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Accueil > Publications

Publications de l’I2BC

2019

  • J. Santolini, « What does "NO-Synthase" stand for ? », Frontiers in Bioscience (Landmark Edition), vol. 24, p. 133-171, janv. 2019.
    Résumé : Mammalian NO-Synthases (NOSs) are the enzymatic sources of Nitric Oxide (NO°), a paradigmatic gasotransmitter involved in many (patho)-physiological processes. The increasing number of available genomes led to the identification of hundreds of new NOS proteins throughout the kingdoms of life, calling for a global investigation of this family of proteins. These new NOSs are commonly believed to share the same structure, functioning and role as mammalian NOSs. The scope of this article is to highlight the singularity of these NOSs and to describe their complex structural and functional diversity. NOS appears as a unique enzymatic machinery that exhibits a complex Structure - Activity - Function relationship. Its sophisticated redox mechanism and enzymatic regulation, coupled to the vast biological chemistry of reactive nitrogen species, leads to a specific cross-talk between NOS catalysis and its biological environment that implies a complex evolution of NOS function. This paper addresses the relationship between structure, function and evolution of NOS proteins using three NOS model families and advocates for an integrative and interdisciplinary approach that combines modelling studies, structural characterization, and in vitro/in vivo functional investigations.
    Mots-clés : B3S, LSOD.

2018


  • N. Abdollahi, A. Albani, E. Anthony, A. Baud, M. Cardon, R. Clerc, D. Czernecki, R. Conte, L. David, A. Delaune, S. Djerroud, P. Fourgoux, N. Guiglielmoni, J. Laurentie, N. Lehmann, C. Lochard, R. Montagne, V. Myrodia, V. Opuu, E. Parey, L. Polit, S. Privé, C. Quignot, M. Ruiz-Cuevas, M. Sissoko, N. Sompairac, A. Vallerix, V. Verrecchia, M. Delarue, R. Guérois, Y. Ponty, S. Sacquin-Mora, A. Carbone, C. Froidevaux, S. Le Crom, O. Lespinet, M. Weigt, S. Abboud, J. Bernardes, G. Bouvier, C. Dequeker, A. Ferré, P. Fuchs, G. Lelandais, P. Poulain, H. Richard, H. Schweke, E. Laine, et A. Lopes, « Meet-U: Educating through research immersion », PLoS computational biology, vol. 14, nᵒ 3, p. e1005992, mars 2018.
    Résumé : We present a new educational initiative called Meet-U that aims to train students for collaborative work in computational biology and to bridge the gap between education and research. Meet-U mimics the setup of collaborative research projects and takes advantage of the most popular tools for collaborative work and of cloud computing. Students are grouped in teams of 4-5 people and have to realize a project from A to Z that answers a challenging question in biology. Meet-U promotes "coopetition," as the students collaborate within and across the teams and are also in competition with each other to develop the best final product. Meet-U fosters interactions between different actors of education and research through the organization of a meeting day, open to everyone, where the students present their work to a jury of researchers and jury members give research seminars. This very unique combination of education and research is strongly motivating for the students and provides a formidable opportunity for a scientific community to unite and increase its visibility. We report on our experience with Meet-U in two French universities with master's students in bioinformatics and modeling, with protein-protein docking as the subject of the course. Meet-U is easy to implement and can be straightforwardly transferred to other fields and/or universities. All the information and data are available at www.meet-u.org.
    Mots-clés : AMIG, B3S, BDG, BIM.

  • A. Abou-Hamdan, L. Belot, A. Albertini, et Y. Gaudin, « Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition », Journal of Molecular Biology, avr. 2018.
    Mots-clés : conformational change, glycoprotein, Influenza, membrane fusion, RHABDO, Vesiculovirus, VIRO.

  • P. Abranyi-Balogh, L. Petri, T. Imre, P. Szijj, A. Scarpino, M. Hrast, A. Mitrovic, U. P. Fonovic, K. Nemeth, H. Barreteau, D. I. Roper, K. Horvati, G. G. Ferenczy, J. Kos, J. Ilas, S. Gobec, et G. M. Keseru, « A road map for prioritizing warheads for cysteine targeting covalent inhibitors », European Journal of Medicinal Chemistry, vol. 160, p. 94-107, déc. 2018.
    Résumé : Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved.
    Mots-clés : Cathepsin B, Cathepsin X, cathepsin-b, Covalent inhibitors, design, drug discovery, electrophiles, Electrophilic warheads, ENVBAC, fragments, GSH reactivity assay, kinase inhibitors, MICROBIO, MurA, Oligopeptide specificity assay.


  • C. Adam, R. Guérois, A. Citarella, L. Verardi, F. Adolphe, C. Béneut, V. Sommermeyer, C. Ramus, J. Govin, Y. Couté, et V. Borde, « The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes », PLOS Genetics, vol. 14, nᵒ 2, p. e1007223, févr. 2018.

  • N. Agier, S. Delmas, Q. Zhang, A. Fleiss, Y. Jaszczyszyn, E. van Dijk, C. Thermes, M. Weigt, M. Cosentino-Lagomarsino, et G. Fischer, « The evolution of the temporal program of genome replication », Nature Communications, vol. 9, nᵒ 1, p. 2199, juin 2018.
    Résumé : Genome replication is highly regulated in time and space, but the rules governing the remodeling of these programs during evolution remain largely unknown. We generated genome-wide replication timing profiles for ten Lachancea yeasts, covering a continuous evolutionary range from closely related to more divergent species. We show that replication programs primarily evolve through a highly dynamic evolutionary renewal of the cohort of active replication origins. We found that gained origins appear with low activity yet become more efficient and fire earlier as they evolutionarily age. By contrast, origins that are lost comprise the complete range of firing strength. Additionally, they preferentially occur in close vicinity to strong origins. Interestingly, despite high evolutionary turnover, active replication origins remain regularly spaced along chromosomes in all species, suggesting that origin distribution is optimized to limit large inter-origin intervals. We propose a model on the evolutionary birth, death, and conservation of active replication origins.
    Mots-clés : CHERDIR, NGS, PF.

