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10 pages; National audience; Pinna nobilis is the largest bivalve in the Mediterranean Sea. This endemic species lives in the Posidonia meadows,in the form of small sparse populations. It is a heritage species: its byssus has long been collected to be wovenand used to make gloves, hats or stoles. Protected by a European Directive (1992), P. nobilis is closely monitored,nationally and internationally. But since the end of 2016, an epidemic, which origin seems to be a protozoan parasite,decimates populations (up to 100% mortality). This event began on the Spanish coast and spreads now all around theMediterranean. Although conservation measures have been taken, the next few years could see the extinction of thisemblematic bivalve. Apart from its symbolic value, P. nobilis is a species worthy of biotechnological interest. Its shellcomprises two mineralized layers, one of which, external, calcitic, consists of large monocrystalline prisms, consideredas a model in biomineralization. Understanding their formation and growth is a challenge in biomimetics. Its byssusforms very fi ne remarkably solid fi laments, with ultrastructural properties different from those of the mussel byssus.Until now, the chemistry of this byssus remains mysterious. Understanding it is also a challenge in material physics.These different points are discussed in this article.; La Grande nacre Pinna nobilis est le plus grand bivalve de Méditerranée. Espèce endémique, elle est inféodée auxherbiers de posidonies, vivant sous forme de petites populations clairsemées. C’est une espèce patrimoniale : sonbyssus a longtemps été collecté pour être utilisé à la confection de gants, bonnets ou étoles. Protégée par uneDirective Européenne (1992), P. nobilis fait l’objet d’une surveillance étroite au plan national et international. Or depuisfi n 2016, une épidémie, dont l’origine semble être un parasite protozoaire, décime les populations (jusqu’à 100 %de mortalité). Détectée au départ sur les côtes espagnoles, elle s’étend sur tout le pourtour méditerranéen.Bien que des mesures aient été prises, les prochaines années pourraient voir l’extinction de ce bivalve emblématique.Hormis sa valeur symbolique, P. nobilis est une espèce digne d’intérêt en science des matériaux et composites bioinspirés.En effet, sa coquille comprend deux couches minéralisées, dont l’une, externe, est constituée de grandsprismes calcitiques d’aspect monocristallin, simples en apparence, mais présentant une structuration multi-échelletrès complexe. Comprendre leur formation et leur croissance est un challenge en biomimétique. Son byssus formedes fi laments très fi ns remarquablement solides, aux propriétés ultrastructurales différentes de celles du byssus demoule. Jusqu’à présent, la chimie de ce byssus reste mystérieuse. L’appréhender est aussi une gageure en physiquedes matériaux. Ces différents points sont abordés dans cet article.

Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated β-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.

Background Cis-regulatory elements control gene expression over large distances through the formation of chromatin loops, which allow contact between enhancers and gene promoters. Alterations in cis-acting regulatory systems could be linked to human genetic diseases. Here, we analyse the spatial organization of a large region spanning the polycystic kidney disease 2 (PKD2) gene, one of the genes responsible of autosomal dominant polycystic kidney disease (ADPKD). Results By using chromosome conformation capture carbon copy (5C) technology in primary human renal cyst epithelial cells, we identify novel contacts of the PKD2 promoter with chromatin regions, which display characteristics of regulatory elements. In parallel, by using functional analysis with a reporter assay, we demonstrate that three DNAse I hypersensitive sites regions are involved in the regulation of PKD2 gene expression. Conclusions Finally, through alignment of CCCTC-binding factor (CTCF) sites, we suggest that these novel enhancer elements are brought to the PKD2 promoter by chromatin looping via the recruitment of CTCF. Electronic supplementary material The online version of this article (10.1186/s12864-018-4892-6) contains supplementary material, which is available to authorized users.

International audience; Metformin inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which is frequently upregulated in hepatocellular carcinoma (HCC). Metformin has also been shown to induce apoptosis in this cancer. Here, we investigate whether metformin-induced apoptosis in HCC is mediated by the downstream mTORC1 effectors eukaryotic initiation factor 4E and (eIF4E)-binding proteins (4E-BPs). Further, we ask whether changes in 4E-BPs activity during metformin treatment negatively regulate translation of the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) mRNA. A genetic HCC mouse model was employed to assess the ability of metformin to reduce tumor formation, induce apoptosis, and control 4E-BP1 activation and Mcl-1 protein expression. In parallel, the HCC cell line Huh7 was transduced with scrambled shRNA (control) or shRNAs targeting 4E-BP1 and 4E-BP2 (4E-BP knock-down (KD)) to measure differences in mRNA translation, apoptosis, and Mcl-1 protein expression after metformin treatment. In addition, immunohistochemical staining of eIF4E and 4E-BP1 protein levels was addressed in a HCC patient tissue microarray. We found that metformin decreased HCC tumor burden, and tumor tissues showed elevated apoptosis with reduced Mcl-1 and phosphorylated 4E-BP1 protein levels. In control but not 4E-BP KD Huh7 cells, metformin induced apoptosis and repressed Mcl-1 mRNA translation and protein levels. Immunostaining of HCC patient tumor tissues revealed a varying ratio of eIF4E/4E-BP1 expression. Our results propose that metformin induces apoptosis in mouse and cellular models of HCC through activation of 4E-BPs, thus tumors with elevated expression of 4E-BPs may display improved clinical chemopreventive benefit of metformin.

