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International audience; More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of β-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.

International audience; OBJECTIVE:The reasons for failure of surgical treatment for mesial temporal lobe epilepsy (MTLE) associated with hippocampal sclerosis (HS) remain unclear. This retrospective study analyzed seizure, cognitive, and psychiatric outcomes, searching for factors associated with seizure relapse or cognitive and psychiatric deterioration after MTLE-HS surgery.METHODS:Seizure, cognitive, and psychiatric outcomes were reviewed after 389 surgeries performed between 1990 and 2015 on patients aged 15-67 years at a tertiary center. Three surgical approaches were used: anterior temporal lobectomy (ATL; n = 209), transcortical selective amygdalohippocampectomy (SAH; n = 144), and transsylvian SAH (n = 36).RESULTS:With an average follow-up of 8.7 years (range = 1.0-25.2), seizure outcome was classified as Engel I in 83.7% and Engel Ia in 57.1% of patients. The histological classification of HS was type 1 for 75.3% of patients, type 2 for 18.7%, and type 3 for 1.2%. Two factors were significantly associated with seizure recurrence: past history of status epilepticus and preoperative intracranial electroencephalographic recording. In contrast, neither HS type, the presence of a dual pathology, nor surgical approach was associated with seizure outcome. Risk of cognitive impairment was 3.12 (95% confidence interval = 1.27-7.70), greater in patients after ATL than in patients after transcortical SAH. A presurgical psychiatric history and postoperative cognitive impairment were associated with poor psychiatric outcome.SIGNIFICANCE:The SAH and ATL approaches have similar beneficial effects on seizure control, whereas transcortical SAH tends to minimize cognitive deterioration after surgery. Variation in postsurgical outcome with the class of HS should be investigated further.

International audience; At the nuclear envelope, the inner nuclear membrane protein emerin contributes to the interface between the nucleoskeleton and the chromatin. Emerin is an essential actor of the nuclear response to a mechanical signal. Genetic defects in emerin cause Emery-Dreifuss muscular dystrophy. It was proposed that emerin oligomerization regulates nucleoskeleton binding , and impaired oligomerization contributes to the loss of function of emerin disease-causing mutants. We here report the first structural characterization of emerin oligomers. We identified an N-terminal emerin region from amino acid 1 to amino acid 132 that is necessary and sufficient for formation of long curvilinear filaments. In emerin monomer, this region contains a globular LEM domain and a fragment that is intrinsically disordered. Solid-state nuclear magnetic resonance analysis identifies the LEM b-fragment as part of the oligomeric structural core. However, the LEM domain alone does not self-assemble into filaments. Additional residues forming a b-structure are observed within the filaments that could correspond to the unstructured region in emerin monomer. We show that the delK37 mutation causing muscular dystrophy triggers LEM domain unfolding and increases emerin self-assembly rate. Similarly, inserting a disulfide bridge that stabilizes the LEM folded state impairs emerin N-terminal region self-assembly, whereas reducing this disulfide bridge triggers self-assembly. We conclude that the LEM domain, responsible for binding to the chromatin protein BAF, undergoes a conformational change during self-assembly of emerin N-terminal region. The consequences of these structural rearrangement and self-assembly events on emerin binding properties are discussed. Abbreviations BAF, barrier-to-autointegration factor; DTT, dithiothreitol; EDMD, Emery-Dreifuss muscular dystrophy; LEM, Lap2-emerin-Man1; LINC, linker of nucleoskeleton and cytoskeleton; NMR, nuclear magnetic resonance; SDS/PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ThT, thioflavin T.

International audience; Human emerin is an inner nuclear membrane protein involved in the response of the nucleus to mechanical stress. It contributes to the physical connection between the cytoskeleton and the nucleoskeleton. It is also involved in chromatin organization. Its N-terminal region is nucleoplasmic and comprises a globular LEM domain from residue 1 to residue 43. The three-dimensional structure of this LEM domain in complex with the chromatin BAF protein was solved from NMR data. Apart from the LEM domain, the nucleoplasmic region of emerin, from residue 44 to residue 221, is predicted to be intrinsically disordered. Mutations in this region impair binding to several emerin partners as lamin A, actin or HDAC3. However the molecular details of these recognition defects are unknown. Here we report (1)H, (15)N, (13)CO, (13)Cα and (13)Cβ NMR chemical shift assignments of the emerin fragment from residue 67 to residue 170, which is sufficient for nuclear localization and involved in lamin A binding. Chemical shift analysis confirms that this fragment is intrinsically disordered in 0 and 8 M urea.

International audience; We present a hybrid, multi-method, computational scheme for protein/ligand systems well suited to be used on modern and forthcoming massively parallel computing systems. The scheme relies on a multi-scale polarizable Molecular Modeling, MM, approach to perform MD simulations and on an efficient DFT Linear Scaling method to post-process simulation snapshots. We use this scheme to investigate recent α-ketoamide inhibitors targeting the main protease of the SARS-CoV-2 virus. We assessed the reliability and the coherence of the hybrid scheme in particular by checking the ability of MM and DFT to reproduce results from high-end ab initio computations regarding such inhibitors. The DFT approach enables an a posterior fragmentation of the system and an investigation into the strength of interaction among identified fragment pairs.

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal-phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein-DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein-protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.

International audience; The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the β-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its β-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable β-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40-56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria.