  • L. Ahmad, S. Plancqueel, V. Dubosclard, N. Lazar, W. Ghattas, I. Li de la Sierra-Gallay, H. van Tilbeurgh, et L. Salmon, « Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-d-arabinonhydrazide », FEBS letters, vol. 592, nᵒ 10, p. 1667-1680, mai 2018.
    Résumé : Type I phosphomannose isomerases (PMIs) are zinc-dependent monofunctional metalloenzymes catalysing the reversible isomerization of d-mannose 6-phosphate to d-fructose 6-phosphate. 5-Phospho-d-arabinonhydrazide (5PAHz), designed as an analogue of the enediolate high-energy intermediate, strongly inhibits PMI from Candida albicans (CaPMI). In this study, we report the 3D crystal structure of CaPMI complexed with 5PAHz at 1.85 Å resolution. The high-resolution structure suggests that Glu294 is the catalytic base that transfers a proton between the C1 and C2 carbon atoms of the substrate. Bidentate coordination of the inhibitor explains the stereochemistry of the isomerase activity, as well as the absence of both anomerase and C2-epimerase activities for Type I PMIs. A detailed mechanism of the reversible isomerization is proposed.
    Mots-clés : B3S, Candida albicans, enzyme mechanism, FAAM, inhibitor, phosphomannose isomerase, zinc metalloenzyme.

  • G. Annio, T. L. Jennings, O. Tagit, et N. Hildebrandt, « Sensitivity Enhancement of Förster Resonance Energy Transfer Immunoassays by Multiple Antibody Conjugation on Quantum Dots », Bioconjugate Chemistry, vol. 29, nᵒ 6, p. 2082-2089, juin 2018.
    Résumé : Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (AB) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many AB per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the AB on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of AB per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both AB and QDs can significantly influence the assay performance.
    Mots-clés : B3S, NANO.

  • M. Ansaldi, L. Debarbieux, S. Gandon, M. - A. Petit, P. Tavares, et P. Boulanger, « "French Phage Network"-Third Meeting Report », Viruses, vol. 10, nᵒ 3, mars 2018.
    Résumé : In its third year of existence, the French Phage Network (Phages.fr) is pursuing its expansion. With more than 25 groups, mostly based in France, working on the various aspects of phage research, the network has increased its visibility, interactivity, and activity. The third meeting of the Phages.fr network, held on November 2017 at the Gif-sur-Yvette Centre National de la Recherche Scientifique (CNRS) campus, was a great opportunity for many young scientists to present their work and interact with more senior scientists, amongst which several were invited from abroad. Here we provide a summary of the work presented at this occasion during the oral presentations and poster sessions.
    Mots-clés : bacteria, bacteriophage, co-evolution, France, genomics, infection, PHAG+, phage therapy, resistance, structural biology, T5PHAG, VIRO, virulence.

  • J. - M. Arbona, A. Goldar, O. Hyrien, A. Arneodo, et B. Audit, « The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation », eLife, vol. 7, juin 2018.
    Résumé : The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris. We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics.
    Mots-clés : BDG, chromosomes, computational biology, gene expression, GTR, human, S. cerevisiae, systems biology.


  • T. Avin-Wittenberg, F. Baluška, P. V. Bozhkov, P. H. Elander, A. R. Fernie, G. Galili, A. Hassan, D. Hofius, E. Isono, R. Le Bars, C. Masclaux-Daubresse, E. A. Minina, H. Peled-Zehavi, N. S. Coll, L. M. Sandalio, B. Satiat-Jeunemaitre, A. Sirko, P. S. Testillano, et H. Batoko, « Autophagy-related approaches for improving nutrient use efficiency and crop yield protection », Journal of Experimental Botany, vol. 69, nᵒ 6, p. 1335-1353, mars 2018.
    Mots-clés : BIOCELL, CYTO, DYNBSJ, PF, PHOT.

  • T. Avin-Wittenberg, F. Baluška, P. V. Bozhkov, P. H. Elander, A. R. Fernie, G. Galili, A. Hassan, D. Hofius, E. Isono, R. Le Bars, C. Masclaux-Daubresse, E. A. Minina, H. Peled-Zehavi, N. S. Coll, L. M. Sandalio, B. Satiat-Jeunemaitre, A. Sirko, P. S. Testillano, et H. Batoko, « Corrigendum: Autophagy-related approaches for improving nutrient use efficiency and crop yield protection », Journal of Experimental Botany, vol. 69, nᵒ 12, p. 3173, mai 2018.
    Mots-clés : BIOCELL, CYTO, DYNBSJ, PF, PHOT.

