41 results on '"Eiler S"'
Search Results
2. Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
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Alzari, Pedro M., Berglund, H., Berrow, N. S., Blagova, E., Busso, D., Cambillau, C., Campanacci, V., Christodoulou, E., Eiler, S., Fogg, M. J., Folkers, G., Geerlof, A., Hart, D., Haouz, A., Herman, M. D., Macieira, S., Nordlund, Pär, Perrakis, A., Quevillon-Cheruel, S., Tarandeau, F., van Tilbeurgh, H., Unger, T., Luna-Vargas, M. P. A., Velarde, M., Willmanns, M., Owens, Raymond J., Alzari, Pedro M., Berglund, H., Berrow, N. S., Blagova, E., Busso, D., Cambillau, C., Campanacci, V., Christodoulou, E., Eiler, S., Fogg, M. J., Folkers, G., Geerlof, A., Hart, D., Haouz, A., Herman, M. D., Macieira, S., Nordlund, Pär, Perrakis, A., Quevillon-Cheruel, S., Tarandeau, F., van Tilbeurgh, H., Unger, T., Luna-Vargas, M. P. A., Velarde, M., Willmanns, M., and Owens, Raymond J.
- Abstract
The implementation of high- throughput ( HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe ( SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non- ligation- based cloning techniques used, namely Gateway, ligation- indendent cloning of PCR products ( LIC- PCR) and In- Fusion, with LIC- PCR emerging as the most cost- effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library- based screening for soluble constructs and parallel small- scale high- density fermentation.
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- 2006
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3. Crystal structure of ERa LBD in complex with RU100132
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Osz, J., primary, Brelivet, Y., additional, Peluso-Iltis, C., additional, Cura, V., additional, Eiler, S., additional, Ruff, M., additional, Bourguet, W., additional, Rochel, N., additional, and Moras, D., additional
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- 2012
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4. Crystal structure of RARb LBD homodimer in complex with TTNPB
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Osz, J., primary, Brelivet, Y., additional, Peluso-Iltis, C., additional, Cura, V., additional, Eiler, S., additional, Ruff, M., additional, Bourguet, W., additional, Rochel, N., additional, and Moras, D., additional
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- 2012
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5. Crystal structure of RARb LBD in complex with 9cis retinoic acid
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Osz, J., primary, Br livet, Y., additional, Peluso-Iltis, C., additional, Cura, V., additional, Eiler, S., additional, Ruff, M., additional, Bourguet, W., additional, Rochel, N., additional, and Moras, D., additional
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- 2012
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6. Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE
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Alzari, Pedro M., primary, Berglund, H., additional, Berrow, N. S., additional, Blagova, E., additional, Busso, D., additional, Cambillau, C., additional, Campanacci, V., additional, Christodoulou, E., additional, Eiler, S., additional, Fogg, M. J., additional, Folkers, G., additional, Geerlof, A., additional, Hart, D., additional, Haouz, A., additional, Herman, M. D., additional, Macieira, S., additional, Nordlund, P., additional, Perrakis, A., additional, Quevillon-Cheruel, S., additional, Tarandeau, F., additional, van Tilbeurgh, H., additional, Unger, T., additional, Luna-Vargas, M. P. A., additional, Velarde, M., additional, Willmanns, M., additional, and Owens, Raymond J., additional
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- 2006
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7. Crystal Structure of the E. coli Aspartyl-tRNA Synthetase:Yeast tRNAasp:aspartyl-Adenylate Complex
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Moulinier, L., primary, Eiler, S., additional, Eriani, G., additional, Gangloff, J., additional, Thierry, J.C., additional, Gabriel, K., additional, McClain, W.H., additional, and Moras, D., additional
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- 2001
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8. The structure of an AspRS–tRNAAsp complex reveals a tRNA-dependent control mechanism
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Moulinier, L., primary, Eiler, S., additional, Eriani, G., additional, Gangloff, J., additional, Thierry, J.-C., additional, Gabriel, K., additional, McClain, W.H., additional, and Moras, D., additional
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- 2001
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9. MUTANT ESTROGEN NUCLEAR RECEPTOR LIGAND BINDING DOMAIN COMPLEXED WITH ESTRADIOL
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Ruff, M., primary, Gangloff, M., additional, Eiler, S., additional, Duclaud, S., additional, Wurtz, J.M., additional, and Moras, D., additional
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- 2000
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10. An intermediate step in the recognition of tRNA Asp by aspartyl-tRNA synthetase 1 1Edited by J. Doudna
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Briand, C., primary, Poterszman, A., additional, Eiler, S., additional, Webster, G., additional, Thierry, J.-C., additional, and Moras, D., additional
