma, Transferrin, Vacuoles.

  • D. Hamdane, C. Velours, D. Cornu, M. Nicaise, M. Lombard, and M. Fontecave, “A chemical chaperone induces inhomogeneous conformational changes in flexible proteins”, Physical chemistry chemical physics: PCCP, vol. 18, no. 30, p. 20410-20421, 2016.
    Abstract: Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the conformation of the marginally stable LPPs to avoid their inappropriate protein/protein interactions and aggregation.
    Tags: PF, PIM, SICAPS.

  • P. Maisonnasse, E. Bouguyon, G. Piton, A. Ezquerra, C. Urien, C. Deloizy, M. Bourge, J. - J. Leplat, G. Simon, C. Chevalier, S. Vincent-Naulleau, E. Crisci, M. Montoya, I. Schwartz-Cornil, and N. Bertho, “The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model”, Mucosal Immunology, vol. 9, no. 4, p. 835-849, 2016.

  • L. Marty, A. Vigouroux, M. Aumont-Nicaise, Y. Dessaux, D. Faure, and S. Moréra, “Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens”, The Journal of Biological Chemistry, vol. 291, no. 43, p. 22638-22649, 2016.
    Abstract: Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.
    Tags: ABC transporter, B3S, Crystal Structure, DFG, host-pathogen interaction, isothermal titration calorimetry (ITC), mannopine, MESB3S, opine, periplasmic binding protein, PF, PIM, plant pathogen, x-ray crystallography.

  • J. A. Pedroza-Garcia, S. Domenichini, C. Mazubert, M. Bourge, C. White, E. Hudik, R. Bounon, Z. Tariq, E. Delannoy, I. del Olmo, M. Piñeiro, J. A. Jarillo, C. Bergounioux, M. Benhamed, and C. Raynaud, “Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis”, Nucleic Acids Research, p. gkw449, May 2016.

  • N. Petryk, M. Kahli, Y. d'Aubenton-Carafa, Y. Jaszczyszyn, Y. Shen, M. Silvain, C. Thermes, C. - L. Chen, and O. Hyrien, “Replication landscape of the human genome”, Nature Communications, vol. 7, p. 10208, Jan. 2016.

  • A. Polesskaya, G. Pinna, Y. Sassi, M. Vandamme, A. Bigot, V. Mouly, N. Morozova, A. Harel-Bellan, and C. Degerny, “Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation”, Biochimica Et Biophysica Acta (BBA) -Molecular Cell Research, vol. 1863, no. 2, p. 263-270, 2016.
    Abstract: CNOT6L is a deadenylase subunit belonging to the CCR4-NOT complex, a major deadenylase complex in eukaryotes involved at multiple levels in regulation of gene expression. While CNOT6L is expressed in skeletal muscle cells, its specific functions in this tissue are still largely unknown. Our previous work highlighted the functional of CNOT6L in skeletal muscle cell differentiation. To further explore how CNOT6L regulates myogenesis, we used here gene expression analysis to identify CNOT6L mRNA targets in human myoblasts. Among these novel targets, IL-8 (interleukin 8) mRNA was the most upregulated in CNOT6L knock-down (KD) cells. Biochemical approaches and poly (A) tail length assays showed that IL-8 mRNA is a direct target of CNOT6L, and further investigations by loss- and gain-of-function assays pointed out that IL-8 is an important effector of myogenesis. Therefore, we have characterized CNOT6L-IL-8 as a new signaling axis that regulates myogenesis.
    Tags: Adult, Animals, Blotting, Western, Cell Differentiation, Cell Line, Cells, Cultured, CNOT6L, DBG, Differentiation, Gene Expression Profiling, Humans, IL-8, Interleukin-8, Microscopy, Fluorescence, Muscle Development, Muscle Fibers, Skeletal, Muscle, Skeletal, Myoblasts, Myogenesis, Oligonucleotide Array Sequence Analysis, PARI, PF, Post-transcriptional regulation, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleases, RNA Interference, RNA, Messenger, RPTEG, Signal Transduction, Transcription, Genetic.