  • M. Bakail, S. Rodriguez-Marin, Z. Hegedüs, M. E. Perrin, F. Ochsenbein, et A. J. Wilson, « Recognition of ASF1 Using Hydrocarbon Constrained Peptides », Chembiochem: A European Journal of Chemical Biology, déc. 2018.
    Résumé : Inhibition of the histone H3-ASF1 (anti-silencing function 1) protein-protein interaction (PPI) represents a potential approach for treatment of numerous cancers. As an α-helix mediated PPI, constraint of the key histone H3 helix (residues 118-135) represents a strategy through which chemical probes might be elaborated to test this hypothesis. In this work variant H3118-135 peptides bearing pentenyl glycine residues at i and i + 4 positions were constrained by olefin metathesis. Biophysical analyses revealed that promotion of a bioactive helical conformation depends on the position at which the constraint is introduced, but that potency of binding towards ASF1 is unaffected by the constraint and instead that enthalpy-entropy compensation occurs.
    Mots-clés : AMIG, B3S, protein-protein interactions histone chaperonnes constrained peptides protein surface recognition chemical biology.

  • A. Barwinska-Sendra, A. Baslé, K. J. Waldron, et S. Un, « A charge polarization model for the metal-specific activity of superoxide dismutases », Physical chemistry chemical physics: PCCP, janv. 2018.
    Résumé : The pathogenicity of Staphylococcus aureus is enhanced by having two superoxide dismutases (SODs): a Mn-specific SOD and another that can use either Mn or Fe. Using 94 GHz electron-nuclear double resonance (ENDOR) and electron double resonance detected (ELDOR)-NMR we show that, despite their different metal-specificities, their structural and electronic similarities extend down to their active-site 1H- and 14N-Mn(ii) hyperfine interactions. However these interactions, and hence the positions of these nuclei, are different in the inactive Mn-reconstituted Escherichia coli Fe-specific SOD. Density functional theory modelling attributes this to a different angular position of the E. coli H171 ligand. This likely disrupts the Mn-H171-E170' triad causing a shift in charge and in metal redox potential, leading to the loss of activity. This is supported by the correlated differences in the Mn(ii) zero-field interactions of the three SOD types and suggests that the triad is important for determining metal specific activity.
    Mots-clés : B3S, BHFMR.

  • E. L. Bastow, V. S. Garcia de la Torre, A. E. Maclean, R. T. Green, S. Merlot, S. Thomine, et J. Balk, « Vacuolar iron stores gated by NRAMP3 and NRAMP4 are the primary source of iron in germinating seeds », Plant Physiology, vol. 177, nᵒ 3, p. 1267-1276, mai 2018.
    Résumé : During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100 and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1, SAPX), Fe homeostasis (FER1, SUFB) and chlorophyll metabolism (HEMA1, NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4. Together these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids but not mitochondria.
    Mots-clés : BIOCELL, MINION.

  • A. Belyy, U. Mechold, L. Renault, et D. Ladant, « ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview », Toxicon: Official Journal of the International Society on Toxinology, vol. 149, p. 65-71, juill. 2018.
    Résumé : ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin.
    Mots-clés : ACTIN.

  • A. Belyy, I. Santecchia, L. Renault, B. Bourigault, D. Ladant, et U. Mechold, « The extreme C-terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin », The Journal of Biological Chemistry, oct. 2018.
    Résumé : Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyse the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilising yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays and confocal microscopy, we demonstrate that the last 9 amino acids of the C-terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY, that had been fused to a carrier protein, bind to F-actin, and that synthetic peptides corresponding to the extreme ExoY C-terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbour ExoY variants with a mutation altering the C-terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.
    Mots-clés : ACTIN, B3S, ExoY, Pseudomonas aeruginosa (P. aeruginosa).

  • M. Benchouaia, H. Ripoche, M. Sissoko, A. Thiébaut, J. Merhej, T. Delaveau, L. Fasseu, S. Benaissa, G. Lorieux, L. Jourdren, S. Le Crom, G. Lelandais, E. Corel, et F. Devaux, « Comparative Transcriptomics Highlights New Features of the Iron Starvation Response in the Human Pathogen Candida glabrata », Frontiers in Microbiology, vol. 9, p. 2689, 2018.
    Résumé : In this work, we used comparative transcriptomics to identify regulatory outliers (ROs) in the human pathogen Candida glabrata. ROs are genes that have very different expression patterns compared to their orthologs in other species. From comparative transcriptome analyses of the response of eight yeast species to toxic doses of selenite, a pleiotropic stress inducer, we identified 38 ROs in C. glabrata. Using transcriptome analyses of C. glabrata response to five different stresses, we pointed out five ROs which were more particularly responsive to iron starvation, a process which is very important for C. glabrata virulence. Global chromatin Immunoprecipitation and gene profiling analyses showed that four of these genes are actually new targets of the iron starvation responsive Aft2 transcription factor in C. glabrata. Two of them (HBS1 and DOM34b) are required for C. glabrata optimal growth in iron limited conditions. In S. cerevisiae, the orthologs of these two genes are involved in ribosome rescue by the NO GO decay (NGD) pathway. Hence, our results suggest a specific contribution of NGD co-factors to the C. glabrata adaptation to iron starvation.
    Mots-clés : Aft, ascomycete fungi, BDG, BIM, ChIP-seq, comparative genomics, evolution, gene regulatory networks, glutathione transferase, messenger-rna, monothiol glutaredoxins, NO GO decay, oxidative stress-response, schistosoma-mansoni, selenite toxicity, yeast, yeast saccharomyces-cerevisiae.

