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International audience; Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.

International audience; In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.

International audience; 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.

International audience; 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.

Pyrococcus abyssivirus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssistrain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genusSalterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

Recently, we observed the first example of a left‐handed G‐quadruplex structure formed by natural DNA, named Z‐G4 . We analysed the Z‐G4 structure and inspected its primary 28‐nt sequence in order to identify motifs that convey the unique left‐handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X‐ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left‐handed DNA G‐quadruplex, which is found to be highly abundant in the human genome. A systematic analysis of thymine loop mutations revealed a moderate sequence tolerance, which would further broaden the space of sequences prone to left‐handed G‐quadruplex formation. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.

Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S. cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation.

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., Nous proposons un ensemble de méthodes pour la conception de systèmes moléculaires. Notre stratégie consiste à utiliser comme modèle des machines naturellement optimisées, les protéines. Les protéines peuvent être des briques structurales, des transporteurs d'informations ou des catalyseurs chimiques. Nous utilisons ici des approches computationnelles, complémentaires aux voies expérimentales, pour concevoir de tels systèmes.Nous avons d'abord entièrement redessiné un domaine PDZ impliqué dans des voies métaboliques. Nous utilisons une approche physics-based basée sur la mécanique moléculaire, un modèle de solvant implicite et un échantillonnage Monte Carlo. Parmi plusieurs milliers de variants prédits pour adopter le repliement PDZ, trois ont été sélectionnés et montrent un repliement correct. Deux ont une affinité détectable pour les ligands peptidiques naturels.Nous avons ensuite re-dessiné le site actif de l'enzyme méthionyl-ARNt synthétase (MetRS). En utilisant un algorithme de type Monte Carlo adaptatif, nous avons sélectionné des variants pour l'affinité MetRS/méthionine (Met). Sur 17 variants testés expérimentalement, 17 sont actifs. La méthode a été ensuite appliquée à l'état de transition pour sélectionner des variants directement sur leur efficacité catalytique.Nous avons étudié la possibilité de modifier la MetRS pour étendre son activitéaux acides aminés β, afin d'étendre le code génétique. Ces acides aminésnon-naturels permettraient d'enrichir le répertoire structural des protéines. 20variants MetRS obtenus à partir de prédictions d'affinité MetRS/β-Met ont ététestés. Aucun n'augmente l'activité mais trois ont amélioré la sélectivité enfaveur de la β-Met. Nous avons implémenté une méthode de sélection de positionsd'intérêt et production de variants pour β-Met et β-Val. Une vingtaine deprédictions sont en cours de tests expérimentaux.Enfin, la modification de protéines in vivo pose la question de leur dérive génétique. Nous introduisons ici une méthode de conception de paires de gènes chevauchants pour des domaines PDZ. Ce codage permettrait de limiter la dérive génétique. Nous avons produit près de 2000 paires de domaines PDZ au codage chevauchant, dont une a été validées par 2 microsecondes de dynamique moléculaire. Des tests expérimentaux sont en cours.

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.; Nous proposons un ensemble de méthodes pour la conception de systèmes moléculaires. Notre stratégie consiste à utiliser comme modèle des machines naturellement optimisées, les protéines. Les protéines peuvent être des briques structurales, des transporteurs d'informations ou des catalyseurs chimiques. Nous utilisons ici des approches computationnelles, complémentaires aux voies expérimentales, pour concevoir de tels systèmes.Nous avons d'abord entièrement redessiné un domaine PDZ impliqué dans des voies métaboliques. Nous utilisons une approche physics-based basée sur la mécanique moléculaire, un modèle de solvant implicite et un échantillonnage Monte Carlo. Parmi plusieurs milliers de variants prédits pour adopter le repliement PDZ, trois ont été sélectionnés et montrent un repliement correct. Deux ont une affinité détectable pour les ligands peptidiques naturels.Nous avons ensuite re-dessiné le site actif de l'enzyme méthionyl-ARNt synthétase (MetRS). En utilisant un algorithme de type Monte Carlo adaptatif, nous avons sélectionné des variants pour l'affinité MetRS/méthionine (Met). Sur 17 variants testés expérimentalement, 17 sont actifs. La méthode a été ensuite appliquée à l'état de transition pour sélectionner des variants directement sur leur efficacité catalytique.Nous avons étudié la possibilité de modifier la MetRS pour étendre son activitéaux acides aminés β, afin d'étendre le code génétique. Ces acides aminésnon-naturels permettraient d'enrichir le répertoire structural des protéines. 20variants MetRS obtenus à partir de prédictions d'affinité MetRS/β-Met ont ététestés. Aucun n'augmente l'activité mais trois ont amélioré la sélectivité enfaveur de la β-Met. Nous avons implémenté une méthode de sélection de positionsd'intérêt et production de variants pour β-Met et β-Val. Une vingtaine deprédictions sont en cours de tests expérimentaux.Enfin, la modification de protéines in vivo pose la question de leur dérive génétique. Nous introduisons ici une méthode de conception de paires de gènes chevauchants pour des domaines PDZ. Ce codage permettrait de limiter la dérive génétique. Nous avons produit près de 2000 paires de domaines PDZ au codage chevauchant, dont une a été validées par 2 microsecondes de dynamique moléculaire. Des tests expérimentaux sont en cours.

International audience; 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.

International audience; Computational protein design (CPD) aims to predict amino acid sequences that fold to specific structures and perform desired functions. CPD depends on a rotamer library, an energy function, and an algorithm to search the sequence/conformation space. Variable neighborhood search (VNS) with cost function networks is a powerful framework that can provide tight upper bounds on the global minimum energy. We propose a new CPD heuristic based on VNS in which a subset of the solution space (a “neighborhood”) is explored, whose size is gradually increased with a dedicated probabilistic heuristic. The algorithm was tested on 99 protein designs with fixed backbones involving nine proteins from the SH2, SH3, and PDZ families. The number of mutating positions was 20, 30, or all of the amino acids, while the rest of the protein explored side-chain rotamers. VNS was more successful than Monte Carlo (MC), replica-exchange MC, and a heuristic steepest-descent energy minimization, providing solutions with equal or lower best energies in most cases. For complete protein redesign, it gave solutions that were 2.5 to 11.2 kcal/mol lower in energy than those obtained with the other approaches. VNS is implemented in the toulbar2 software. It could be very helpful for large and/or complex design problems.