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- 2000
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11. Synthesis of aspartyl-tRNAAsp in Escherichia coli—a snapshot of the second step
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Eiler, S., primary, Dock-Bregeon, A.-C., additional, Moulinier, L., additional, Thierry, J.-C., additional, and Moras, D., additional
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- 1999
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12. Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant
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Martin, F, primary, Sharples, G J, additional, Lloyd, R G, additional, Eiler, S, additional, Moras, D, additional, Gangloff, J, additional, and Eriani, G, additional
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- 1997
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13. tRNA recognition by procaryotic aspartyl tRNA-synthetases
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Moras, D., primary, Moulinier, L., additional, Briand, C., additional, Eiler, S., additional, Poterzmann, A., additional, and Thierry, J. C., additional
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- 1996
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14. Crystallization of aspartyl-tRNA synthetase-tRNAAsp complex from Escherichia coli and first crystallographic results
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Eiler, S., primary, Boeglin, M., additional, Martin, F., additional, Eriani, G., additional, Gangloff, J., additional, Thierry, J.C., additional, and Moras, D., additional
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- 1992
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15. The structure of an AspRS-tRNAAsp complex reveals a tRNA-dependent control mechanism.
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Moulinier, L., Eiler, S., Eriani, G., Gangloff, J., Thierry, J.-C., Gabriel, K., McClain, W.H., and Moras, D.
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- *
RNA , *ESCHERICHIA coli , *DIMERS , *AMINO acids , *ADENOSINES , *ADENINE - Abstract
The 2.6 Å resolution crystal structure of an inactive complex between yeast tRNAASP and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone-protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism. [ABSTRACT FROM AUTHOR]
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- 2001
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16. Synthesis of aspartyl-tRNAAsp in Escherichia coli-a snapshot of the second step.
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Eiler, S., Dock-Bregeon, A. C., Moulinier, L., Thierry, J. C., and Moras, D.
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RNA-protein interactions , *AMINO acids , *ESCHERICHIA coli , *TRANSFER RNA , *LIGASES , *ENZYMES - Abstract
The 2.4 Å crystal structure of the Escherichia coli espartyl-tRNA synthetase (AspRS)-tRNA Asp-espartyladenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3′-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterialspecific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal β-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme. [ABSTRACT FROM AUTHOR]
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- 1999
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17. An intermediate step in the recognition of tRNAAspby aspartyl-tRNA synthetase11Edited by J. Doudna
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Briand, C., Poterszman, A., Eiler, S., Webster, G., Thierry, J.-C., and Moras, D.
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The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNAAspfrom either T. thermophilusor Escherichia colireveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 °C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.
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- 2000
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18. Econometrics of Incomplete Cross-Section/Time-Series Data: Consumer Demand in Norwegian Households, 1975-1977
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Erik Biorn, Anup K. Chanda, and Eiler S. Jansen
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Statistics and Probability ,Cross section (physics) ,Consumer demand ,language ,Econometrics ,Economics ,Norwegian ,Statistics, Probability and Uncertainty ,Time series ,language.human_language - Published
- 1984
19. Structural basis for HIV-1 DNA integration in the human genome
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Moras Dino, Benarous Richard, Emiliani Stéphane, Nazabal Alexis, Gottikh Marina, Mouscadet Jean-François, Granger Florence, Eiler Sylvia, Michel Fabrice, Schultz Patrick, and Ruff Marc
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Immunologic diseases. Allergy ,RC581-607 - Published
- 2009
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20. Crystallization of aspartyl-tRNA synthetase-tRNA complex from and first crystallographic results
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EILER, S
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- 1992
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21. Inhibitors of UHRF1 base flipping activity showing cytotoxicity against cancer cells.
