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Publications Département B3S


  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • J. Fischböck-Halwachs, S. Singh, M. Potocnjak, G. Hagemann, V. Solis-Mezarino, S. Woike, M. Ghodgaonkar-Steger, F. Weissmann, L. D. Gallego, J. Rojas, J. Andreani, A. Köhler, et F. Herzog, « The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore », eLife, vol. 8, p. e42879, mai 2019.
    Résumé : Kinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1CENP-U/Q heterodimer, which forms the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1INCENP/Aurora-B core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affected chromosome segregation fidelity in Sli15 wild-type cells. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating CPC-binding and accurate chromosome segregation.
    Mots-clés : AMIG, aurora B, aurora b kinase, B3S, biochemistry, cell biology, cenp-a nucleosomes, centromeric nucleosome, chemical biology, chemical crosslinking, chromosomal passenger complex, chromosome segregation, error correction, feedback control, kinetochore, mass spectrometry, mitotic checkpoint, molecular-basis, protein complex, rwd domain, S. cerevisiae, saccharomyces-cerevisiae, spindle.

  • P. Frit, V. Ropars, M. Modesti, J. - B. Charbonnier, et P. Calsou, « Plugged into the Ku-DNA hub: The NHEJ network », Progress in Biophysics and Molecular Biology, mars 2019.
    Résumé : In vertebrates, double-strand breaks in DNA are primarily repaired by Non-Homologous End-Joining (NHEJ). The ring-shaped Ku heterodimer rapidly senses and threads onto broken DNA ends forming a recruiting hub. Through protein-protein contacts eventually reinforced by protein-DNA interactions, the Ku-DNA hub attracts a series of specialized proteins with scaffolding and/or enzymatic properties. To shed light on these dynamic interplays, we review here current knowledge on proteins directly interacting with Ku and on the contact points involved, with a particular accent on the different classes of Ku-binding motifs identified in several Ku partners. An integrated structural model of the core NHEJ network at the synapsis step is proposed.
    Mots-clés : B3S, Conserved binding motifs, DNA damage, DNA repair machineries, Double-strand breaks, INTGEN, Ku, NHEJ, Protein interactions network.

  • J. Gantner, J. Ordon, C. Kretschmer, R. Guerois, et J. Stuttmann, « An EDS1-SAG101 Complex is Essential for TNL-mediated Immunity in Nicotiana benthamiana », The Plant Cell, juill. 2019.
    Résumé : Heterodimeric complexes containing the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) are regarded as central regulators of plant innate immunity. In this context, a complex of EDS1 with PHYTOALEXIN DEFICIENT4 (PAD4) is required for basal resistance and signaling downstream of immune receptors containing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs) in Arabidopsis thaliana. Here we analyze EDS1 functions in the model Solanaceous plant Nicotiana benthamiana (Nb). Stable Nb mutants deficient in EDS1 complexes are not impaired in basal resistance, a finding which contradicts a general role for EDS1 in immunity. In Nb, PAD4 demonstrated no detectable immune functions, but TNL-mediated resistance responses required EDS1 complexes incorporating a SENESCENCE ASSOCIATED GENE101 (SAG101) isoform. Intriguingly, SAG101 is restricted to those genomes also encoding TNL receptors, and we propose it may be required for TNL-mediated immune signaling in most plants, except the Brassicaceae. Transient complementation in Nb was used for accelerated mutational analyses while avoiding complex biotic interactions. We identify a large surface essential for EDS1-SAG101 immune functions, which extends from the N-terminal lipase domains to the C-terminal EP domains and might mediate interaction partner recruitment. Further, this work demonstrates the value of genetic resources in Nb, which will facilitate elucidation of EDS1 functions.
    Mots-clés : AMIG, B3S.

  • A. Gelzinis, R. Augulis, V. Butkus, B. Robert, et L. Valkunas, « Two-dimensional spectroscopy for non-specialists », Biochimica Et Biophysica Acta-Bioenergetics, vol. 1860, nᵒ 4, p. 271-285, avr. 2019.
    Résumé : Detailed studies of the excitation dynamics in photosynthetic pigment-proteins require an application of a wide range of spectroscopic methods. From the later part of the previous century, pump probe and time-resolved fluorescence spectroscopy provided an impressive amount of information. Being simple to grasp, these methods are well-understood and widely used by the photosynthesis research community. In the last fifteen years, two-dimensional (2D) spectroscopy was developed. It has significant advantages over other methods, in particular higher temporal resolution available and higher signal-to-noise ratio. Even though it provides considerable opportunities in research, both its experimental realization and theoretical description are rather complicated, making it somewhat difficult to understand and apply. This makes an unfortunate gap in the community, with spectroscopy experts being able to use the technique, but sometimes lacking the relevant biological knowledge, while biologists having that knowledge are dubious about 2D spectroscopy due to the complexity of the approach. This publication is an attempt to fill this gap by providing an accessible introduction to the concepts, principles and possible applications of the 2D spectroscopy, aimed at the biologically trained members of the photosynthesis research community.
    Mots-clés : B3S, complex, dynamics, electronic spectroscopy, Energy transfer, energy-transfer, LBMS, optical spectroscopy, protein, Pump-probe spectroscopy, quantum coherence, reaction centers, Two-dimensional spectroscopy, uv.

