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Theillet F.-X.,Leibniz Institute for Molecular Pharmacology | Theillet F.-X.,CNRS Institute of Integrative Biology | Binolfi A.,Leibniz Institute for Molecular Pharmacology | Binolfi A.,Max Planck Institute of Biophysics | And 9 more authors.
Nature | Year: 2016

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells. © 2016 Macmillan Publishers Limited. All rights reserved. Source

Machnicka M.A.,International Institute of Molecular and Cell Biology | Olchowik A.,International Institute of Molecular and Cell Biology | Grosjean H.,University Paris - Sud | Grosjean H.,CNRS Institute of Integrative Biology | And 2 more authors.
RNA Biology | Year: 2014

Functional tRNA molecules always contain a wide variety of post-transcriptionally modified nucleosides. These modifications stabilize tRNA structure, allow for proper interaction with other macromolecules and fine-tune the decoding of mRNAs during translation. Their presence in functionally important regions of tRNA is conserved in all domains of life. However, the identities of many of these modified residues depend much on the phylogeny of organisms the tRNAs are found in, attesting for domain-specific strategies of tRNA maturation. In this work we present a new tool, tRNAmodviz web server (http://genesilico.pl/trnamodviz) for easy comparative analysis and visualization of modification patterns in individual tRNAs, as well as in groups of selected tRNA sequences. We also present results of comparative analysis of tRNA sequences derived from 7 phylogenetically distinct groups of organisms: Gram-negative bacteria, Gram-positive bacteria, cytosol of eukaryotic single cell organisms, Fungi and Metazoa, cytosol of Viridiplantae, mitochondria, plastids and Euryarchaeota. These data update the study conducted 20 y ago with the tRNA sequences available at that time. © 2014 Taylor & Francis Group, LLC. Source

The natural history of tuberculosis may be tackled by various means, among which the record of molecular scars that have been registered by the Mycobacterium tuberculosis complex (MTBC) genomes transmitted from patient to patient for tens of thousands years and possibly more. Recently discovered polymorphic loci, the CRISPR sequences, are indirect witnesses of the historical phage-bacteria struggle, and may be related to the time when the ancestor of today's tubercle bacilli were environmental bacteria, i.e. before becoming intracellular parasites. In this article, we present what are CRISPRs and try to summarize almost 20 years of research results obtained using the genetic diversity of the CRISPR loci in MTBC as a perspective for studying new models. We show that the study of the diversity of CRISPR sequences, thanks to «spoligotyping», has played a great role in our global understanding of the population structure of MTBC. © 2015 Elsevier Ltd. All rights reserved. Source

Boussac A.,CNRS Institute of Integrative Biology | Rutherford A.W.,Imperial College London | Sugiura M.,Ehime University | Sugiura M.,Japan Science and Technology Agency
Biochimica et Biophysica Acta - Bioenergetics | Year: 2015

The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (YZ). Cl- is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S = 5/2) and low spin (S = 1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S = 3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ)′ split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S = 5/2) form of S2 shows a marked heterogeneity with a g = 4.3 form generated at low temperature that converts to a relaxed form at g = 4.9 at higher temperatures. The high spin g = 4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180 K whereas the low spin (S = 1/2) S2-state can only advance to the S3 state at temperatures 235 K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at 235 K but not at 198 K. Since both S2 configurations are formed at 198 K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s). ©2015 Elsevier B.V. All rights reserved. Source

Midonet C.,CNRS Institute of Integrative Biology | Das B.,Translational Health Science and Technology Institute | Sherratt D.J.,University of Oxford
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXφ, the cholera toxin phage. Here, we show that in contrast, integration of TLCφ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXφ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCφ, along with any CTXφ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXφ copies can be eliminated, as occurred in the second wave of the current cholera pandemic. Source

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