Publications

2020

• Physics-Based Computational Protein Design: An Update
  
  • Mignon David
  • Druart Karen
  • Michael Eleni
  • Opuu Vaitea
  • Polydorides Savvas
  • Villa Francesco
  • Gaillard Thomas
  • Panel Nicolas
  • Archontis Georgios
  • Simonson Thomas
  Journal of Physical Chemistry A, American Chemical Society, 2020, 124 (51), pp.10637-10648. We describe methods for physics-based protein design and some recent applications from our work. We present the physical interpretation of a MC simulation in sequence space and show that sequences and conformations form a well-defined statistical ensemble, explored with Monte Carlo and Boltzmann sampling. The folded state energy combines molecular mechanics for solutes with continuum electrostatics for solvent. We usually assume one or a few fixed protein backbone structures and discrete side chain rotamers. Methods based on molecular dynamics, which introduce additional backbone and side chain flexibility, are under development. The redesign of a PDZ domain and an aminoacyl-tRNA synthetase enzyme were successful. We describe a versatile, adaptive, Wang–Landau MC method that can be used to design for substrate affinity, catalytic rate, catalytic efficiency, or the specificity of these properties. The methods are transferable to all biomolecules, can be systematically improved, and give physical insights. (10.1021/acs.jpca.0c07605)
  DOI : 10.1021/acs.jpca.0c07605
• Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation
  
  • Coureux Pierre-Damien
  • Lazennec-Schurdevin Christine
  • Bourcier Sophie
  • Mechulam Yves
  • Schmitt Emmanuelle
  Communications Biology, Nature Publishing Group, 2020, 3 (1). Archaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNAiMet. Here, we determine the cryo-EM structure of a 30S:mRNA:aIF1A:aIF2:GTP:Met-tRNAiMet complex from Pyrococcus abyssi at 3.2 Å resolution. It highlights archaeal features in ribosomal proteins and rRNA modifications. We find an aS21 protein, at the location of eS21 in eukaryotic ribosomes. Moreover, we identify an N-terminal extension of archaeal eL41 contacting the P site. We characterize 34 N4-acetylcytidines distributed throughout 16S rRNA, likely contributing to hyperthermostability. Without aIF1, the 30S head is stabilized and initiator tRNA is tightly bound to the P site. A network of interactions involving tRNA, mRNA, rRNA modified nucleotides and C-terminal tails of uS9, uS13 and uS19 is observed. Universal features and domain-specific idiosyncrasies of translation initiation are discussed in light of ribosomal structures from representatives of each domain of life. (10.1038/s42003-020-0780-0)
  DOI : 10.1038/s42003-020-0780-0
• CYFIP2 containing WAVE complexes inhibit cell migration
  
  • Polesskaya Anna
  • Boutillon Arthur
  • Wang Yanan
  • Lavielle Marc
  • Vacher Sophie
  • Schnitzler Anne
  • Molinie Nicolas
  • Rocques Nathalie
  • Fokin Artem
  • Bièche Ivan
  • David Nicolas B.
  • Gautreau Alexis
  , 2020. Branched actin networks polymerized by the Arp2/3 complex are critical for cell migration. The WAVE complex is the major Arp2/3 activator at the leading edge of migrating cells. However, multiple distinct WAVE complexes can be assembled in a cell, due to the combinatorial complexity of paralogous subunits. When systematically analyzing the contribution of each WAVE complex subunit to the metastasis-free survival of breast cancer patients, we found that overexpression of the CYFIP2 subunit was surprisingly associated with good prognosis. Gain and loss of function experiments in transformed and untransformed mammary epithelial cells revealed that cell migration was always inversely related to CYFIP2 levels. The role of CYFIP2 was systematically opposite to the role of the paralogous subunit CYFIP1 or of the NCKAP1 subunit. The specific CYFIP2 function in inhibiting cell migration was related to its unique ability to down-regulate classical pro-migratory WAVE complexes. The anti-migratory function of CYFIP2 was also revealed in migration of prechordal plate cells during gastrulation of the zebrafish embryo, indicating that the unique function of CYFIP2 is critically important in both physiological and pathophysiological migrations. (10.1101/2020.07.02.184655)
  DOI : 10.1101/2020.07.02.184655
• The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis
  
  • Lacoux Caroline
  • Wacheul Ludivine
  • Saraf Kritika
  • Pythoud Nicolas
  • Huvelle Emmeline
  • Figaro Sabine
  • Graille Marc
  • Carapito Christine
  • Lafontaine Denis L J
  • Heurgué-Hamard Valérie
  Nucleic Acids Research, Oxford University Press, 2020, 48 (21), pp.gkaa972. The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination. (10.1093/nar/gkaa972)
  DOI : 10.1093/nar/gkaa972
• Structural and functional insights into $Archaeoglobus\ fulgidus$ m$^2$G$_{10}$ tRNA methyltransferase Trm11 and its Trm112 activator
  