  • M. Genera, D. Samson, B. Raynal, A. Haouz, B. Baron, C. Simenel, R. Guerois, N. Wolff, et C. Caillet-Saguy, « Structural and functional characterization of the PDZ domain of the human phosphatase PTPN3 and its interaction with the human papillomavirus E6 oncoprotein », Scientific Reports, vol. 9, nᵒ 1, p. 7438, mai 2019.
    Résumé : The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a PDZ (PSD-95/Dlg/ZO-1) domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers. Interestingly, the high-risk genital human papillomavirus (HPV) types 16 and 18 target the PDZ domain of PTPN3. The presence of a PDZ binding motif (PBM) on E6 confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential. Here, we report the molecular basis of interaction between the PDZ domain of PTPN3 and the PBM of the HPV E6 protein. We combined biophysical, NMR and X-ray experiments to investigate the structural and functional properties of the PDZ domain of PTPN3. We showed that the C-terminal sequences from viral proteins encompassing a PBM interact with PTPN3-PDZ with similar affinities to the endogenous PTPN3 ligand MAP kinase p38γ. PBM binding stabilizes the PDZ domain of PTPN3. We solved the X-ray structure of the PDZ domain of PTPN3 in complex with the PBM of the HPV E6 protein. The crystal structure and the NMR chemical shift mapping of the PTPN3-PDZ/peptide complex allowed us to pinpoint the main structural determinants of recognition of the C-terminal sequence of the E6 protein and the long-range perturbations induced upon PBM binding.
    Mots-clés : AMIG, B3S, binding, complex, proteins, receptor, system.

  • J. Godau, L. P. Ferretti, A. Trenner, E. Dubois, C. von Aesch, A. Marmignon, L. Simon, A. Kapusta, R. Guérois, M. Bétermier, et A. A. Sartori, « Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair », DNA repair, vol. 77, p. 96-108, mars 2019.
    Résumé : DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.
    Mots-clés : AMIG, B3S, CtIP, ctp1, damage response, DBG, DNA double-strand breaks, DNA end resection, end-resection, endonuclease, gene, Homologous recombination, human ctip, Meiosis, MICMAC, mre11 complex, Paramecium tetraurelia, protein, rad32(mre11) nuclease, sae2.

  • H. H. Gorris, T. Soukka, A. Bednarkiewicz, J. Perez-Prieto, et N. Hildebrandt, « A new forum for upconversion research: The UPCON conference », Methods and Applications in Fluorescence, juin 2019.
    Résumé : The Conference and Spring School on Properties, Design and Applications of Upconversion Nanomaterials (UPCON) provides a new forum for all experts and newcomers in the field of upconversion research. On the occasion of the second UPCON 2018 in Valencia (Spain), we are pleased to present a collection of 12 reviews and research articles that reflect recent advances in upconversion materials, their unique luminescent properties and many applications spanning from nanoscale thermometry to biomedicine.
    Mots-clés : B3S, background-free, luminescence, NANO, nanomaterials, upconversion.

  • P. Gotico, B. Boitrel, R. Guillot, M. Sircoglou, A. Quaranta, Z. Halime, W. Leibl, et A. Aukauloo, « Second-Sphere Biomimetic Multipoint Hydrogen-Bonding Patterns to Boost CO2 Reduction of Iron Porphyrins », Angewandte Chemie (International Ed. in English), vol. 58, nᵒ 14, p. 4504-4509, mars 2019.
    Résumé : Inspired by nature's orchestra of chemical subtleties to activate and reduce CO2 , we have developed a family of iron porphyrin derivatives in to which we have introduced urea groups functioning as multipoint hydrogen-bonding pillars on the periphery of the porphyrinic ring. This structure closely resembles the hydrogen-bond stabilization scheme of the carbon dioxide (CO2 ) adduct in the carbon monoxide dehydrogenase (CODH). We found that such changes to the second coordination sphere significantly lowered the overpotential for CO2 reduction in this family of molecular catalysts and importantly increased the CO2 binding rate while maintaining high turnover frequency (TOF) and selectivity. Entrapped water molecules within the molecular clefts were found to be the source of protons for the CO2 reduction.
    Mots-clés : B3S, binding, carbon dioxide reduction, carbon-dioxide, co2-to-co electrochemical conversion, complexes, efficient, fe, hydrogen bonding, iron, LPB, models, molecular catalysis, picket-fence porphyrins, porphyrins, urea.