  • A. Berto, J. Yu, S. Morchoisne-Bolhy, C. Bertipaglia, R. Vallee, J. Dumont, F. Ochsenbein, R. Guerois, et V. Doye, « Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores », EMBO reports, vol. 19, nᵒ 5, mai 2018.
    Résumé : Cenp-F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp-F C-terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp-F targeting to kinetochores. By combining in silico structural modeling and yeast two-hybrid assays, we generate an interaction model between a conserved helix within the Nup133 β-propeller and a short leucine zipper-containing dimeric segment of Cenp-F. We thereby create mutants affecting the Nup133/Cenp-F interface and show that they prevent Cenp-F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp-F KT-core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT-core binding partner of Cenp-F. Finally, we show that Cenp-E redundantly contributes together with Bub1 to the recruitment of Cenp-F to kinetochores.
    Mots-clés : AMIG, B3S, Cenp‐F, in silico modeling, kinetochores, mitosin, nuclear pore.

  • S. Bhullar, C. D. Wilkes, O. Arnaiz, M. Nowacki, L. Sperling, et E. Meyer, « A mating-type mutagenesis screen identifies a zinc-finger protein required for specific DNA excision events in Paramecium », Nucleic Acids Research, vol. 46, nᵒ 18, p. 9550-9562, oct. 2018.
    Résumé : In the ciliate Paramecium tetraurelia, functional genes are reconstituted during development of the somatic macronucleus through the precise excision of similar to 45 000 single-copy Internal Eliminated Sequences (IESs), thought to be the degenerate remnants of ancient transposon insertions. Like introns, IESs are marked only by a weak consensus at their ends. How such a diverse set of sequences is faithfully recognized and precisely excised remains unclear: specialized small RNAs have been implicated, but in their absence up to similar to 60% of IESs are still correctly excised. To get further insight, we designed a mutagenesis screen based on the hypersensitivity of a specific excision event in the mtA gene, which determines mating types. Unlike most IES-containing genes, the active form of mtA is the unexcised one, allowing the recovery of hypomorphic alleles of essential IES recognition/excision factors. Such is the case of one mutation recovered in the Piwi gene PTIWI09, a key player in small RNA-mediated IES recognition. Another mutation identified a novel protein with a C2H2 zinc finger, mtGa, which is required for excision of a small subset of IESs characterized by enrichment in a 5-bp motif. The unexpected implication of a sequence-specific factor establishes a new paradigm for IES recognition and/or excision.
    Mots-clés : ANGE, BDG, developmental excision, differentiation, discovery, genetic-analysis, GTR, internal eliminated sequences, macronuclear development, piwi proteins, rearrangements, rna pathways, tetraurelia.

  • J. Bischerour, S. Bhullar, C. Denby Wilkes, V. Régnier, N. Mathy, E. Dubois, A. Singh, E. Swart, O. Arnaiz, L. Sperling, M. Nowacki, et M. Bétermier, « Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium », eLife, vol. 7, sept. 2018.
    Résumé : The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.
    Mots-clés : BDG, chromosomes, ciliates, DNA elimination, gene expression, GTR, IES, MICMAC, piggyBacv, transposées domestication.

  • J. Bischerour, S. Bhullar, C. D. Wilkes, V. Regnier, N. Mathy, E. Dubois, A. Singh, E. Swart, O. Arnaiz, L. Sperling, M. Nowacki, et M. Betermier, « Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium », Elife, vol. 7, p. e37927, sept. 2018.
    Résumé : The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.
    Mots-clés : ANGE, BDG, genes, genome rearrangements, interference, macronucleus, MICMAC, precise excision, sequences, tetrahymena-thermophila, tetraurelia, transposition, v(d)j recombination.


  • S. Blanchet, D. Cornu, I. Hatin, H. Grosjean, P. Bertin, et O. Namy, « Deciphering the reading of the genetic code by near-cognate tRNA », Proceedings of the National Academy of Sciences, vol. 115, nᵒ 12, p. 3018-3023, mars 2018.

  • M. Blondeau, M. Sachse, C. Boulogne, C. Gillet, J. - M. Guigner, F. Skouri-Panet, M. Poinsot, C. Ferard, J. Miot, et K. Benzerara, « Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria », Frontiers in Microbiology, vol. 9, p. 1768, 2018.
    Résumé : The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature ultramicrotomy and (ii) high-pressure freezing followed by cryo-ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.
    Mots-clés : amorphous calcium carbonate, bacterial microcompartment, biomineralization, ca, calcification, carboxysome, CEMOVIS, MET, PF.

  • E. Bordet, F. Blanc, M. Tiret, E. Crisci, E. Bouguyon, P. Renson, P. Maisonnasse, M. Bourge, J. - J. Leplat, E. Giuffra, L. Jouneau, I. Schwartz-Cornil, O. Bourry, et N. Bertho, « Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells », Frontiers in Immunology, vol. 9, p. 2299, oct. 2018.
    Résumé : Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFN gamma secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.
    Mots-clés : blood, cDc1, cellular response, CYTO, dendritic cells, differentiation, dust mite allergen, europe, Lena, lung, macrophages, monocytes, PF, pigs, prrsv, prrsv strain, sialoadhesin, swine, Th1 response.

  • S. Bouffard, E. Dambroise, A. Brombin, S. Lempereur, I. Hatin, M. Simion, R. Corre, F. Bourrat, J. - S. Joly, et F. Jamen, « Fibrillarin is essential for S-phase progression and neuronal differentiation in zebrafish dorsal midbrain and retina », Developmental Biology, févr. 2018.
    Résumé : Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.
    Mots-clés : BDG, Cell cycle regulation, Danio rerio, Differentiation, GST, Neural progenitors, Optic tectum, Ribosome biogenesis.