  • Wang Can
  • Van tran Nhan
  • Jactel Vincent
  • Guérineau Vincent
  • Graille Marc
  Nucleic Acids Research, Oxford University Press, 2020, 48 (19), pp.11068 - 11082. tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N$^2$-methylguanosine (m$^2$G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the $Archaeoglobus\ fulgidus$ archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin. (10.1093/nar/gkaa830)
  DOI : 10.1093/nar/gkaa830
• Computational design of proteins and enzymes
  
  • Opuu Vaitea
  , 2020. We propose a set of methods to design molecular systems. We start from naturally optimized components, namely proteins. Proteins can act as structural components, information transporters, or catalysts. We use computational methods to complement experiments and design protein systems.First, we fully redesigned a PDZ domain involved in metabolic pathways. We used a physics-based approach combining molecular mechanics, continuum electrostatics, and Monte Carlo sampling. Thousands of variants predicted to adopt the PDZ fold were selected. Three were validated experimentally. Two showed binding of the natural peptide ligand.Next, we redesigned the active site of the methionyl-tRNA synthetase enzyme (MetRS). We used an adaptive Monte Carlo method to select variants for methionine (Met) binding. Out of 17 predicted variants that were tested experimentally, 17 were found to be active. We extended the method to transition state binding to select mutants directly according to their catalytic power.We redesigned the MetRS binding site to obtain activity towards two β-amino acids, in order to expand the genetic code. These unnatural amino acids can enhance the structural repertoire of proteins. 20 predicted mutants were tested. Although none had increased β-Met activity, three had a gain in selectivity for β-Met. We then implemented a method to select optimal positions for design and applied it to β-Met and β-Val. Around 20 variants are being experimental tested.Finally, in vivo protein modifications raise the question of their eventual drift away from the original design. We introduce here a design approach for overlapping genes coding PDZ domains. This overlap would reduce genetic drift and provide bio-confinement. We computationally produced almost 2000 pairs of overlapping PDZ domains. One was validated by 2 microsecond molecular dynamic simulations. Experiments are underway.
• Recent Advances in Archaeal Translation Initiation
  
  • Schmitt Emmanuelle
  • Coureux Pierre-Damien
  • Kazan Ramy
  • Bourgeois Gabrielle
  • Lazennec-Schurdevin Christine
  • Mechulam Yves
  Frontiers in Microbiology, Frontiers Media, 2020, 11. Translation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed. However, even if the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes, and archaea and may also vary depending on the mRNA. Because TI mechanisms have evolved lately, their studies bring important information about the evolutionary relationships between extant organisms. In this context, recent structural data on ribosomal complexes and genome-wide studies are particularly valuable. This review focuses on archaeal translation initiation highlighting its relationships with either the eukaryotic or the bacterial world. Eukaryotic features of the archaeal small ribosomal subunit are presented. Ribosome evolution and TI mechanisms diversity in archaeal branches are discussed. Next, the use of leaderless mRNAs and that of leadered mRNAs having Shine-Dalgarno sequences is analyzed. Finally, the current knowledge on TI mechanisms of SD-leadered and leaderless mRNAs is detailed. (10.3389/fmicb.2020.584152)
  DOI : 10.3389/fmicb.2020.584152
• Using NMR for the structural and kinetic characterization of peptide - liposome associations to support formulation design
  
  • Doyen Camille
  • Larquet Eric
  • Coureux Pierre-Damien
  • Frances Oriane
  • Herman Frédéric
  • Sablé Serge
  • Burnouf Jean-Pierre
  • Sizun Christina
  • Lescop Ewen
  , 2020.
• Application of Various Molecular Modelling Methods in the Study of Estrogens and Xenoestrogens
  
