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Publications de l’I2BC

I2BC is ranked in the Nature Index 2017-2018


  • F. Acosta, J. Agapito, A. M. Cabibbe, T. Cáceres, C. Sola, L. Pérez-Lago, E. Abascal, M. Herranz, E. Meza, B. Klotoe, P. Muñoz, G. M. Rossolini, A. Bartoloni, E. Tortoli, D. M. Cirillo, E. Gotuzzo, et D. García de Viedma, « Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru », Emerging Infectious Diseases, vol. 25, nᵒ 3, p. 596-598, mars 2019.
    Résumé : We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.
    Mots-clés : antimicrobial resistance, bacteria, Europe, exportation, Florence, IGEPE, Italy, Lima, Madrid, MDR, MICROBIO, migration, MIRU-VNTR, molecular epidemiology, mycobacterium, Mycobacterium tuberculosis, Peru, single-nucleotide polymorphism, Spain, spoligotype, transmission, tuberculosis, tuberculosis and other mycobacteria.

  • A. Alard, C. Marboeuf, B. Fabre, C. Jean, Y. Martineau, F. Lopez, P. Vende, D. Poncet, R. J. Schneider, C. Bousquet, et S. Pyronnet, « Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome », Frontiers in Genetics, vol. 10, p. 254, mars 2019.
    Résumé : The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress.
    Mots-clés : 20s proteasome, DAP5, NQO1, Nrf2, Oxidative Stress, Pest, Proteasome, ROTA, VIRO.
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  • I. Altinoglu, C. J. Merrifield, et Y. Yamaichi, « Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline », Scientific Reports, vol. 9, nᵒ 1, p. 6680, avr. 2019.
    Résumé : Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.
    Mots-clés : BIOCELL, DBG, EQYY.

  • A. - A. Arteni, A. M. LaFountain, M. T. A. Alexandre, M. Fradot, M. M. Mendes-Pinto, J. - A. Sahel, S. Picaud, H. A. Frank, B. Robert, et A. A. Pascal, « Carotenoid composition and conformation in retinal oil droplets of the domestic chicken », PloS One, vol. 14, nᵒ 5, p. e0217418, 2019.
    Résumé : Carotenoid-containing oil droplets in the avian retina act as cut-off filters to enhance colour discrimination. We report a confocal resonance Raman investigation of the oil droplets of the domestic chicken, Gallus gallus domesticus. We show that all carotenoids present are in a constrained conformation, implying a locus in specific lipid binding sites. In addition, we provide proof of a recent conclusion that all carotenoid-containing droplets contain a mixture of all carotenoids present, rather than only a subset of them-a conclusion that diverges from the previously-held view. Our results have implications for the mechanism(s) giving rise to these carotenoid mixtures in the differently-coloured droplets.
    Mots-clés : B3S, LBMS.

  • J. Astier, A. Mounier, J. Santolini, S. Jeandroz, et D. Wendehenne, « The evolution of nitric oxide signalling diverges between the animal and the green lineages », Journal of Experimental Botany, mars 2019.
    Résumé : Nitric oxide (NO) is a ubiquitous signalling molecule with widespread distribution in prokaryotes and eukaryotes where it is involved in countless physiological processes. While the mechanisms governing NO synthesis and signalling are well established in animals, the situation is less clear in the green lineage. Recent investigations have shown that NO synthase (NOS), the major enzymatic source for NO in animals, is absent in land plants but present in a limited number of algae. First detailed analysis highlighted that these new NOSs are functional but display specific structural features and probably original catalytic activities. Completing this picture, analyses were undertaken in order to investigate whether major components of the prototypic NO/cyclic GMP signalling cascades mediating many physiological effects of NO in animals were also present in plants. Only few homologues of soluble guanylate cyclases, cGMP-dependent protein kinases, cyclic nucleotide-gated channels and cGMP-regulated phosphodiesterases, were identified in some algal species and their presence did not correlate with that of NOSs. In contrast, GSNO reductase, a critical regulator of S-nitrosothiols, was recurrently found. Overall, these findings highlight that plants do not mediate NO signalling through the classical NO/cGMP-signalling module and support the concept that S-nitrosation is a ubiquitous NO-dependent signalling mechanism.
    Mots-clés : Algae, B3S, cGMP, cGMP-dependent protein kinase, cyclic nucleotide-gated channel, Guanylate cyclase, LSOD, Nitric oxide, Nitric oxide synthase, phosphodiesterase, Plant, Signalling.

  • C. Aubry, J. - L. Pernodet, et S. Lautru, « A set of modular and integrative vectors for synthetic biology in Streptomyces », Applied and Environmental Microbiology, juin 2019.
    Résumé : With the development of synthetic biology in the field of (actinobacteria) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed.We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FRT sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains, when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalysed excision were all confirmed.To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the Biocrick assembly method. We also used the seamless Ligase Chain Reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain.IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites, or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacteria chromosome. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for protein production or genetic complementation, for example.
    Mots-clés : ACTINO, MICROBIO.

  • M. Bakail, S. Rodriguez-Marin, Z. Hegedues, M. E. Perrin, F. Ochsenbein, et A. J. Wilson, « Recognition of ASF1 by Using Hydrocarbon-Constrained Peptides », Chembiochem, vol. 20, nᵒ 7, p. 891-895, avr. 2019.
    Résumé : Inhibiting the histone H3-ASF1 (anti-silencing function 1) protein-protein interaction (PPI) represents a potential approach for treating numerous cancers. As an alpha-helix-mediated PPI, constraining the key histone H3 helix (residues 118-135) is a strategy through which chemical probes might be elaborated to test this hypothesis. In this work, variant H3(118-135) peptides bearing pentenylglycine residues at the 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 the potency of binding towards ASF1 is unaffected by the constraint and instead that enthalpy-entropy compensation occurs.
    Mots-clés : AMIG, B3S, chemical biology, complex, constrained peptides, helix, histone chaperones, modulators, protein surface recognition, protein-protein interactions histone chaperonnes constrained peptides protein surface recognition chemical biology, protein-protein interactions, replication, stapled peptides, structural basis.

  • A. Baroin-Tourancheau, Y. Jaszczyszyn, X. Benigni, et L. Amar, « Evaluating and Correcting Inherent Bias of microRNA Expression in Illumina Sequencing Analysis », Frontiers in Molecular Biosciences, vol. 6, p. 17, 2019.
    Résumé : microRNA (miRNA) expression profiles based on the highly powerful Illumina sequencing technology rely on the construction of cDNA libraries in which adaptor ligation is known to deeply favor some miRNAs over others. This introduces erroneous measurements of the miRNA abundances and relative miRNA quantities in biological samples. Here, by using the commercial miRXplore Universal Reference that contains an equimolar mixture of 963 animal miRNAs and TruSeq or bulged adaptors, we describe a method for correcting ligation biases in expression profiles obtained with standard protocols of cDNA library construction and provide data for quantifying the true miRNA abundances in biological samples. Ligation biases were evaluated at three ratios of miRNA to 3'-adaptor and four numbers of polymerase chain reaction amplification cycles by calculating efficiency captures/correcting factors for each miRNA. We show that ligation biases lead to over- or under-expression covering a 105 amplitude range. We also show that, at each miRNA:3'-adaptor ratio, coefficients of variation (CVs) of efficiency captures calculated over the four number of amplification cycles using sliding windows of 10 values ranged from 0.1 for the miRNAs of high expression to 0.6 for the miRNAs of low expression. Efficiency captures of miRNAs of high and low expression in profiles are therefore differently impacted by the number of amplification cycles. Importantly, we observed that at a given number of amplification cycles, CVs of efficiency captures calculated over the three miRNA:3'-adaptor ratios displayed a steady value of 0.3 +/- 0.05 STD for miRNAs of high and low expression. This allows, at a given number of amplification cycles, accurate comparison of miRNA expression between biological samples over a substantial expression range. Finally we provide tables of correcting factors that allow to measure the abundances of 963 miRNAs in biological samples from TruSeq-based expression profiles and, an example of their use by characterizing miRNAs of the let-7, miR-26, miR-29, and miR-30 families as the more abundant miRNAs of the rat adult cerebellum.
    Mots-clés : cerebellum, high-throughput sequencing, Illumina technology, ligation bias, miRNA abundance, miRNA expression profile, NGS, PF.