International audience; MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nu-cleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 ˚ A resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure , but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 het-eromeric complex. Thermodynamic analysis of the quaternary complex together with structural model-ing support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.

Toxoplasma gondii is a member of Apicomplexa phylum, which is charaterzied by the presence of three parasite’s specific secretory organelles named micronemes, rhoptries and dense granules. While some Apicomplexa parasites can infect either only animals or humans cells, Toxoplasma gondii infects both animals and humans to induce toxoplasmosis. Like other Apicomplexa parasites such as Plasmodium SP, Cryptosporidium SP..., the virulence and intracellular survival of T. gondii depend on the MICs, ROPs/RONs and GRAs proteins that are the virulence factors contained in micronemes, rhoptries and dense granules respectively. The trafficking of this factors for the biogenesis of the parasite organelles as well as their secretion during invasion depend on other essential factors such as TgVPS35 and TgSORT which are partners of each other and which allowed to identify TgREMIND (T. gondii REgulator of Membrane Interacting Domain) which is the project of my thesis. The goal of this thesis is to elucidate the biological functions of the TgREMIND protein in intracellular trafficking and biogenesis of specific secretory organelles in T. gondii. We first showed by bioinformatics that TgREMIND has a N-terminus F-BAR domain known to bind membranes by inducing vesicle formation and a novel C-terminus domain that we named REMIND for Regulator of Membrane Interacting Domain. We subsequently showed by Lipid Overlay assay that TgREMIND binds several phosphatidylinositol phosphate (PIP2 species). These data confirm the predicted interaction of TgREMIND with the membrane. We also identified by GST-pull down TgREMIND partners, such as some trafficking factors like TgVPS35, TgSORT, SECs etc..., some lipid factors as START domaining containing protein and phosphatidylinositol kinases, and dense granules proteins and rhoptries proteins, which are in the majority of all partners. Moreover, by testing the anti-TgREMIND antibody on wild type parasites, we first identified by confocal microscopy the signal of TgREMIND which is localized as punctated siganls in the cytoplasm of the parasites and includes the golgi and post-golgi compartments, endosomal and at the apical end where rhoptries are located. Then we determined by western blot, TgREMIND native size that is about 140 kDa. Moreover, we created an inducible knock-out mutant of TgREMIND and showed that in the absence of TgREMIND protein, there are morphological changes of the rhoptries, disappearance of dense granules and inhibition of the secretion of these organelles. This severely affected the motility of the parasites resulting in their inability to invade mammalian cells to cause the infection. These defects were restored by complementation of the TgREMIND mutants. We thus conclude that TgREMIND protein is probably involved in the formation of membrane vesicules that are required for the biogenesis of the specific secretory organelles of T. gondii. The second part of my thesis focused on the TgSTART protein, lipid partner of TgREMIND. We have characterized TgSTART from transgenic parasites and the Knockdown mutant of the TgSTART gene tagged with the HA epitope.; Toxoplasma gondii est membre des parasites Apicomplexa qui sont caractérisés non seulement par un développement intracellulaire obligatoire, mais aussi par trois organites sécrétoires spécifiques à savoir les micronèmes, rhoptries et les granules denses. Pendant que certains parasites Apicomplexa sont capables d’infecter soit uniquement les animaux ou les humains, Toxoplasma gondii infecte à la fois les animaux et les humains en causant la toxoplasmose. Tout comme les autres parasites Apicomplexa (Plasmodium SP, Cryptosporidium SP…) la virulence et la survie intracellulaire de T. gondii dépendent des facteurs de virulence MICs, ROPs/RONs et GRAs que renferment respectivement les micronèmes, les rhoptries et les granules denses. Le trafic des facteurs pour la biogénèse des organites du parasite ainsi que leur sécrétion lors de l’invasion dépendent d’autres facteurs indispensables dont TgVPS35 et TgSORT qui sont partenaires l’une de l’autre et qui ont permis d’identifier TgREMIND (T. gondii REgulator of Membrane Interacting Domain) qui a fait l’objet de ma thèse. Le but principal de cette thèse était d’élucider les fonctions biologiques de la protéine TgREMIND dans le trafic intracellulaire et la biogénèse des organites sécrétoires spécifiques chez T. gondii. Nous avons dans un premier temps montré par bio-informatique que TgREMIND possède un premier domaine F-BAR connu pour fixer les membranes en induisant la formation de vésicules et un second domaine original de fonctions inconnues que nous avons appelé REMIND (Regulator of Membrane Interacting Domain). Ce domaine a donc donné le nom de la protéine entière TgREMIND. Nous avons montré par la suite grâce aux tests de Lipid Overlay que TgREMIND fixe plusieurs phosphoinositols (PIP2) confirmant ainsi l’interaction prédite de TgREMIND avec la membrane. Nous avons aussi identifié par GST-pull down les partenaires de TgREMIND, comme certains facteurs du trafic (TgVPS35, TgSORT, SECs etc…), certains facteurs lipidiques (START domaining containing protein et des phosphatidylinositols kinases) et des protéinesdes granules denses et celles des rhoptries qui sont majoritaires de toutes les partenaires. Par ailleurs, en testant l’anticorps anti TgREMIND sur les parasites sauvages, nous avons d’abord identifié en microscopie confocale le signal de TgREMIND qui est un signal ponctiforme cytoplasmique dans différents compartiments subcellulaires y compris les compartiments golgiens et post-golgiens, endosomal et à l’extrémité apical du parasite donc à proximité des rhoptries. Nous avons déterminé par western blots que la taille native est d’environ 140 kDa de TgREMIND. Ensuite, nous avons créé un mutant knock-out inductible de TgREMIND à partir duquel nous avons montré qu’en absence de TgREMIND chez le mutant, qu’il y a des changements morphologiques des rhoptries, disparition des granules denses et absence de la sécrétion de leur contenu. Cela a affecté sévèrement la motilité des parasites, ce qui a pour conséquence une incapacité sévère des parasites à envahir les cellules de mammifères pour provoquer une infection toxoplasmique. Les différents défauts ont été corrigés par complémentation du mutant TgREMIND. Nous avons ainsi conclu que la protéine TgREMIND est probablement impliquée dans la formation de la membrane vésiculaire requise pour la biogenèse des organites sécrétoires spécifiques de T. gondii. La deuxième partie de ma thèse était portée sur la protéine TgSTART, partenaire lipidique de TgREMIND. Nous avons caractérisé TgSTART à partir des parasites transgéniques et du mutant Knockdown du gène TgSTART taggués avec l’épitope HA.