Une correction de l'article est publiée sur le site de l'éditeur : https://pubs.acs.org/doi/abs/10.1021/acs.jpca.8b12337; International audience; Phosphate groups are essential components of nucleic acids and proteins, whose interactions with solvent, metal ions, and ionic side chains help control folding and binding. Methyl phosphate (MP) represents a simple analog of phosphate moieties that are post-translation modifications in proteins and present at the termini of nucleic acids, among other environments. In the present study, we optimized parameters for use in polarizable molecular dynamics simulations of MP in its mono- and dianionic forms, MP- ≡ CH3HPO4- and MP2- ≡ CH3PO42-, along with P i2- ≡ HPO42-, in the context of the classical Drude oscillator model. Parameter optimization was done in a manner consistent with the remainder of the Drude molecular mechanics force field, choosing atomic charges and polarizabilities to reproduce molecular properties from quantum mechanics as well as experimental hydration free energies. Optimized parameters were similar to existing dimethyl phosphate parameters, with a few significant differences. The developed parameters were then used to compute magnesium binding affinities in aqueous solution, using alchemical molecular dynamics free energy simulations. Good agreement with experiment was obtained, and outer sphere binding was shown to be predominant for MP- and MP2-.

International audience; One proposed scenario for the emergence of biochemical oscillations is that they may have provided the basic mechanism behind cellular self-replication by growth and division. However, alternative scenarios not requiring any chemical oscillation have also been proposed. Each of the various protocell models proposed to support one or another scenario comes with its own set of specific assumptions, which makes it difficult to ascertain whether chemical oscillations are required or not for cellular self-replication. This article compares these two cases within a single whole-cell model framework. This model relies upon a membrane embedding a chemical reaction network (CRN) synthesizing all the cellular constituents, including the membrane, by feeding from an external nutrient. Assuming the osmolarity is kept constant, the system dynamics are governed by a set of nonlinear differential equations coupling the chemical concentrations and the surface-area-to-volume ratio. The resulting asymptotic trajectories are used to determine the cellular shape by minimizing the membrane bending energy (within an approximate predefined family of shapes). While the stationary case can be handled quite generally, the oscillatory one is investigated using a simple oscillating CRN example, which is used to identify features that are expected to hold for any network. It is found that cellular self-replication can be reached with or without chemical oscillations, and that a requirement common to both stationary and oscillatory cases is that a minimum spontaneous curvature of the membrane is required for the cell to divide once its area and volume are both doubled. The oscillatory case can result in a greater variety of cellular shape trajectories but raises additional constraints for cellular division and self-replication: (i) the ratio of doubling time to oscillation period should be an integer, and (ii) if the oscillation amplitude is sufficiently high, then the spontaneous curvature must be below a maximum value to avoid early division before the end of the cycle. Because of these additional stringent constraints, it is likely that early protocells did not rely upon chemical oscillations. Biochemical oscillations typical of modern evolved cells may have emerged later through evolution for other reasons (e.g., metabolic advantage) and must have required additional feedback mechanisms for such a self-replicating system to be robust against even slight environmental variations (e.g., temperature fluctuations).

International audience; Condensed-phase simulations commonly use periodic boundary conditions (PBCs) to represent the thermodynamic limit. For the vapor to liquid transfer of an ion, the gas/liquid boundary and its associated potential change are then missing. Furthermore, the electric potential and field at a given point are given by conditionally convergent infinite series, for which different summation schemes give different results. Nevertheless, standard simulation protocols can be used to compute experimental quantities unambiguously. In particular, using an auxiliary test particle and a multistep solvation path, we show that particle-based, Ewald, and common molecule-based summation schemes for the potential and field are all essentially equivalent. However, all methods require prior knowledge of the gas/liquid boundary potential to compute ionic solvation free energies using PBC protocols for both force-field and quantum-mechanical models.

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.

Living cells use 20 canonical α amino acids during ribosomal protein synthesis, although in rare cases, other amino acids such as selenocysteine or pyrrolysine can be used. The repertoire of usable amino acids is therefore quite limited. This limits the ability to build proteins having new properties. A dominant trend in the field of protein engineering is the construction of systems capable of incorporating non-canonical amino acids during in vivo protein synthesis. These studies have been very successful, but all amino acids incorporated until the 2010s were α-amino acids. Incorporation of β-amino acids at discrete sites would create unprecedented flexibility in the main chain, which would increase the potential for new protein folds. It has recently been shown using an in vitro protein synthesis system that it is possible to incorporate β-amino acids at specific positions (Katoh and Suga, 2018). In order to transpose this system in vivo, the main limitation is the aminoacylation of tRNAs with β -amino acids. It has recently been proposed that some aminoacyl-tRNA synthetases can use β-amino acids, but this capacity remains limited. The aim of this thesis is to incorporate β-methionine in polypeptides in vivo. For this purpose, we have characterized the recognition and use of L-β-homomethionine by methionyl-tRNA synthetase (MetRS) from E. coli. In particular, we have determined a high-resolution crystallographic structure of the MetRS:β-Met complex. Using fluorescence spectroscopy and mass spectroscopy, we were able to demonstrate the activation of -Met into adenylate as well as its esterification onto tRNAMet. However, the measured efficiencies are very small compared to what was published. Contamination of commercial β-Met with methionine may explain these differences. An in silico study based on the structure of the MetRS:β-Met complex was conducted in collaboration with the laboratory's bioinformatics team in order to search for mutant enzymes more efficient in the use of β-amino acids. Finally, we initiated the implementation of a directed evolution method to improve the efficiency of incorporation of amino acids in vivo.; Les cellules vivantes utilisent 20 acides α aminés canoniques lors de la synthèse ribosomale des protéines, même si dans de rares occasions, d’autres acides aminés (selenocysteine ou pyrrolysine) peuvent être utilisés. Le répertoire des acides aminés utilisables est donc assez restreint. Cela limite la possibilité de construire des protéines ayant de nouvelles propriétés. Une tendance forte dans le domaine de l’ingénierie des protéines est la construction de systèmes permettant d’incorporer des acides aminés non canoniques pendant la biosynthèse in vivo. Ces travaux ont connu d’importants succès, mais tous les acides aminés incorporés jusqu'aux années 2010 étaient des acides aminés α. L’incorporation d’acides aminés β à des sites spécifiques créerait une flexibilité supérieure à celle des acides aminés α dans la chaîne principale, ce qui augmenterait les possibilités de repliement de la protéine. Il a été montré récemment en utilisant un système de traduction in vitro de synthèse protéique qu’il était possible d’incorporer des acides aminés β à des positions spécifiques (Katoh and Suga, 2018). Pour parvenir à transposer ce système in vivo, la principale limitation est l’aminoacylation des ARNt avec les acides aminés β. Il a été récemment proposé que certaines aminoacyl-ARNt synthetases étaient capables d’utiliser les acides aminées β, mais cette capacité reste limitée.Le but de cette thèse est d’incorporer la β-méthionine dans des polypeptides in vivo. Pour cela nous avons caractérisé la reconnaissance et l’utilisation de la L-β-homométhionine par la méthionyl-ARNt synthetase (MetRS) d’E. coli. Nous avons en particulier déterminé une structure cristallographique à haute résolution du complexe MetRS: β -Met. En utilisant la spectroscopie de fluorescence ainsi que la spectroscopie de masse, nous avons pu mettre en évidence l’activation de la -Met en adénylate ainsi que son estérification à un ARNtMet. Toutefois, les efficacités mesurées sont très petites par rapport à ce qui avait été publié. Une contamination de la -Met commerciale par de la méthionine pourrait expliquer ces différences. Une étude in silico basée sur la structure du complexe MetRS:β-Met a été menée en collaboration avec l’équipe de Bio-informatique du laboratoire afin de rechercher des enzymes mutantes plus efficaces vis à vis des acides aminés β. Enfin, nous avons amorcé la mise en place d’une méthode d’évolution dirigée destinée à améliorer l’efficacité d’incorporation des acides aminés β in vivo.