  • H. Barreteau, M. Vandervennet, L. Guedon, V. Point, S. Canaan, S. Rebuffat, J. Peduzzi, et A. Carre-Mlouka, « Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment », International Journal of Systematic and Evolutionary Microbiology, vol. 69, nᵒ 3, p. 732-738, mars 2019.
    Résumé : A halophilic organism, SWO25(T) , was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25(T) - grew optimally at 35-45 degrees C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25(T) to the genus Haloarcula. The DNA G+C content was 61.1 mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25(T) is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarcula sebkhae sp. nov. is proposed. The type strain is SWO25(T)(=CIP 110583(T) =JCM 19018(T)).
    Mots-clés : 16s ribosomal-rna, dna hybridization, ENVBAC, gen. nov., genera, haloarchaea, Haloarcula, halophilic archaeon, heterogeneity, hypersaline environments, MICROBIO, mukohataei, organic-solvent tolerance, rapid method, salt lake, sebkha, sequence.

  • E. M. Bayer, T. Calì, F. Giordano, A. Hamacher-Brady, et L. Pellegrini, « EMBO Workshop: Membrane Contact Sites in Health and Disease », Contact, vol. 2, p. 2515256419825931, janv. 2019.

  • P. Béguin, Y. Chekli, G. Sezonov, P. Forterre, et M. Krupovic, « Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei », Nucleic Acids Research, mai 2019.
    Résumé : Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.
    Mots-clés : ARCHEE, MICROBIO.

  • K. Ben Ouirane, Y. Boulard, et S. Bressanelli, « The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif », The Journal of Biological Chemistry, vol. 294, nᵒ 19, p. 7573-7587, mai 2019.
    Résumé : RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modelling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of the RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that some at least of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.
    Mots-clés : B3S, catalysis, complex, crystal-structure, fidelity, IMAPP, insights, mechanism, molecular dynamics, nucleoside, nucleoside/nucleotide transport, nucleotide transport, positive-sense RNA virus, protein motif, RNA virus, RNA-dependent RNA polymerase (RdRp), simulations, Single-stranded, Single-stranded, positive-sense RNA virus, structural basis, structural biology, viral polymerase.

  • L. Benkaidali, F. André, G. Moroy, B. Tangour, F. Maurel, et M. Petitjean, « Four Major Channels Detected in the Cytochrome P450 3A4: A Step toward Understanding Its Multispecificity », International Journal of Molecular Sciences, vol. 20, nᵒ 4, p. 987, févr. 2019.
    Résumé : We computed the network of channels of the 3A4 isoform of the cytochrome P450 (CYP) on the basis of 16 crystal structures extracted from the Protein Data Bank (PDB). The calculations were performed with version 2 of the CCCPP software that we developed for this research project. We identified the minimal cost paths (MCPs) output by CCCPP as probable ways to access to the buried active site. The algorithm of calculation of the MCPs is presented in this paper, with its original method of visualization of the channels. We found that these MCPs constitute four major channels in CYP3A4. Among the many channels proposed by Cojocaru et al. in 2007, we found that only four of them open in 3A4. We provide a refined description of these channels together with associated quantitative data.
    Mots-clés : access channels, active site access channels, B3S, buried active-site, cavities boundaries, cavities boundaries, crystal-structure, CYP3A4, LSOD.

  • E. Bordet, M. Frétaud, E. Crisci, E. Bouguyon, S. Rault, J. Pezant, A. Pleau, P. Renson, E. Giuffra, T. Larcher, M. Bourge, O. Bourry, O. Boulesteix, C. Langevin, I. Schwartz-Cornil, et N. Bertho, « Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus », Frontiers in Immunology, vol. 10, p. 953, 2019.
    Résumé : Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.
    Mots-clés : antibodies, antigen, B cell, BCL6, CYTO, PF, PRRSV.

  • C. Bou-Nader, P. Barraud, L. Pecqueur, J. Perez, C. Velours, W. Shepard, M. Fontecave, C. Tisne, et D. Hamdane, « Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2 », Nucleic Acids Research, vol. 47, nᵒ 6, p. 3117-3126, avr. 2019.
    Résumé : Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouridine within the complex elbow structure of tRNA. We here unveil the molecular mechanism by which hDus2 dsRBD recognizes a tRNA ligand. By solving the crystal structure of this dsRBD in complex with a dsRNA together with extensive characterizations of its interaction with tRNA using mutagenesis, NMR and SAXS, we establish that while hDus2 dsRBD retains a conventional dsRNA recognition capability, the presence of an N-terminal extension appended to the canonical domain provides additional residues for binding tRNA in a structure-specific mode of action. Our results support that this extension represents a feature by which the dsRBD specializes in tRNA biology and more broadly highlight the importance of structural appendages to canonical domains in promoting the emergence of functional diversity.
    Mots-clés : complex reveals, evolution, extended dsrbd, mechanism, motif, nmr, PF, PIM, proteins, structural insights, trna(3)(lys).

  • A. Boussac, « Temperature dependence of the high-spin S-2 to S-3 transition in Photosystem II: Mechanistic consequences », Biochimica Et Biophysica Acta-Bioenergetics, vol. 1860, nᵒ 6, p. 508-518, juin 2019.
    Résumé : The Mn4CaO5-cluster in Photosystem II advances through five oxidation states, S-0 to S-4, before water is oxidized and O-2 is generated. The S-2-state exhibits either a low-spin, S = 1/2 (S-2(LS)), or a high-spin state, S = 5/2 (S-2(HS)). Increasing the pH favors the S-2(HS) Sens configuration and mimics the formation of Tyr(z)center dot in the S-2(LS)-state at lower pH values (Boussac et al. Biochim. Biophys. Acta 1859 (2018) 342). Here, the temperature dependence of the S-2(HS) to S-3 transition was studied by EPR spectroscopy at pH 8.6. The present data strengthened the involvement of S2I Us as a transient state in the S(2)(LS)Tyr(z)center dot S(2)(HS)Tyr(z) -> S(3)Tyr(z) transition. Depending on the temperature, the S-2(HS) progresses to S-3 states exhibiting different EPR properties. One S-3-state with a S = 3 signal, supposed to have a structure with the water molecule normally inserted in S-2 to S-3 transition, can be formed at temperatures as low as 77 K. This suggests that this water molecule is already bound in the S-2(HS) state at pH 8.6. The nature of the EPR invisible S-3 state, formed down to 4.2 K from a S-2(HS) is state, and that of the EPR detectable S3 state formed down to 77 K are discussed. It is proposed that in the S-2(LS) to S-3 transition, at pH < 8.6, the proton release (Sugiura et al. Biochim. Biophys. Acta 1859 (2018) 1259), the S-2(LS) to S-2(HS) conversion and the binding of the water molecule are all triggered by the formation of Tyr(z)center dot.
    Mots-clés : active-site, B3S, bond formation, camn4o5 cluster, electron-paramagnetic-res, EPR, Mn(4)CaO(5) cluster, Mn4CaO5 cluster, Oxygen evolution, Photosystem II, PS2, Spin state.

  • G. Briassoulis, P. Briassoulis, M. Miliaraki, S. Ilia, M. Parlato, F. Philippart, A. Rouquette, V. Moucadel, J. - M. Cavaillon, B. Misset, et Combined Approach for The eArly diagnosis of INfection in sepsis (CAPTAIN) study group, « Biomarker cruises in sepsis: who is the CAPTAIN? Discussion on "Circulating biomarkers may be unable to detect infection at the early phase of sepsis in ICU patients: the CAPTAIN prospective multicenter cohort study" », Intensive Care Medicine, janv. 2019.