  • Mazurek Anna Helena
  • Szeleszczuk Łukasz
  • Simonson Thomas
  • Pisklak Dariusz Maciej
  International Journal of Molecular Sciences, MDPI, 2020, 21 (17), pp.6411. In this review, applications of various molecular modelling methods in the study of estrogens and xenoestrogens are summarized. Selected biomolecules that are the most commonly chosen as molecular modelling objects in this field are presented. In most of the reviewed works, ligand docking using solely force field methods was performed, employing various molecular targets involved in metabolism and action of estrogens. Other molecular modelling methods such as molecular dynamics and combined quantum mechanics with molecular mechanics have also been successfully used to predict the properties of estrogens and xenoestrogens. Among published works, a great number also focused on the application of different types of quantitative structure-activity relationship (QSAR) analyses to examine estrogen's structures and activities. Although the interactions between estrogens and xenoestrogens with various proteins are the most commonly studied, other aspects such as penetration of estrogens through lipid bilayers or their ability to adsorb on different materials are also explored using theoretical calculations. Apart from molecular mechanics and statistical methods, quantum mechanics calculations are also employed in the studies of estrogens and xenoestrogens. Their applications include computation of spectroscopic properties, both vibrational and Nuclear Magnetic Resonance (NMR), and also in quantum molecular dynamics simulations and crystal structure prediction. The main aim of this review is to present the great potential and versatility of various molecular modelling methods in the studies on estrogens and xenoestrogens. (10.3390/ijms21176411)
  DOI : 10.3390/ijms21176411
• Hybrid MC/MD for protein design
  
  • Michael Eleni
  • Polydorides Savvas
  • Simonson Thomas
  • Archontis Georgios
  The Journal of Chemical Physics, American Institute of Physics, 2020, 153 (5), pp.054113. Computational protein design relies on simulations of a protein structure, where selected amino acids can mutate randomly, and mutations are selected to enhance a target property, such as stability. Often, the protein backbone is held fixed and its degrees of freedom are modeled implicitly to reduce the complexity of the conformational space. We present a hybrid method where short molecular dynamics (MD) segments are used to explore conformations and alternate with Monte Carlo (MC) moves that apply mutations to side chains. The backbone is fully flexible during MD. As a test, we computed side chain acid/base constants or pKa’s in five proteins. This problem can be considered a special case of protein design, with protonation/deprotonation playing the role of mutations. The solvent was modeled as a dielectric continuum. Due to cost, in each protein we allowed just one side chain position to change its protonation state and the other position to change its type or mutate. The pKa’s were computed with a standard method that scans a range of pH values and with a new method that uses adaptive landscape flattening (ALF) to sample all protonation states in a single simulation. The hybrid method gave notably better accuracy than standard, fixed-backbone MC. ALF decreased the computational cost a factor of 13. (10.1063/5.0013320)
  DOI : 10.1063/5.0013320
• A physics-based energy function allows the computational redesign of a PDZ domain
  
  • Opuu Vaitea
  • Sun Young Joo
  • Hou Titus
  • Panel Nicolas
  • Fuentes Ernesto
  • Simonson Thomas
  Scientific Reports, Nature Publishing Group, 2020, 10 (1), pp.11150. Computational protein design (CPD) can address the inverse folding problem, exploring a large space of sequences and selecting ones predicted to fold. CPD was used previously to redesign several proteins, employing a knowledge-based energy function for both the folded and unfolded states. We show that a PDZ domain can be entirely redesigned using a "physics-based" energy for the folded state and a knowledge-based energy for the unfolded state. Thousands of sequences were generated by Monte Carlo simulation. Three were chosen for experimental testing, based on their low energies and several empirical criteria. All three could be overexpressed and had native-like circular dichroism spectra and 1D-NMR spectra typical of folded structures. Two had upshifted thermal denaturation curves when a peptide ligand was present, indicating binding and suggesting folding to a correct, PDZ structure. Evidently, the physical principles that govern folded proteins, with a dash of empirical post-filtering, can allow successful whole-protein redesign. (10.1038/s41598-020-67972-w)
  DOI : 10.1038/s41598-020-67972-w
• The 18S ribosomal RNA m 6 A methyltransferase Mettl5 is required for normal walking behavior in Drosophila
  
  • Leismann Jessica
  • Spagnuolo Mariangela
  • Pradhan Mihika
  • Wacheul Ludivine
  • Vu Minh Anh
  • Musheev Michael
  • Mier Pablo
  • Andrade‐navarro Miguel
  • Graille Marc
  • Niehrs Christof
  • Lafontaine Denis Lj
  • Roignant Jean‐yves
  EMBO Reports, EMBO Press, 2020, 21 (7), pp.e49443. RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6 A methyltransferases were identified, uncovering the specificity of m6 A deposition by structurally distinct enzymes. In order to discover additional m6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior. (10.15252/embr.201949443)
  DOI : 10.15252/embr.201949443
• Pby1 is a direct partner of the Dcp2 decapping enzyme
  