  • M. Grabsztunowicz, P. Mulo, F. Baymann, R. Mutoh, G. Kurisu, P. Sétif, P. Beyer, et A. Krieger-Liszkay, « Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L », The Plant Journal: For Cell and Molecular Biology, mars 2019.
    Résumé : During daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b6 f complex, ATP synthase and several isoforms of ferredoxin-NADP+ oxidoreductase (FNR) and of ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b6 f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b6 f complex, FNR and redox-inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd, and cytochrome bh of the cytochrome b6 f complex and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both, NADH and NADPH, as electron donor. FNR and Fd are not involved in this pathway. The NDH-complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration which may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b6 f complex during cyclic electron transport in chloroplasts. This article is protected by copyright. All rights reserved.
    Mots-clés : B3S, chromoplast, chromorespiration, cytochrome b6f complex, electron transport chain, MROP, Narcissus pseudonarcissus, NDH, PTOX.

  • J. Guo, C. Mingoes, X. Qiu, et N. Hildebrandt, « Simple, Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction », Analytical Chemistry, vol. 91, nᵒ 4, p. 3101-3109, févr. 2019.
    Résumé : The hybridization chain reaction (HCR) is a simple and sensitive method for quantifying nucleic acids. Current approaches cannot combine a washing-free sensing format with multiplexed target quantification at low concentrations, which would be highly desirable for detection both in solution and in situ. Here, we demonstrate the implementation of time-gated Förster resonance energy transfer (TG-FRET) between terbium donors and dye acceptors into HCR for multiplexed quantification of microRNAs (miR-20a and miR-21) and their DNA analogues. HCR-TG-FRET provided washing-free nucleic acid quantification with very low limits of detection down to 240 amol (1.7 pM) of microRNA and 123 amol (0.88 pM) of DNA. Efficient distinction from very homologous microRNAs demonstrated high target specificity. Multiplexing with a single measurement, a single excitation wavelength, and a single FRET pair allowed for a simultaneous quantification of miR-20a and miR-21 at concentrations between 30 and 300 pM from the same sample. HCR-TG-FRET showed similar performance for serum-free and serum-containing samples without the use of RNase inhibitors. Our results present a significant improvement in current HCR approaches regarding simplicity, sensitivity, and multiplexing. The versatile diagnostic performance of HCR-TG-FRET even in challenging biological environments presents an important advantage for advanced nucleic acid biosensing.
    Mots-clés : B3S, construction, diagnostic assay, dna, in-situ, isothermal amplification, NANO, sensitive detection, signal amplification, strand displacement amplification.

  • J. Guo, X. Qiu, C. Mingoes, J. R. Deschamps, K. Susumu, I. L. Medintz, et N. Hildebrandt, « Conformational Details of Quantum Dot-DNA Resolved by Forster Resonance Energy Transfer Lifetime Nanoruler », Acs Nano, vol. 13, nᵒ 1, p. 505-514, janv. 2019.
    Résumé : DNA-nanoparticle conjugates are important tools in nanobiotechnology. Knowing the orientation, function, and length of DNA on nanoparticle surfaces at low nanomolar concentrations under physiological conditions is therefore of great interest. Here, we investigate the conformation of a 31 nucleotides (nt) long DNA attached to a semiconductor quantum dot (QD) via Forster resonance energy transfer (FRET) from Tb-DNA probes hybridized to different positions on the QD-DNA. Precise Tb-to-QD distance determination from 7 to 14 nm along 26 nt of the peptide-appended QD-DNA was realized by time-resolved FRET spectroscopy. The FRET nanoruler measured linear single-stranded (ssDNA) and double-stranded (dsDNA) extensions of similar to 0.15 and similar to 0.31 nm per base, reflecting the different conformations. Comparison with biomolecular modeling confirmed the denser conformation of ssDNA and a possibly more flexible orientation on the QD surface, whereas the dsDNA was fully extended with radial orientation. The temporally distinct photoluminescence decays of the different DNA-FRET configurations were used for prototypical DNA hybridization assays that demonstrated the large potential for extended temporal multiplexing. The extensive experimental and theoretical analysis of 11 different distances/configurations of the same QD-DNA conjugate provided important information on DNA conformation on nanoparticle surfaces and will be an important benchmark for the development and optimization of DNA-nanobiosensors.
    Mots-clés : B3S, conformation, fret, ligands, molecular ruler, NANO, nanoparticles, platform, quantum dots, semiconductor, sensing, single-stranded-dna, terbium, transfer fret.