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X 4 1–157 -Cernunnos 1–224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X 4 1–157 and Cernunnos 1–224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4–Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4–Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

International audience; Aerosol particles are known to have a major impact on the climate by directly absorbing and scattering solar radiation and indirectly by acting as cloud condensation nuclei. The main contribution to aerosol emission comes from the ocean through the production of sea spray aerosols (SSA), which consist of organic and inorganic species, formed in different sizes and shapes, depending on their mixing ratios. The chemical composition of those SSA will be naturally impacted by the chemical composition of the air/water interface. Through complex experiments, it has been shown how surface activity impacts the selective transfer of species from solution to the aerosol phase. While experiments offer a general picture, it is interesting to probe the properties through a systematic and decoupled approach at the molecular level. The present study provides indeed theoretical results to be confronted to reported experimental observations by means of molecular level simulations. We chose a series of linear mono- and dicarboxylates (containing 4, 6, or 8 carbon atoms) being identified as a large fraction of the SSA surface composition, with the possible counterions (Na+, K+, Ca2+, Mg2+). Classical molecular dynamics is employed to model the distribution of these ions in a water slab using an ab initio based polarizable force-field model. These calculations allow us to derive some trends on the enrichment of inorganic and organic ions at the liquid–air interface compared to their eventual partitioning in the bulk phase. Indeed, medium to long chain mono- or dicarboxylates predominantly remain at the surface of the aerosol, with inorganic cations accumulating underneath, neutralizing the surface charge. For shorter chains, solvation in the bulk appears more favorable. K+ and the dications show a strong ion pairing with the organics, this ion pairing driving their surface vs bulk distribution. In addition, surface tension has been evaluated for the long chain, i.e., having 8 carbons, mono- and dicarboxylates in the presence of the various cations. The surface propensity of the organic chains leads in general to a reduction of surface tension compared to neat water, while mixtures of dicarboxylates and dications give higher values. Finally, these outcomes are discussed in comparison with previous work and within the context of SSA chemistry.

International audience; In mammals, the majority of DNA double-strand breaks are processed by the Non Homologous End-Joining pathway (NHEJ), composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA Ligase IV (L4) and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. In order to improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent 3D structures of this factor. Ten out of 27 screened mutants were non functional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg$^{64}$, Leu$^{65}$, and Leu$^{115}$) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

International audience; Artemis is a key factor of the nonhomologous end-joining (NHEJ) pathway, which is critical for DNA double-strand break (DSB) repair in eukaryotic cells. It belongs to the β-CASP family of nucleases, forming a distinct group within the metallo-β-lactamase superfamily. Proteins of this group are specific for nucleic acids and contain an original domain, the β-CASP domain, which serves as a cap covering the active site displayed by the metallo-β-lactamase domain. Here, we have identified in the highly divergent sequences of the β-CASP domains from DNA-specific nucleases two conserved residues (Artemis E213 and H254), which are not present in RNA-specific enzymes, and shown that H254 plays a key role in the Artemis function, as it is critical for its full activity in vitro. Moreover, inherited mutation of H254 results in radiosensitive severe combined immune deficiency (RS-SCID) in humans. This residue might play a key role in specificity towards DNA, if not directly in zinc binding.

Pyridoxal-5′-phosphate (PLP) is a cofactor in the reactions of over 160 enzymes, several of which are implicated in diseases. Methionine γ-lyase (MGL) is of interest as a therapeutic protein for cancer treatment. It binds PLP covalently through a Schiff base linkage and digests methionine, whose depletion is damaging for cancer cells but not normal cells. To improve MGL activity, it is important to understand and engineer its PLP binding. We develop a simulation model for MGL, starting with force field parameters for PLP in four main states: two phosphate protonation states and two tautomeric states, keto or enol for the Schiff base moiety. We used the force field to simulate MGL complexes with each form, and showed that those with a fully-deprotonated PLP phosphate, especially keto, led to the best agreement with MGL structures in the PDB. We then confirmed this result through alchemical free energy simulations that compared the keto and enol forms, confirming a moderate keto preference, and the fully-deprotonated and singly-protonated phosphate forms. Extensive simulations were needed to adequately sample conformational space, and care was needed to extrapolate the protonation free energy to the thermodynamic limit of a macroscopic, dilute protein solution. The computed phosphate pKa was 5.7, confirming that the deprotonated, −2 form is predominant. The PLP force field and the simulation methods can be applied to all PLP enzymes and used, as here, to reveal fine details of structure and dynamics in the active site.