N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid., SCOPUS: ar.j, info:eu-repo/semantics/published

Summary: cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation. : Ros et al. developed SponGee, a genetically encoded cGMP chelator that enables the manipulation of this second messenger in single cells with subcellular specificity. SponGee alters the migration of developing cortical neurons in vivo. Lipid raft targeting of SponGee prevents axon repulsion, in contrast to exclusion from this subcellular compartment. Keywords: cGMP buffer, subcellular compartment, single cell pharmacology, axon guidance, neuronal migration, lipid grafts, genetically encoded, FRET, ThPDE5VV, PKG

Analogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex. The structure reveals a stacking interaction between two G-quadruplex blocks with different helical orientations and displays features of both right- and left-handed G-quadruplexes. An analysis of loop mutations suggests that single-nucleotide loops are preferred or even required for the left-handed G-quadruplex formation. The discovery of a right- and left-handed hybrid G-quadruplex further expands the polymorphism of G-quadruplexes and is potentially useful in designing a left-to-right junction in G-quadruplex engineering. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases., SCOPUS: ar.j, info:eu-repo/semantics/published

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM., Placental sequestration of malaria-infected erythrocytes is mediated by the interaction between parasite VAR2CSA and the placental receptor chondroitin sulfate A; this study shows that phosphorylation of VAR2CSA plays a major role in this process, with implications for protecting pregnant women and their babies against placental malaria.

The actin cytoskeleton generates and senses forces. Here we report that branched actin networks from the cell cortex depend on ARPC1B-containing Arp2/3 complexes and that they are specifically monitored by type I coronins to control cell cycle progression in mammary epithelial cells. Cortical ARPC1B-dependent branched actin networks are regulated by the RAC1/WAVE/ARPIN pathway and drive lamellipodial protrusions. Accordingly, we uncover that the duration of the G1 phase scales with migration persistence in single migrating cells. Moreover, cortical branched actin more generally determines S-phase entry by integrating soluble stimuli such as growth factors and mechanotransduction signals, ensuing from substratum rigidity or stretching of epithelial monolayers. Many tumour cells lose this dependence for cortical branched actin. But the RAC1-transformed tumour cells stop cycling upon Arp2/3 inhibition. Among all genes encoding Arp2/3 subunits, ARPC1B overexpression in tumours is associated with the poorest metastasis-free survival in breast cancer patients. Arp2/3 specificity may thus provide diagnostic and therapeutic opportunities in cancer.

International audience; Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5' untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome.

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We developed SpiCee, a versatile and genetically-encoded chelator combining low and high affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically-controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

Living cells use 20 canonical α amino acids during ribosomal protein synthesis, although in rare cases, other amino acids such as selenocysteine or pyrrolysine can be used. The repertoire of usable amino acids is therefore quite limited. This limits the ability to build proteins having new properties. A dominant trend in the field of protein engineering is the construction of systems capable of incorporating non-canonical amino acids during in vivo protein synthesis. These studies have been very successful, but all amino acids incorporated until the 2010s were α-amino acids. Incorporation of β-amino acids at discrete sites would create unprecedented flexibility in the main chain, which would increase the potential for new protein folds. It has recently been shown using an in vitro protein synthesis system that it is possible to incorporate β-amino acids at specific positions (Katoh and Suga, 2018). In order to transpose this system in vivo, the main limitation is the aminoacylation of tRNAs with β -amino acids. It has recently been proposed that some aminoacyl-tRNA synthetases can use β-amino acids, but this capacity remains limited. The aim of this thesis is to incorporate β-methionine in polypeptides in vivo. For this purpose, we have characterized the recognition and use of L-β-homomethionine by methionyl-tRNA synthetase (MetRS) from E. coli. In particular, we have determined a high-resolution crystallographic structure of the MetRS:β-Met complex. Using fluorescence spectroscopy and mass spectroscopy, we were able to demonstrate the activation of -Met into adenylate as well as its esterification onto tRNAMet. However, the measured efficiencies are very small compared to what was published. Contamination of commercial β-Met with methionine may explain these differences. An in silico study based on the structure of the MetRS:β-Met complex was conducted in collaboration with the laboratory's bioinformatics team in order to search for mutant enzymes more efficient in the use of β-amino acids. Finally, we initiated the implementation of a directed evolution method to improve the efficiency of incorporation of amino acids in vivo., Les cellules vivantes utilisent 20 acides α aminés canoniques lors de la synthèse ribosomale des protéines, même si dans de rares occasions, d’autres acides aminés (selenocysteine ou pyrrolysine) peuvent être utilisés. Le répertoire des acides aminés utilisables est donc assez restreint. Cela limite la possibilité de construire des protéines ayant de nouvelles propriétés. Une tendance forte dans le domaine de l’ingénierie des protéines est la construction de systèmes permettant d’incorporer des acides aminés non canoniques pendant la biosynthèse in vivo. Ces travaux ont connu d’importants succès, mais tous les acides aminés incorporés jusqu'aux années 2010 étaient des acides aminés α. L’incorporation d’acides aminés β à des sites spécifiques créerait une flexibilité supérieure à celle des acides aminés α dans la chaîne principale, ce qui augmenterait les possibilités de repliement de la protéine. Il a été montré récemment en utilisant un système de traduction in vitro de synthèse protéique qu’il était possible d’incorporer des acides aminés β à des positions spécifiques (Katoh and Suga, 2018). Pour parvenir à transposer ce système in vivo, la principale limitation est l’aminoacylation des ARNt avec les acides aminés β. Il a été récemment proposé que certaines aminoacyl-ARNt synthetases étaient capables d’utiliser les acides aminées β, mais cette capacité reste limitée.Le but de cette thèse est d’incorporer la β-méthionine dans des polypeptides in vivo. Pour cela nous avons caractérisé la reconnaissance et l’utilisation de la L-β-homométhionine par la méthionyl-ARNt synthetase (MetRS) d’E. coli. Nous avons en particulier déterminé une structure cristallographique à haute résolution du complexe MetRS: β -Met. En utilisant la spectroscopie de fluorescence ainsi que la spectroscopie de masse, nous avons pu mettre en évidence l’activation de la -Met en adénylate ainsi que son estérification à un ARNtMet. Toutefois, les efficacités mesurées sont très petites par rapport à ce qui avait été publié. Une contamination de la -Met commerciale par de la méthionine pourrait expliquer ces différences. Une étude in silico basée sur la structure du complexe MetRS:β-Met a été menée en collaboration avec l’équipe de Bio-informatique du laboratoire afin de rechercher des enzymes mutantes plus efficaces vis à vis des acides aminés β. Enfin, nous avons amorcé la mise en place d’une méthode d’évolution dirigée destinée à améliorer l’efficacité d’incorporation des acides aminés β in vivo.