Coronaviruses are a family of enveloped single-stranded positive-sense RNA viruses causing respiratory, enteric, and neurologic diseases in mammals and fowl. Human coronaviruses are recognized to cause up to a third of common colds and are suspected to be involved in enteric and neurologic diseases. Coronavirus replication involves the generation of nested subgenomic mRNAs (sgmRNAs) with a common capped 5′ leader sequence. The translation of most of the sgmRNAs is thought to be cap dependent and displays a requirement for eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex needed for the recruitment of 40S ribosomes. We recently reported on an ultrahigh-throughput screen to discover compounds that inhibit eIF4F activity by blocking the interaction of two of its subunits (R. Cencic et al., Proc. Natl. Acad. Sci. U. S. A. 108: 1046–1051, 2011). Herein we describe a molecule from this screen that prevents the interaction between eIF4E (the cap-binding protein) and eIF4G (a large scaffolding protein), inhibiting cap-dependent translation. This inhibitor significantly decreased human coronavirus 229E (HCoV-229E) replication, reducing the percentage of infected cells and intra- and extracellular infectious virus titers. Our results support the strategy of targeting the eIF4F complex to block coronavirus infection.

The involvement of macrophages (Mvarphis) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mvarphi protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)(2)H(4-x)]((1+x)-) as the species responsible for the specific inhibition of Mvarphi PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)(2)](5-) targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K–AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (PtenFV), found in human cancer. Despite having reduced levels of PTEN protein, homozygous PtenFV/FV embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous PtenFV/+ mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

International audience; Genetic deletion of both 4E-BP1 and 4E-BP2 was found to protect cells against viral infections. Here we demonstrate that the individual loss of either 4E-BP1 or 4E-BP2 in mouse embryonic fibroblasts (MEFs) is sufficient to confer viral resistance. shRNA-mediated silencing of 4E-BP1 or 4E-BP2 renders MEFs resistant to viruses, and compared to wild type cells, MEFs knockout for either 4E-BP1 or 4E-BP2 exhibit enhanced translation of Irf-7 and consequently increased innate immune response to viruses. Accordingly, the replication of vesicular stomatitis virus, encephalomyocarditis virus, influenza virus and Sindbis virus is markedly suppressed in these cells. Importantly, expression of either 4E-BP1 or 4E-BP2 in double knockout or respective single knockout cells diminishes their resistance to viral infection. Our data show that loss of 4E-BP1 or 4E-BP2 potentiates innate antiviral immunity. These results provide further evidence for translational control of innate immunity and support targeting translational effectors as an antiviral strategy.

International audience; More than a simple carrier of the genetic information, messenger RNA (mRNA) coding regions can also harbor functional elements that evolved to control different post-transcriptional processes, such as mRNA splicing, localization and translation. Functional elements in RNA molecules are often encoded by secondary structure elements. In this aticle, we introduce Structural Profile Assignment of RNA Coding Sequences (SPARCS), an efficient method to analyze the (secondary) structure profile of protein-coding regions in mRNAs. First, we develop a novel algorithm that enables us to sample uniformly the sequence landscape preserving the dinucleotide frequency and the encoded amino acid sequence of the input mRNA. Then, we use this algorithm to generate a set of artificial sequences that is used to estimate the Z-score of classical structural metrics such as the sum of base pairing probabilities and the base pairing entropy. Finally, we use these metrics to predict structured and unstructured regions in the input mRNA sequence. We applied our methods to study the structural profile of the ASH1 genes and recovered key structural elements. A web server implementing this discovery pipeline is available at http://csb.cs.mcgill.ca/sparcs together with the source code of the sampling algorithm.

International audience; Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-β (IFN-β). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Hsp90 stabilizes and prevents degradation of cytoplasmic activation-induced deaminase., Activation-induced deaminase (AID) is the mutator enzyme that initiates somatic hypermutation and isotype switching of the antibody genes in B lymphocytes. Undesired byproducts of AID function are oncogenic mutations. AID expression levels seem to correlate with the extent of its physiological and pathological functions. In this study, we identify AID as a novel Hsp90 (heat shock protein 90 kD) client. We find that cytoplasmic AID is in a dynamic equilibrium regulated by Hsp90. Hsp90 stabilizes cytoplasmic AID, as specific Hsp90 inhibition leads to cytoplasmic polyubiquitination and proteasomal degradation of AID. Consequently, Hsp90 inhibition results in a proportional reduction in antibody gene diversification and off-target mutation. This evolutionarily conserved regulatory mechanism determines the functional steady-state levels of AID in normal B cells and B cell lymphoma lines. Thus, Hsp90 assists AID-mediated antibody diversification by stabilizing AID. Hsp90 inhibition provides the first pharmacological means to down-regulate AID expression and activity, which could be relevant for therapy of some lymphomas and leukemias.