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Ciaco S, Mazzoleni V, Javed A, Eiler S, Ruff M, Mousli M, Mori M, and Mély Y
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- Humans, Ubiquitin-Protein Ligases metabolism, DNA Methylation, DNA chemistry, Models, Molecular, CCAAT-Enhancer-Binding Proteins chemistry, CCAAT-Enhancer-Binding Proteins genetics, CCAAT-Enhancer-Binding Proteins metabolism, Neoplasms genetics
- Abstract
Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein overexpressed in numerous human cancer types. We previously disclosed the anthraquinone derivative UM63 that inhibits UHRF1-SRA domain base-flipping activity, although having DNA intercalating properties. Herein, based on the UM63 structure, new UHRF1-SRA inhibitors were identified through a multidisciplinary approach, combining molecular modelling, biophysical assays, molecular and cell biology experiments. We identified AMSA2 and MPB7, that inhibit UHRF1-SRA mediated base flipping at low micromolar concentrations, but do not intercalate into DNA, which is a key advantage over UM63. These molecules prevent UHRF1/DNMT1 interaction at replication forks and decrease the overall DNA methylation in cells. Moreover, both compounds specifically induce cell death in numerous cancer cell lines, displaying marginal effect on non-cancer cells, as they preferentially affect cells with high level of UHRF1. Overall, these two compounds are promising leads for the development of anti-cancer drugs targeting UHRF1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2023
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22. Effectiveness and cost-effectiveness of a web-based routine assessment with integrated recommendations for action for depression and anxiety (RehaCAT+): protocol for a cluster randomised controlled trial for patients with elevated depressive symptoms in rehabilitation facilities.
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Knauer J, Terhorst Y, Philippi P, Kallinger S, Eiler S, Kilian R, Waldmann T, Moshagen M, Bader M, and Baumeister H
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- Anxiety therapy, Cost-Benefit Analysis, Humans, Internet, Randomized Controlled Trials as Topic, Depression psychology, Quality of Life
- Abstract
Introduction: The integration of a web-based computer-adaptive patient-reported outcome test (CAT) platform with persuasive design optimised features including recommendations for action into routine healthcare could provide a promising way to translate reliable diagnostic results into action. This study aims to evaluate the effectiveness and cost-effectiveness of such a platform for depression and anxiety (RehaCAT+) compared with the standard diagnostic system (RehaCAT) in cardiological and orthopaedic health clinics in routine care., Methods and Analysis: A two-arm, pragmatic, cluster-randomised controlled trial will be conducted. Twelve participating rehabilitation clinics in Germany will be randomly assigned to a control (RehaCAT) or experimental group (RehaCAT+) in a 1:1 design. A total sample of 1848 participants will be recruited across all clinics. The primary outcome, depression severity at 12 months follow-up (T3), will be assessed using the CAT Patient-Reported Outcome Measurement Information System Emotional Distress-Depression Item set. Secondary outcomes are depression at discharge (T1) and 6 months follow-up (T2) as well as anxiety, satisfaction with participation in social roles and activities, pain impairment, fatigue, sleep, health-related quality of life, self-efficacy, physical functioning, alcohol, personality and health economic-specific general quality of life and socioeconomic cost and benefits at T1-3. User behaviour, acceptance, facilitating and hindering factors will be assessed with semistructured qualitative interviews. Additionally, a smart sensing substudy will be conducted, with daily ecological momentary assessments and passive collection of smartphone usage variables. Data analysis will follow the intention-to-treat principle with additional per-protocol analyses. Cost-effectiveness analyses will be conducted from a societal perspective and the perspective of the statutory pension insurance., Ethics and Dissemination: The study will be conducted according to the Declaration of Helsinki. The Ethics Committee of Ulm University, has approved the study (on 24 February 2021 ref. 509/20). Written informed consent will be obtained for all participants. Results will be published via peer-reviewed journals., Trial Registration Number: DRKS00027447., Competing Interests: Competing interests: Authors of the manuscript were partly involved in the development of RehaCAT(+). HB has been the beneficiary of study support (third party funding) from several public funding organisations in the context of research on computer-adaptive testing and patient-reported outcome systems., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2022
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23. The relative effects of pace of life-history and habitat characteristics on the evolution of sexual ornaments: A comparative assessment.