  • G. Bourgeois, J. Seguin, M. Babin, P. Belin, M. Moutiez, Y. Mechulam, M. Gondry, et E. Schmitt, « Structural basis for partition of the cyclodipeptide synthases into two subfamilies », Journal of Structural Biology, vol. 203, nᵒ 1, p. 17-26, juill. 2018.
    Résumé : Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
    Mots-clés : Aminoacyl-tRNA synthetases, BIOSYN, Cyclodipeptides, Diketopiperazines, MICROBIO, Non-ribosomal peptide synthesis, Rossmann fold, Transfer RNA.

  • A. Boussac, I. Ugur, A. Marion, M. Sugiura, V. R. I. Kaila, et A. W. Rutherford, « The low spin - high spin equilibrium in the S2-state of the water oxidizing enzyme », Biochimica Et Biophysica Acta, vol. 1859, nᵒ 5, p. 342-356, févr. 2018.
    Résumé : In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0to S4) before water is oxidized and O2is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S2using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S2LSand S2HSis pH dependent, with a pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5and pKa ≈ 7.5 (n ≈ 1) for Mn4SrO5. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pKaupon the Ca/Sr exchange. EPR also showed that NH3addition reversed the effect of high pH, NH3-S2LSbeing present at all pH values studied. Absorption spectroscopy indicates that NH3is no longer bound in the S3TyrZstate, consistent with EPR data showing minor or no NH3-induced modification of S3and S0. In both Ca-PSII and Sr-PSII, S2HSwas capable of advancing to S3at low temperature (198 K). This is an experimental demonstration that the S2LSis formed first and advances to S3via the S2HSstate without detectable intermediates. We discuss the nature of the changes occurring in the S2LSto S2HStransition which allow the S2HSto S3transition to occur below 200 K. This work also provides a protocol for generating S3in concentrated samples without the need for saturating flashes.
    Mots-clés : B3S, DFT, EPR, Mn(4)CaO(5) cluster, Oxygen evolution, Photosystem II, PS2, Spin state.

  • L. Brottier, C. Chaintreuil, P. Simion, C. Scornavacca, R. Rivallan, P. Mournet, L. Moulin, G. P. Lewis, J. Fardoux, S. C. Brown, M. Gomez-Pacheco, M. Bourges, C. Hervouet, M. Gueye, R. Duponnois, H. Ramanankierana, H. Randriambanona, H. Vandrot, M. Zabaleta, M. DasGupta, A. D'Hont, E. Giraud, et J. - F. Arrighi, « A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses », BMC plant biology, vol. 18, nᵒ 1, p. 333, déc. 2018.
    Résumé : BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. RESULTS: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. CONCLUSIONS: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses.
    Mots-clés : Aeschynomene, CHERDIR, Genetics, Legumes, Nodulation, Phylogenetics, Polyploidy, Symbiosis.

  • M. Byrdin, C. Duan, D. Bourgeois, et K. Brettel, « A Long-Lived Triplet State Is the Entrance Gateway to Oxidative Photochemistry in Green Fluorescent Proteins », Journal of the American Chemical Society, vol. 140, nᵒ 8, p. 2897-2905, févr. 2018.
    Résumé : Though ubiquitously used as selective fluorescence markers in cellular biology, fluorescent proteins (FPs) still have not disclosed all of their surprising properties. One important issue, notably for single-molecule applications, is the nature of the triplet state, suggested to be the starting point for many possible photochemical reactions leading to phenomena such as blinking or bleaching. Here, we applied transient absorption spectroscopy to characterize dark states in the prototypical enhanced green fluorescent protein (EGFP) of hydrozoan origin and, for comparison, in IrisFP, a representative phototransformable FP of anthozoan origin. We identified a long-lived (approximately 5 ms) dark state that is formed with a quantum yield of approximately 1% and has pronounced absorption throughout the visible-NIR range (peak at around 900 nm). Detection of phosphorescence emission with identical kinetics and excitation spectrum allowed unambiguous identification of this state as the first excited triplet state of the deprotonated chromophore. This triplet state was further characterized by determining its phosphorescence emission spectrum, the temperature dependence of its decay kinetics and its reactivity toward oxygen and electron acceptors and donors. It is suggested that it is this triplet state that lies at the origin of oxidative photochemistry in green FPs, leading to phenomena such as so-called "oxidative redding", "primed photoconversion", or, in a manner similar to that previously observed for organic dyes, redox induced blinking control with the reducing and oxidizing system ("ROXS").
    Mots-clés : B3S, LPB.

  • T. Candelli, D. Challal, J. - B. Briand, J. Boulay, O. Porrua, J. Colin, et D. Libri, « High-resolution transcription maps reveal the widespread impact of roadblock termination in yeast », The EMBO journal, janv. 2018.
    Résumé : Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.
    Mots-clés : BDG, DBG, pervasive transcription, Rap1, roadblock termination, TENOR, transcription readthrough, transcription termination mechanism.

  • N. Canu, P. Belin, R. Thai, I. Correia, O. Lequin, J. Seguin, M. Moutiez, et M. Gondry, « Incorporation of Non-canonical Amino Acids into 2,5-Diketopiperazines by Cyclodipeptide Synthases », Angewandte Chemie (International Ed. in English), vol. 57, nᵒ 12, p. 3118-3122, mars 2018.
    Résumé : The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5-diketopiperazines (2,5-DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an in vivo method for incorporating non-canonical amino acids (ncAAs) into 2,5-DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5-DKP biosynthetic pathways. CDPSs use aminoacyl-tRNAs as substrates. We exploited the natural ability of aminoacyl-tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non-canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5-DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide-tailoring enzymes found in 2,5-DKP biosynthetic pathways.
    Mots-clés : biocatalysis, BIOSYN, biosynthesis, Cyclodipeptide synthases, cyclodipeptides, Diketopiperazines, MICROBIO, Natural product engineering, Non-canonical amino acid.