International audience; Although Merlin function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt or Hippo pathways is well documented,in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora A kinase during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in Hela cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates Ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with Neurofibromatosis type 2 that display an aberant phosphorylation profile along with defective α-tubulin binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and Ezrin suggest a potential role for Merlin in cell cycle progression.

In eukaryotes, the elimination of the m7GpppN mRNA cap, a process known as decapping, is a critical, largely irreversible and highly regulated step of mRNA decay that withdraws the targeted mRNAs from the pool of translatable templates. The decapping reaction is catalysed by a multi-protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor, a holoenzyme that is poorly active on its own and needs several accessory proteins (Lsm1–7 complex, Pat1, Edc1–2, Edc3 and/or EDC4) to be fully efficient. Here, we discuss the several crystal structures of Dcp2 domains bound to various partners (proteins or small molecules) determined in the last couple of years that have considerably improved our current understanding of how Dcp2, assisted by its various activators, is recruited to its mRNA targets and adopts its active conformation upon substrate recognition. We also describe how, over the years, elegant integrative structural biology approaches combined to biochemistry and genetics led to the identification of the correct structure of the active Dcp1–Dcp2 holoenzyme among the many available conformations trapped by X-ray crystallography.This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

International audience; Cyclodipeptide synthases (CDPSs) form various cyclodipeptides from two aminoacyl tRNAs via a stepwise mechanism with the formation of a dipeptidyl enzyme intermediate. As a final step of the catalytic reaction, the dipeptidyl group undergoes intramolecular cyclization to generate the target cyclodipeptide product. In this work, we investigated the cyclization reaction in the cyclodipeptide synthase AlbC using QM/MM methods and free energy simulations. The results indicate that the catalytic Y202 residue is in its neutral protonated form, and thus, is not likely to serve as a general base during the reaction. We further demonstrate that the reaction relies on the conserved residue Y202 serving as a proton relay, and the direct proton transfer from the amino group to S37 of AlbC is unlikely. Calculations reveal that the hydroxyl group of tyrosine is more suitable for the proton transfer than hydroxyl groups of other amino acids, such as serine and threonine. Results also show that the residues E182, N40, Y178 and H203 maintain the correct conformation of the dipeptide needed for the cyclization reaction. The mechanism discovered in this work relies on the amino groups conserved among the entire CDPS family and, thus is expected to be universal among CDPSs. Cyclodipeptide synthases (CDPSs) are a family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) to synthetize cyclodipeptides, which are precursors of many secondary metabolites with diverse biological functions 1,2. The first member of this family was identified in 2002 during characterization of the albonoursin biosynthetic pathway in Streptomyces noursei and called AlbC 3. Three CDPSs are structurally characterized and all three share a common architecture, consisting of a monomer containing a Rossmann fold domain 4-7. The CDPSs display strong structural similarity to the catalytic domains of class Ic aminoacyl tRNA synthetases (aaRSs), suggesting that CDPSs evolved from the class Ic of aaRSs 1. However, there are several significant differences between CDPSs and class Ic aaRSs. The ATP-binding motifs present in aaRSs are not present in CDPSs, since there is no need to activate amino acids in CDPSs, and CDPSs are active as monomers in contrast to the TyrRS and TrpRS that function as homodimers. The catalytic mechanism has been extensively studied experimentally for the structurally characterized CDPSs, and a structure mimicking a reaction intermediate was obtained for AlbC 7. It was demonstrated that AlbC uses Phe-tRNA Phe and Leu-tRNA Leu (or a second Phe-tRNA Phe) as substrates for a ping-pong mechanism involving the formation of two successive acyl-enzyme intermediates 1. The catalytic reaction starts with the binding of the first aa-tRNA and the transfer of its aminoacyl moiety to a conserved serine residue leading to the formation of an aminoacyl enzyme intermediate. For the second step, the tRNA Phe part of the first substrate dissociates from AlbC and a second aa-tRNA binds to the enzyme. The phenylalanyl-AlbC reacts with the second aa-tRNA to form a dipeptidyl-AlbC intermediate. In the final step, the target cyclodipeptide is obtained through intramolecular cyclization. Residues important for the reaction in AlbC were identified through site-directed mutagenesis and chemical biology studies 5,7. These residues, apart from S37, the conserved residue that accepts the aminoacyl group, are Y202, Y178, E182, N40, and H203. The residues Y178 and E182 are involved in the stabilization of the aminoacyl moiety of the first substrate (named Phe1) throughout the catalytic cycle as suggested by the crystal structure of the diphenylalanyl-enzyme intermediate mimic 7. E182 was also suggested to act as a general catalytic base during the formation of the dipeptidyl-enzyme by deprotonating the ammonium group of the aminoacyl-enzyme

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., Cell polarity is marked by re-orientation of the centrosome, but the mechanisms governing centrosome polarization are poorly understood. Here Obino et al. show that in lymphocytes centrosome-associated Arp2/3 nucleates actin that tethers the centrosome to the nucleus; activation depletes Arp2/3 from the centrosome and frees it from the nucleus.

International audience; More than 100 naturally occurring modified nucleotides have been found in RNA molecules, in particular in tRNAs. We have determined molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field for all these modifications using the CHARMM force field parametrization strategy. Emphasis was placed on fine tuning of the partial atomic charges and torsion angle parameters. Quantum mechanics calculations on model compounds provided the initial set of target data, and extensive molecular dynamics simulations of nucleotides and oligonucleotides in aqueous solutions were used for further refinement against experimental data. The presented parameters will allow for computational studies of a wide range of RNAs containing modified nucleotides, including the ribosome and transfer RNAs. (C) 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

International audience; We report an intramolecular Diels-Alder reaction efficiently accelerated by Schreiner's thiourea, to build a functionalized cytochalasin scaffold (periconiasin series) amenable to biological purpose. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end-game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines, while preserving actin polymerization.