International audience; The mammalian Slc11a1 and Slc11a2 proteins define a large family of secondary metal transporters. Slc11a1 and Slc11a2 function as pH-dependent divalent cation transporters that play a critical role in host defenses against infections and in Fe2+ homeostasis, respectively. The position and polarity of individual transmembrane domains (TMD) of Slc11a2 were studied by an epitope tagging method based on the insertion of small antigenic hemagglutinin A (HA) peptides (YPYDVPDYAS) in predicted intra- or extracellular loops of the protein. The tagged proteins were expressed in transfected LLC-PK1 kidney cells and tested for transport activity, and the polarity of inserted tags with respect to the plasma membrane was determined by immunofluorescence in intact and permeabilized cells. HA epitope tags were inserted at positions 1, 98, 131, 175, 201, 243, 284, 344, 403, 432, 468, 504, and 561. Insertions at positions 98, 131, 175, 403, and 432 abrogated metal transport by Slc11a2, while insertions at positions 1, 201, 243, 284, 344, 468, 504, and 561 resulted in functional proteins. Topology mapping in functional HA-tagged Slc11a2 proteins indicated that the N-terminus (1), as well as loops delineated by TMD4-5 (201), TMD6-7 (284), and TMD10-11 (468), and C-terminus (561) are intracellular, while loops separating TMD5-6 (243), TMD7-8 (344), and TMD11-12 (504) are extracellular. These results are compatible with a topology of 12 transmembrane domains, with intracellular amino and carboxy termini. Structural models constructed by homology threading support this 12TMD topology and show 2-fold structural symmetry in the arrangement of membrane helices for TM1-5 and TM6-10 (conserved Slc11 hydrophobic core).

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-ΔfimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.

International audience; mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

Pinna nobilis is the largest bivalve in the Mediterranean Sea. This endemic species lives in the Posidonia meadows,in the form of small sparse populations. It is a heritage species: its byssus has long been collected to be wovenand used to make gloves, hats or stoles. Protected by a European Directive (1992), P. nobilis is closely monitored,nationally and internationally. But since the end of 2016, an epidemic, which origin seems to be a protozoan parasite,decimates populations (up to 100% mortality). This event began on the Spanish coast and spreads now all around theMediterranean. Although conservation measures have been taken, the next few years could see the extinction of thisemblematic bivalve. Apart from its symbolic value, P. nobilis is a species worthy of biotechnological interest. Its shellcomprises two mineralized layers, one of which, external, calcitic, consists of large monocrystalline prisms, consideredas a model in biomineralization. Understanding their formation and growth is a challenge in biomimetics. Its byssusforms very fi ne remarkably solid fi laments, with ultrastructural properties different from those of the mussel byssus.Until now, the chemistry of this byssus remains mysterious. Understanding it is also a challenge in material physics.These different points are discussed in this article., La Grande nacre Pinna nobilis est le plus grand bivalve de Méditerranée. Espèce endémique, elle est inféodée auxherbiers de posidonies, vivant sous forme de petites populations clairsemées. C’est une espèce patrimoniale : sonbyssus a longtemps été collecté pour être utilisé à la confection de gants, bonnets ou étoles. Protégée par uneDirective Européenne (1992), P. nobilis fait l’objet d’une surveillance étroite au plan national et international. Or depuisfi n 2016, une épidémie, dont l’origine semble être un parasite protozoaire, décime les populations (jusqu’à 100 %de mortalité). Détectée au départ sur les côtes espagnoles, elle s’étend sur tout le pourtour méditerranéen.Bien que des mesures aient été prises, les prochaines années pourraient voir l’extinction de ce bivalve emblématique.Hormis sa valeur symbolique, P. nobilis est une espèce digne d’intérêt en science des matériaux et composites bioinspirés.En effet, sa coquille comprend deux couches minéralisées, dont l’une, externe, est constituée de grandsprismes calcitiques d’aspect monocristallin, simples en apparence, mais présentant une structuration multi-échelletrès complexe. Comprendre leur formation et leur croissance est un challenge en biomimétique. Son byssus formedes fi laments très fi ns remarquablement solides, aux propriétés ultrastructurales différentes de celles du byssus demoule. Jusqu’à présent, la chimie de ce byssus reste mystérieuse. L’appréhender est aussi une gageure en physiquedes matériaux. Ces différents points sont abordés dans cet article.