  • A. Briquet, R. Vong, J. - B. Roseau, E. Javelle, N. Cazes, F. Rivière, M. Aletti, M. - P. Otto, C. Ficko, S. Duron, M. Fabre, C. Pourcel, F. Simon, et C. Soler, « Clinical features of Mycobacterium canettii infection: a retrospective study of 20 cases among French soldiers and relatives », Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, févr. 2019.
    Résumé : Background: Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. M. canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Our study aims to describe M. canettii infections among French military or their families between 1998 and 2015. Methods: This retrospective study relied on 3 sources of data: the reference centre for mycobacteria in the Biology Department at Percy military hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families. Results: 20 cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; one case was observed one month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6) and 3 atypical forms are described. All patients had stayed in Djibouti. Conclusions: Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments were comparable to tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • E. M. S. Brito, V. M. Romero-Núñez, C. A. Caretta, P. Bertin, J. C. Valerdi-Negreros, R. Guyoneaud, et M. Goñi-Urriza, « The bacterial diversity on steam vents from Paricutín and Sapichu volcanoes », Extremophiles: Life Under Extreme Conditions, févr. 2019.
    Résumé : Vapor steam vents are prevailing structures on geothermal sites in which local geochemical conditions allow the development of extremophilic microorganisms. We describe the structure of the prokaryotic community able to grow on the walls and rocks of such microecosystems in two terrestrial Mexican volcanoes: Paricutín (PI and PII samples) and its satellite Sapichu (S sample). The investigated samples showed similar diversity indices, with few dominant OTUs (abundance > 1%): 21, 16 and 23, respectively for PI, PII and S. However, each steam vent showed a particular community profile: PI was dominated by photosynthetic bacteria (Cyanobacteria and Chloroflexia class), PII by Actinobacteria and Proteobacteria, and S by Ktedonobacteria class, Acidobacteria and Cyanobacteria phyla. Concerning the predicted metabolic potential, we found a dominance of cellular pathways, especially the ones for energy generation with metabolisms for sulfur respiration, nitrogen fixation, methanogenesis, carbon fixation, photosynthesis, and metals, among others. We suggest a different maturity stage for the three studied fumaroles, from the youngest (PI) to the oldest (S and PII), also influenced by the temperature and other geochemical parameters. Furthermore, four anaerobic strains were isolated, belonging to Clostridia class (Clostridium sphenoides, C. swellfunanium and Anaerocolumna cellulosilytica) and to Bacilli class (Paenibacillus azoreducens).
    Mots-clés : Anaerobic bacteria, DBG, Extreme environment, GST, Microbial biodiversity, Predictive metagenomics profiling, Volcanic fumaroles.

  • A. Cabrie, O. Guittet, R. Tomasini, P. Vincendeau, et M. Lepoivre, « Crosstalk between TAp73 and TGF-beta in fibroblast regulates iNOS expression and Nrf2-dependent gene transcription », Free Radical Biology and Medicine, vol. 134, p. 617-629, avr. 2019.
    Résumé : Inducible nitric oxide synthase (iNOS) activity produces anti-tumor and anti-microbial effects but also promotes carcinogenesis through mutagenic, immunosuppressive and pro-angiogenic mechanisms. The tumor suppressor p53 contributes to iNOS downregulation by repressing induction of the NOS2 gene encoding iNOS, thereby limiting NO-mediated DNA damages. This study focuses on the role of the p53 homologue TAp73 in the regulation of iNOS expression. Induction of iNOS by immunological stimuli was upregulated in immortalized MEFs from TAp73(-/-) mice, compared to TAp73(+/+) fibroblasts. This overexpression resulted both from increased levels of NOS2 transcripts, and from an increased stability of the protein. Limitation of iNOS expression by TAp73 in wild-type cells is alleviated by TGF-beta receptor I inhibitors, suggesting a cooperation between TAp73 and TGF-beta in suppression of iNOS expression. Accordingly, downregulation of iNOS expression by exogenous TGF-beta 1 was impaired in TAp73(-/-) fibroblasts. Increased NO production in these cells resulted in a stronger, NO-dependent induction of Nrf2 target genes, indicating that the Nrf2-dependent adaptive response to nitrosative stress in fibroblasts is proportional to iNOS activity. NO-dependent induction of two HIF-1 target genes was also stronger in TAp73-deficient cells. Finally, the antimicrobial action of NO against Trypanosoma musculi parasites was enhanced in TAp73(-/-) fibroblasts. Our data indicate that tumor suppressive TAp73 isoforms cooperate with TGF-beta to control iNOS expression, NO-dependent adaptive responses to stress, and pathogen proliferation.
    Mots-clés : activation, B3S, cells, growth, immunity, Inducible nitric oxide synthase, innate, LBMS, macrophages, Nitric oxide, nitric-oxide production, Nuclear factor erythroid 2-related factor 2, suppression, Transforming growth factor beta, Transforming growth factor beta, transforming growth-factor-beta-1, tumorigenesis.

  • P. I. Calzadilla, F. Muzzopappa, P. Setif, et D. Kirilovsky, « Different roles for ApcD and ApcF in Synechococcus elongatus and Synechocystis sp. PCC 6803 phycobilisomes », Biochimica Et Biophysica Acta-Bioenergetics, vol. 1860, nᵒ 6, p. 488-498, juin 2019.
    Résumé : The phycobilisome, the cyanobacterial light harvesting complex, is a huge phycobiliprotein containing extra membrane complex, formed by a core from which rods radiate. The phycobilisome has evolved to efficiently absorb sun energy and transfer it to the photosystems via the last energy acceptors of the phycobilisome, ApcD and ApcE. ApcF also affects energy transfer by interacting with ApcE. In this work we studied the role of ApcD and ApcF in energy transfer and state transitions in Synechococcus elongatus and Synechocystis PCC6803. Our results demonstrate that these proteins have different roles in both processes in the two strains. The lack of ApcD and ApcF inhibits state transitions in Synechocystis but not in S. elongatus. In addition, lack of ApcF decreases energy transfer to both photosystems only in Synechocystis, while the lack of ApcD alters energy transfer to photosystem I only in S. elongatus. Thus, conclusions based on results obtained in one cyanobacterial strain cannot be systematically transferred to other strains and the putative role(s) of phycobilisomes in state transitions need to be reconsidered.
    Mots-clés : anacystis-nidulans, B3S, chlamydomonas-reinhardtii, Cyanobacteria, Energy transfer, excitation-energy transfer, light, MROP, orange carotenoid protein, photosystem-ii fluorescence, Phycobilisome, porphyridium-cruentum, quenching mechanisms, red alga, State transition, state transitions.

  • P. I. Calzadilla, J. Zhan, P. Sétif, C. Lemaire, D. Solymosi, N. Battchikova, Q. Wang, et D. Kirilovsky, « The cytochrome b6f complex is not involved in cyanobacterial state transitions », The Plant Cell, vol. 31, nᵒ 4, p. 911-931, avr. 2019.
    Résumé : Photosynthetic organisms need to sense and respond to fluctuating environmental conditions to avoid the formation of dangerous reactive oxygen species. The excitation energy arriving at each photosystem permanently changes due to variations of intensity and spectral properties of the absorbed light. Cyanobacteria, like plants and algae, have developed a mechanism, named state transitions, that sense and respond to these fluctuating conditions. In this work, we characterize the role of the cytochrome b6f and phosphorylation reactions in cyanobacterial state transitions using Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803. A large Photosystem II fluorescence quenching was observed in State II which seems not to be related to spillover. This membrane-associated process was inhibited by betaine, sucrose and high concentrations of phosphate. Then, using different chemicals affecting the PQ pool redox state and the activity of the cytochrome b6f, we demonstrated that this complex is not involved in S. elongatus and Synechocystis PCC6803 state transitions. Finally, by constructing and characterizing 21 kinase and phosphatase mutants and using chemical inhibitors, it was clearly shown that phosphorylation reactions are not essential in cyanobacterial state transitions. Thus, signal transduction is completely different in cyanobacteria and plant (green alga) state transitions.
    Mots-clés : B3S, MROP.