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International audience; Selenium (Se) is an essential trace element for mammals. It is involved in redox functions as the amino acid selenocysteine, translationally inserted in the active site of a few proteins. However, at high doses it is toxic and the mechanisms underlying this toxicity are poorly understood. Because of the high level of conservation of its genes and pathways with those of higher organisms and the powerful genetic techniques that it offers, Saccharomyces cerevisiae is an attractive model organism to study the molecular basis of Se toxicity. High-throughput technologies developed in this yeast include genome-wide screening of bar-coded systematic deletion sets, as well as whole-transcriptome, -proteome, and -metabolome analysis.This chapter focuses on the contribution of S. cerevisiae to the understanding of the mechanisms of selenocompound toxicity, combining results from classical biochemistry with genome-wide analyses and more detailed gene-by-gene approaches. Experimental data demonstrate that toxicity is compound specific. Inorganic Se induces DNA damage whereas selenoamino acids cause proteotoxicity.

Protein-protein interactions (PPIs) regulate complex signaling networks in eukaryotic cells. Many binding events between several protein domains transfer information through communication pathways. Disrupting or altering the equilibrium between PPIs plays an important role inseveral diseases and the inibition of targeted PPIs is a recognized strategy for computational drug design. In the present thesis we focused on PDZ domains, which are among the most widespread signaling domains. PDZs recognize the 4-10 C-terminal amino acids of their target proteins as well as the corresponding peptides in isolation. We studied PDZ:peptide binding for the Tiam1 protein, which is a Rac GTP exchange factor involved in neuronal protrusion and axon guidance. Tiam1 activity modulates signaling for cell proliferation and migration, whose dysregulation increases growth of metastatic cancers. Its natural binder peptide is Syndecan1 (Sdc1), composed of 8 amino acids. Its last 5 Cter residues drive interactions in the binding pocket. Experimental affinities for several mutants of Sdc1 and in the protein domain constitute a complete dataset to study many ionic interactions with molecular simulations. These calculations are still challenging, despite the dramatic improvement of biomolecular modelling in the 1990's and 2000's. Upon binding, residues are transferred from a solvent-exposed environment to a solvent-poor one. This is expected to change the electron distribution within residues and nearby solvent molecules. Comparing ligands that differ by one or more ionic side-chain mutations, more sophisticated force fields where electronic polarizability is treated explicitly may be required. We developed and tested both Computational Protein Design (CPD) models and more precise free energy calculation methods based on polarizable molecular dynamics. We developed a general, high-througtput CPD protocol to optimize protein:peptide binding. The model has been implemented in on our in-house CPD package Proteus ( Simonson et al, 2014) and has been tested computing relative binding affinities for many variants of the Tiam1:Sdc1 complex. Monte Carlo sampling of equilibrium distributions of protein sequences is performed using an adaptive bias potential which flattens the energy landscape in sequence space and allows to estimate binding affinities for thousands of protein variants in limited CPU time (~1hour). We also improved our CPD implicit solvent model, implementing a more realistic description of the solute-solvent dielectric boundary. The new method, called Fluctuating Dielectric Boundary (FDB) showed a systematic improvement in the prediction of acid:base constants of several proteins. Promising results were also obtained for the complete sequence redesign of three PDZ domains. In the second part of this work we studied Tiam1:peptide affinities with more sophisticated models, based on free energy simulations with the Drude Polarizable Force field (DrudeFF). We first computed relative binding free energies for charge mutations in the Tiam1:Sdc1 complex, obtaining a clear improvement respect to equivalent calculations performed using two additive force fields. We applied the well-enstablished Dual Topology Approach: to our knowledge, this was the first example of such a calculation for a protein:peptide complex with uses the DrudeFF. Then we went on, developing the Drude polarizable models for methyl phosphate (MP) and phospho tyrosine (pTyr). We were interested in the change in binding affinity associated with phosphorylation of a Tyrosine residue of Sdc1, but Drude pTyr parameters were not yet developed. We tested our new phosphate parameters studying standard binding free energies between MP and magnesium (Mg2+) in water solution. Results showed a good agreement with experiment, improving previous calculations performed using additive force field; Les interactions protéine-protéine (IPPs) médient la signalisation cellulaire. Leur ingénierie peut fournir des informations et conduire au développement de molécules thérapeutiques. Les domaines PDZ sont des médiateurs importants de IPPs. Elles lient les 4--10 résidus C-terminaux de protéines cibles. Elles lient aussi les peptides correspondants, qui peuvent servir de systèmes modèles ou d'inhibiteurs. Nous avons développé deux approches computationnelles et les avons appliquées au domaine PDZ de la protéine Tiam1, un facteur d'échange pour la protéine Rac, impliqué dans la protrusion neuronale. Sa cible est la protéine Syndecan1. Des affinités expérimentales sont connues pour le peptide C-terminal, noté Sdc1, et plusieurs mutants; elles ont servi pour tester les calculs. Nous avons d'abord développé une méthode de dessin computationnel haut débit. Une simulation Monte Carlo est faite où les chaines latérales de la protéine et du peptide peuvent changer de conformères et certaines positions peuvent muter. Le solvant est implicite. Le paysage énergétique est aplati par la méthode adaptative de Wang-Landau, de sorte qu'un vaste ensemble de variantes est échantillonné. Effectuant des simulations distinctes du complexe et du peptide seul nous avons obtenu les énergies libres relatives d'association de 75,000 variantes en heure CPU sur une machine de bureau. Les valeurs sont compatibles avec les quelques données expérimentales disponibles. Ensuite, nous avons développé une approche beaucoup plus détaillée et réaliste. Soluté et solvant sont décrits par un champ de force atomique, qui représente explicitement la polarisation électronique: le champ de force Drude de Charmm. La polarisabilité peut être importante car les résidus de l'interface PDZ:peptide passent, lors de l'association, d'un environnement riche en solvant à un autre pauvre en solvant. Nous avons fait des simulations alchimiques d'énergie libre pour comparer quatre variantes du peptide qui diffèrent par une ou deux chaines latérales ioniques. Les résultats sont en bon accord avec l'expérience. Les champs de force additifs Charmm et Amber, qui représentent la polarisabilité implicitement, donnent un moins bon accord. Ces calculs sont le premier exemple de simulations alchimiques d'énergies libre d'association relatives protéine: ligand avec un champ de force polarisable. Enfin, pour une modélisation future de peptides phosphorylés, nous avons étendu le champ de force Drude pour inclure le méthyl phosphate et la phospho tyrosine. Il en résulte un excellent accord avec les affinités expérimentales phosphate: magnésium.