  • D. Harris, A. Wilson, F. Muzzopappa, N. N. Sluchanko, T. Friedrich, E. G. Maksimov, D. Kirilovsky, et N. Adir, « Structural Rearrangements in the C-Terminal Domain Homolog of Orange Carotenoid Protein are Crucial for Carotenoid Transfer », Biophysical Journal, vol. 116, nᵒ 3, p. 47A-47A, févr. 2019.

  • I. K. Ilev, Y. Chen, F. Bartoli, G. Popescu, K. Dholakia, A. Fales, et N. Hildebrandt, « Introduction to the JSTQE Issue on Nanobiophotonics », Ieee Journal of Selected Topics in Quantum Electronics, vol. 25, nᵒ 2, p. 0200204, avr. 2019.

  • R. Končitíková, A. Vigouroux, M. Kopečná, M. Šebela, S. Moréra, et D. Kopečný, « Kinetic and structural analysis of human ALDH9A1 », Bioscience Reports, vol. 39, p. BSR20190558, mars 2019.
    Résumé : Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes, which detoxify aldehydes produced in various metabolic pathways to the corresponding carboxylic acids. Among the 19 human ALDHs, the cytosolic ALDH9A1 has so far never been fully enzymatically characterized and its structure is still unknown. Here, we report complete molecular and kinetic properties of human ALDH9A1 as well as three crystal forms at 2.3 Å, 2.9 Å and 2.5 Å resolution. We show that ALDH9A1 exhibits wide substrate specificity to aminoaldehydes, aliphatic and aromatic aldehydes with a clear preference for γ -trimethylaminobutyraldehyde (TMABAL). The structure of ALDH9A1 reveals that the enzyme assembles as a tetramer. Each ALDH monomer displays a typical ALDHs fold composed of an oligomerization domain, a coenzyme domain, a catalytic domain and an inter-domain linker highly conserved in amino-acid sequence and folding. Nonetheless, structural comparison reveals a position and a fold of the inter-domain linker of ALDH9A1 never observed in any other ALDH so far. This unique difference is not compatible with the presence of a bound substrate and a large conformational rearrangement of the linker up to 30 Å has to occur to allow the access of the substrate channel. Moreover, the αβE region consisting of an α-helix and a β-strand of the coenzyme domain at the dimer interface are disordered, likely due to the loss of interactions with the inter-domain linker, which leads to incomplete NAD+ binding pocket.
    Mots-clés : 3-aminopropionaldehyde, 4-n-trimethylaminobutyraldehyde dehydrogenase, 4-trimethylaminobutyraldehyde, aldehyde dehydrogenase, B3S, betaine aldehyde dehydrogenase, carnitine, cloning, diamine oxidase, Homo sapiens, involvement, MESB3S, metabolism, oxidation, purification, structure-function, superfamily, X‐ray crystallography.

  • P. E. Konold, I. H. M. van Stokkum, F. Muzzopappa, A. Wilson, M. - L. Groot, D. Kirilovsky, et J. T. M. Kennis, « Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein », Journal of the American Chemical Society, vol. 141, nᵒ 1, p. 520-530, janv. 2019.
    Résumé : The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid beta 1-ring in picoseconds occurs at a low yield of <1%, whereby the beta 1-ring retains a trans configuration with respect to the conjugated pi-electron chain. This event initiates structural changes at the N-terminal domain in 1 mu s, which allow the carotenoid to translocate into the N-terminal domain in 10 mu s. We identified infrared signatures of helical elements that dock on the C-terminal domain beta-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.
    Mots-clés : B3S, deactivation, domain, energy-transfer, femtosecond, MROP, photoprotection, phycobilisome, proton-transfer, rearrangements, reveals, ultrafast spectroscopy.

  • D. A. Korasick, R. Koncitikova, M. Kopecna, E. Hajkova, A. Vigouroux, S. Morera, D. F. Becker, M. Sebela, J. J. Tanner, et D. Kopecny, « Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants », Journal of Molecular Biology, vol. 431, nᵒ 3, p. 576-592, févr. 2019.
    Résumé : Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD +-dependent glutamate gamma-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate gamma-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-ofdimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-A resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family. (C) 2018 Elsevier Ltd. All rights reserved.
    Mots-clés : ALDH12, B3S, MESB3S, Physcomitrella patens, Zea mays.