Funder: LABEX DYNAMO ANR-11-LABEX-0011-01, Funder: ANR HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01, Funder: ANR HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01 ANR LABEX DYNAMO ANR-11-LABEX-0011-01, ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB’s are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.

Mycolic acids are key components of the complex cell envelope of Corynebacteriales . These fatty acids, conjugated to trehalose or to arabinogalactan form the backbone of the mycomembrane. While mycolic acids are essential to the survival of some species, such as Mycobacterium tuberculosis , their absence is not lethal for Corynebacterium glutamicum, which has been extensively used as a model to depict their biosynthesis. Mycolic acids are first synthesized on the cytoplasmic side of the inner membrane and transferred onto trehalose to give trehalose monomycolate (TMM). TMM is subsequently transported to the periplasm by dedicated transporters and used by mycoloyltransferase enzymes to synthesize all the other mycolate-containing compounds. Using a random transposition mutagenesis, we recently identified a new uncharacterized protein (Cg1246) involved in mycolic acid metabolism. Cg1246 belongs to the DUF402 protein family that contains some previously characterized nucleoside phosphatases. In this study, we performed a functional and structural characterization of Cg1246. We showed that absence of the protein led to a significant reduction in the pool of TMM in C. glutamicum , resulting in a decrease in all other mycolate-containing compounds. We found that, in vitro, Cg1246 has phosphatase activity on organic pyrophosphate substrates but is most likely not a nucleoside phosphatase. Using a computational approach, we identified important residues for phosphatase activity and constructed the corresponding variants in C. glutamicum . Surprisingly complementation with these non-functional proteins fully restored the defect in TMM of the Δcg1246 mutant strain, suggesting that in vivo, the phosphatase activity is not involved in mycolic acid biosynthesis.

Genomic instability and mutations underlie the hallmarks of cancer—genetic alterations determine cancer cell fate by affecting cell proliferation, apoptosis and immune response, and increasing data show that mutations are involved in metastasis, a crucial event in cancer progression and a life-threatening problem in cancer patients. Invasion is the first step in the metastatic cascade, when tumour cells acquire the ability to move, penetrate into the surrounding tissue and enter lymphatic and blood vessels in order to disseminate. A role for genetic alterations in invasion is not universally accepted, with sceptics arguing that cellular motility is related only to external factors such as hypoxia, chemoattractants and the rigidity of the extracellular matrix. However, increasing evidence shows that mutations might trigger and accelerate the migration and invasion of different types of cancer cells. In this review, we summarise data from published literature on the effect of chromosomal instability and genetic mutations on cancer cell migration and invasion.

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.

A powerful approach to understand protein structure and evolution is to perform computer simulations that mimic aspects of evolution. In particular, structure-based computational protein design (CPD) can address the inverse folding problem, exploring a large space of amino acid sequences and selecting ones predicted to adopt a given fold. Previously, CPD has been used to entirely redesign several proteins: all or most of the protein sequence was allowed to mutate freely; among sampled sequences, those with low computed folding energy were selected, and a few percent of them did indeed adopt the correct fold. Those studies used an energy function that was partly or largely knowledge-based, with several empirical terms. Here, we show that a PDZ domain can be entirely redesigned using a "physics-based" energy function that combines standard molecular mechanics and a recent, continuum electrostatic solvent model. Many thousands of sequences were generated by Monte Carlo simulation. Among the lowest-energy sequences, three were chosen for experimental testing. All three could be overexpressed and had native-like circular dichroism and 1D NMR spectra. Two exhibited an upshift of their thermal denaturation curves when a peptide ligand was present, indicating they were able to bind and were most likely correctly folded. Evidently, the physical principles that govern molecular mechanics and continuum electrostatics are sufficient to perform whole-protein redesign. This is encouraging, since these methods provide physical insights, can be systematically improved, and are transferable to other biopolymers and ligands of medical or technological interest.

Most eukaryotic mRNAs harbor a characteristic 5′ m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5′-3′ exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1–Dcp2–Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1–Dcp2 holoenzyme.

Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein–RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein–RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems.

International audience; Liposomal formulations represent attractive biocompatible and tunable drug delivery systems for peptide drugs. Among the tools to analyze their physicochemical properties, nuclear magnetic resonance (NMR) spectroscopy, despite being an obligatory technique to characterize molecular structure and dynamics in chemistry as well as in structural biology, yet appears to be rather sparsely used to study drug-liposome formulations. In this work, we exploited several facets of liquid-state NMR spectroscopy to characterize liposomal delivery systems for the apelinderived K14P peptide and K14P modified by Nα-fatty acylation. Various liposome compositions and preparation modes were analyzed. Using NMR, in combination with cryo-electron microscopy and dynamic light scattering, we determined structural, dynamic, and self-association properties of these peptides in solution and probed their interactions with liposomes. Using 31 P and 1 H NMR, we characterized membrane fluidity and thermotropic phase transitions in empty and loaded liposomes. Based on diffusion and 1 H NMR experiments, we localized and quantified peptides with respect to the interior/exterior of liposomes and changes over time and upon thermal treatments. Finally, we assessed the release kinetics of several solutes and compared various formulations. Taken together, this work shows that NMR has the potential to assist the design of peptide/liposome systems and more generally drug delivery systems.