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The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. Recently, an inactivator of the Arp2/3 complex at the lamellipodium tip, a small protein, Arpin, was discovered and characterized. On its C-terminus, Arpin possesses an acidic (A) motif, which is homologous to the A-motif of various Nucleation Promoting Factors (NPFs). It was predicted that Arpin can bind at two binding sites to the Arp2/3 complex, similar to VCA domains of NPFs. Here, we used single particle electron microscopy to obtain a 3D reconstruction of the Arp2/3 complex bound to Arpin at a 25Å resolution. We showed that the binding of Arpin causes the standard open conformational of the Arp2/3 complex. We confirmed that there are two binding sites on the Arp2/3 complex for Arpin: one on the back of the Arp3 subunit, and the second is located between Arp2 and ARPC1 subunits. The distance between the Arp2/3 complex and Arpin (5 nm) supports the view that Arpin interacts with its partner via its unstructured C-terminal acidic tail.Next, using the pull-down assay, we identified the new Arpin binding partners, Tankyrases1/2. Interestingly, Tankyrases and the Arp2/3 complex possess overlapping amino acid sequences at Arpin binding sites. Hence, we demonstrated a competition between the ARC4 domain of Tankyrase1 and the Arp2/3 complex in a dose-dependent manner.To understand the principles of Tankyrases-Arpin interaction, we created a mutant Arpin (ArpinG218D) that lacks its ability to interact with Tankyrases, but not with the Arp2/3 complex in vitro. Interestingly, ArpinG218D was not able to inhibit the Arp2/3 complex in vivo, suggesting that Tankyrase may be necessary for Arpin-Arp2/3 complex interaction. Arpin is the turning factor of migrating cells, so we performed a migration analysis of MCF10-A cells expressing either wild type Arpin (ArpinWT) or mutant ArpinG218D in parallel with the depletion of endogenous Arpin. Cells expressing ArpinG218D had higher directional persistence, similar to the cells where the endogenous Arpin was knocked down. Thus, we suggested that mutant ArpinG218D cannot inactivate the Arp2/3 complex since it is not present at the lamellipodial tip. We compared the amount of protein for both ArpinWT and ArpinG218D in the membrane fraction of the migrating cells. A significant difference (44%) in the amount of ArpinWT and Arpin G218D was consistent with our hypothesis.Tankyrases are therapeutic targets in a variety of cancers, but currently there is no structural model available for these large and flexible proteins. In this work, we obtained for the first time two 3D reconstructions of full-length Tankyrase1 and Tankyrase1 bound to Arpin using single particle electron microscopy. The achieved resolution (27Å) was enough to detect a dramatic conformational change in Tankyrase SAM and PARP domains upon binding of Arpin molecules. In our reconstruction, three Arpins were bound to the ARC1, ARC4 and ARC5 domains of Tankyrase1. ARC5 was shown to be the most flexible part of the ARC cluster.Based on the obtained data, we suggested a model of regulation of the activity of Arpin by Tankyrases. According to our model, Tankyrases bind Arpin in the cytoplasm, change their conformational state and bring Arpin closer to the membrane in the lamellipodia. Deciphering the extracellular signals, Rac GTPase activates Arpin, which sequentially inactivates the Arp2/3 complex, while Tankyrases are released.; Le complexe Arp2/3, conservé sur le plan évolutif, joue un rôle central dans la nucléation d’actine branchée, qui entraîne la migration cellulaire, l’endocytose et d’autres processus cellulaire. Récemment, une petite protéine, Arpin, qui inhibe le complexe Arp2/3 au front du lamellipode a été découverte et caractérisée. Sur sa partie C-terminale, Arpin possède un motif acide (A), qui est homologue au motif A des différents NPF (Nucleation Promoting Factor). Il a été prédit qu’Arpin peut se lier à deux sites de liaison au complexe Arp2/3, similaire aux domaines VCA des NPF. Ici, nous utilisons la microscopie électronique de particules uniques pour obtenir une reconstruction 3D du complexe Arp2/3 lié à Arpin, à une résolution de 25 Å. Nous avons montré que la liaison d’Arpin induit la conformation ouverte, standard, du complexe Arp2/3. Nous avons confirmé qu’il y a deux sites de liaison sur le complexe Arp2/3 pour Arpin : un à l’arrière de la sous-unité Arp3, et le second localisé entre les sous-unités Arp2 et ARPC1. La distance entre le complexe Arp2/3 et Arpin (5nm) confirme qu’Arpin interagit avec son partenaire via sa queue acide C-terminale non structurée.Nous avons, ensuite, identifié Tankyrases1/2, comme un nouveau partenaire qui se lie à Arpin, par « pull-down ». De façon intéressante, les sites de liaisons d’Arpin aux Tankyrases et à Arp2/3 se chevauchent. Nous avons, par conséquent, démontré qu’il y a une compétition dose-dépendante entre le domaine ARC4 de Tankyrase1 et le complexe Arp2/3.Pour comprendre les principes de l’interaction entre Arpin et Tankyrases, nous avons créé un mutant d’Arpin (ArpinG218D) qui, in vitro, se lie toujours au complexe Arp2/3, mais plus aux Tankyrases. In vivo, ArpinG218D n’est pas capable d’inhiber le complexe Arp2/3, ce qui suggère que Tankyrase pourrait être nécessaire pour l’interaction entre Arpin et le complexe Arp2/3. Arpin est le facteur responsable du changement de direction des cellules migrantes. Nous avons donc analysé, la migration de cellules MCF10A exprimant soit la forme sauvage d’Arpin (ArpinWT) soit son mutant ArpinG218D en parallèle de la déplétion d’Arpin endogène. Les cellules exprimant ArpinG218D ont une persistance de migration supérieure, similaire à celles déplétées d’Arpin endogène. Nous avons, ainsi, fait l’hypothèse que le mutant ArpinG218D ne peut pas inactiver le complexe Arp2/3 car il n’est pas présent au niveau du lamellipode. Nous avons donc comparé la quantité de protéine d’ArpinWT et d’ArpinG218D dans la fraction membranaire de cellules migrantes. Une différence significative (44%) dans la quantité d’ArpinWT et d’ArpinG218D a confirmé notre hypothèse.Les Tankyrases sont des cibles thérapeutiques dans de nombreux cancers, mais il n’existe pas de modèle structural pour ces protéines grandes et flexibles. Dans ce travail, nous avons, pour la première fois, obtenu deux reconstructions 3D de Tankyrase1 et Tankyrase2 complètes liées à Arpin en utilisant la microscopie électronique de particules uniques. La résolution obtenue (27 Å) a été suffisante pour détecter un changement de conformation dramatique des domaines SAM et PARP de Tankyrase après fixation d’Arpin. Dans notre reconstruction, trois molécules d’Arpin se lient aux domaines ARC1, ARC4 et ARC5 de Tankyrase1. ARC5 a été montré pour être la partie le plus flexible de l’ensemble des domaines ARC.Grâce aux données que nous avons obtenues, nous avons suggéré un modèle de régulation de l’activité d’Arpin par les Tankyrases. Selon notre modèle, les Tankyrases se lient à Arpin dans le cytoplasme, changent sa conformation et amènent Arpin au niveau de la membrane dans le lamellipode. Traduisant les signaux extracellulaires, la GTPase Rac active Arpin, qui séquentiellement inactive le complexe Arp2/3, tandis que les Tankyrases sont libérées.