  • D. A. Kretov, M. - J. Clement, G. Lambert, D. Durand, D. N. Lyabin, G. Bollot, C. Bauvais, A. Samsonova, K. Budkina, R. C. Maroun, L. Hamon, A. Bouhss, E. Lescop, F. Toma, P. A. Curmi, A. Maucuer, L. P. Ovchinnikov, et D. Pastre, « YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis », Nucleic Acids Research, vol. 47, nᵒ 6, p. 3127-3141, avr. 2019.
    Résumé : The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.
    Mots-clés : B3S, cold-shock protein, dynamics, FAAM, genome-wide analysis, helicases, model, phosphorylation, progression, reveals, translation, y-box protein.

  • A. Krieger-Liszkay, K. Krupinska, et G. Shimakawa, « The impact of photosynthesis on initiation of leaf senescence », Physiologia Plantarum, vol. 166, nᵒ 1, p. 148-164, janv. 2019.
    Résumé : Senescence is the last stage of leaf development preceding the death of the organ, and it is important for nutrient remobilization and for feeding sink tissues. There are many reports on leaf senescence but the mechanisms initiating leaf senescence are still poorly understood. Leaf senescence is affected by many environmental factors and seems to vary in different species and even varieties of plants, which makes it difficult to generalize the mechanism. Here, we give an overview on studies reporting about alterations in the composition of the photosynthetic electron transport chain in chloroplasts during senescence. We hypothesize that alternative electron flow and related generation of the proton motive force required for ATP synthesis become increasingly important during progression of senescence. We address the generation of reactive oxygen species (ROS) in chloroplasts in the initiation of senescence, retrograde signaling from the chloroplast to the nucleus and ROS-dependent signaling associated with leaf senescence. Finally, differences between natural senescence and dark-induced senescence are pointed out and a few ideas for increasing crop yields by increasing the chloroplast lifespan are presented. This article is protected by copyright. All rights reserved.
    Mots-clés : a/b-binding proteins, arabidopsis-thaliana, B3S, cyclic electron flow, harvesting complex-ii, hydrogen-peroxide, MROP, oxidative stress, photooxidative-stress, plant photosystem-i, singlet oxygen, superoxide-dismutase.

  • S. Leach, N. C. Jones, S. V. Hoffmann, et S. Un, « Isoquinoline gas-phase absorption spectrum in the vacuum ultraviolet between 3.7 and 10.7 eV. New valence and Rydberg electronic states », Rsc Advances, vol. 9, nᵒ 9, p. 5121-5141, févr. 2019.
    Résumé : VUV photons from a synchrotron source were used to record the gas-phase absorption spectrum of isoquinoline over the range 3.5 to 10.7 eV. The rich spectrum exhibits both broad and sharp features, of varying intensities, that are analyzed into eight valence and eight Rydberg transitions. Previous data on the valence transitions of isoquinoline were essentially limited to solution spectra up to 5.4 eV. Our study increases their number considerably. The features in the 3.96 eV region are discussed in terms of vibronic coupling between the n pi* 1(1)A '' and pi pi*2(1)A' valence electronic states. The intensities of some spectral features are augmented by collective p-electron modes considered to be of plasmon-type. Assignments of the valence transitions were facilitated by our DFT calculations and by earlier Pariser-Parr-Pople MO calculations. The calculation results are compared and their relative value is discussed. The DFT calculations reproduce very well a number of experimentally determined properties of the ground state of isoquinoline, in particular its bond distances and angles, rotational constants, vibrational frequencies and dipole moment. No Rydberg series of isoquinoline have previously been observed. Three of the newly observed Rydberg series converge to the D-0 electronic ground state of the ion, while two converge to the D-1 and three to the D-3 excited electronic states of the cation. Astrophysical applications of the VUV absorption spectrum of isoquinoline, in particular the measured absorption cross-sections, are briefly discussed. A comparison between the absorption spectra of isoquinoline and quinoline highlights their similarities and differences, related to their respective molecular orbitals.
    Mots-clés : B3S, BHFMR, density-functional theory, excitation-energies, infrared-spectroscopy, ionization-potentials, molecules, photoelectron-spectroscopy, polycyclic aromatic-hydrocarbons, quinoline, radiationless transitions, vibrational-spectra.