  • V. Campanacci, A. Urvoas, S. Cantos-Fernandes, M. Aumont-Nicaise, A. - A. Arteni, C. Velours, M. Valerio-Lepiniec, B. Dreier, A. Plückthun, A. Pilon, C. Poüs, P. Minard, et B. Gigant, « Insight into microtubule nucleation from tubulin-capping proteins », Proceedings of the National Academy of Sciences of the United States of America, vol. 116, nᵒ 20, p. 9859-9864, avr. 2019.
    Résumé : Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or β-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.
    Mots-clés : artificial binding proteins, B3S, CopN, CRYOEM, MIKICA, MIP, PF, PIM.

  • V. Campanacci, A. Urvoas, T. Consolati, S. Cantos-Fernandes, M. Aumont-Nicaise, M. Valerio-Lepiniec, T. Surrey, P. Minard, et B. Gigant, « Selection and Characterization of Artificial Proteins Targeting the Tubulin alpha Subunit », Structure, vol. 27, nᵒ 3, p. 497-+, mars 2019.
    Résumé : Microtubules are cytoskeletal filaments of eukaryotic cells made of alpha beta-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an alpha Rep library that are specific to alpha-tubulin. Turbidity experiments indicate that these alpha Reps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (-) end. Structural data indicate that they do so by targeting the alpha-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the alpha subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These alpha-tubulin-specific alpha Reps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation.
    Mots-clés : artificial protein, B3S, beta-tubulin, complex, depolymerization, design, dynamic instability, in vitro selection, microtubule, microtubule plus, MIKICA, MIP, overexpression, purification, stathmin, structural basis, tubulin, αRep.

  • M. Chan-Yao-Chong, C. Deville, L. Pinet, C. van Heijenoort, D. Durand, et T. Ha-Duong, « Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data », Biophysical Journal, vol. 116, nᵒ 7, p. 1216-1227, avr. 2019.
    Résumé : Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.
    Mots-clés : alpha-synuclein, angle scattering data, atomic-resolution, B3S, c-13' chemical-shifts, FAAM, force-field, fuzzy complexes, intrinsically disordered proteins, molecular recognition features, quantum-mechanics, unstructured proteins.

  • M. Chan-Yao-Chong, D. Durand, et T. Ha-Duong, « Molecular Dynamics Simulations Combined with Nuclear Magnetic Resonance and/or Small-Angle X-ray Scattering Data for Characterizing Intrinsically Disordered Protein Conformational Ensembles », Journal of Chemical Information and Modeling, vol. 59, nᵒ 5, p. 1743-1758, mai 2019.
    Résumé : The concept of intrinsically disordered proteins (IDPs) has emerged relatively slowly, but over the past 20 years, it has become an intense research area in structural biology. Indeed, because of their considerable flexibility and structural heterogeneity, the determination of IDP conformational ensemble is particularly challenging and often requires a combination of experimental measurements and computational approaches. With the improved accuracy of all-atom force fields and the increasing computing performances, molecular dynamics (MD) simulations have become more and more reliable to generate realistic conformational ensembles. And the combination of MD simulations with experimental approaches, such as nuclear magnetic resonance (NMR) and/or small-angle X-ray scattering (SAXS) allows one to converge toward a more accurate and exhaustive description of IDP structures. In this Review, we discuss the state of the art of MD simulations of IDP conformational ensembles, with a special focus on studies that back-calculated and directly compared theoretical and experimental NMR or SAXS observables, such as chemical shifts (CS), 3J-couplings (3Jc), residual dipolar couplings (RDC), or SAXS intensities. We organize the review in three parts. In the first section, we discuss the studies which used NMR and/or SAXS data to test and validate the development of force fields or enhanced sampling techniques. In the second part, we explore different methods for the refinement of MD-derived structural ensembles, such as NMR or SAXS data-restrained MD simulations or ensemble reweighting to better fit experiments. Finally, we survey some recent studies combining MD simulations with NMR and/or SAXS measurements to investigate the relationship between IDP conformational ensemble and biological activity, as well as their implication in human diseases. From this review, we noticed that quite a few studies compared MD-generated conformational ensembles with both NMR and SAXS measurements to validate IDP structures at both local and global levels. Yet, beside the IDP propensity to form local secondary structures, their dynamic extension or compactness also appears important for their activity. Thus, we believe that a close synergy between MD simulations, NMR, and SAXS experiments would be greatly appropriate to address the challenges of characterizing the disordered structures of proteins and their complexes, relative to their biological functions.
    Mots-clés : alpha-synuclein, atomic-level characterization, B3S, chemical-shifts, FAAM, folding simulations, force-field, generalized born model, replica exchange, structural ensembles, unfolded states, water model.

  • F. Chauffour, M. Bailly, F. Perreau, G. Cueff, H. Suzuki, B. Collet, A. Frey, G. Clément, L. Soubigou-Taconnat, T. Balliau, A. Krieger-Liszkay, L. Rajjou, et A. Marion-Poll, « Multi-omics analysis reveals sequential roles for ABA during seed maturation », Plant Physiology, vol. 180, nᵒ 2, p. 1198-1218, avr. 2019.
    Résumé : Abscisic acid (ABA) is an important hormone for seed development and germination whose physiological action is modulated by its endogenous levels. Cleavage of carotenoid precursors by 9-cis epoxycarotenoid dioxygenase (NCED) and inactivation of ABA by ABA 8'-hydroxylase (CYP707A) are key regulatory metabolic steps. In Arabidopsis (Arabidopsis thaliana), both enzymes are encoded by multigene families, having distinctive expression patterns. To evaluate the genome-wide impact of ABA deficiency in developing seeds at the maturation stage when dormancy is induced, we used a nced2569 quadruple mutant in which ABA deficiency is mostly restricted to seeds, thus limiting the impact of maternal defects on seed physiology. ABA content was very low in nced2569 seeds, similar to the severe mutant aba2; unexpectedly, ABA glucose ester was detected in aba2 seeds, suggesting the existence of an alternative metabolic route. Hormone content in nced2569 seeds compared with nced259 and wild-type strongly suggested that specific expression of NCED6 in the endosperm is mainly responsible for ABA production. In accordance, transcriptome analyses revealed broad similarities in gene expression between nced2569 and either wild type or nced259 developing seeds. Gene ontology enrichments revealed a large spectrum of ABA activation targets involved in reserve storage and desiccation tolerance, and repression of photosynthesis and cell cycle. Proteome and metabolome profiles in dry nced2569 seeds, compared with wild-type and cyp707a1a2 seeds, also highlighted an inhibitory role of ABA on remobilisation of reserves, ROS production, and protein oxidation. Down-regulation of these oxidative processes by ABA may have an essential role in dormancy control.
    Mots-clés : 9-cis-epoxycarotenoid dioxygenase, abscisic-acid biosynthesis, arabidopsis seeds, B3S, dormancy, drought tolerance, genome-wide analysis, mass-spectrometry, metabolism, MROP, protein oxidation, signaling networks.

  • C. Chen, B. Corry, L. Huang, et N. Hildebrandt, « FRET-Modulated Multihybrid Nanoparticles for Brightness-Equalized Single-Wavelength Barcoding », Journal of the American Chemical Society, juin 2019.
    Résumé : Semiconductor quantum dots (QDs) are the most versatile fluorophores for Förster resonance energy transfer (FRET) because they can function as both donors and acceptors for a multitude of fluorophores. However, a complete understanding of multidonor-multiacceptor FRET networks on QDs and their full employment into advanced fluorescence sensing and imaging have not been accomplished. Here, we provide a holistic photophysical analysis of such multidonor-QD-multiacceptor FRET systems using time-resolved and steady-state photoluminescence (PL) spectroscopy and Monte Carlo simulations. Multiple terbium complex (Tb) donors (1-191 units) and Cy5.5 dye acceptors (1-60 units) were attached to a central QD, and the entire range of combinations of FRET pathways was investigated by Tb, QD, and Cy5.5 PL. Experimental and simulation results were in excellent agreement and could disentangle the distinct contributions of hetero-FRET, homo-FRET, and dye dimerization. The FRET efficiency was independent of the number of Tb donors and dependent on the number of Cy5.5 acceptors, which could be used to independently adapt the PL intensity by the number of Tb donors and the PL lifetime by the number of Cy5.5 acceptors. We used this unique tuning capability to prepare Tb-QD-Cy5.5 conjugates with distinct QD PL lifetimes but similar QD PL intensities. These brightness-equalized multihybrid FRET nanoparticles were applied to optical barcoding via three time-gated PL intensity detection windows, which resulted in simple RGB ratios. Direct applicability was demonstrated by an efficient RGB distinction of different nanoparticle-encoded microbeads within the same field of view with both single-wavelength excitation and detection on a standard fluorescence microscope.
    Mots-clés : B3S, NANO.