The translation of an mRNA template into the corresponding protein is a highly complex and regulated choreography performed by ribosomes, tRNAs, and translation factors. Most RNAs involved in this process are decorated by multiple chemical modifications (known as epitranscriptomic marks) contributing to the efficiency, the fidelity, and the regulation of the mRNA translation process. Many of these epitranscriptomic marks are written by holoenzymes made of a catalytic subunit associated with an activating subunit. These holoenzymes play critical roles in cell development. Indeed, several mutations being identified in the genes encoding for those proteins are linked to human pathologies such as cancers and intellectual disorders for instance. This review describes the structural and functional properties of RNA methyltransferase holoenzymes, which when mutated often result in brain development pathologies. It illustrates how structurally different activating subunits contribute to the catalytic activity of these holoenzymes through common mechanistic trends that most likely apply to other classes of holoenzymes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Processing > Capping and 5' End Modifications.

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.

International audience; The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

International audience; The Arp2/3 complex generates branched actin networks at different locations of the cell. The WASH and WAVE Nucleation Promoting Factors (NPFs) activate the Arp2/3 complex at the surface of endosomes or at the cell cortex, respectively. In this review, we will discuss how these two NPFs are controlled within distinct, yet related, multiprotein complexes. These complexes are not spontaneously assembled around WASH and WAVE, but require cellular assembly factors. The centrosome, which nucleates microtubules and branched actin, appears to be a privileged site for WASH complex assembly. The actin and microtubule cytoskeletons are both responsible for endosome shape and membrane remodeling. Motors, such as dynein, pull endosomes and extend membrane tubules along microtubule tracks, whereas branched actin pushes onto the endosomal membrane. It was recently uncovered that WASH assembles a super complex with dynactin, the major dynein activator, where the Capping Protein (CP) is exchanged from dynactin to the WASH complex. This CP swap initiates the first actin filament that primes the autocatalytic nucleation of branched actin at the surface of endosomes. Possible coordination between pushing and pulling forces in the remodeling of endosomal membranes is discussed.

Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra- or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1-containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs., In fission yeast, the antagonistic RNA-binding proteins Mmi1 and Mei2 respectively promote and inhibit meiotic mRNA degradation during mitotic growth. Here the authors show that the lncRNA mamRNA scaffolds Mmi1 and Mei2 proteins to enable their mutual controls.

Dendritic actin networks grow in an autocatalytic manner starting from the uncapped minifilament of dynactin., Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

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.

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version Singapore National Research Foundation Investigatorship [NRF-NRFI2017-09]; Singapore Ministry of Education Academic Research Fund Tier 2 [MOE2015-T2- 1092]; Nanyang Technological University (NTU Singapore) grants (to A.T.P.). Funding for open access charge: Nanyang Technological University.

A recent study on the left-handed G-quadruplex (LHG4) DNA revealed a 12-nt minimal motif GTGGTGGTGGTG with the ability to independently form an LHG4 and to drive an adjacent sequence to LHG4 formation. Here we have identified a second LHG4-forming motif, GGTGGTGGTGTG, and determined the X-ray crystal structure of an LHG4 involving this motif. Our structural analysis indicated the role of split guanines and single thymine loops in promoting LHG4 formation. Ministry of Education (MOE) National Research Foundation (NRF) Accepted version NRF-NRFI2017-09

International audience; Selenium (Se) is an essential trace element of considerable interest in humans from both a nutritional and a toxicological perspective because of the narrow margin between intakes that result in efficacy and toxicity. It is used as selenocysteine in a few selenoproteins with important physiological functions. Moreover, at supranutritional doses, Se-containing compounds have attracted interest as potential anticancer agents with high efficacy and selectivity against cancer cells. Thus, Se is becoming a widely used dietary supplement. However, accumulating evidence indicate that adverse health effects are associated with excess dietary supplementation. Therefore, characterizing the toxicity of Se metabolic intermediates are important steps to better understand both the beneficial and toxic mechanisms of Se. This review focuses on the metabolism of Se and the biological mechanisms explaining the toxicity of important Se-metabolites in the yeast Saccharomyces cerevisiae, which can be used as a model system to understand the mode of action and the biological effects of supranutritional Se in higher eukaryotes.