  • Charenton Clément
  • Gaudon-Plesse Claudine
  • Back Régis
  • Ulryck Nathalie
  • Cosson Loreline
  • Séraphin Bertrand
  • Graille Marc
  Nucleic Acids Research, Oxford University Press, 2020, 48 (11). Most eukaryotic mRNAs harbor a characteristic 5 m 7 GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5-3 exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 de-capping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation , both stemming from its interaction with the Dcp1-Dcp2 holoenzyme. (10.1093/nar/gkaa337)
  DOI : 10.1093/nar/gkaa337
• Meeting Report – Workshop ‘Actin-based mechanosensation and force generation in health and disease’
  
  • Polesskaya Anna
  • Vicente-Manzanares Miguel
  Journal of Cell Science, Company of Biologists, 2020, 133 (6), pp.jcs244319. International experts in the fields of cellular motility, force generation and mechanosensation met in Baeza, a UNESCO World Heritage city, from the 10th to the 13th of November, 2019. The meeting, part of the 'Current Trends in Biomedicine' series, took place at the 'Sede Antonio Machado', a beautiful 17th century building turned into a conference center of the Universidad Internacional de Andalucía (UNIA), which sponsored the event. The meeting was organized by Alexis Gautreau, Pekka Lappalainen and Miguel Vicente-Manzanares, with the support of the European Molecular Biology Organization (EMBO) and the Spanish-based company IMPETUX. Fifty scientists presented recent results during the talks, poster sessions and thematic discussions. As Baeza itself served as a crossroads of medieval Christian, Moorish and Jewish cultures, the meeting brought together cell biologists, biochemists, biophysicists and engineers from around the world that provided an integrated vision of the role of the actin cytoskeleton, force generation and mechanosensation in diverse physiological processes and pathologies. (10.1242/jcs.244319)
  DOI : 10.1242/jcs.244319
• ERH proteins: connecting RNA processing to tumorigenesis?
  
  • Graille Marc
  • Rougemaille Mathieu
  Current Genetics, Springer Verlag, 2020, 66 (4), pp.689-692. With the development of -omics approaches, the scientific community is now submerged by a wealth of information that can be used to analyze various parameters: the degree of protein sequence conservation, protein 3D structures as well as RNA and protein expression levels in various benign and tumor tissues, during organism development or upon exposure to chemicals such as endocrine disrupters. However, if such information can be used to identify genes with potentially important biological function, additional studies are needed to deeply characterize their cellular function in model organisms. Here, we discuss the case of such a gene: ERH, encoding a highly conserved homodimeric protein found in unicellular eukaryotes, plants and metazoan, of yet unknown biological function, which might be linked to mRNA metabolism and that is emerging as important for cell migration and metastasis. (10.1007/s00294-020-01065-z)
  DOI : 10.1007/s00294-020-01065-z
• Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression
  
  • Hazra Ditipriya
  • Andrić Vedrana
  • Palancade Benoit
  • Rougemaille Mathieu
  • Graille Marc
  Scientific Reports, Nature Publishing Group, 2020, 10 (1), pp.1034. Timely and accurate expression of the genetic information relies on the integration of environmental cues and the activation of regulatory networks involving transcriptional and post-transcriptional mechanisms. In fission yeast, meiosis-specific transcripts are selectively targeted for degradation during mitosis by the EMC complex, composed of Erh1, the ortholog of human ERH, and the YTH family RNA-binding protein Mmi1. Here, we present the crystal structure of Erh1 and show that it assembles as a homodimer. Mutations of amino acid residues to disrupt Erh1 homodimer formation result in loss-of-function phenotypes, similar to erh1∆ cells: expression of meiotic genes is derepressed in mitotic cells and meiosis progression is severely compromised. Interestingly, formation of Erh1 homodimer is dispensable for interaction with Mmi1, suggesting that only fully assembled EMC complexes consisting of two Mmi1 molecules bridged by an Erh1 dimer are functionally competent. We also show that Erh1 does not contribute to Mmi1-dependent down-regulation of the meiosis regulator Mei2, supporting the notion that Mmi1 performs additional functions beyond EMC. Overall, our results provide a structural basis for the assembly of the EMC complex and highlight its biological relevance in gametogenic gene silencing and meiosis progression. (10.1038/s41598-020-57872-4)
  DOI : 10.1038/s41598-020-57872-4
• Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power
  
  • Opuu Vaitea
  • Nigro Giuliano
  • Gaillard Thomas
  • Schmitt Emmanuelle
  • Mechulam Yves
  • Simonson Thomas
  PLoS Computational Biology, PLOS, 2020, 16 (1), pp.e1007600. Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design. (10.1371/journal.pcbi.1007600)
  DOI : 10.1371/journal.pcbi.1007600
• Actin polymerization downstream of integrins: signaling pathways and mechanotransduction
  