  • C. Léger, T. Di Meo, M. Aumont-Nicaise, C. Velours, D. Durand, I. Li de la Sierra-Gallay, H. van Tilbeurgh, N. Hildebrandt, M. Desmadril, A. Urvoas, M. Valerio-Lepiniec, et P. Minard, « Ligand-induced conformational switch in an artificial bidomain protein scaffold », Scientific Reports, vol. 9, nᵒ 1, p. 1178, févr. 2019.
    Résumé : Artificial proteins binding any predefined "target" protein can now be efficiently generated using combinatorial libraries based on robust protein scaffolds. αRep is such a family of artificial proteins, based on an α-solenoid protein repeat scaffold. The low aggregation propensity of the specific "binders" generated from this library opens new protein engineering opportunities such as the creation of biosensors within multidomain constructions. Here, we have explored the properties of two new types of artificial bidomain proteins based on αRep structures. Their structural and functional properties are characterized in detail using biophysical methods. The results clearly show that both bidomain proteins adopt a closed bivalve shell-like conformation, in the ligand free form. However, the presence of ligands induces a conformational transition, and the proteins adopt an open form in which each domain can bind its cognate protein partner. The open/closed equilibria alter the affinities of each domain and induce new cooperative effects. The binding-induced relative domain motion was monitored by FRET. Crystal structures of the chimeric proteins indicate that the conformation of each constituting domain is conserved but that their mutual interactions explain the emergent properties of these artificial bidomain proteins. The ligand-induced structural transition observed in these bidomain proteins should be transferable to other αRep proteins with different specificity and could provide the basis of a new generic biosensor design.
    Mots-clés : alpha-rep, B3S, binding-protein, biosensors, design principles, FAAM, MIP, NANO, PIM, repeat proteins, sedimentation-velocity.

  • S. - Z. Li, A. Vigouroux, M. Ahmar, A. El Sahili, L. Soulere, L. Sago, D. Cornu, S. Morera, et Y. Queneau, « Synthesis of a non-natural glucose-2-phosphate ester able to dupe the acc system of Agrobacterium fabrum », Organic & Biomolecular Chemistry, vol. 17, nᵒ 5, p. 1090-1096, févr. 2019.
    Résumé : The first non-natural derivative of the rare D-glucose-2-phosphate (G2P), namely glucose-2-(O-lactic acid phosphate) (G2LP), has been synthesized. When used as sole carbon source, G2LP enables bacterial growth of the plant pathogenic strain Agrobacterium fabrum C58 (formerly referred to as Agrobacterium tumefaciens). X-ray crystallography and affinity measurements investigations reveal that G2LP binds the periplasmic binding protein (PBP) AccA similarly to the natural compounds and with the same affinity. Moreover, enzymatic assays show that it is able to serve as substrate of the phosphodiesterase AccF. The properties found for G2LP demonstrate that the very unusual glucose-2-phosphoryl residue, present in G2LP, can be used as structural feature for designing non-natural systems fully compatible with the Acc cascade of A. fabrum.
    Mots-clés : B3S, diesters, glycogen, hydrolysis, MESB3S, opines, PF, phosphate, plasmids, SICAPS.

  • G. Lippens et B. Gigant, « Elucidating Tau function and dysfunction in the era of cryo-EM », The Journal of Biological Chemistry, vol. 294, nᵒ 24, p. 9316-9325, mai 2019.
    Résumé : Tau is a microtubule-associated protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms fibrils that are molecular hallmarks of neurological disorders known as tauopathies. In the last two years, cryo-EM has given unprecedented high-resolution views of Tau in both physiological and pathological conditions. We review here these new findings and put them into the context of the knowledge about Tau before this structural breakthrough. The first structures of Tau fibrils, a molecular hallmark of Alzheimer's disease (AD), were based on fibrils from the brain of an individual with AD and, along with similar patient-derived structures, have set the gold standard for the field. Cryo-EM structures of Tau fibers in three distinct diseases, AD, Pick's disease and Chronic Traumatic Encephalopathy, represent the end-points of Tau's molecular trajectory. We propose that the recent Tau structures may call for a re-examination of databases that link different Tau variants to various forms of dementia. We also address the question how this structural information may link Tau's functional and pathological aspects. Because this structural information on Tau was obtained in a very short period, the new structures should be viewed in light of earlier structural observations and past and present functional data to shed additional light on Tau function and dysfunction.
    Mots-clés : amyloid, B3S, cryo-electron microscopy, microtubule, MIKICA, Tau protein (Tau), tauopathy, tubulin.

