Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.Peer Reviewe

Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria

Successful treatment of human tuberculosis requires 6–9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms – organized communities of surface-attached cells – but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment

Première étude structurale et dynamique d'une peroxyrédoxine par RMN

Les cellules luttent en permanence contre les pro-oxydants et leurs effets néfastes. Les hydroperoxydes en sont les plus courants. La cystéine nucléophile du site actif confère aux peroxyrédoxines (Prx) une activité peroxydasique, réductrice des hydroperoxydes dépendante notamment de thiorédoxines. Pour la première fois, une Prx, Ahp1 de Saccharomyces cerevisiae, a fait l'objet d'une étude structurale par RMN triple résonance (1H/13C/15N) sur un échantillon uniformément marqué [13C/15N/50 % 2H] en milieu aqueux réducteur. Sa topologie est semblable à celle des autres Prx. L'analyse des données de relaxation RMN 15N a émontré que Ahp1 est un homodimère 2x19,4kDa en solution, ce qui a été confirmé par ultracentrifugation analytique et par des calculs d'hydrodynamique. La forme suroxydée, préparée in vitro par un excès de tBuOOH, a été cractérisée grâce à un marquage sélectif Cys[b-13C]. Les résultats montrent que la cystéine du site actif est alors oxydée en sulfonateLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Influence of the pH on the Condensation of Tetravalent Cerium Cations in Association with [α-SiW9O34]10- Leading to the Formation of a Ce6O4 (OH)4 Core

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NMR Reveals the Interplay among the AMSH SH3 Binding Motif, STAM2, and Lys63-Linked Diubiquitin.

We are indebted to the Research Federation FRABio (Univ. Lille, CNRS, FR 3688, FRABio, Biochmie Structurale et Fonctionnelle des Assemblages Biomoleculaires) for providing the scientific and technical environment conducive to achieving this work. We acknowledge Pr. Guy Lippens for helpful discussion related to the choice of the different AMSH variant peptides and Dr. Bernhard Brutscher for providing us a modified HNCO experiment to assign unambiguous NH2 side chain of asparagine.International audienceAMSH [associated molecule with a Src homology 3 domain of signal transducing adaptor molecule (STAM)] is one of the deubiquitinating enzymes associated in the regulation of endocytic cargo trafficking. It shows an exquisite selectivity for Lys63-linked polyubiquitin chains that are the main chains involved in cargo sorting. The first step requires the ESCRT-0 complex that comprises the STAM and hepatocyte growth factor-regulated substrate (Hrs) proteins. Previous studies have shown that the presence of the STAM protein increases the efficiency of Lys63-linked polyubiquitin chain cleavage by AMSH, one of the deubiquitinating enzyme involved in lysosomal degradation. In the present study, we are seeking to understand if a particular structural organization among these three key players is responsible for the stimulation of the catalytic activity of AMSH. To address this question, we first monitored the interaction between the ubiquitin interacting motif (UIM)-SH3 construct of STAM2 and the Lys63-linked diubiquitin (Lys63-Ub2) chains by means of NMR. We show that Lys63-Ub2 is able to bind either the UIM or the SH3 domain without any selectivity. We further demonstrate that the SH3 binding motif (SBM) of AMSH (AMSH-SBM) outcompetes Lys63-Ub2 for binding SH3. Additionally, we show how different AMSH-SBM variants, modified by their sequence and length, exhibit similar equilibrium dissociation constants when binding SH3 but significantly differ in their dissociation rate constants. Finally, we report the solution NMR structure of the AMSH-SBM/SH3 complex and propose a structural organization where the AMSH-SBM interacts with the STAM2-SH3 domain and contributes to the correct positioning of AMSH prior to polyubiquitin chains' cleavage

1H, 13C and 15N chemical shift backbone resonance NMR assignment of tobacco calmodulin 2

International audienceCalcium is a ubiquitous second messenger regulating numbers of cellular processes in living organisms. It encodes and transmits information perceived by cells to downstream sensors, including calmodulin (CaM), that initiate cellular responses. In plants, CaM has been involved in the regulation of plant responses to biotic and abiotic environmental cues. Plant CaMs possess a cysteine residue in their first calcium-binding motif EF-hand, which is not conserved in other eucaryotic organisms. In this work, we report the near-complete backbone chemical shift assignment of tobacco CaM2 with calcium. These results will be useful to study the impact of this particular EF-hand domain regarding CaM interaction with partners involved in stress responses

Molecular niobium(V) complexes with mononuclear {Nb1} and dinuclear oxo species {Nb2O} connected through aryl di- or tetra-carboxylate linker

International audienceSeveral aryl polycarboxylic ligands (pyromellitic acid, 4,4′-azobenzenedicarboxylic acid and 3,3′,5,5′- azobenzenetetracarboxylic acid) have been used to react with the pentavalent ethoxide niobium(V) precursor Nb(OEt)5. Three new crystalline molecular compounds (1 with pyromellitate, 2 with 4,4′- azobenzenedicarboxylate and 3 with 3,3′,5,5′-azobenzenetetracarboxylate) were isolated from slowcrystallization at room temperature from the mother liquid containing the precursor Nb(OEt)5. They are composed of one polycarboxylate ligand fully coordinated to mononuclear or dinuclear octahedrally- coordinated niobium(V) units. For the dimers, the two adjacent niobium centers are bridged by one μ-O and one μ-OEt groups and complexed by only one of the syn–syn bidentate carboxylate function arms from the polytopic ligand. Compounds 1–3 were characterized in solid state by single-crystal and powder X-ray diffraction analysis. Insight into the formation of the three compounds was supported by the help of infrared and liquid 1H NMR spectroscopy.Plusieurs ligands aryl polycarboxyliques (acide pyromellitique, acide 4,4′-azobenzènedicarboxylique et acide 3,3′,5,5′-azobenzènetétracarboxylique) ont été utilisés pour réagir avec l'éthoxyde pentavalent niobium(V) précurseur Nb(OEt)5. Trois nouveaux composés moléculaires cristallins (1 avec du pyromellitate, 2 avec du 4,4′-azobenzènedicarboxylate et 3 avec du 3,3′,5,5′-azobenzènetétracarboxylate) ont été isolés à partir decristallisation à température ambiante à partir du liquide mère contenant le précurseur Nb(OEt)5. Ils sont composés d'un ligand polycarboxylate entièrement coordonné à des unités de niobium (V) octaédriquement coordonnées mononucléaires ou dinucléaires. Pour les dimères, les deux centres de niobium adjacents sont pontés par un groupe μ-O et un groupe μ-OEt et complexés par un seul des bras de la fonction carboxylate syn – syn ​​bidentate du ligand polytopique. Les composés 1 à 3 ont été caractérisés à l'état solide par analyse par diffraction des rayons X sur monocristal et sur poudre. La compréhension de la formation des trois composés a été étayée par l'aide de la spectroscopie RMN 1H infrarouge et liquide

Azobenzene: a Visible‐Light Chemical Actinometer for the Characterization of Fluidic Photosystems

International audience(E)-Azobenzene is introduced as a suitable chemical actinometer in the visible spectral range (440–540 nm) for photon flux determination of fluidic microphotoreactors or for assessing efficiency of visible light photo-induced reactions, its evaluation is straightforward without tedious analytics. Photoisomerization quantum yields (ΦE→Z) of (E)-azobenzene were accurately determined upon irradiation at several wavelengths and in different solvents based on well-known diarylethene

Bumim][PF6] ionic liquid as reactant for uranium (IV) oxide fluorination

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