  • M. Cardoso Dos Santos, J. Goetz, H. Bartenlian, K. - L. Wong, L. J. Charbonnière, et N. Hildebrandt, « Autofluorescence-Free Live-Cell Imaging Using Terbium Nanoparticles », Bioconjugate Chemistry, févr. 2018.


  • D. Carmona-Gutierrez, M. A. Bauer, A. Zimmermann, A. Aguilera, N. Austriaco, K. Ayscough, R. Balzan, S. Bar-Nun, A. Barrientos, P. Belenky, M. Blondel, R. J. Braun, M. Breitenbach, W. C. Burhans, S. Buettner, D. Cavalieri, M. Chang, K. F. Cooper, M. Côrte-Real, V. Costa, C. Cullin, I. Dawes, J. Dengjel, M. B. Dickman, T. Eisenberg, B. Fahrenkrog, N. Fasel, K. - U. Froehlich, A. Gargouri, S. Giannattasio, P. Goffrini, C. W. Gourlay, C. M. Grant, M. T. Greenwood, N. Guaragnella, T. Heger, J. Heinisch, E. Herker, J. M. Herrmann, S. Hofer, A. Jiménez-Ruiz, H. Jungwirth, K. Kainz, D. P. Kontoyiannis, P. Ludovico, S. Manon, E. Martegani, C. Mazzoni, L. A. Megeney, C. Meisinger, J. Nielsen, T. Nystroem, H. D. Osiewacz, T. F. Outeiro, H. - O. Park, T. Pendl, D. Petranovic, S. Picot, P. Polčic, T. Powers, M. Ramsdale, M. Rinnerthaler, P. Rockenfeller, C. Ruckenstuhl, R. Schaffrath, M. Segovia, F. F. Severin, A. Sharon, S. J. Sigrist, C. Sommer-Ruck, M. J. Sousa, J. M. Thevelein, K. Thevissen, V. Titorenko, M. B. Toledano, M. Tuite, F. - N. Voegtle, B. Westermann, J. Winderickx, S. Wissing, S. Woelfl, Z. J. Zhang, R. Y. Zhao, B. Zhou, L. Galluzzi, G. Kroemer, et F. Madeo, « Guidelines and recommendations on yeast cell death nomenclature », Microbial Cell, vol. 5, nᵒ 1, p. 4-31, janv. 2018.

  • B. Castrec, C. Dian, S. Ciccone, C. L. Ebert, W. V. Bienvenut, J. - P. Le Caer, J. - M. Steyaert, C. Giglione, et T. Meinnel, « Structural and genomic decoding of human and plant myristoylomes reveals a definitive recognition pattern », Nature Chemical Biology, juin 2018.
    Résumé : An organism's entire protein modification repertoire has yet to be comprehensively mapped. N-myristoylation (MYR) is a crucial eukaryotic N-terminal protein modification. Here we mapped complete Homo sapiens and Arabidopsis thaliana myristoylomes. The crystal structures of human modifier NMT1 complexed with reactive and nonreactive target-mimicking peptide ligands revealed unexpected binding clefts and a modifier recognition pattern. This information allowed integrated mapping of myristoylomes using peptide macroarrays, dedicated prediction algorithms, and in vivo mass spectrometry. Global MYR profiling at the genomic scale identified over a thousand novel, heterogeneous targets in both organisms. Surprisingly, MYR involved a non-negligible set of overlapping targets with N-acetylation, and the sequence signature marks for a third proximal acylation-S-palmitoylation-were genomically imprinted, allowing recognition of sequences exhibiting both acylations. Together, the data extend the N-end rule concept for Gly-starting proteins to subcellular compartmentalization and reveal the main neighbors influencing protein modification profiles and consequent cell fate.
    Mots-clés : BDG, PROMTI.

  • R. Catchpole, A. Gorlas, J. Oberto, et P. Forterre, « A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors », Extremophiles: Life Under Extreme Conditions, vol. 22, nᵒ 4, p. 591-598, juill. 2018.
    Résumé : Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli-T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli-T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.
    Mots-clés : Archaea, ARCHEE, Cloning, Gene cloning and expression, Genetics of extremophiles, Hyperthermophiles, MICROBIO, Molecular biology, Molecular biology of archaea.

  • F. Celli, A. Petitalot, C. Samson, F. - X. Theillet, et S. Zinn-Justin, « H-1, C-13 and N-15 backbone resonance assignment of the lamin C-terminal region specific to prelamin A », Biomolecular Nmr Assignments, vol. 12, nᵒ 2, p. 225-229, oct. 2018.
    Résumé : Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson-Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR H-1, N-15, (CO)-C-13, C-13 and C-13 chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR H-1, N-15, (CO)-C-13, C-13 and C-13 chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated -helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.
    Mots-clés : B3S, INTGEN, Intrinsically disordered protein, mutations, networks, NMR spectroscopy, NMR spectroscopy, Nuclear envelope, Nucleoskeleton.


  • C. Chaintreuil, X. Perrier, G. Martin, J. Fardoux, G. P. Lewis, L. Brottier, R. Rivallan, M. Gomez-Pacheco, M. Bourges, L. Lamy, B. Thibaud, H. Ramanankierana, H. Randriambanona, H. Vandrot, P. Mournet, E. Giraud, et J. - F. Arrighi, « Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics », BMC Plant Biology, vol. 18, nᵒ 1, 2018.