  • 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, vol. 139, nᵒ 1-3, p. 281-293, mars 2019.
    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 : angstrom resolution, B3S, c nitrite reductase, c554, conservation, Crystal structure, crystal-structure, Cytochrome c, Electron transfer, environment, genes, Multiheme, proteins, PS2, Purple sulfur bacteria, spectroscopy, subunit, Thermochromatium tepidum.

  • D. Ciardo, A. Goldar, et K. Marheineke, « On the Interplay of the DNA Replication Program and the Intra-S Phase Checkpoint Pathway », Genes, vol. 10, nᵒ 2, p. 94, janv. 2019.
    Résumé : DNA replication in eukaryotes is achieved by the activation of multiple replication origins which needs to be precisely coordinated in space and time. This spatio-temporal replication program is regulated by many factors to maintain genome stability, which is frequently threatened through stresses of exogenous or endogenous origin. Intra-S phase checkpoints monitor the integrity of DNA synthesis and are activated when replication forks are stalled. Their activation leads to the stabilization of forks, to the delay of the replication program by the inhibition of late firing origins, and the delay of G2/M phase entry. In some cell cycles during early development these mechanisms are less efficient in order to allow rapid cell divisions. In this article, we will review our current knowledge of how the intra-S phase checkpoint regulates the replication program in budding yeast and metazoan models, including early embryos with rapid S phases. We sum up current models on how the checkpoint can inhibit origin firing in some genomic regions, but allow dormant origin activation in other regions. Finally, we discuss how numerical and theoretical models can be used to connect the multiple different actors into a global process and to extract general rules.
    Mots-clés : ataxia-telangiectasia, ATR, Chk1, DBG, DNA replication, DYNREP, GTR, MBT.

  • E. C. Conceição, G. Refregier, H. M. Gomes, X. Olessa-Daragon, F. Coll, N. H. Ratovonirina, V. Rasolofo-Razanamparany, M. L. Lopes, D. van Soolingen, L. Rutaihwa, S. Gagneux, V. R. Bollela, P. N. Suffys, R. S. Duarte, K. V. B. Lima, et C. N. Sola, « Mycobacterium tuberculosis lineage 1 genetic diversity in Pará, Brazil, suggests common ancestry with east-African isolates potentially linked to historical slave trade », Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, vol. 73, p. 337-341, juin 2019.
    Résumé : Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sub lineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48, sp. 40) and L1/EAI6-BGD1 (SIT591, sp. 23) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th-18th slave trade period to Northern Brazil.
    Mots-clés : IGEPE, Lineage 1, MICROBIO, Molecular evolution, Mycobacterium tuberculosis complex, Single-nucleotide polymorphisms, Spoligotyping, Whole genome sequencing.

  • I. Corcoles-Saez, J. - L. Ferat, M. Costanzo, C. M. Boone, et R. S. Cha, « Functional link between mitochondria and Rnr3, the minor catalytic subunit of yeast ribonucleotide reductase », Microbial Cell (Graz, Austria), vol. 6, nᵒ 6, p. 286-294, mai 2019.
    Résumé : Ribonucleotide reductase (RNR) is an essential holoenzyme required for de novo synthesis of dNTPs. The Saccharomyces cerevisiae genome encodes for two catalytic subunits, Rnr1 and Rnr3. While Rnr1 is required for DNA replication and DNA damage repair, the function(s) of Rnr3 is unknown. Here, we show that carbon source, an essential nutrient, impacts Rnr1 and Rnr3 abundance: Non-fermentable carbon sources or limiting concentrations of glucose down regulate Rnr1 and induce Rnr3 expression. Oppositely, abundant glucose induces Rnr1 expression and down regulates Rnr3. The carbon source dependent regulation of Rnr3 is mediated by Mec1, the budding yeast ATM/ATR checkpoint response kinase. Unexpectedly, this regulation is independent of all currently known components of the Mec1 DNA damage response network, including Rad53, Dun1, and Tel1, implicating a novel Mec1 signalling axis. rnr3Δ leads to growth defects under respiratory conditions and rescues temperature sensitivity conferred by the absence of Tom6, a component of the mitochondrial TOM (translocase of outer membrane) complex responsible for mitochondrial protein import. Together, these results unveil involvement of Rnr3 in mitochondrial functions and Mec1 in mediating the carbon source dependent regulation of Rnr3.
    Mots-clés : atr, autophagy, carbon source, cell-cycle, checkpoint, DBG, dna-damage response, dNTP, EMC2, gene, kinase, Mec1, Rnr1, Rnr3.

  • M. S. Deffieu, T. D. Alayi, C. Slomianny, et S. Tomavo, « The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport », Biology Open, vol. 8, nᵒ 3, p. UNSP bio039818, mars 2019.
    Résumé : After entry into the host cell, the intracellular parasite Toxoplasma gondii resides within a membrane-bound compartment, the parasitophorous vacuole (PV). The PV defines an intracellular, parasite-specific niche surrounded by host organelles, including the Golgi apparatus. The mechanism by which T. gondii hijacks the host Golgi and subverts its functions remains unknown. Here, we present evidence that the dense granule protein TgGRA3 interacts with host Golgi, leading to the formation of tubules and the entry of host Golgi material into the PV. Targeted disruption of the TgGRA3 gene delays this engulfment of host Golgi. We also demonstrate that TgGRA3 oligomerizes and binds directly to host Golgi membranes. In addition, we show that TgGRA3 dysregulates anterograde transport in the host cell, thereby revealing one of the mechanisms employed by T. gondii to recruit host organelles and divert their functions. This article has an associated First Person interview with the first author of the paper.
    Mots-clés : Anterograde transport, Anterograde transport, association, BMCT, cell endoplasmic-reticulum, cholesterol, Dense granules, domains, gra3, Host Golgi, invasion, nanotubular network, parasitophorous vacuole membrane, Secretion, Toxoplasma gondii, VIRO.

  • A. Demené, L. Legrand, J. Gouzy, R. Debuchy, G. Saint-Jean, O. Fabreguettes, et C. Dutech, « Whole-genome sequencing reveals recent and frequent genetic recombination between clonal lineages of Cryphonectria parasitica in western Europe », Fungal genetics and biology: FG & B, juin 2019.
    Résumé : Changes in the mode of reproduction are frequently observed in invasive fungal populations. The ascomycete Cryphonectria parasitica, which causes Chestnut Blight, was introduced to Europe from North America and Asia in the 20th century. Previous genotyping studies based on ten microsatellite markers have identified several clonal lineages which have spread throughout western Europe, suggesting that asexuality was the main reproductive mode of this species during colonization, although occasional sexual reproduction is not excluded. Based on the whole-genome sequences alignment of 46 C. parasitica isolates from France, North America and Asia, genealogy and population structure analyses mostly confirmed these lineages as clonal. However, one of these clonal lineages showed a signal of strong recombination, suggesting different strategies of reproduction in western Europe. Signatures of several recent recombination events within all the French clonal lineages studied here were also identified, indicating that gene flow is regular between these lineages. In addition, haplotype identification of seven French clonal lineages revealed that emergences of new clonal lineages during colonization were the result of hybridization between the main expanding clonal lineages and minor haplotypes non-sequenced in the present study. This whole-genome sequencing study underlines the importance of recombination events in the invasive success of these clonal populations, and suggests that sexual reproduction may be more frequent within and between the western European clonal lineages of C. parasitica than previously assumed using few genetic markers.
    Mots-clés : Bayesian inferences, clonal evolution, DBG, intra-haploid mating, MRP, recombination rates, whole genome sequencing.