  • J. Lisboa, L. Celmal, D. Sanchez, M. Marquis, J. Andreani, R. Guerois, F. Ochsenbein, D. Durand, S. Marsin, P. Cuniasse, J. P. Radicella, et S. Queyillon-Cheruel, « The C-terminal domain of HpDprA is a DNA-binding winged helix domain that does not bind double-stranded DNA », Febs Journal, vol. 286, nᵒ 10, p. 1941-1958, mai 2019.
    Résumé : DNA-processing protein A, a ubiquitous multidomain DNA-binding protein, plays a crucial role during natural transformation in bacteria. Here, we carried out the structural analysis of DprA from the human pathogen Helicobacter pylori by combining data issued from the 1.8-angstrom resolution X-ray structure of the Pfam02481 domain dimer (RF), the NMR structure of the carboxy terminal domain (CTD), and the low-resolution structure of the full-length DprA dimer obtained in solution by SAXS. In particular, we sought a molecular function for the CTD, a domain that we show here is essential for transformation in H.pylori. Albeit its structural homology to winged helix DNA-binding motifs, we confirmed that the isolated CTD does not interact with ssDNA nor with dsDNA. The key R52 and K137 residues of RF are crucial for these two interactions. Search for sequences harboring homology to either HpDprA or Rhodopseudomonas palustris DprA CTDs led to the identification of conserved patches in the two CTD. Our structural study revealed the similarity of the structures adopted by these residues in RpDprA CTD and HpDprA CTD. This argues for a conserved, but yet to be defined, CTD function, distinct from DNA binding.
    Mots-clés : AMIG, B3S, bacillus-subtilis dpra, DprA, FAAM, Helicobacter pylori, helicobacter-pylori, INTGEN, natural transformation, nmr structure determination, protein, reca, recognition, recombination, sequence, WH DNA binding motif, WH DNA-binding motif, xplor-nih.

  • M. Margier, X. Collet, C. le May, C. Desmarchelier, F. André, C. Lebrun, C. Defoort, A. Bluteau, P. Borel, A. Lespine, et E. Reboul, « ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux », Faseb Journal, vol. 33, nᵒ 2, p. 2084-2094, févr. 2019.
    Résumé : Efficient intestinal absorption of dietary vitamin D is required in most people to ensure an adequate status. Thus, we investigated the involvement of ATP binding cassette subfamily B member 1 (ABCB1) in vitamin D intestinal efflux. Both cholecalciferol (D3) and 25-hydroxycholecalciferol [25(OH)D3] apical effluxes were decreased by chemical inhibition of ABCB1 in Caco-2 cells and increased by ABCB1 overexpression in Griptites or Madin-Darby canine kidney type II cells. Mice deficient for the 2 murine ABCB1s encoded by Abcb1a and Abcb1b genes ( Abcb1-/-) displayed an accumulation of 25(OH)D3 in plasma, intestine, brain, liver, and kidneys, together with an increased D3 postprandial response after gavage compared with controls. 25(OH)D3 efflux through Abcb1-/- intestinal explants was markedly decreased compared with controls. This reduction of 25(OH)D3 transfer from plasma to lumen was further confirmed in vivo in intestine-perfused mice. Docking experiments established that both D3 and 25(OH)D3 could bind with high affinity to Caenorhabditis elegans P-glycoprotein, used as an ABCB1 model. Finally, in a group of 39 healthy male adults, a single-nucleotide polymorphism (SNP) in ABCB1 (rs17064) was significantly associated with the fasting plasma 25(OH)D3 concentration. Thus, we showed here for the first time that ABCB1 is involved in neo-absorbed vitamin D efflux by the enterocytes and that it also contributes to vitamin D transintestinal excretion and likely impacts vitamin D status.-Margier, M., Collet, X., le May, C., Desmarchelier, C., André, F., Lebrun, C., Defoort, C., Bluteau, A., Borel, P., Lespine, A., Reboul, E. ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.
    Mots-clés : 25-hydroxycholecalciferol, B3S, bioavailability, caco-2, cells, cholecalciferol, cholesterol excretion, enterocyte, expression, fatty-acids, LSOD, MDR1.

  • L. Marty, A. Vigouroux, M. Aumont-Nicaise, F. Pelissier, T. Meyer, C. L. Lavire, Y. Dessaux, et S. Moréra, « Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens », The Biochemical Journal, vol. 476, p. 165-178, janv. 2019.
    Résumé : Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid binding mode through three crystal structures at 1.4, 1.74 and 1.9 Å resolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Å resolution defines a different agropinic acid binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.
    Mots-clés : affinity, agrobacterium tumefaciens, B3S, catabolism, crown-gall, crystal structures, cyclase, degradation, gene, mannopinic acid, MESB3S, MICROBIO, octopine, opine, PBI, periplasmic binding protein, PF, PIM, region, ti-plasmid, transformed plants.

  • A. Mezzetti, M. T. A. Alexandre, A. Thurotte, A. Wilson, M. Gwizdala, et D. Kirilovsky, « Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time Resolved FTIR Spectroscopy », The Journal of Physical Chemistry. B, vol. 123, nᵒ 15, p. 3259-3266, avr. 2019.
    Résumé : The Orange Carotenoid Protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved FTIR difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times the disappearance of a non-solvent exposed α-helix (negative 1651 cm-1 band) is observed. At longer times, a 1644 cm-1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of - at least - two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.
    Mots-clés : B3S, domain, forms, MROP, reorganization, water.

