Backbone chemical shift assignments of human 14-3-3σ\sigma

14-3-3 proteins are a group of seven dimeric adapter proteins that exert their biological function by interacting with hundreds of phosphorylated proteins, thus influencing their sub-cellular localization, activity or stability in the cell. Due to this remarkable interaction network, 14-3-3 proteins have been associated with several pathologies and the protein-protein interactions established with a number of partners are now considered promising drug targets. The activity of 14-3-3 proteins is often isoform specific and to our knowledge only one out of seven isoforms, 14-3-3$\zeta$, has been assigned. Despite the availability of the crystal structures of all seven isoforms of 14-3-3, the additional NMR assignments of 14-3-3 proteins are important for both biological mechanism studies and chemical biology approaches. Herein, we present a robust backbone assignment of 14-3-3$\sigma$, which will allow advances in the discovery of potential therapeutic compounds. This assignment is now being applied to the discovery of both inhibitors and stabilizers of 14-3-3 protein-protein interactions

Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

International audienceHepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance

Etude in vivo de l'activation d'une pro-drogue par des mycobactéries à l'aide de la RMN 1H HRMAS (analyse structurale et biochimique de OPgG de Escherichia coli, une protéine requise pour la biosynthèse des glucanes périplasmiques osmorégulés)

L'éthionamide (ETH) est un antituberculeux de seconde ligne, très efficace dans le traitement des patients infectés par des souches de Mycobacterium tuberculosis multirésistantes aux molécules de première ligne. L'ETH est une pro-drogue, elle nécessite donc de subir un processus d'activation afin d'acquérir son activité antibiotique dirigée contre la biosynthèse de l'enveloppe cellulaire de M. tuberculosis. Une cible identifie e de l'ETH est InhA, une enzyme impliquée dans la synthèse des acides mycoliques, un constituant majeur de la paroi des mycobactéries. Un activateur de l'ETH a été identifié chez M. tuberculosis, il s'agit de la protéine EthA, une monooxygénase. La surexpression de cette protéine engendre une hypersensibilité des mycobactéries vis-à-vis de l'ETH. A l'inverse, la surexpression de EthR, un répresseur transcriptionnel de ethA, rend les bactéries résistantes à l'ETH. Dans le but de mieux appréhender le rôle de l'activateur EthA, nous avons surexprimé et partiellement purifié cette protéine. En solution, EthA forme des agrégats de hauts poids moléculaires, ce qui ne nous a pas permis d'aller plus en avant dans l'étude structurale de la protéine que nous avions envisagée. Nous avons alors utilisé une technique originale : la RMN 1H HRMAS afin de caractériser in vivo l'activation de l'ETH par des mycobactéries. Cette technique nous a permis de détecter un métabolite, ETH*, dérivé de l'ETH et formé par les mycobactéries surexprimant EthA. Le métabolite ETH* s'accumule dans les cellules tandis que deux autres métabolites de l'ETH, respectivement ses dérivés S-oxyde et alcool, sont présents uniquement dans le milieu de culture. Nous avons partiellement caractérisé, par RMN, la structure de ETH* qui comprend un cycle 2-éthyl-pyridine ainsi qu'un groupement en position 4 qui contient un atome de carbone quaternaire. La nature exacte de cette fonction exo-cyclique reste à préciser. La technique RMN 1H HRMAS nous a également permis de détecter, ex-vivo, les métabolites de l'ETH présents dans un foie d'une souris ayant reçu de l'ETH. L'ensemble de ces travaux s'inscrit dans la démarche d'une meilleure compréhension de l'activation de l'ETH chez les mycobactéries afin de concevoir in fine des versions améliorées de la drogue. Ces études ont également permis de mettre en évidence les bénéfices de l'utilisation de la RMN 1H HRMAS dans l'étude d'un processus de métabolisation in vivo. Les OPGs (Glucanes Périplasmiques Omorégulés) sont des molécules glucosidiques très largement répandues parmi les Protéobactéries. Les OPGs sont synthétisés en réponse à un environnement où l'osmolarité est faible. Leur concentration périplasmique devient alors très élevée et les OPGs peuvent représenter jusqu'à 5 % du poids sec des cellules de E. coli. Le rôle physiologique des OPGs demeure incertain mais ces molécules semblent être impliquées dans un processus d'osmoprotection et pourraient servir de signal indiquant aux bactéries d'adapter leur métabolisme aux conditions environnementales. Les OPGs jouent un rôle, direct ou indirect, dans la pathogénicité. En effet, en inhibant leur biosynthèse chez Erwinia chrysanthemi, Agrobacterium tumefasciens ou encore Pseudomonas aeruginosa, ces bactéries sont avirulentes. Les OPGs de E. coli sont des oligomères linéaires de glucose liés en -1,2 et ramifiés par des résidus de glucose en position -1,6. Ces OPGs sont substitués par des groupements phosphoglycérol, phosphoéthanolamine et succinate. Chez E. coli, deux protéines, OpgH et OpgG, sont strictement nécessaires à la biosynthèse du squelette glycosidique des OPGs. La première est une glycosyltransférase membranaire tandis que la deuxième est une protéine périplasmique dont la fonction est inconnue. Dans le but de comprendre le rôle de OpgG dans la biosynthèse des OPGs nous avons réalisé une étude structurale et biochimique de la protéine. OpgG de E. coli a été cristallisée puis sa structure résolue à 2,5 Å par diffraction des rayons X. OpgG est organisée en deux domaines formés par des feuillets . Le domaine N-terminal comprend une large cavité qui représente un site actif potentiel et présente des homologies de structure avec de nombreuses protéines reliées au métabolisme de composés glycosidiques. Le domaine C-terminal a une structure similaire à celle d'un domaine de type immunoglobuline. Nous avons ensuite investigué une potentielle interaction entre OpgG et la glycosyltransférase membranaire OpgH. Pour ce faire, une boucle périplasmique de OpgH a été clonée puis exprimée en fusion au domaine B1 de la protéine G streptococcale. L'interaction entre les deux partenaires à été étudiée par RMN et confirmée par chromatographie d'exclusion. Dans une dernière partie, nous avons optimisé les conditions expérimentales permettant d'obtenir des spectres RMN de qualité sur la protéine OpgG malgré sa masse moléculaire élevée de 56 kDa. Des résultats prometteurs ont été obtenus en utilisant des séquences de type TROSY sur la protéine OpgG triplement enrichie [2H, 15N, 13C].LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies