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Sowersby W, Eckerström-Liedholm S, Rowiński PK, Balogh J, Eiler S, Upstone JD, Gonzalez-Voyer A, and Rogell B
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- Animal Fins, Animals, Ecosystem, Male, Sexual Behavior, Animal, Selection, Genetic, Sex Characteristics
- Abstract
Selection may favor greater investment into sexual ornaments when opportunities for future reproduction are limited, for example, under high adult mortality. However, predation, a key driver of mortality, typically selects against elaborate sexual ornaments. Here, we examine the evolution of sexual ornaments in killifishes, which have marked contrasts in life-history strategy among species and inhabit environments that differ in accessibility to aquatic predators. We first assessed if the size of sexual ornaments (unpaired fins) influenced swimming performance. Second, we investigated whether the evolution of larger ornamental fins is driven primarily by the pace of life-history (investment into current vs. future reproduction) or habitat type (a proxy for predation risk). We found that larger fins negatively affected swimming performance. Further, males from species inhabiting ephemeral habitats, with lower predation risk, had larger fins and greater sexual dimorphism in fin size, compared to males from more accessible permanent habitats. We show that enlarged ornamental fins, which impair locomotion, evolve more frequently in environments that are less accessible to predators, without clear associations to life-history strategy. Our results provide a rare link between the evolution of sexual ornaments, effects on locomotion performance, and natural selection on ornament size potentially through habitat differences in predation risk., (© 2021 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)
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- 2022
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24. A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.
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Zaayter L, Mori M, Ahmad T, Ashraf W, Boudier C, Kilin V, Gavvala K, Richert L, Eiler S, Ruff M, Botta M, Bronner C, Mousli M, and Mély Y
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- 5-Methylcytosine chemistry, Binding Sites, DNA (Cytosine-5-)-Methyltransferase 1 chemistry, Drug Evaluation, Preclinical methods, HeLa Cells, Humans, Kinetics, Methylation, Molecular Docking Simulation, Molecular Structure, Protein Binding, Protein Conformation, Structure-Activity Relationship, Transfection methods, Ubiquitin-Protein Ligases, Anthraquinones chemistry, CCAAT-Enhancer-Binding Proteins antagonists & inhibitors, Enzyme Inhibitors chemistry
- Abstract
During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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25. An atlas of larval organogenesis in the European shore crab Carcinus maenas L. (Decapoda, Brachyura, Portunidae).
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Spitzner F, Meth R, Krüger C, Nischik E, Eiler S, Sombke A, Torres G, and Harzsch S
- Abstract
Background: The life history stages of brachyuran crustaceans include pelagic larvae of the Zoea type which grow by a series of moults from one instar to the next. Zoeae actively feed and possess a wide range of organ systems necessary for autonomously developing in the plankton. They also display a rich behavioural repertoire that allows for responses to variations in environmental key factors such as light, hydrostatic pressure, tidal currents, and temperature. Brachyuran larvae have served as distinguished models in the field of Ecological Developmental Biology fostering our understanding of diverse ecophysiological aspects such as phenotypic plasticity, carry-over effects on life-history traits, and adaptive mechanisms that enhance tolerance to fluctuations in environmental abiotic factors. In order to link such studies to the level of tissues and organs, this report analyses the internal anatomy of laboratory-reared larvae of the European shore crab Carcinus maenas . This species has a native distribution extending across most European waters and has attracted attention because it has invaded five temperate geographic regions outside of its native range and therefore can serve as a model to analyse thermal tolerance of species affected by rising sea temperatures as an effect of climate change., Results: Here, we used X-ray micro-computed tomography combined with 3D reconstruction to describe organogenesis in brachyuran larvae. We provide a detailed atlas of the larval internal organization to complement existing descriptions of its external morphology. In a multimethodological approach, we also used cuticular autofluorescence and classical histology to analyse the anatomy of selected organ systems., Conclusions: Much of our fascination for the anatomy of brachyuran larvae stems from the opportunity to observe a complex organism on a single microscopic slide and the realization that the entire decapod crustacean bauplan unfolds from organ anlagen compressed into a miniature organism in the sub-millimetre range. The combination of imaging techniques used in the present study provides novel insights into the bewildering diversity of organ systems that brachyuran larvae possess. Our analysis may serve as a basis for future studies bridging the fields of evolutionary developmental biology and ecological developmental biology., Competing Interests: The research presented in this paper complies with the guidelines from the directives 2010/63/EU of the European parliament and of the Council of 22nd September 2010 on the protection of animals used for scientific purposes.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Published
- 2018
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26. Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.