The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. Recently, an inactivator of the Arp2/3 complex at the lamellipodium tip, a small protein, Arpin, was discovered and characterized. On its C-terminus, Arpin possesses an acidic (A) motif, which is homologous to the A-motif of various Nucleation Promoting Factors (NPFs). It was predicted that Arpin can bind at two binding sites to the Arp2/3 complex, similar to VCA domains of NPFs. Here, we used single particle electron microscopy to obtain a 3D reconstruction of the Arp2/3 complex bound to Arpin at a 25Å resolution. We showed that the binding of Arpin causes the standard open conformational of the Arp2/3 complex. We confirmed that there are two binding sites on the Arp2/3 complex for Arpin: one on the back of the Arp3 subunit, and the second is located between Arp2 and ARPC1 subunits. The distance between the Arp2/3 complex and Arpin (5 nm) supports the view that Arpin interacts with its partner via its unstructured C-terminal acidic tail.Next, using the pull-down assay, we identified the new Arpin binding partners, Tankyrases1/2. Interestingly, Tankyrases and the Arp2/3 complex possess overlapping amino acid sequences at Arpin binding sites. Hence, we demonstrated a competition between the ARC4 domain of Tankyrase1 and the Arp2/3 complex in a dose-dependent manner.To understand the principles of Tankyrases-Arpin interaction, we created a mutant Arpin (ArpinG218D) that lacks its ability to interact with Tankyrases, but not with the Arp2/3 complex in vitro. Interestingly, ArpinG218D was not able to inhibit the Arp2/3 complex in vivo, suggesting that Tankyrase may be necessary for Arpin-Arp2/3 complex interaction. Arpin is the turning factor of migrating cells, so we performed a migration analysis of MCF10-A cells expressing either wild type Arpin (ArpinWT) or mutant ArpinG218D in parallel with the depletion of endogenous Arpin. Cells expressing ArpinG218D had higher directional persistence, similar to the cells where the endogenous Arpin was knocked down. Thus, we suggested that mutant ArpinG218D cannot inactivate the Arp2/3 complex since it is not present at the lamellipodial tip. We compared the amount of protein for both ArpinWT and ArpinG218D in the membrane fraction of the migrating cells. A significant difference (44%) in the amount of ArpinWT and Arpin G218D was consistent with our hypothesis.Tankyrases are therapeutic targets in a variety of cancers, but currently there is no structural model available for these large and flexible proteins. In this work, we obtained for the first time two 3D reconstructions of full-length Tankyrase1 and Tankyrase1 bound to Arpin using single particle electron microscopy. The achieved resolution (27Å) was enough to detect a dramatic conformational change in Tankyrase SAM and PARP domains upon binding of Arpin molecules. In our reconstruction, three Arpins were bound to the ARC1, ARC4 and ARC5 domains of Tankyrase1. ARC5 was shown to be the most flexible part of the ARC cluster.Based on the obtained data, we suggested a model of regulation of the activity of Arpin by Tankyrases. According to our model, Tankyrases bind Arpin in the cytoplasm, change their conformational state and bring Arpin closer to the membrane in the lamellipodia. Deciphering the extracellular signals, Rac GTPase activates Arpin, which sequentially inactivates the Arp2/3 complex, while Tankyrases are released.; Le complexe Arp2/3, conservé sur le plan évolutif, joue un rôle central dans la nucléation d’actine branchée, qui entraîne la migration cellulaire, l’endocytose et d’autres processus cellulaire. Récemment, une petite protéine, Arpin, qui inhibe le complexe Arp2/3 au front du lamellipode a été découverte et caractérisée. Sur sa partie C-terminale, Arpin possède un motif acide (A), qui est homologue au motif A des différents NPF (Nucleation Promoting Factor). Il a été prédit qu’Arpin peut se lier à deux sites de liaison au complexe Arp2/3, similaire aux domaines VCA des NPF. Ici, nous utilisons la microscopie électronique de particules uniques pour obtenir une reconstruction 3D du complexe Arp2/3 lié à Arpin, à une résolution de 25 Å. Nous avons montré que la liaison d’Arpin induit la conformation ouverte, standard, du complexe Arp2/3. Nous avons confirmé qu’il y a deux sites de liaison sur le complexe Arp2/3 pour Arpin : un à l’arrière de la sous-unité Arp3, et le second localisé entre les sous-unités Arp2 et ARPC1. La distance entre le complexe Arp2/3 et Arpin (5nm) confirme qu’Arpin interagit avec son partenaire via sa queue acide C-terminale non structurée.Nous avons, ensuite, identifié Tankyrases1/2, comme un nouveau partenaire qui se lie à Arpin, par « pull-down ». De façon intéressante, les sites de liaisons d’Arpin aux Tankyrases et à Arp2/3 se chevauchent. Nous avons, par conséquent, démontré qu’il y a une compétition dose-dépendante entre le domaine ARC4 de Tankyrase1 et le complexe Arp2/3.Pour comprendre les principes de l’interaction entre Arpin et Tankyrases, nous avons créé un mutant d’Arpin (ArpinG218D) qui, in vitro, se lie toujours au complexe Arp2/3, mais plus aux Tankyrases. In vivo, ArpinG218D n’est pas capable d’inhiber le complexe Arp2/3, ce qui suggère que Tankyrase pourrait être nécessaire pour l’interaction entre Arpin et le complexe Arp2/3. Arpin est le facteur responsable du changement de direction des cellules migrantes. Nous avons donc analysé, la migration de cellules MCF10A exprimant soit la forme sauvage d’Arpin (ArpinWT) soit son mutant ArpinG218D en parallèle de la déplétion d’Arpin endogène. Les cellules exprimant ArpinG218D ont une persistance de migration supérieure, similaire à celles déplétées d’Arpin endogène. Nous avons, ainsi, fait l’hypothèse que le mutant ArpinG218D ne peut pas inactiver le complexe Arp2/3 car il n’est pas présent au niveau du lamellipode. Nous avons donc comparé la quantité de protéine d’ArpinWT et d’ArpinG218D dans la fraction membranaire de cellules migrantes. Une différence significative (44%) dans la quantité d’ArpinWT et d’ArpinG218D a confirmé notre hypothèse.Les Tankyrases sont des cibles thérapeutiques dans de nombreux cancers, mais il n’existe pas de modèle structural pour ces protéines grandes et flexibles. Dans ce travail, nous avons, pour la première fois, obtenu deux reconstructions 3D de Tankyrase1 et Tankyrase2 complètes liées à Arpin en utilisant la microscopie électronique de particules uniques. La résolution obtenue (27 Å) a été suffisante pour détecter un changement de conformation dramatique des domaines SAM et PARP de Tankyrase après fixation d’Arpin. Dans notre reconstruction, trois molécules d’Arpin se lient aux domaines ARC1, ARC4 et ARC5 de Tankyrase1. ARC5 a été montré pour être la partie le plus flexible de l’ensemble des domaines ARC.Grâce aux données que nous avons obtenues, nous avons suggéré un modèle de régulation de l’activité d’Arpin par les Tankyrases. Selon notre modèle, les Tankyrases se lient à Arpin dans le cytoplasme, changent sa conformation et amènent Arpin au niveau de la membrane dans le lamellipode. Traduisant les signaux extracellulaires, la GTPase Rac active Arpin, qui séquentiellement inactive le complexe Arp2/3, tandis que les Tankyrases sont libérées.