In the fission yeast S. pombe, a subset of meiosis-specific genes is constitutively transcribed during the mitotic cell cycle. To prevent untimely expression of the meiotic program and premature initiation of sexual differentiation, cells have evolved an RNA degradation system that selectively eliminates the corresponding meiotic transcripts. This process requires the YTH-family RNA-binding protein Mmi1, which recognizes cis-elements within RNA molecules (UNAAAC motifs) and targets them for degradation by the nuclear exosome. At the onset of meiosis, Mmi1 is sequestered in a ribonucleoparticle composed of the RNA-binding protein Mei2 and the long non-coding RNA (lncRNA) meiRNA, thereby allowing expression of meiotic genes and meiosis progression. My PhD work consisted in studying the mechanisms by which Mmi1 promotes the degradation of meiotic transcripts and how its activity is regulated during both the mitotic and meiotic cell cycles. During vegetative growth, Mmi1 tightly associates with the evolutionarily conserved Erh1 protein to form the heterotetrameric Erh1-Mmi1 complex (EMC) that is essential for the degradation of meiotic transcripts. Using biochemical and structural approaches, we have shown that Erh1 assembles as a homodimer in vitro and in vivo, consistent with recent analyses. Mutations that disrupt Erh1 homodimerization but preserve interaction with Mmi1 result in the accumulation of meiotic transcripts due to inefficient binding of Mmi1 to its RNA targets. Erh1 homodimerization is also required for Mmi1 luring by the Mei2-meiRNA complex and meiosis progression. Thus, EMC assembly is essential for the recognition and degradation of meiotic transcripts by Mmi1 in mitotic cells and contributes to Mmi1 inactivation at meiosis onset. Previous work showed that, during vegetative growth, Mmi1 recruits the conserved Ccr4-Not complex to ubiquitinylate and downregulate a pool of its own inhibitor Mei2, thereby maintaining its activity in meiotic RNA degradation. We have identified a lncRNA, different from meiRNA and termed mamRNA (Mmi1- and Mei2-associated RNA), to which Mmi1 associates to target Mei2 to the Ccr4-Not complex. Conversely, when Mei2 downregulation is impaired, mamRNA is necessary for Mmi1 inactivation by increased Mei2 levels. Single molecule RNA FISH experiments also indicated that mamRNA localizes to a nuclear body enriched in Mmi1, suggesting that the mutual control of Mmi1 and Mei2 is spatially confined. mamRNA can also take over meiRNA to inhibit Mmi1 and promote meiosis progression. Therefore, mamRNA emerges as a critical regulator of Mmi1 and Mei2 activities to fine tune meiotic RNA degradation and shape the mitosis to meiosis transition.; Chez la levure fissipare Schizosaccharomyces pombe, un sous-ensemble de gènes méiotiques est transcrit de manière constitutive au cours de la mitose. Afin d’éviter l'expression prématurée du programme méiotique et l'initiation de la différenciation sexuelle, les cellules ont développé un système de dégradation de l'ARN qui élimine sélectivement les transcrits méiotiques correspondants. Ce processus nécessite la protéine de liaison à l'ARN Mmi1 (à domaine YTH), qui reconnaît en cis les molécules d'ARN (motifs UNAAAC) et les cible pour la dégradation par l'exosome nucléaire. Au début de la méiose, Mmi1 est séquestrée au sein d’une particule ribonucléoprotéique composée de la protéine de liaison à l'ARN Mei2 et du long ARN non-codant (lncRNA) meiRNA, permettant ainsi l'expression des gènes méiotiques et le déroulement de la méiose. Mon travail de thèse a consisté à étudier les mécanismes par lesquels Mmi1 assure la dégradation des transcrits méiotiques et qui régulent son activité au cours des cycles mitotiques et méiotiques. Pendant la croissance végétative, Mmi1 s'associe étroitement à la protéine conservée Erh1 pour former le complexe hétérotétramérique Erh1-Mmi1 (EMC) qui est essentiel pour la dégradation des transcrits méiotiques. Par des approches de biologie structurale et de biochimie, nous avons montré qu'Erh1 s'assemble en homodimère in vitro et in vivo, en accord avec des analyses récentes. Des mutations qui empêchent l'homodimérisation d'Erh1 mais préservent son interaction avec Mmi1 entraînent l'accumulation de transcrits méiotiques en raison d'un défaut de liaison de Mmi1 à ses cibles ARN. L'homodimérisation d’Erh1 est également nécessaire pour séquestrer Mmi1 dans le complexe Mei2-meiRNA et assurer la progression de la méiose. Ainsi, l'assemblage d’EMC est essentiel pour la reconnaissance et la dégradation des transcrits méiotiques par Mmi1 dans les cellules mitotiques et contribue à l'inactivation de cette dernière au début de la méiose. Des travaux antérieurs ont montré que, pendant la croissance végétative, Mmi1 recrute le complexe Ccr4-Not pour ubiquitinyler et limiter l’accumulation de son propre inhibiteur Mei2, maintenant ainsi son activité dans la dégradation des ARNs méiotiques. Nous avons identifié un lncRNA, différent de meiRNA et appelé mamRNA (Mmi1- and Mei2-associated RNA), qui sert de plateforme à Mmi1 pour cibler Mei2 vers le complexe Ccr4-Not. Réciproquement, lorsque cette régulation négative de Mei2 est défectueuse, mamRNA est nécessaire pour l'inactivation de Mmi1 par les niveaux élevés de Mei2. Des expériences d’hybridation in situ par fluorescence en molécules uniques (smFISH) ont également montré que mamRNA est localisé dans un corps nucléaire contenant Mmi1, suggérant que le contrôle mutuel de Mmi1 et Mei2 est confiné dans l’espace. mamRNA peut également relayer meiRNA pour inhiber Mmi1 et favoriser la progression de la méiose. mamRNA apparait donc comme un régulateur critique des activités de Mmi1 et Mei2 pour ajuster la dégradation des ARNs méiotiques et modeler la transition de la mitose vers la méiose.

Archaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNAiMet. Here, we determine the cryo-EM structure of a 30S:mRNA:aIF1A:aIF2:GTP:Met-tRNAiMet complex from Pyrococcus abyssi at 3.2 Å resolution. It highlights archaeal features in ribosomal proteins and rRNA modifications. We find an aS21 protein, at the location of eS21 in eukaryotic ribosomes. Moreover, we identify an N-terminal extension of archaeal eL41 contacting the P site. We characterize 34 N4-acetylcytidines distributed throughout 16S rRNA, likely contributing to hyperthermostability. Without aIF1, the 30S head is stabilized and initiator tRNA is tightly bound to the P site. A network of interactions involving tRNA, mRNA, rRNA modified nucleotides and C-terminal tails of uS9, uS13 and uS19 is observed. Universal features and domain-specific idiosyncrasies of translation initiation are discussed in light of ribosomal structures from representatives of each domain of life., Coureux et al. describe a cryo-EM structure of a 30S initiation complex of Pyrococcus abyssi at 3.2Å resolution. The structure uncovers a novel archaeal ribosomal protein aS21, N-terminal extension of eL41 and brings insights into base modifications of the rRNA.

Branched actin networks polymerized by the Arp2/3 complex are critical for cell migration. The WAVE complex is the major Arp2/3 activator at the leading edge of migrating cells. However, multiple distinct WAVE complexes can be assembled in a cell, due to the combinatorial complexity of paralogous subunits. When systematically analyzing the contribution of each WAVE complex subunit to the metastasis-free survival of breast cancer patients, we found that overexpression of the CYFIP2 subunit was surprisingly associated with good prognosis. Gain and loss of function experiments in transformed and untransformed mammary epithelial cells revealed that cell migration was always inversely related to CYFIP2 levels. The role of CYFIP2 was systematically opposite to the role of the paralogous subunit CYFIP1 or of the NCKAP1 subunit. The specific CYFIP2 function in inhibiting cell migration was related to its unique ability to down-regulate classical pro-migratory WAVE complexes. The anti-migratory function of CYFIP2 was also revealed in migration of prechordal plate cells during gastrulation of the zebrafish embryo, indicating that the unique function of CYFIP2 is critically important in both physiological and pathophysiological migrations.

We describe methods for physics-based protein design and some recent applications from our work. We present the physical interpretation of a MC simulation in sequence space and show that sequences and conformations form a well-defined statistical ensemble, explored with Monte Carlo and Boltzmann sampling. The folded state energy combines molecular mechanics for solutes with continuum electrostatics for solvent. We usually assume one or a few fixed protein backbone structures and discrete side chain rotamers. Methods based on molecular dynamics, which introduce additional backbone and side chain flexibility, are under development. The redesign of a PDZ domain and an aminoacyl-tRNA synthetase enzyme were successful. We describe a versatile, adaptive, Wang-Landau MC method that can be used to design for substrate affinity, catalytic rate, catalytic efficiency, or the specificity of these properties. The methods are transferable to all biomolecules, can be systematically improved, and give physical insights.

International audience; Translation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed. However, even if the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes, and archaea and may also vary depending on the mRNA. Because TI mechanisms have evolved lately, their studies bring important information about the evolutionary relationships between extant organisms. In this context, recent structural data on ribosomal complexes and genome-wide studies are particularly valuable. This review focuses on archaeal translation initiation highlighting its relationships with either the eukaryotic or the bacterial world. Eukaryotic features of the archaeal small ribosomal subunit are presented. Ribosome evolution and TI mechanisms diversity in archaeal branches are discussed. Next, the use of leaderless mRNAs and that of leadered mRNAs having Shine-Dalgarno sequences is analyzed. Finally, the current knowledge on TI mechanisms of SD-leadered and leaderless mRNAs is detailed.

We performed 10 ns scale molecular dynamics simulations of 6 SARS-CoV-2 main protease/alpha-ketoamide inhibitor complexes in aqueous solution, in the phase before the inhibitor covalently binds to the protease's catalytic cysteine, using a polarizable multi-scale molecular modeling approach. For each simulation, 100 Mpro/inhibitor snapshots(about 4 800 atoms) were extracted along the last 2 ns simulation segments. They were post processed using a fully quantum mechanical O(N) approach to decompose the protease in sets of fragments from which we computed the mean local interaction energies between the inhibitors and the different pockets of the protease catalytic domain. Contrary to earlier results, our analysis shows that the protease pocket S2 to be a key anchoring site able to lock within the catalytic domain an alpha-ketoamide inhibitor even before covalent bonding to the protease catalytic cysteine occurs. To target that pocket our computations suggest to consider hydrophobic groups, like cyclo-propyl or cyclo-hexyl.

International audience; In this review, applications of various molecular modelling methods in the study of estrogens and xenoestrogens are summarized. Selected biomolecules that are the most commonly chosen as molecular modelling objects in this field are presented. In most of the reviewed works, ligand docking using solely force field methods was performed, employing various molecular targets involved in metabolism and action of estrogens. Other molecular modelling methods such as molecular dynamics and combined quantum mechanics with molecular mechanics have also been successfully used to predict the properties of estrogens and xenoestrogens. Among published works, a great number also focused on the application of different types of quantitative structure-activity relationship (QSAR) analyses to examine estrogen's structures and activities. Although the interactions between estrogens and xenoestrogens with various proteins are the most commonly studied, other aspects such as penetration of estrogens through lipid bilayers or their ability to adsorb on different materials are also explored using theoretical calculations. Apart from molecular mechanics and statistical methods, quantum mechanics calculations are also employed in the studies of estrogens and xenoestrogens. Their applications include computation of spectroscopic properties, both vibrational and Nuclear Magnetic Resonance (NMR), and also in quantum molecular dynamics simulations and crystal structure prediction. The main aim of this review is to present the great potential and versatility of various molecular modelling methods in the studies on estrogens and xenoestrogens.