  • T. Denecker, W. Durand, J. Maupetit, C. Hébert, J. - M. Camadro, P. Poulain, et G. Lelandais, « Pixel: a content management platform for quantitative omics data », PeerJ, vol. 7, p. e6623, 2019.
    Résumé : Background: In biology, high-throughput experimental technologies, also referred as "omics" technologies, are increasingly used in research laboratories. Several thousands of gene expression measurements can be obtained in a single experiment. Researchers are routinely facing the challenge to annotate, store, explore and mine all the biological information they have at their disposal. We present here the Pixel web application (Pixel Web App), an original content management platform to help people involved in a multi-omics biological project. Methods: The Pixel Web App is built with open source technologies and hosted on the collaborative development platform GitHub ( It is written in Python using the Django framework and stores all the data in a PostgreSQL database. It is developed in the open and licensed under the BSD 3-clause license. The Pixel Web App is also heavily tested with both unit and functional tests, a strong code coverage and continuous integration provided by CircleCI. To ease the development and the deployment of the Pixel Web App, Docker and Docker Compose are used to bundle the application as well as its dependencies. Results: The Pixel Web App offers researchers an intuitive way to annotate, store, explore and mine their multi-omics results. It can be installed on a personal computer or on a server to fit the needs of many users. In addition, anyone can enhance the application to better suit their needs, either by contributing directly on GitHub (encouraged) or by extending Pixel on their own. The Pixel Web App does not provide any computational programs to analyze the data. Still, it helps to rapidly explore and mine existing results and holds a strategic position in the management of research data.
    Mots-clés : BIM, candida-glabrata, Data cycle analyses, DBG, Omics, Open source, Pixel Web App.

  • A. Devigne, L. Meyer, C. B. de la Tour, N. Eugenie, S. Sommer, et P. Servant, « The absence of the RecN protein suppresses the cellular defects of Deinococcus radiodurans irradiated cells devoid of the PprA protein by Cheek tot limiting recombinational repair of DNA lesions », Dna Repair, vol. 73, p. 144-154, janv. 2019.
    Résumé : The Deinococcus radiodurans bacterium is one of the most radioresistant organisms known. It can repair hundreds of radiation-induced DNA double-strand breaks without loss of viability and reconstitute an intact genome through RecA-dependent and RecA-independent DNA repair pathways. Among the Deinococcus specific proteins required for radioresistance, the PprA protein was shown to play a major role for accurate chromosome segregation and cell division after completion of DNA repair. Here, we analyzed the cellular role of the deinococcal RecN protein belonging to the SMC family and, surprisingly, observed that the absence of the RecN protein suppressed the sensitivity of cells devoid of the PprA protein to gamma- and UV-irradiation and to treatment with MMC or DNA gyrase inhibitors. This suppression was not observed when Delta pprA cells were devoid of SMC or SbcC, two other proteins belonging to the SMC family. The absence of RecN also alleviated the DNA segregation defects displayed by Delta pprA cells recovering from y-irradiation. When exposed to 5 kGy gamma-irradiation, Delta pprA, Delta recN and Delta pprA Delta recN cells repaired their DNA with a delay of about one hour, as compared to the wild type cells. After irradiation, the absence of RecN reduced recombination between chromosomal and plasmid DNA, indicating that the deinococcal RecN protein is important for recombinational repair of DNA lesions. The transformation efficiency of genomic DNA was also reduced in the absence of the RecN protein. Here, we propose a model in which RecN, via its cohesin activity, might favor recombinational repair of DNA double strand breaks. This might increase, in irradiated cells, DNA constraints with PprA protein being required to resolve them via its ability to recruit DNA gyrase and to stimulate its decatenation activity.
    Mots-clés : chromosomes, complex, DBG, Deinococcus radiodurans, DNA segregation, DNA segregation, double-strand breaks, dynamics, Homologous recombination, identification, PprA, RBA, RecN, recruitment, replication, segregation, smc protein, structural maintenance.

  • T. Di Meo, K. Kariyawasam, W. Ghattas, M. Valerio-Lepiniec, G. Sciortino, J. - D. Marechal, P. Minard, J. - P. Mahy, A. Urvoas, et R. Ricoux, « Functionalized Artificial Bidomain Proteins Based on an alpha-Solenoid Protein Repeat Scaffold: A New Class of Artificial Diels-Alderases », Acs Omega, vol. 4, nᵒ 2, p. 4437-4447, févr. 2019.
    Résumé : alpha Rep is a family of entirely artificial repeat proteins. Within the previously described alpha Rep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called alpha Rep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3' domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels-Alder (D-A) reaction. This allowed us to obtain four new artificial Diels-Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV-vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D-A reaction. They were found to be able to catalyze the enantioselective D-A reaction of azachalcone with cyclopentadiene with up to 38% yield and 52% enantiomeric excess, which validates the proposed strategy. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggest that artificial metalloenzymes based on bidomain A3_A3 proteins modified with nitrogen donor ligands may be suitable for further catalyst optimization and may constitute valuable tools toward more efficient and selective artificial biocatalysts.
    Mots-clés : B3S, biocatalyst, cavity, construction, design, genetic algorithm, metalloenzymes, MIP.

  • C. Djediat, K. Feilke, A. Brochard, L. Caramelle, S. K. Tiam, P. Sétif, T. Gauvrit, C. Yéprémian, A. Wilson, L. Talbot, B. Marie, D. Kirilovsky, et C. Bernard, « Light stress in green and red Planktothrix strains: The orange carotenoid protein and its related photoprotective mechanism », Biochimica Et Biophysica Acta. Bioenergetics, juin 2019.
    Résumé : Photosynthetic organisms need to sense and respond to fluctuating environmental conditions, to perform efficient photosynthesis and avoid the formation of harmful reactive oxygen species. Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranal light-harvesting antenna. This mechanism is triggered by the photoactive orange carotenoid protein (OCP). In this study, we characterized OCP and the related photoprotective mechanism in non-stressed and light-stressed cells of three different strains of Planktothrix that can form impressive blooms. In addition to changing lake ecosystemic functions and biodiversity, Planktothrix blooms can have adverse effects on human and animal health as they produce toxins (e.g., microcystins). Three Planktothrix strains were selected: two green strains, PCC 10110 (microcystin producer) and PCC 7805 (non-microcystin producer), and one red strain, PCC 7821. The green strains colonize shallow lakes with higher light intensities while red strains proliferate in deep lakes. Our study allowed us to conclude that there is a correlation between the ecological niche in which these strains proliferate and the rates of induction and recovery of OCP-related photoprotection. However, differences in the resistance to prolonged high-light stress were correlated to a better replacement of damaged D1 protein and not to differences in OCP photoprotection. Finally, microcystins do not seem to be involved in photoprotection as was previously suggested.
    Mots-clés : B3S, Cyanobacteria, Fluorescence, Microcystin, MROP, Orange carotenoid protein, Planktothrix.

