A novel mechanism of RNase L inhibition: Theiler\u27s virus L* protein prevents 2-5A from binding to RNase L

<div><p>The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L <i>in vivo</i>. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.</p></div

Redox controls reca protein activity via reversible oxidation of its methionine residues

Reactive oxygen species (ROS) cause damage to DNA and proteins. Here we report that the RecA recombinase is itself oxidized by ROS. Genetic and biochemical analyses revealed that oxidation of RecA altered its DNA repair and DNA recombination activities. Mass spectrometry analysis showed that exposure to ROS converted 4 out of 9 Met residues of RecA to methionine sulfoxide. Mimicking oxidation of Met35 by changing it for Gln caused complete loss of function whereas mimicking oxidation of Met164 resulted in constitutive SOS activation and loss of recombination activity. Yet, all ROS-induced alterations of RecA activity were suppressed by methionine sulfoxide reductases MsrA and MsrB. These findings indicate that under oxidative stress, MsrA/B is needed for RecA homeostasis control. The implication is that, besides damaging DNA structure directly, ROS prevent repair of DNA damage by hampering RecA activity.Agence Nationale de la Re-cherche ANR-10-LABX-62-IBEIDFondation pour la Recherche Medicale FRM - FDT20150532554National Institute of General Medical Sciences GM3233

An unbiased immunization strategy results in the identification of enolase as a potential marker for nanobody-based detection of Trypanosoma evansi

Trypanosoma evansi is a widely spread parasite that causes the debilitating disease “surra” in several types of ungulates. This severely challenges livestock rearing and heavily weighs on the socio-economic development in the affected areas, which include countries on five continents. Active case finding requires a sensitive and specific diagnostic test. In this paper, we describe the application of an unbiased immunization strategy to identify potential biomarkers for Nanobody (Nb)-based detection of T. evansi infections. Alpaca immunization with soluble lysates from different T. evansi strains followed by panning against T. evansi secretome resulted in the selection of a single Nb (Nb11). By combining Nb11-mediated immuno-capturing with mass spectrometry, the T. evansi target antigen was identified as the glycolytic enzyme enolase. Four additional anti-enolase binders were subsequently generated by immunizing another alpaca with the recombinant target enzyme. Together with Nb11, these binders were evaluated for their potential use in a heterologous sandwich detection format. Three Nb pairs were identified as candidates for the further development of an antigen-based assay for Nb-mediated diagnosis of T. evansi infection

Удосконалення комерційної діяльності як фактор підвищення конкурентоспроможності підприємства

Additional file 5. ELISA to assess the interaction between Campylobacter -specific nanobodies and purified MOMP. The saturation binding curve of the interaction between coated MOMP (1 µg/mL) and a His-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) was obtained via ELISA. The dose-dependent inhibitory effect of a strep-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) on the interaction between His-tagged Nb84 (5.10−2 µg/mL) and MOMP (1 µg/mL), is demonstrated in the competition binding curve. Inhibition by strep-tagged (A) Nb5, (B) Nb22, (C) Nb23, (D) Nb24, (E) Nb49, (F) 84, (G) Nb15, (H) Nb32, (I) Nb34, (J) Nb45, (K) Nb48 and (L) Nb63, was assessed. The ELISA was developed with mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase. The error bars represent the standard deviations

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo

Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression.Comparative StudyJournal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases

Wheat germ in vitro translation to produce one of the most toxic sodium channel specific toxins

Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD50 (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD50 of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice

Substrate binding sites in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase

La 6-phosphofructo-2-kinase/fructose-2,6-biphosphate (PFK-2/FBPase-2) est l’enzyme bifonctionnelle qui catalyse la synthèse et la dégradation du fructose-2,6-biphosphate, un puissant régulateur de la glycolyse. Au cours de ce travail, nous avons tenté d’élucider les mécanismes moléculaires gouvernant la catalyse du domaine PFK-2. Nous avons d’abord démontré que, contraitement à ce qui avait été postulé, le mécanisme catalytique de la PFK-2 n’était en aucun point comparable à celui de la 6-phosphofructo-1-kinase (PFK-1). Nous avons dès lors réalisé une analyse théorique du domaine PFK-2 par alignements de séquences. Ceci a permis l’élaboration d’un modèle tridimentionnel de la PFK-2 en analogie avec l’adenylate kinase. Cette approche théorique nous a permis de repérer les résidus potentiellement impliqués dans le processus catalytique et/ou la liaison des substrats. Nous avons vérifié expérimentalement par mutagène dirigé que certains parmi ces acides aminés sont de fiat impliqués dans la liaison des deux substrats de la PFK-2. Cette approche expérimentale a confirmé la modèle tridimensionnel. Finalement, l’association de ces approches théoriques et pratiques nous a permis de proposer un modèle catalytique de la PFK-2The bifunctional enzyme 6-phosphofructo-2-kinase/furctose-2,6-biphosphate (PFK-2/FBPase-2) catalyses the sunthesis and breakdown of fructose-2,6biphosphate, a potent regulator of glycolysis. In this work, we have attempted to elucidate the mechanisms involved in the PFK-2 reaction at the molecular level. We showed that the structure of the PFK-2 domain is unrelated to that of 6-phosphofructo-1-kinase (PFK1), on which a 3D structure model of the PFK-2 domain had been made previously, and that the catalytic mechanism of the two reactions is probably different. Secondly, we analysed the PFK-2 domain by multiple sequence alignment. This analysis led to a structural model of the PFK-2 domain based on adenylate kinase. This theoretical study allowed residues potentially involved in catalysis or substrate binding to be predicted. The role of some of these residues was tested by site-directed mutagenenis and showed that, indeed, several were involved in binding the two substrates of PFK-2. This experimental approach validated the structure model. Finally, the combination of theoretical and practical studies allowed us to propose a catalytic mechanism for the PFK-2 reaction.Thèse de doctorat en sciences biomédicales (biochimie) -- UCL, 199