Hepatitis C virus (HCV) envelope glycoprotein heterodimer, E1E2, plays an essential role in virus entry and assembly. Furthermore, due to their exposure at the surface of the virion, these proteins are the major targets of anti-HCV neutralizing antibodies. Their ectodomain are heavily glycosylated with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycans. Thus, one-third of the molecular mass of E1E2 heterodimer corresponds to glycans. Despite the high sequence variability of E1 and E2, N-glycosylation sites of these proteins are generally conserved among the seven major HCV genotypes. N-glycans have been shown to be involved in E1E2 folding and modulate different functions of the envelope glycoproteins. Indeed, site-directed mutagenesis studies have shown that specific glycans are needed for virion assembly and infectivity. They can notably affect envelope protein entry functions by modulating their affinity for HCV receptors and their fusion activity. Importantly, glycans have also been shown to play a key role in immune evasion by masking antigenic sites targeted by neutralizing antibodies. It is well known that the high mutational rate of HCV polymerase facilitates the appearance of neutralization resistant mutants, and occurrence of mutations leading to glycan shifting is one of the mechanisms used by this virus to escape host humoral immune response. As a consequence of the importance of the glycan shield for HCV immune evasion, the deletion of N-glycans also leads to an increase in E1E2 immunogenicity and can induce a more potent antibody response against HCV

NMR and circular dichroism data for domain 2 of the HCV NS5A protein phosphorylated by the Casein Kinase II

International audienc

Interaction study between HCV NS5A-D2 and NS5B using 19F NMR

The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a F-19-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented. The dissociation constants (K-D) between NS5A-D2 and NS5B obtained with these two methods are in good agreement, and yield values comparable to those derived previously from a surface plasmon resonance study. We compare benefits and limitations of both labeling methods to study the interaction between an intrinsically disordered protein and a large molecular target by F-19 NMR

Graphical interpretation of Boolean operators for protein NMR assignments

We have developed a graphics based algorithm for semi-automated protein NMR assignments. Using the basic sequential triple resonance assignment strategy, the method is inspired by the Boolean operators as it applies "AND"-, "OR"- and "NOT"-like operations on planes pulled out of the classical three-dimensional spectra to obtain its functionality. The method's strength lies in the continuous graphical presentation of the spectra, allowing both a semi-automatic peaklist construction and sequential assignment. We demonstrate here its general use for the case of a folded protein with a well-dispersed spectrum, but equally for a natively unfolded protein where spectral resolution is minimal

Sandwich-ELISE NMR: Reducing the sample volume of NMR samples

We present Sandwich-ELISE, a concatenated version of our previously proposed Experimental LIquid SEaling (ELISE) protocol, in which an aqueous sample is effectively sealed by the addition of a small layer of mineral oil, or, alternatively, a chloroform sample was sealed by a water layer. With Sandwich-ELISE, a triple layered geometry composed of deuterated chloroform/aqueous buffer/mineral oil can be used to limit the sample to the active coil volume, effectively replacing the popular Shigemi tubes. Importantly, this procedure is readily applicable to smaller diameter tubes, for which no Shigemi tubes are available. We further present spectra of a 1 microl protein sample sandwiched between the chloroform and Nujol phases in a 1mm tube, demonstrating thereby that the volume of the aqueous phase of interest can be reduced even further

Hepatitis E Virus RNA‐Dependent RNA Polymerase is Involved in RNA Replication and Infectious Particle Production

International audienceBackground and Aims: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and known as the viral replicase. The RNA-dependent RNA polymerase (RdRp) domain is responsible for the synthesis of viral RNA.Approach and Results: Here, we identified a highly conserved α-helix located in the RdRp thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RdRp palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release.Conclusions: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RdRp with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RdRp thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus productio