  • P. Donate-Macian, E. Alvarez-Marimon, F. Sepulcre, J. L. Vazquez-Ibar, et A. Peralvarez-Marin, « The Membrane Proximal Domain of TRPV1 and TRPV2 Channels Mediates Protein-Protein Interactions and Lipid Binding In Vitro », International Journal of Molecular Sciences, vol. 20, nᵒ 3, p. 682, févr. 2019.
    Résumé : Constitutive or regulated membrane protein trafficking is a key cell biology process. Transient receptor potential channels are somatosensory proteins in charge of detecting several physical and chemical stimuli, thus requiring fine vesicular trafficking. The membrane proximal or pre-S1 domain (MPD) is a highly conserved domain in transient receptor potential channels from the vanilloid (TRPV) subfamily. MPD shows traits corresponding to protein-protein and lipid-protein interactions, and protein regulatory regions. We have expressed MPD of TRPV1 and TRPV2 as green fluorescente protein (GFP)-fusion proteins to perform an in vitro biochemical and biophysical characterization. Pull-down experiments indicate that MPD recognizes and binds Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptors (SNARE). Synchrotron radiation scattering experiments show that this domain does not self-oligomerize. MPD interacts with phosphatidic acid (PA), a metabolite of the phospholipase D (PLD) pathway, in a specific manner as shown by lipid strips and Trp fluorescence quenching experiments. We show for the first time, to the best of our knowledge, the binding to PA of an N-terminus domain in TRPV channels. The presence of a PA binding domain in TRPV channels argues for putative PLD regulation. Findings in this study open new perspectives to understand the regulated and constitutive trafficking of TRPV channels exerted by protein-protein and lipid-protein interactions.
    Mots-clés : activation, B3S, biophysics, exocytosis, fusion, lipid-protein interactions, LPSM, protein-protein interactions, protein–protein interactions, snap-25, synaptotagmin, syntaxin, Transient Receptor Potential (TRP) channels.

  • M. C. Dos Santos, A. Runser, H. Bartenlian, A. M. Nonat, L. J. Charbonniere, A. S. Klymchenko, N. Hildebrandt, et A. Reisch, « Lanthanide-Complex-Loaded Polymer Nanoparticles for Background-Free Single-Particle and Live-Cell Imaging », Chemistry of Materials, vol. 31, nᵒ 11, p. 4034-4041, juin 2019.
    Résumé : Imaging single molecules and nanoparticles in complex biological media is highly challenging notably due to autofluorescence of cells and tissues. Lanthanides and lanthanide complexes, with their particularly long luminescence lifetimes, offer the possibility to perform time-gated imaging and thus to strongly reduce the autofluorescence background. However, their very low brightness and photon flux have limited their use in single-molecule imaging. Here, we encapsulate high amounts of Europium complexes into poly(methyl methacrylate)-based particles of 10, 20, and 30 nm size. The resulting particles contain up to 5000 copies of the complex with a quantum yield of >= 0.2, resulting in a per particle brightness of up to 4 x 10(7) M-1 cm(-1). They can be imaged at the single-particle level using low illumination intensities (0.24 W cm(-2)) and low acquisition times (300 ms) and internalize well into living cells, where they can be monitored through time-gated imaging at illumination conditions compatible with living specimen. These Eu-complex-loaded nanoparticles can thus be applied for highly sensitive and autofluorescence-free imaging and have the potential to become very performant probes for fast intracellular tracking of single biomolecules.
    Mots-clés : agents, B3S, bright, dyes, emission, in-vitro, luminescence, NANO, photoluminescence, quantum dots, time-resolved fluorescence, tracking.

  • M. Duarte, P. Vende, A. Charpilienne, M. Gratia, C. Laroche, et D. Poncet, « Rotavirus Infection Alters Splicing of the Stress-Related Transcription Factor XBP1 », Journal of Virology, vol. 93, nᵒ 5, p. e01739-18, mars 2019.
    Résumé : XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA ( XBP1es) that corresponded to exon skipping in the XBP1 pre-RNA is induced depending on the rotavirus strain used. We show that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using monoreassortant between ES+ ("skipping") and ES-("nonskipping") strains of rotavirus, we show that gene 7 encoding the viral translation enhancer NSP3 is involved in this phenomenon and that exon skipping parallels the nuclear relocalization of cytoplasmic PABP. We further show, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype is linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP. IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA translation enhancer NSP3. Since XBP1 is involved in cellular stress and immune responses and since the XBP1 protein made from the alternatively spliced RNA is an active transcription factor, our observations raise the question of whether alternative splicing is a cellular response to rotavirus infection.
    Mots-clés : consensus sequence, endoplasmic-reticulum stress, ifn-beta induction, immune response, messenger-rna, nsp3, nuclear import, nuclear transport, nucleocytoplasmic transport, pabp, poly(a) binding-protein, poly(a)-binding protein, reverse genetics, ROTA, rotavirus, splicing, stress response, unfolded protein response, VIRO, xbp1.

  • E. Dubois, A. De Muyt, J. L. Soyer, K. Budin, M. Legras, T. Piolot, R. Debuchy, N. Kleckner, D. Zickler, et E. Espagne, « Building bridges to move recombination complexes », Proceedings of the National Academy of Sciences of the United States of America, mai 2019.
    Résumé : A central feature of meiosis is pairing of homologous chromosomes, which occurs in two stages: coalignment of axes followed by installation of the synaptonemal complex (SC). Concomitantly, recombination complexes reposition from on-axis association to the SC central region. We show here that, in the fungus Sordaria macrospora, this critical transition is mediated by robust interaxis bridges that contain an axis component (Spo76/Pds5), DNA, plus colocalizing Mer3/Msh4 recombination proteins and the Zip2-Zip4 mediator complex. Mer3-Msh4-Zip2-Zip4 colocalizing foci are first released from their tight axis association, dependent on the SC transverse-filament protein Sme4/Zip1, before moving to bridges and thus to a between-axis position. Ensuing shortening of bridges and accompanying juxtaposition of axes to 100 nm enables installation of SC central elements at sites of between-axis Mer3-Msh4-Zip2-Zip4 complexes. We show also that the Zip2-Zip4 complex has an intrinsic affinity for chromosome axes at early leptotene, where it localizes independently of recombination, but is dependent on Mer3. Then, later, Zip2-Zip4 has an intrinsic affinity for the SC central element, where it ultimately localizes to sites of crossover complexes at the end of pachytene. These and other findings suggest that the fundamental role of Zip2-Zip4 is to mediate the recombination/structure interface at all post-double-strand break stages. We propose that Zip2-Zip4 directly mediates a molecular handoff of Mer3-Msh4 complexes, from association with axis components to association with SC central components, at the bridge stage, and then directly mediates central region installation during SC nucleation.
    Mots-clés : chromosome structure, DBG, interaxis bridges, meiotic recombination, MRP, synaptonemal complex, Zip2-Zip4.

  • P. Dubois, I. Correia, F. Le Chevalier, S. Dubois, I. Jacques, N. Canu, M. Moutiez, R. Thai, M. Gondry, O. Lequin, et P. Belin, « Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids », Scientific Reports, vol. 9, nᵒ 1, p. 9208, juin 2019.
    Résumé : Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. In the context of synthetic biology, efficient means for their biological production would increase their chemical diversification and the discovery of novel bioactive compounds. Here, we prove the suitability of the Escherichia coli chassis for the production of prenylated indole DKP alkaloids. We used enzyme combinations not found in nature by co-expressing bacterial cyclodipeptide synthases (CDPSs) that assemble the DKP ring and fungal prenyltransferases (PTs) that transfer the allylic moiety from the dimethylallyl diphosphate (DMAPP) to the indole ring of tryptophanyl-containing cyclodipeptides. Of the 11 tested combinations, seven resulted in the production of eight different prenylated indole DKP alkaloids as determined by LC-MS/MS and NMR characterization. Two were previously undescribed. Engineering E. coli by introducing a hybrid mevalonate pathway for increasing intracellular DMAPP levels improved prenylated indole DKP alkaloid production. Purified product yields of 2-26 mg/L per culture were obtained from culture supernatants. Our study paves the way for the bioproduction of novel prenylated indole DKP alkaloids in a tractable chassis that can exploit the cyclodipeptide diversity achievable with CDPSs and the numerous described PT activities.
    Mots-clés : BIOSYN, MICROBIO.