  • H. - J. Chang, P. Mayonove, A. Zavala, A. De Visch, P. Minard, M. Cohen-Gonsaud, et J. Bonnet, « A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria », ACS synthetic biology, vol. 7, nᵒ 1, p. 166-175, janv. 2018.
    Résumé : Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-binding domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.
    Mots-clés : B3S, MIP.

  • G. Chararalambidis, S. Das, A. Trapali, A. Quaranta, M. Orio, Z. Halime, P. Fertey, R. Guillot, A. Coutsolelos, W. Leibl, A. Aukauloo, et M. Sircoglou, « Water Molecules Gating a Photoinduced One Electron Two Protons Transfer in a Tyr/His model of Photosystem II », Angewandte Chemie (International Ed. in English), mai 2018.
    Résumé : In this report, we investigate on a biomimetic model of a H-bonded TyrZ/His190 pair covalently attached to a porphyrin sensitizer. Laser flash photolysis in presence of an external electron acceptor reveals the need of water molecules to unlock the light-induced oxidation of the phenol through an intramolecular pathway. Kinetics monitoring encompasses two fast phases with distinct spectral properties. The first phase is related to one-electron transfer from the phenol to the porphyrin radical cation coupled with a domino two-proton transfer leading to the ejection of a proton from the imidazole-phenol pair. The second phase concerns the convoy of the released proton to the porphyrin N4 coordinating cavity. Importantly, our study provides an unprecedented example of light induced electron transfer process in a TyrZ/His190 model of Photosystem II, evidencing the movement of both the phenol and imidazole protons along an isoenergetic pathway.
    Mots-clés : artificial photosynthesis, B3S, LPB, Proton Coupled Electron Transfer, TyrZ-His190 model.

  • M. Chaumorcel, M. Lussignol, L. Mouna, Y. Cavignac, K. Fahie, J. Cotte-Laffitte, A. Geballe, W. Brune, I. Beau, P. Codogno, et A. Esclatine, « Correction for Chaumorcel et al., "The Human Cytomegalovirus Protein TRS1 Inhibits Autophagy via Its Interaction with Beclin 1" », Journal of Virology, vol. 92, nᵒ 9, 2018.

  • C. Chen, L. Ao, Y. - T. Wu, V. Cifliku, M. C. Dos Santos, E. Bourrier, M. Delbianco, D. Parker, J. M. Zwier, L. Huang, et N. Hildebrandt, « Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots », Angewandte Chemie-International Edition, vol. 57, nᵒ 41, p. 13686-13690, oct. 2018.
    Résumé : Fluorescence barcoding based on nanoparticles provides many advantages for multiparameter imaging. However, creating different concentration-independent codes without mixing various nanoparticles and by using single-wavelength excitation and emission for multiplexed cellular imaging is extremely challenging. Herein, we report the development of quantum dots (QDs) with two different SiO2 shell thicknesses (6 and 12nm) that are coated with two different lanthanide complexes (Tb and Eu). FRET from the Tb or Eu donors to the QD acceptors resulted in four distinct photoluminescence (PL) decays, which were encoded by simple time-gated (TG) PL intensity detection in three individual temporal detection windows. The well-defined single-nanoparticle codes were used for live cell imaging and a one-measurement distinction of four different cells in a single field of view. This single-color barcoding strategy opens new opportunities for multiplexed labeling and tracking of cells.
    Mots-clés : B3S, biomolecules, biosensors, bright, europium complexes, FRET, Lanthanides, Lifetime, NANO.

  • C. Chen, C. Midelet, S. Bhuckory, N. Hildebrandt, et M. H. V. Werts, « Nanosurface Energy Transfer from Long-Lifetime Terbium Donors to Gold Nanoparticles », Journal of Physical Chemistry C, vol. 122, nᵒ 30, p. 17566-17574, août 2018.
    Résumé : Photoluminescence (PL) quenching by gold nanoparticles (AuNPs) is a frequently applied principle in nanobiosensing. The quenching is most often explained in terms of the Forster resonance energy-transfer (FRET) mechanism, and more rarely in terms of the nanosurface energy-transfer (NSET) mechanism. Although both consider nonradiative resonance energy transfer, there are significant differences in predictions of the strength and the distance-dependence of the quenching. Here, we investigate the energy transfer to AuNPs from a terbium(III)-complex (Tb) with a long (millisecond) PL decay time with the aim to provide a better understanding of the underlying energy-transfer process. The binding of Tb-labeled streptavidin (Tb-sAv) to biotinylated AuNPs (biot-AuNPs) was studied using light-scattering spectroscopy. Quenching of the PL of Tb-sAv upon binding to biotAuNPs of different diameters (5, 30, 50, 80 nm) was studied by time-resolved PL spectroscopy. Energy-transfer efficiencies were found to be practically independent of the AuNP size. Analysis according to FRET theory yielded donor-acceptor distances that were inconsistent and far beyond the expected Tb-AuNP distance. In contrast, the NSET model yielded a good agreement between the Tb-to-AuNP surface distance estimated from the geometry of the Tb-sAv/biotin-AuNP assembly (4.5 nm) and those calculated from PL lifetime analysis, which range from 4.0 to 6.3 nm. Our findings strongly suggest that NSET (and not FRET) is the operational mechanism in PL quenching by AuNPs, which is important information for the development, characterization, and application of nanobiosensors based on PL quenching by AuNPs.
    Mots-clés : B3S, binding, fluorescence, metal, microscopy, modulation, NANO, quantum dots, single, spectroscopy, surface-plasmon resonance.

