Полеміка навколо наукової спадщини Володимира Антоновича в українській історіографії радянської доби

(uk) Проаналізовано стан дослідження наукової спадщини класика української історіографії В.Б. Антоновича в працях істориків різних ідеологічних спрямувань. Указано на відмінності теоретичних узагальнень щодо творчості вченого в національно-державницькій та марксистській науці радянської доби.(ru) Проанализировано состояние исследования научного наследия классика украинской историографии В. Б. Антоновича в работах историков различных идеологических направлений. Подчеркнуты различия теоретических обобщений относительно творчества в национально-государственной и марксистской науке советского периода.(en) The state of investigation of V.B. Antonovich\s scientific activity in the researches of historians of different ideological direction was analyzed. It is pointed out the differences of theoretical generalization about activity of scientist in national state and marxist historical science of Soviet epoch

Études structurales des interactions virus-hôte à travers deux exemples : le récepteur du système immunitaire inne RIG-I et le domaine endonucléase de l'ARN polymérase du virus de la grippe

The first line of defense against invading pathogens in the human body is the innate im- mune system. Astonishingly, with only a handful of different pathogen recognition recep- tors (around 50), the innate immune system is able to detect a remarkably broad variety of pathogen specific molecules to trigger protective pathways and to activate the adaptive immune system. In the case of intruding viruses, two families of pattern recognition re- ceptors (PRRs) are active: retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and Toll-like receptors (TLRs). The family of RIG-I like receptors includes the proteins RIG-I, MDA5 and LGP2, which recognize viral RNA in the cytosol. In the first part of this thesis, aspects of the RIG-I pathway are discussed: With which RNA does RIG-I interact and how? Does the oligomeric state of RIG-I change upon RNA binding in or- der to trigger signaling? How is RIG-I regulated by ubiquitin and its E3 ubiquitin ligase TRIM25? The structure of the PRYSPRY domain of TRIM25, its putative RIG-I binding domain, will be presented. Furthermore, preliminary work on the second receptor MDA5 in complex with parainfluenza virus V, which inhibits the MDA5 pathway, protein will be shown. One of the activators of the receptor RIG-I is the RNA of influenza virus. Influenza viruses belong to the family of Orthomyxoviridae that affect birds and mammals and spread in seasonal epidemics. In pandemic years, this can result in up to millions of deaths worldwide, underlining the need for research on efficient novel anti-viral drugs. In the second part of the thesis (appended as an article manuscript), the A/California/04/2009- H1N1 "swine flu" influenza RNA polymerase will be investigated as a novel antiviral drug target. Crystal structures of the endonuclease domain (PA-Nter) of the polymerase with four different inhibitors are presented. Moreover, the atomic structures of H1N1 PA-Nter with rUMP and dTMP, elements of the nucleic acid substrate, in the active site are discussed. These high resolution structures will serve as a basis for structure based inhibitor optimization.Le système immunitaire inné constitue la première barrière de défense du corps humain contre les agents infectieux. Constitue d'une poignée de récepteurs seulement (approximativement 50), ce système est capable de détecter la plupart des agents pathogènes et d'activer le système immunitaire adaptatif. Dans le cas d'une infection virale, deux classes de récepteurs sont mobilisées : les RLRs (Retinoic acid Like Récepteurs) et les TLRs (Toll Like Receptors). La famille des RLRs comprend notamment trois protéines, RIG-I, MDA-5 et LGP2, qui reconnaissent la présence d'ARN viral dans le cytosol. Dans la première partie de cette thèse, nous discuterons de la voie d'activation de RIG-I : avec quels type d'ARN et de quelle manière RIG-I interagit-il ? L'etat d'oligomerisation de RIG-I change-t-il lors de l'interaction avec l'ARN ? De quelle manière RIG-I est-il influence par l'ubiquitine et sa ligase E3, TRIM25 ? Nous présenterons egalement la structure du domaine PRYSPRY de TRIM25, domaine minimum nécessaire à l'interaction avec RIG-I. Nous d'écrirons également les travaux préliminaires obtenus sur un complexe constitue de MDA5 et une molecule d'inhibition virale. L'ARN des virus de la grippe est un des activateurs du récepteur RIG-I. Les virus de la grippe appartiennent 'a la famille des Orthomyxoviridae qui touche les mammifères et les aves créant des épidémies saisonnières. Ces dernières peuvent causer jusqu'à plusieurs millions de morts lors de pandémies, soulignant le besoin de trouver de nouvelles solutions thérapeutiques. Dans la seconde partie de ma thèse, nous nous intéresserons à l'activité endonucléase de l'ARN polymérase du virus A/California/04/2009-H1N1 de la grippe porcine comme une cible pour de nouvelles molécules antivirales. Les structures de quatre inhibiteurs en complexe avec ce domaine seront présentés. Nous présenterons également la structure de PA-Nter avec les substrats rUMP et dTMP co-cristallisés dans le site actif. Toutes ces structures atomiques forment une base pour l'optimisation et la synthèse d'inhibiteurs

RIG-I

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D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not

Interconversion of D-ribose-5-phosphate (R5P) and D-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally

RIG-I self-oligomerization is either dispensable or very transient for signal transduction

International audienceEffective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5'ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient

Structural Basis for the Activation of Innate Immune Pattern-Recognition Receptor RIG-I by Viral RNA

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Structural Analysis of Specific Metal Chelating Inhibitor Binding to the Endonuclease Domain of Influenza pH1N1 (2009) Polymerase

<div><p>It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.</p> </div

Analysis of RIG-I oligomerization <i>in cellula</i> as determined by co-immunoprecipitation 18 hours after stimulation by a cognate RNA ligand.

<p>(A) Similar expression of Flag-RIG-I and cl25-RIG-I constructs in 293T cells as revealed by western blot. (B) Efficiency of Flag-RIG-I and Cl25-RIG-I to activate the IFN-β promoter after Poly(I:C) transfection. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108770#pone-0108770-g002" target="_blank">figure 2</a> legend for details. (C, D) Lack of co-immunoprecipitation of Cl25-RIG-I with Flag-RIG-I after their co-transfection in Huh7.5 cells and stimulation with Poly(I:C), ^5′pppssRNA(62-mer) or ^5′pppdsRNA(62-mer) (C) or MeV infection (MOI 1) (D) as detected by western blot. (E) Nonsensical co-immunoprecipitation of Cl25-RIG-I with Flag-RIG-I expressed in 293T cells and after transfection of 1 µg or 50 ng of ^5′pppds(or ss)RNA(62-mer) or MeV infection (MOI 0.5).</p

RIG-I binding to synthetic RNA and activation of IFN-β promoter.

<p>(A) Expression of Flag-RIG-I in Huh7.5 cells two days after transfection and analysed by western blot as revealed with Flag-specific antibody. (B) Luciferase expression driven under the control of the IFN-β promoter measured 24 h after transfection with 20 ng of synthetic RNA in Huh7.5 cells expressing or not Flag-RIG-I. (C, D) Immunoprecipitation of RIG-I:RNA complexes formed <i>in cellula</i>. Synthetic RNA were transfected in Huh7.5 cells previously transfected or not with Flag-RIG-I or Flag-RIG-I^ko 24 h before. Cells were harvested 6 hours after RNA transfection and RIG-I:RNA complexes were eluted from anti-Flag antibody immobilized on beads with a Flag peptide. (C) Specific immunoprecipitation of Flag-RIG-I as analysed by western blot. (D) RNA immunoprecipitated with Flag-RIG-I and analysed by RT-PCR.</p