  • E. Durand, I. Gagnon-Arsenault, J. Hallin, I. Hatin, A. K. Dube, L. Nielly-Thibault, O. Namy, et C. R. Landry, « Turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations », Genome Research, vol. 29, nᵒ 6, p. 932-943, juin 2019.
    Résumé : Little is known about the rate of emergence of de novo genes, what their initial properties are, and how they spread in populations. We examined wild yeast populations (Saccharomyces paradoxus) to characterize the diversity and turnover of intergenic ORFs over short evolutionary timescales. We find that hundreds of intergenic ORFs show translation signatures similar to canonical genes, and we experimentally confirmed the translation of many of these ORFs in laboratory conditions using a reporter assay. Compared with canonical genes, intergenic ORFs have lower translation efficiency, which could imply a lack of optimization for translation or a mechanism to reduce their production cost. Translated intergenic ORFs also tend to have sequence properties that are generally close to those of random intergenic sequences. However, some of the very recent translated intergenic ORFs, which appeared <110 kya, already show gene-like characteristics, suggesting that the raw material for functional innovations could appear over short evolutionary timescales.
    Mots-clés : DBG, drosophila-yakuba, evolution, genome, GST, in-vivo, map, origin, sequences, translation.

  • P. Durand, D. De Luca, et P. Tissieres, « What's new in lung ultrasound in the critically ill or injured child », Intensive Care Medicine, vol. 45, nᵒ 4, p. 508-511, avr. 2019.
    Mots-clés : chest radiography, ESHR, infants, MICROBIO, pneumonia, ultrasonography, ventilation.

  • M. Esposito, S. Hermann-Le Denmat, et A. Delahodde, « Contribution of ERMES subunits to mature peroxisome abundance », PloS One, vol. 14, nᵒ 3, p. e0214287, 2019.
    Résumé : Eukaryotic organelles share different components and establish physical contacts to communicate throughout the cell. One of the best-recognized examples of such interplay is the metabolic cooperation and crosstalk between mitochondria and peroxisomes, both organelles being functionally and physically connected and linked to the endoplasmic reticulum (ER). In Saccharomyces cerevisiae, mitochondria are linked to the ER by the ERMES complex that facilitates inter-organelle calcium and phospholipid exchanges. Recently, peroxisome-mitochondria contact sites (PerMit) have been reported and among Permit tethers, one component of the ERMES complex (Mdm34) was shown to interact with the peroxin Pex11, suggesting that the ERMES complex or part of it may be involved in two membrane contact sites (ER-mitochondria and peroxisome- mitochondria). This opens the possibility of exchanges between these three membrane compartments. Here, we investigated in details the role of each ERMES subunit on peroxisome abundance. First, we confirmed previous studies from other groups showing that absence of Mdm10 or Mdm12 leads to an increased number of mature peroxisomes. Secondly, we showed that this is not simply due to respiratory function defect, mitochondrial DNA (mtDNA) loss or mitochondrial network alteration. Finally, we present evidence that the contribution of ERMES subunits Mdm10 and Mdm12 to peroxisome number involves two different mechanisms.
    Mots-clés : BIOCELL, biogenesis, complex, dna, endoplasmic-reticulum, er, FDMITO, inheritance, mitochondrial morphology, outer-membrane protein, smp domains, yeast.

  • N. Essawy, C. Samson, A. Petitalot, S. Moog, A. Bigot, I. Herrada, A. Marcelot, A. - A. Arteni, C. Coirault, et S. Zinn-Justin, « An Emerin LEM-Domain Mutation Impairs Cell Response to Mechanical Stress », Cells, vol. 8, nᵒ 6, juin 2019.
    Résumé : Emerin is a nuclear envelope protein that contributes to genome organization and cell mechanics. Through its N-terminal LAP2-emerin-MAN1 (LEM)-domain, emerin interacts with the DNA-binding protein barrier-to-autointegration (BAF). Emerin also binds to members of the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Mutations in the gene encoding emerin are responsible for the majority of cases of X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). Most of these mutations lead to an absence of emerin. A few missense and short deletion mutations in the disordered region of emerin are also associated with X-EDMD. More recently, missense and short deletion mutations P22L, ∆K37 and T43I were discovered in emerin LEM-domain, associated with isolated atrial cardiac defects (ACD). Here we reveal which defects, at both the molecular and cellular levels, are elicited by these LEM-domain mutations. Whereas K37 mutation impaired the correct folding of the LEM-domain, P22L and T43I had no impact on the 3D structure of emerin. Surprisingly, all three mutants bound to BAF, albeit with a weaker affinity in the case of K37. In human myofibroblasts derived from a patient's fibroblasts, emerin ∆K37 was correctly localized at the inner nuclear membrane, but was present at a significantly lower level, indicating that this mutant is abnormally degraded. Moreover, SUN2 was reduced, and these cells were defective in producing actin stress fibers when grown on a stiff substrate and after cyclic stretches. Altogether, our data suggest that the main effect of mutation K37 is to perturb emerin function within the LINC complex in response to mechanical stress.
    Mots-clés : actin, atrial cardiac defects, B3S, BAF, emerin, INTGEN, mechano-transduction.

  • C. Essoh, J. - P. Vernadet, G. Vergnaud, A. Coulibaly, A. Kakou-N'Douba, A. S. - P. N'Guetta, G. Resch, et C. Pourcel, « Complete Genome Sequences of Five Acinetobacter baumannii Phages from Abidjan, Côte d'Ivoire », Microbiology Resource Announcements, vol. 8, nᵒ 1, janv. 2019.
    Résumé : Five bacteriophages of Acinetobacter baumannii were isolated from sewage water in Abidjan, Côte d'Ivoire. Phages Aci01-1, Aci02-2, and Aci05 belong to an unclassified genus of the Myoviridae family, with double-stranded DNA (dsDNA) genomes, whereas Aci07 and Aci08 belong to the Fri1virus genus of the Podoviridae family of phages.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • P. Fernández Varela, C. Velours, M. Aumont-Niçaise, B. Pineau, P. Legrand, et I. Poquet, « Biophysical and structural characterization of a zinc-responsive repressor of the MarR superfamily », PloS One, vol. 14, nᵒ 2, p. e0210123, 2019.
    Résumé : The uptake of zinc, which is vital in trace amounts, is tightly controlled in bacteria. For this control, bacteria of the Streptococcaceae group use a Zn(II)-binding repressor named ZitR in lactococci and AdcR in streptococci, while other bacteria use a Zur protein of the Ferric uptake regulator (Fur) superfamily. ZitR and AdcR proteins, characterized by a winged helix-turn-helix DNA-binding domain, belong to the multiple antibiotic resistance (MarR) superfamily, where they form a specific group of metallo-regulators. Here, one such Zn(II)-responsive repressor, ZitR of Lactococcus lactis subspecies cremoris strain MG1363, is characterized. Size Exclusion Chromatography-coupled to Multi Angle Light Scattering, Circular Dichroism and Isothermal Titration Calorimetry show that purified ZitR is a stable dimer complexed to Zn(II), which is able to bind its two palindromic operator sites on DNA fragments. The crystal structure of ZitR holo-form (Zn(II)4-ZitR2), has been determined at 2.8 Å resolution. ZitR is the fourth member of the MarR metallo-regulator subgroup whose structure has been determined. The folding of ZitR/AdcR metallo-proteins is highly conserved between both subspecies (cremoris or lactis) in the Lactococcus lactis species and between species (Lactococcus lactis and Streptococcus pneumoniae or pyogenes) in the Streptococcaceae group. It is also similar to the folding of other MarR members, especially in the DNA-binding domain. Our study contributes to better understand the biochemical and structural properties of metallo-regulators in the MarR superfamily.
    Mots-clés : PF, PIM.

  • A. Ferrandi, F. Gastoni, M. Pitaro, S. Tagliaferri, C. B. de la Tour, R. Alduina, S. Sommer, M. Fasano, P. Barbieri, M. Mancini, et I. M. Bonapace, « Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage », Biochimica Et Biophysica Acta-General Subjects, vol. 1863, nᵒ 1, p. 118-129, janv. 2019.
    Résumé : Background: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes. Methods: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coif and DR. slip knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette. Results: DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm(+) genomic DNA, and to cytosine unmethylated DR and E. coli dcm(-) genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced > 100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors. Conclusions: The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage. General significance: Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.
    Mots-clés : cytosine, DBG, DNA cytosine-methylation, DNA damage, DR1533 locus, features, Genotoxic agents, manganese(ii), Mn2+, oxidation, perspective, RBA, recognition, repair, resistance, SRA domain, uhrf1.

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