Publications

2015

• Trm112, a hub protein interacting with and activating four methyltransferases involved in mRNA translation
  
  • Graille Marc
  , 2015.
• Actin nucleation at the centrosome controls lymphocyte polarity.
  
  • Obino Dorian
  • Farina Francesca
  • Malbec Odile
  • Sáez Pablo J
  • Maurin Mathieu
  • Gaillard Jérémie
  • Dingli Florent
  • Loew Damarys
  • Gautreau Alexis
  • Yuseff Maria-Isabel
  • Blanchoin Laurent
  • Théry Manuel
  • Lennon-Duménil Ana-Maria
  Nature Communications, Nature Publishing Group, 2015, 7, pp.10969. Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome-regulated by the availability of the Arp2/3 complex-determines its capacity to polarize in response to external stimuli. (10.1038/ncomms10969)
  DOI : 10.1038/ncomms10969
• Far infrared spectra of solid state l-serine, l-threonine, l-cysteine, and l-methionine in different protonation states
  
  • Gaillard Thomas
  • Trivella Aurélien
  • Stote Roland H
  • Hellwig Petra
  Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy [1994-...], Elsevier, 2015, 150, pp.301-307. In this study, experimental far infrared measurements of L-serine, L-threonine, L-cysteine, and L-methionine are presented showing the spectra for the 1.0-13.0 pH range. In parallel, solid state DFT calculations were performed on the amino acid zwitterions in the crystalline form. We focused on the lowest frequency far infrared normal modes, which required the most precision and convergence of the calculations. Analysis of the computational results, which included the potential energy distribution of the vibrational modes, permitted a detailed and almost complete assignment of the experimental spectrum. In addition to characteristic signals of the two main acid-base couples, CO2H/CO2- and NH3+/NH2, specific side chain contributions for these amino acids, including CCO and CCS vibrational modes were analyzed. This study is in line with the growing application of FIR measurements to biomolecules. (c) 2015 Elsevier B.V. All rights reserved. (10.1016/j.saa.2015.05.031)
  DOI : 10.1016/j.saa.2015.05.031
• Cdc123, a Cell Cycle Regulator Needed for eIF2 Assembly, Is an ATP-Grasp Protein with Unique Features
  
  • Panvert Michel
  • Dubiez Etienne
  • Arnold Lea
  • Perez Javier
  • Mechulam Yves
  • Seufert Wolfgang
  • Schmitt Emmanuelle
  Structure, Elsevier (Cell Press), 2015, 23 (9), pp.1596-1608. Eukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2 gamma. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg2+, and conserved residues contacting ATP-Mg2+ are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2 alpha gamma onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2 gamma to eIF2 alpha. (10.1016/j.str.2015.06.014)
  DOI : 10.1016/j.str.2015.06.014
• Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor.
  
  • Basquin Cyril
  • Trichet Michaël
  • Vihinen Helena
  • Malardé Valérie
  • Lagache Thibault
  • Ripoll Léa
  • Jokitalo Eija
  • Olivo-Marin Jean-Christophe
  • Gautreau Alexis
  • Sauvonnet Nathalie
  EMBO Journal, EMBO Press, 2015, 34 (16), pp.2147-61. Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions. (10.15252/embj.201490788)
  DOI : 10.15252/embj.201490788
• Specific GFP-binding artificial proteins ( Rep): a new tool for in vitro to live cell applications
  
  • Chevrel Anne
  • Urvoas Agathe
  • Li de La Sierra-Gallay Ines
  • Aumont-Nicaise Magali
  • Moutel Sandrine
  • Desmadril Michel
  • Perez Franck
  • Gautreau Alexis
  • van Tilbeurgh Herman
  • Minard Philippe
  • Valerio-Lepiniec Marie
  Bioscience Reports, Portland Press, 2015, 35 (4), pp.e00223. A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes. (10.1042/BSR20150080)
  DOI : 10.1042/BSR20150080
• The Arp2/3 inhibitory protein arpin induces cell turning by pausing cell migration
  
  • Gorelik Roman
  • Gautreau Alexis
  Cytoskeleton, 2015, 72 (7), pp.362-371. Branched actin networks generated by the Arp2/3 complex provide the driving force for leading edge protrusion in migrating cells. We recently identified Arpin, a protein that inhibits the Arp2/3 complex in lamellipodia. Arpin is activated by the small GTPase Rac, which triggers lamellipodium formation, and thus Arpin renders protrusions unstable. A conserved role of Arpin is to induce migrating cells to turn in different migration models. Here we investigated the mechanism by which Arpin controls directional persistence. For this analysis, we segmented migration trajectories into alternating phases of active migration and pauses, based on a speed threshold. Regardless of the threshold value, Arpin induced more frequent pausing, during which the cell was more likely to change the direction of its migration. Arpin simultaneously acts on cell speed and directional persistence, which are strongly coupled parameters. Induction of frequent pausing by Arpin is consistent with Arpin circuitry: by inhibiting the Arp2/3 complex as a response to Rac activation, Arpin antagonizes a positive feedback loop that sustains protrusions at the leading edge and maintains active migration. We propose the duration of active migration' as a useful proxy to measure feedbacks associated with cell migration. (c) 2015 Wiley Periodicals, Inc. (10.1002/cm.21233)
  DOI : 10.1002/cm.21233
• Electrostatic free energies in translational GTPases: Classic allostery and the rest
  