International audience; 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 introduce 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.

RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6A methyltransferases were identified, uncovering the specificity of m6A deposition by structurally distinct enzymes. In order to discover additional m6A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; With the development of -omics approaches, the scientific community is now submerged by a wealth of information that can be used to analyze various parameters: the degree of protein sequence conservation, protein 3D structures as well as RNA and protein expression levels in various benign and tumor tissues, during organism development or upon exposure to chemicals such as endocrine disrupters. However, if such information can be used to identify genes with potentially important biological function, additional studies are needed to deeply characterize their cellular function in model organisms. Here, we discuss the case of such a gene: ERH, encoding a highly conserved homodimeric protein found in unicellular eukaryotes, plants and metazoan, of yet unknown biological function, which might be linked to mRNA metabolism and that is emerging as important for cell migration and metastasis.

In the context of nuclear fuel recycling and environmental issues, the understanding of the properties of radio-elements with various approaches remains a challenge regarding their dangerousness. Moreover, experimentally, it is imperative to work at sufficiently high concentrations to reach the sensitivities of the analysis tools, which often leads to precipitation for some of them, and stabilizing of specific oxidation states of some actinides remains a challenge, thus making it difficult to extract general trends across the actinide series. Complementary to experiments, modelling can be used to unbiasedly probe the actinide's properties in aquatic environment and offers a predictive tool. We report the first molecular dynamics simulations based on homogeneously built force fields for the whole series of the tetravalent actinides in aqueous phase from $\mathrm{Th^{IV}}$ to $\mathrm{Bk^{IV}}$ and including $\mathrm{Pu^{IV}}$. The force fields used to model the interactions among the constituents include polarization and charge donation microscopic effects. They are built from an automated iterative \textit{ab initio} based engine, the core element of a future machine learning procedure devoted to generate accurate force fields. The comparison of our simulated hydrated actinide properties to available experimental data show the model robustness and the relevance of our parameter assignment engine. Moreover our simulated structural, dynamical and hydration free energy data show that, apart from $\mathrm{Am^{IV}}$ and $\mathrm{Cm^{IV}}$, the actinides properties change progressively along the series., Comment: 7 pages, 4 figures

Summary Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5′ cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-β production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-β induction, prompting a negative feedback regulatory mechanism that represses IFN-β expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design., Author summary Designed enzymes are of major interest. Experimental directed evolution still has significant limitations, and computational approaches are another route. Enzymes must be stable, bind substrates, and be powerful catalysts. It is challenging to design for all these properties. A method to design substrate binding was proposed recently. It used an adaptive Monte Carlo method to explore mutations of a few amino acids near the substrate. A bias energy was gradually “learned” such that, in the absence of the ligand, the simulation visited most of the possible protein mutations with comparable probabilities. Remarkably, a simulation of the protein:ligand complex, including the bias, will then preferentially sample tight-binding sequences. We generalized the method to design binding specificity. We tested it for the methionyl-tRNA synthetase enzyme, which has been engineered in order to expand the genetic code. We redesigned the enzyme to obtain variants with low activation free energies for the catalytic step. The variants proposed by the simulations were shown experimentally to be active, and the predicted activation free energies were in reasonable agreement with the experimental values. We expect the new method will become the paradigm for computational enzyme design.

International experts in the fields of cellular motility, force generation and mechanosensation met in Baeza, a UNESCO World Heritage city, from the 10th to the 13th of November, 2019. The meeting, part of the ‘Current Trends in Biomedicine’ series, took place at the ‘Sede Antonio Machado’, a beautiful 17th century building turned into a conference center of the Universidad Internacional de Andalucía (UNIA), which sponsored the event. The meeting was organized by Alexis Gautreau, Pekka Lappalainen and Miguel Vicente-Manzanares, with the support of the European Molecular Biology Organization (EMBO) and the Spanish-based company IMPETUX. Fifty scientists presented recent results during the talks, poster sessions and thematic discussions. As Baeza itself served as a crossroads of medieval Christian, Moorish and Jewish cultures, the meeting brought together cell biologists, biochemists, biophysicists and engineers from around the world that provided an integrated vision of the role of the actin cytoskeleton, force generation and mechanosensation in diverse physiological processes and pathologies., EMBO and IMPETUX for their support, and speakers for critical reading of the manuscript. The meeting was funded by UNIA with support from EMBO.

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination., info:eu-repo/semantics/published

WOS:000448616200009; The major challenge to Exaflop computing, and more generally, efficient high-end computing, is in finding the best "matches" between advanced hardware capabilities and the software used to program applications, so that top performance will be achieved. Several benchmarks show very disappointing performance progress over the last decade, clearly indicating a mismatch between hardware and software. To remedy this problem, it is important that key performance enablers at the software level-autotuning, performance analysis tools, full application optimization-are understood. For each area, we highlight major limitations and most promising approaches to reaching better performance and energy levels. Finally, we conclude by analyzing hardware and software design, trying to pave the way for more tightly integrated hardware and software codesign.