  • Romero Stéphane
  • Le Clainche Christophe
  • Gautreau Alexis
  Biochemical Journal, Portland Press, 2020, 477 (1), pp.1--21. A cell constantly adapts to its environment. Cell decisions to survive, to proliferate or to migrate are dictated not only by soluble growth factors, but also through the direct interaction of the cell with the surrounding extracellular matrix (ECM). Integrins and their connections to the actin cytoskeleton are crucial for monitoring cell attachment and the physical properties of the substratum. Cell adhesion dynamics are modulated in complex ways by the polymerization of branched and linear actin arrays, which in turn reinforce ECM-cytoskeleton connection. This review describes the major actin regulators, Ena/VASP proteins, formins and Arp2/3 complexes, in the context of signaling pathways downstream of integrins. We focus on the specific signaling pathways that transduce the rigidity of the substrate and which control durotaxis, i.e. directed migration of cells towards increased ECM rigidity. By doing so, we highlight several recent findings on mechanotransduction and put them into a broad integrative perspective that is the result of decades of intense research on the actin cytoskeleton and its regulation. (10.1042/BCJ20170719)
  DOI : 10.1042/BCJ20170719
• MasterPATH: network analysis of functional genomics screening data
  
  • Rubanova Natalia
  • Pinna Guillaume
  • Kropp Jeremie
  • Campalans Anna
  • Radicella Juan Pablo
  • Polesskaya Anna
  • Harel-Bellan Annick
  • Morozova Nadya
  BMC Genomics, BioMed Central, 2020, 21 (1), pp.632. BACKGROUND: Functional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks. RESULTS: Here we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks - HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers - the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition. CONCLUSION: The current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening. (10.1186/s12864-020-07047-2)
  DOI : 10.1186/s12864-020-07047-2
• Structural basis of the interaction between cyclodipeptide synthases and aminoacylated tRNA substrates
  
  • Bourgeois Gabrielle
  • Seguin Jérôme
  • Babin Morgan
  • Gondry Muriel
  • Mechulam Yves
  • Schmitt Emmanuelle
  RNA, Cold Spring Harbor Laboratory Press, 2020. Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic β2 and β7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis. (10.1261/rna.075184.120)
  DOI : 10.1261/rna.075184.120
• Use of β3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase
  
  • Nigro Giuliano
  • Bourcier Sophie
  • Lazennec-Schurdevin Christine
  • Schmitt Emmanuelle
  • Marlière Philippe
  • Mechulam Yves
  Journal of Structural Biology, Elsevier, 2020, 209 (2), pp.107435. Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds β3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in β3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: β3-Met complex shows that β3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved β3-Met aminoacylation capabilities. (10.1016/j.jsb.2019.107435)
  DOI : 10.1016/j.jsb.2019.107435
• Mutational drivers of cancer cell migration and invasion
  
  • Novikov Nikita M
  • Zolotaryova Sofia y
  • Gautreau Alexis
  • Denisov Evgeny V
  British Journal of Cancer, Cancer Research UK, 2020, 124, pp.102-114. Genomic instability and mutations underlie the hallmarks of cancer—genetic alterations determine cancer cell fate by affecting cell proliferation, apoptosis and immune response, and increasing data show that mutations are involved in metastasis, a crucial event in cancer progression and a life-threatening problem in cancer patients. Invasion is the first step in the metastatic cascade, when tumour cells acquire the ability to move, penetrate into the surrounding tissue and enter lymphatic and blood vessels in order to disseminate. A role for genetic alterations in invasion is not universally accepted, with sceptics arguing that cellular motility is related only to external factors such as hypoxia, chemoattractants and the rigidity of the extracellular matrix. However, increasing evidence shows that mutations might trigger and accelerate the migration and invasion of different types of cancer cells. In this review, we summarise data from published literature on the effect of chromosomal instability and genetic mutations on cancer cell migration and invasion. (10.1038/s41416-020-01149-0)
  DOI : 10.1038/s41416-020-01149-0
• Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3'-end tetrad of a DNA G-quadruplex
  
  • Heddi Brahim
  • Vee Cheong Vee
  • Schmitt Emmanuelle
  • Mechulam Yves
  • Tuân Phan Anh
  Journal of Structural Biology, Elsevier, 2020, 209 (1), pp.S1047-8477(19)30210-2. G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU - a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to the exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to the G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3'-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5'-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads. (10.1016/j.jsb.2019.10.001)
  DOI : 10.1016/j.jsb.2019.10.001