  • J. - H. Chen, L. - J. Yu, A. Boussac, Z. - Y. Wang-Otomo, T. Kuang, et J. - R. Shen, « Properties and structure of a low-potential, penta-heme cytochrome c 552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum », Photosynthesis Research, avr. 2018.
    Résumé : The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c 552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c 552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.
    Mots-clés : B3S, Crystal structure, Cytochrome c, Electron transfer, Multiheme, PS2, Purple sulfur bacteria, Thermochromatium tepidum.

  • M. Chevreuil, D. Law-Hine, J. Chen, S. Bressanelli, S. Combet, D. Constantin, J. Degrouard, J. Möller, M. Zeghal, et G. Tresset, « Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte », Nature Communications, vol. 9, nᵒ 1, p. 3071, août 2018.
    Résumé : The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.
    Mots-clés : B3S, cryoelectron microscopy, disassembly, IMAPP, kinetic pathway, mechanism, Modeling, nanoparticles, pathway, plant-virus, protein, reconstruction, rna, templates, time-resolved small-angle X-ray scattering, Virus, virus-like particles.

  • R. R. Choubeh, E. Wientjes, P. C. Struik, D. Kirilovsky, et H. van Amerongen, « State transitions in the cyanobacterium Synechococcus elongatus 7942 involve reversible quenching of the photosystem II core », Biochimica Et Biophysica Acta-Bioenergetics, vol. 1859, nᵒ 10, p. 1059-1066, oct. 2018.
    Résumé : Cyanobacteria use chlorophyll and phycobiliproteins to harvest light. The resulting excitation energy is delivered to reaction centers (RCs), where photochemistry starts. The relative amounts of excitation energy arriving at the RCs of photosystem I (PSI) and II (PSII) depend on the spectral composition of the light. To balance the excitations in both photosystems, cyanobacteria perform state transitions to equilibrate the excitation energy. They go to state I if PSI is preferentially excited, for example after illumination with blue light (light I), and to state II after illumination with green-orange light (light II) or after dark adaptation. In this study, we performed 77-K time-resolved fluorescence spectroscopy on wild-type Synechococcus elongatus 7942 cells to measure how state transitions affect excitation energy transfer to PSI and PSII in different light conditions and to test the various models that have been proposed in literature. The time-resolved spectra show that the PSII core is quenched in state II and that this is not due to a change in excitation energy transfer from PSII to PSI (spill-over), either direct or indirect via phycobilisomes.
    Mots-clés : B3S, cells, Cyanobacteria, excitation-energy, less mutant, MROP, photosynthesis, Photosystem II, phycobilisome, picosecond fluorescence spectroscopy, porphyridium-cruentum, State transitions, synechococcus sp, synechocystis pcc 6803, Time-resolved fluorescence spectroscopy, wild-type.

  • C. Cohen, A. Corpet, S. Roubille, M. A. Maroui, N. Poccardi, A. Rousseau, C. Kleijwegt, O. Binda, P. Texier, N. Sawtell, M. Labetoulle, et P. Lomonte, « Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis », PLoS pathogens, vol. 14, nᵒ 9, p. e1007313, sept. 2018.
    Résumé : Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.
    Mots-clés : HERPES, VIRO.


  • F. Coll, J. Phelan, G. A. Hill-Cawthorne, M. B. Nair, K. Mallard, S. Ali, A. M. Abdallah, S. Alghamdi, M. Alsomali, A. O. Ahmed, S. Portelli, Y. Oppong, A. Alves, T. B. Bessa, S. Campino, M. Caws, A. Chatterjee, A. C. Crampin, K. Dheda, N. Furnham, J. R. Glynn, L. Grandjean, D. Minh Ha, R. Hasan, Z. Hasan, M. L. Hibberd, M. Joloba, E. C. Jones-López, T. Matsumoto, A. Miranda, D. J. Moore, N. Mocillo, S. Panaiotov, J. Parkhill, C. Penha, J. Perdigão, I. Portugal, Z. Rchiad, J. Robledo, P. Sheen, N. T. Shesha, F. A. Sirgel, C. Sola, E. Oliveira Sousa, E. M. Streicher, P. V. Helden, M. Viveiros, R. M. Warren, R. McNerney, A. Pain, et T. G. Clark, « Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis », Nature Genetics, janv. 2018.

  • M. Comisso, A. Hadchouel, J. de Blic, et M. Mirande, « Mutations in MARS identified in a specific type of pulmonary alveolar proteinosis alter methionyl-tRNA synthetase activity », The FEBS journal, mai 2018.
    Résumé : Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme. The pathophysiological mechanisms involved in MARS-related PAP are currently unknown. In this work, we analyzed the effect of the PAP-related mutations in MARS on the thermal stability and on the catalytic parameters of the MetRS mutants, relative to wild-type. The effect of these mutations on the structural integrity of the enzyme as a member of the cytosolic multisynthetase complex was also investigated. Our results establish that the PAP-related substitutions in MetRS impact the tRNAMet -aminoacylation reaction especially at the level of methionine recognition, and suggest a direct link between the loss of activity of the enzyme and the pathological disorders in PAP.
    Mots-clés : aminoacylation kinetics, BDG, MARS, methionyl-tRNA synthetase, pulmonary alveolar proteinosis.

  • D. Couvin, A. Bernheim, C. Toffano-Nioche, M. Touchon, J. Michalik, B. Néron, E. P. C Rocha, G. Vergnaud, D. Gautheret, et C. Pourcel, « CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins », Nucleic Acids Research, mai 2018.
    Résumé : CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.
    Mots-clés : BDG, LGBMB, MICROBIO, SSFA.

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