  • P. Minard, « The directed evolution of proteins », Medecine Sciences: M/S, vol. 35, nᵒ 2, p. 169-175, févr. 2019.
    Mots-clés : B3S, display, expression, MIP, phage, reshaping human-antibodies.

  • M. Nagel, C. E. Seal, L. Colville, A. Rodenstein, S. Un, J. Richter, H. W. Pritchard, A. Börner, et I. Kranner, « Wheat seed ageing viewed through the cellular redox environment and changes in pH », Free Radical Research, p. 1-341, mai 2019.
    Résumé : To elucidate biochemical mechanisms leading to seed deterioration, we studied 23 wheat genotypes after exposure to seed bank storage for 6-16 years compared to controlled deterioration (CD) at 45 °C and 14 (CD14) and 18% (CD18) moisture content (MC) for up to 32 days. Under two seed bank storage conditions, seed viability was maintained in cold storage (CS) at 0 °C and 9% seed MC, but significantly decreased in ambient storage (AS) at 20 °C and 9% MC. Under AS and CS, organic free radicals, most likely semiquinones, accumulated, detected by electron paramagnetic resonance, while the antioxidant glutathione (GSH) was partly lost and partly converted to glutathione disulphide (GSSG), detected by HPLC. Under AS the glutathione half-cell reduction potential (EGSSG/2GSH) shifted towards more oxidizing conditions, from -186 to -141 mV. In seeds exposed to CD14 or CD18, no accumulation of organic free radicals was observed, GSH and seed viability declined within 32 and 7 days, respectively, GSSG hardly changed (CD14) or decreased (CD18) and EGSSG/2GSH shifted to -116 mV. The pH of extracts prepared from seeds subjected to CS, AS and CD14 decreased with viability, and remained high under CD18. Across all treatments EGSSG/2GSH correlated significantly with seed viability (r = 0.8, p < 0.001). Data are discussed with a view that the cytoplasm is in a glassy state in CS and AS, but during the CD treatments, underwent transition to a liquid state. We suggest that enzymes can be active during CD but not under the seed bank conditions tested. However, upon CD, enzyme-based repair processes were apparently outweighed by deteriorative reactions. We conclude that seed ageing by CD and under seed bank conditions are accompanied by different biochemical reactions.
    Mots-clés : Antioxidants, B3S, BHFMR, EPR, ESR, gene bank, germination, organic radicals, seed longevity.

  • M. Nakamura, A. Boussac, et M. Sugiura, « Consequences of structural modifications in cytochrome b(559) on the electron acceptor side of Photosystem II », Photosynthesis Research, vol. 139, nᵒ 1-3, p. 475-486, mars 2019.
    Résumé : Cytb(559) in Photosystem II is a heterodimeric b-type cytochrome. The subunits, PsbE and PsbF, consist each in a membrane -helix. Mutants were previously designed and studied in Thermosynechococcus elongatus (Sugiura et al., Biochim Biophys Acta 1847:276-285, 2015) either in which an axial histidine ligand of the haem-iron was substituted for a methionine, the PsbE/H23M mutant in which the haem was lacking, or in which the haem environment was modified, the PsbE/Y19F and PsbE/T26P mutants. All these mutants remained active showing that the haem has no structural role provided that PsbE and PsbF subunits are present. Here, we have carried on the characterization of these mutants. The following results were obtained: (i) the Y19F mutation hardly affect the E-m of Cytb(559), whereas the T26P mutation converts the haem into a form with a E-m much below 0mV (so low that it is likely not reducible by Q(B)(-)) even in an active enzyme; (ii) in the PsbE/H23M mutant, and to a less extent in PsbE/T26P mutant, the electron transfer efficiency from Q(A)(-) to Q(B) is decreased; (iii) the lower E-m of the Q(A)/Q(A)(-) couple in the PsbE/H23M mutant correlates with a higher production of singlet oxygen; (iv) the superoxide and/or hydroperoxide formation was not increased in the PsbE/H23M mutant lacking the haem, whereas it was significantly larger in the PsbE/T26P. These data are discussed in view of the literature to discriminate between structural and redox roles for the haem of Cytb(559) in the production of reactive oxygen species.
    Mots-clés : Acceptor side, alpha-subunit, axial-ligands, B3S, b559, Cytb 559, Cytb(559), energetics, Haem axial ligand, Photosystem II, PS2, Redox, redox properties, singlet oxygen production, site-directed mutagenesis, synechocystis pcc 6803, thermosynechococcus-elongatus, water oxidation.

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