International audience; Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.

International audience; For the high throughput design of protein:peptide binding, one must explore a vast space of amino acid sequences in search of low binding free energies. This complex problem is usually addressed with either simple heuristic scoring or expensive sequence enumeration schemes. Far more efficient than enumeration is a recent Monte Carlo approach that adaptively flattens the energy landscape in sequence space of the unbound peptide and provides formally exact binding free energy differences. The method allows the binding free energy to be used directly as the design criterion. We propose several improvements that allow still more efficient sampling and can address larger design problems. They include the use of Replica Exchange Monte Carlo and landscape flattening for both the unbound and bound peptides. We used the method to design peptides that bind to the PDZ domain of the Tiam1 signaling protein and could serve as inhibitors of its activity. Four peptide positions were allowed to mutate freely. Almost 75 000 peptide variants were processed in two simulations of 109 steps each that used 1 CPU hour on a desktop machine. 96% of the theoretical sequence space was sampled. The relative binding free energies agreed qualitatively with values from experiment. The sampled sequences agreed qualitatively with an experimental library of Tiam1-binding peptides. The main assumption limiting accuracy is the fixed backbone approximation, which could be alleviated in future work by using increased computational resources and multi-backbone designs.

International audience; PDZ domains contain 80-100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.

International audience; Protein design aims at conceiving new proteins or modifying existing ones to obtain a given function. Computational approaches are a valuable help for protein design, to rationalize the predictions and guide the experimental tests. Computational protein design (CPD) has sparked important methodological efforts and obtained spectacular successes such as the creation of a protein with a new fold or enzyme active site engineering. The main difficulty of CPD lies in the astronomical number of possible sequences and conformations, of the order of (20x10)^100 for a protein with 100 amino acids. Another key element for CPD success is the energy function used to evaluate and select the sequences and conformations.Our approach of CPD is based on an atomic model of the protein structure and a molecular mechanics energy function. An important aspect is the solvent treatment, represented as a dielectric continuum with a Generalized-Born term, supplemented by a term proportional to the solvent accessible surface area. The key elements of our implementation are: 1) the protein backbone is maintained fixed, 2) the side-chain conformational space is reduced to a discrete library of rotamers, 3) the energy function is decomposed into interaction pairs. The first step consists in calculating a matrix of interactions between each pair of rotamers. In the next step, the sequence-conformation space is explored with an optimization algorithm. Energy evaluations in this second step are fast thanks to the pre-calculation of the energy matrix. The implementation of our CPD procedure is presented, as well as applications to the prediction of side-chain conformations and to the design of full protein sequences.

The actin cytoskeleton generates and mechanotransducts forces. Here we report that the cortical branched actin that depends on RAC1, WAVE and ARPC1B-containing Arp2/3 complexes is specifically monitored by the Coronin1B sensor, WISp39 and the cyclin-CDK inhibitory protein p21, to control cell cycle progression. Accordingly, the duration of the G1 phase scales with the persistence of single cell migration, ensuing from branched actin and lamellipodium protrusion. Cortical branched actin determines the cell decision to enter into S phase by integrating soluble stimulifrom growth factors and mechanotransduced signals, such as substratum rigidity and cell density. The Arp2/3 complex is overall overexpressed in brest cancer. Among its subunits, The ARPC1B isoform overexpression is the strongest prognostic factor for patients. Furthermore, Arp2/3 inhibition prevents the growth of mammary carcinoma and melanoma cell lines transformed by the RAC1 oncogene, for which no targeted therapy is available. The discovery of the cortical branched actin checkpoint thus provides diagnostic and therapeutic opportunities in cancer., Résumé : Le cytosquelette d’actine génère et mécanotransduit des forces. Dans cette étude, nous montrons que l’actine branchée corticale, qui dépend de RAC1, WAVE et des complexes Arp2/3 contenant ARPC1B, est spécifiquement détectée par le senseur Coronin1B, qui signale, via WISp39 et l’inhibiteur de cycline/CDK p21, à la cellule, de progresser dans le cycle cellulaire. En conséquence, la formation d’un lamellipode et la migration persistante des cellules qui en découle, est corrélée à la durée de la phase G1. L’actine branchée corticale détermine l’entrée en phase S des cellules, en intégrant les stimuli solubles des facteurs de croissance et la mécanotransduction desadhérences à la matrice extracellulaire et aux cellules voisines. Le complexe Arp2/3 est globalement sur-exprimé dans le cancer du sein. Parmi ses sous-unités, la sur-expression de l’isoforme ARPC1B est le plus fort facteur prognostique pour les patientes. En outre, l’inhibition du complexe Arp2/3 bloque la prolifération de lignées de carcinomes mammaires et de mélanomes transformées par l’oncogène RAC1, contre laquelle il n’existe pas de thérapie ciblée. La découverte du checkpoint de l’actine branchée corticale apporte ainsi de nouvelles options pronostiques, diagnostiques et thérapeutiques dans les cancers.

International audience

Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.

International audience; The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocy-tosed receptors, such as a5b1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyos-telium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

International audience; In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNA i Met) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using flu-orescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.

The Arp2/3 complex is an evolutionary conserved molecular machine that generates branched actin networks. When activated, the Arp2/3 complex contributes the actin branched junction and thus cross-links the polymerizing actin filaments in a network that exerts a pushing force. The different activators initiate branched actin networks at the cytosolic surface of different cellular membranes to promote their protrusion, movement, or scission in cell migration and membrane traffic. Here we review the structure, function, and regulation of all the direct regulators of the Arp2/3 complex that induce or inhibit the initiation of a branched actin network and that controls the stability of its branched junctions. Our goal is to present recent findings concerning novel inhibitory proteins or the regulation of the actin branched junction and place these in the context of what was previously known to provide a global overview of how the Arp2/3 complex is regulated in human cells. We focus on the human set of Arp2/3 regulators to compare normal Arp2/3 regulation in untransformed cells to the deregulation of the Arp2/3 system observed in patients affected by various cancers. In many cases, these deregulations promote cancer progression and have a direct impact on patient survival.

International audience; The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a twofold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein-and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.