  • A. Rémion, F. Khoder-Agha, D. Cornu, M. Argentini, V. Redeker, and M. Mirande, “Identification of protein interfaces within the multi-aminoacyl-tRNA synthetase complex: the case of lysyl-tRNA synthetase and the scaffold protein p38”, FEBS open bio, vol. 6, no. 7, p. 696-706, 2016.
    Abstract: Human cytoplasmic lysyl-tRNA synthetase (LysRS) is associated within a multi-aminoacyl-tRNA synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC-associated and MSC-dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N-terminal peptide of p38 was reported [Ofir-Birin Y et al. (2013) Mol Cell 49, 30-42]. In order to identify amino acid residues involved in interaction of the two proteins, the non-natural, photo-cross-linkable amino acid p-benzoyl-l-phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross-linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross-linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.
    Tags: cross‐link, DBG, lysyl‐tRNA synthetase, MARS, multisynthetase complex, p38, PF, protein:protein interaction, SICAPS.

  • A. Talagas, L. Fontaine, L. Ledesma-Garca, J. Mignolet, I. Li de la Sierra-Gallay, N. Lazar, M. Aumont-Nicaise, M. J. Federle, G. Prehna, P. Hols, and S. Nessler, “Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS”, PLOS Pathogens, vol. 12, no. 12, p. e1005980, Dec. 2016.

  • V. D. T. Tran, O. Souiai, N. Romero-Barrios, M. Crespi, and D. Gautheret, “Detection of generic differential RNA processing events from RNA-seq data”, RNA Biology, vol. 13, no. 1, p. 59-67, Jan. 2016.

  • M. Wery, M. Descrimes, N. Vogt, A. - S. Dallongeville, D. Gautheret, and A. Morillon, “Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure”, Molecular Cell, vol. 61, no. 3, p. 379-392, 2016.

  • Z. Yi, M. Manil-Ségalen, L. Sago, A. Glatigny, V. Redeker, R. Legouis, and M. - H. Mucchielli-Giorgi, “SAFER, an Analysis Method of Quantitative Proteomic Data, Reveals New Interactors of the C. elegans Autophagic Protein LGG-1”, Journal of Proteome Research, vol. 15, no. 5, p. 1515-1523, 2016.
    Abstract: Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.
    Tags: atg-8/LC3, Autophagy, BIM, BIOCELL, C. elegans, DBG, label free mass spectrometry, OTOFAG, PF, proteomics, SICAPS, statistical methodology.


  • A. - A. Arteni, M. Fradot, D. Galzerano, M. M. Mendes-Pinto, J. - A. Sahel, S. Picaud, B. Robert, and A. A. Pascal, “Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment”, PLOS ONE, vol. 10, no. 8, p. e0135779, Aug. 2015.
    Tags: B3S, Humans, LBMS, Lutein, Macular Pigment, Molecular Conformation, PF, Retinal Pigments, SE, Spectrum Analysis, Raman, Zeaxanthins.

  • M. Bensussan, V. Lefebvre, A. Ducamp, J. Trouverie, E. Gineau, M. - N. Fortabat, A. Guillebaux, A. Baldy, D. Naquin, S. Herbette, C. Lapierre, G. Mouille, C. Horlow, and M. Durand-Tardif, “Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis”, Plant Physiology, vol. 168, no. 2, p. 452-463, 2015.
    Abstract: eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants.
    Tags: Acetylation, Arabidopsis, Arabidopsis Proteins, Biomass, Cell Wall, Cellulose, Ethyl Methanesulfonate, Glucuronidase, Molecular Sequence Data, Mutation, NGS, PF, Phenotype, Phloem, Plant Vascular Bundle, Suppression, Genetic, Water, Xylem.

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