  • Simonson Thomas
  • Aleksandrov Alexey
  • Satpati Priyadarshi
  BBA - Biochimica et Biophysica Acta, Elsevier, 2015, 1850 (5), pp.1006–1016. GTPases typically switch between an inactive, OFF conformation and an active, ON conformation when a GDP ligand is replaced by GTP. Their ON/OFF populations and activity thus depend on the stabilities of four protein complexes, two apo-protein forms, and GTP/GDP in solution. A complete characterization is usually not possible experimentally and poses major challenges for simulations. We review the most important methodological challenges and we review thermodynamic data for two GTPases involved in translation of the genetic code: archaeal Initiation Factors 2 and 5B (aIF2, aIF5B). One main challenge is the multiplicity of states and conformations, including those of GTP/GDP in solution. Another is force field accuracy, especially for interactions of GTP/GDP with co-bound divalent Mg2 + ions. The calculation of electrostatic free energies also poses specific challenges, and requires careful protocols. For aIF2, experiments and earlier simulations showed that it is a “classic” GTPase, with distinct ON/OFF conformations that prefer to bind GTP and GDP, respectively. For aIF5B, we recently proposed a non-classic mechanism, where the ON/OFF states differ only in the protonation state of Glu81 in the nucleotide binding pocket. This model is characterized here using free energy simulations. The methodological analysis should help future studies, while the aIF2, aIF5B examples illustrate the diversity of ATPase/GTPase mechanisms. This article is part of a Special Issue entitled Recent developments of molecular dynamics. (10.1016/j.bbagen.2014.07.006)
  DOI : 10.1016/j.bbagen.2014.07.006
• Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae.
  
  • Lazard Myriam
  • Dauplais Marc
  • Blanquet Sylvain
  • Plateau Pierre
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2015, 290 (17), pp.10741-50. Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity. (10.1074/jbc.M115.640375)
  DOI : 10.1074/jbc.M115.640375
• Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2
  
  • Dubiez Etienne
  • Aleksandrov Alexey
  • Lazennec-Schurdevin Christine
  • Mechulam Yves
  • Schmitt Emmanuelle
  Nucleic Acids Research, Oxford University Press, 2015, 43 (5), pp.2946-2957. Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eu-karyotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In ar-chaea, orthologs of eIF5 are not found and aIF2 GT-Pase activity is thought to be non-assisted. However , no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2␥, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2␥. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2. (10.1093/nar/gkv053)
  DOI : 10.1093/nar/gkv053
• MatrixDB, the extracellular matrix interaction database: updated content, a new navigator and expanded functionalities
  
  • Launay G.
  • Salza R.
  • Multedo D.
  • Thierry-Mieg Nicolas
  • Ricard-Blum S.
  Nucleic Acids Research, Oxford University Press, 2015, 43 (D1), pp.D321 - D327. (10.1093/nar/gku1091)
  DOI : 10.1093/nar/gku1091
• Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure
  
  • Létoquart Juliette
  • van Tran Nhan
  • Caroline Vonny
  • Aleksandrov Alexey
  • Lazar Noureddine
  • van Tilbeurgh Herman
  • Liger Dominique
  • Graille Marc
  Nucleic Acids Research, Oxford University Press, 2015, 43 (22), pp.10989-11002. Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm 5 U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. (10.1093/nar/gkv1009)
  DOI : 10.1093/nar/gkv1009
• Structure of a left-handed DNA G-quadruplex
  
  • Chung Wan Jun
  • Brahim Heddi
  • Schmitt Emmanuelle
  • Lim Kah Wai
  • Mechulam Yves
  • Phan Anh Tuân
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2015, 112 (9), pp.2729–2733. Aside from the well-known double helix, DNA can also adopt an alternative four-stranded structure known as G-quadruplex. Implications of such a structure in cellular processes, as well as its therapeutic and diagnostic applications, have been reported. The G-quadruplex structure is highly polymorphic, but so far, only right-handed helical forms have been observed. Here we present the NMR solution and X-ray crystal structures of a left-handed DNA G-quadruplex. The structure displays unprecedented features that can be exploited as unique recognition elements. (10.1073/pnas.1418718112)
  DOI : 10.1073/pnas.1418718112
• Quantitative analysis of endosome tubulation and microdomain organization mediated by the WASH complex
  
  • Derivery Emmanuel
  • Gautreau Alexis
  , 2015, 130, pp.215-234. Sorting of cargoes in endosomes occurs through their concentration into sorting platforms, called microdomains, from which transport intermediates are formed. The WASH complex localizes to such endosomal microdomains and triggers localized branched actin nucleation by activating the Arp2/3 complex. These branched actin networks are required for both the lateral compartmentalization of endosome membranes into distinct microdomains and for the fission of transport intermediates from these sorting platforms. In this chapter, we provide experimental protocols to study these two aspects of WASH physiology. We first describe how to image the dynamic membrane tubules resulting from the defects of WASH-mediated fission. We then describe how to study quantitatively the microdomain localization of WASH in live and fixed cells. Since microdomains are below the resolution limit of conventional light-microscopy techniques, this required the development of specific image analysis pipelines, which are detailed. The guidelines presented in this chapter can apply to other endomembrane microdomains beyond WASH in order to increase our understanding of trafficking in molecular and quantitative terms. (10.1016/bs.mcb.2015.03.025)
  DOI : 10.1016/bs.mcb.2015.03.025