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Bonnard D, Le Rouzic E, Eiler S, Amadori C, Orlov I, Bruneau JM, Brias J, Barbion J, Chevreuil F, Spehner D, Chasset S, Ledoussal B, Moreau F, Saïb A, Klaholz BP, Emiliani S, Ruff M, Zamborlini A, and Benarous R
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- Allosteric Regulation, Binding Sites, Cell Line, HIV Integrase Inhibitors chemistry, Humans, Molecular Structure, Pyridines chemistry, Pyridines pharmacology, Structure-Activity Relationship, Thiophenes chemistry, Thiophenes pharmacology, HIV Integrase drug effects, HIV Integrase Inhibitors pharmacology, HIV-1 physiology, Virus Assembly drug effects, Virus Integration drug effects
- Abstract
Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2018
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27. Unstable Protein Purification Through the Formation of Stable Complexes.
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Eiler S, Levy N, Maillot B, Batisse J, Aubreton KP, Oladosu O, and Ruff M
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- Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing metabolism, HIV Integrase chemistry, HIV Integrase metabolism, Humans, Nuclear Receptor Coactivator 2 chemistry, Nuclear Receptor Coactivator 2 metabolism, Protein Binding, Receptors, Cytoplasmic and Nuclear chemistry, Receptors, Cytoplasmic and Nuclear metabolism, Receptors, Glucocorticoid chemistry, Receptors, Glucocorticoid metabolism, Transcription Factors chemistry, Transcription Factors metabolism, Adaptor Proteins, Signal Transducing isolation & purification, Chromatography, Affinity methods, HIV Integrase isolation & purification, Nuclear Receptor Coactivator 2 isolation & purification, Protein Interaction Domains and Motifs, Receptors, Cytoplasmic and Nuclear isolation & purification, Receptors, Glucocorticoid isolation & purification, Transcription Factors isolation & purification
- Abstract
Purification of proteins containing disordered regions and participating in transient complexes is often challenging because of the small amounts available after purification, their heterogeneity, instability, and/or poor solubility. To circumvent these difficulties, we set up a methodology that enables the production of stable complexes in large amounts for structural and functional studies. In this chapter, we describe the methodology used to establish the best cell culture conditions and buffer compositions to optimize soluble protein production and their stabilization through protein complex formation. Two examples of challenging protein families are described, namely, the human steroid nuclear receptors and the HIV-1 pre-integration complexes.
- Published
- 2018
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28. Dynamics of Methylated Cytosine Flipping by UHRF1.
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Kilin V, Gavvala K, Barthes NP, Michel BY, Shin D, Boudier C, Mauffret O, Yashchuk V, Mousli M, Ruff M, Granger F, Eiler S, Bronner C, Tor Y, Burger A, and Mély Y
- Subjects
- CCAAT-Enhancer-Binding Proteins chemistry, Cytosine chemistry, DNA chemistry, DNA Methylation, DNA Replication, Fluorescence, Humans, Kinetics, Molecular Structure, Ubiquitin-Protein Ligases, CCAAT-Enhancer-Binding Proteins metabolism, Cytosine metabolism, DNA metabolism, Thermodynamics
- Abstract
DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (
th G), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.- Published
- 2017
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29. Production of unstable proteins through the formation of stable core complexes.
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Levy N, Eiler S, Pradeau-Aubreton K, Maillot B, Stricher F, and Ruff M
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- Cell Line, HIV-1 metabolism, Humans, Multiprotein Complexes isolation & purification, Protein Stability, Receptors, Cytoplasmic and Nuclear metabolism, Solubility, Solvents, Multiprotein Complexes metabolism
- Abstract
Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes.
- Published
- 2016
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30. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.
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Le Rouzic E, Bonnard D, Chasset S, Bruneau JM, Chevreuil F, Le Strat F, Nguyen J, Beauvoir R, Amadori C, Brias J, Vomscheid S, Eiler S, Lévy N, Delelis O, Deprez E, Saïb A, Zamborlini A, Emiliani S, Ruff M, Ledoussal B, Moreau F, and Benarous R
- Subjects
- Cell Line, Crystallography, X-Ray, HIV Integrase chemistry, HIV Integrase Inhibitors chemistry, Humans, Intercellular Signaling Peptides and Proteins chemistry, Protein Binding, Protein Conformation, HIV Integrase metabolism, HIV Integrase Inhibitors pharmacology, HIV-1 drug effects, HIV-1 physiology, Intercellular Signaling Peptides and Proteins metabolism, Virus Integration drug effects, Virus Replication drug effects
- Abstract
Background: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction., Results: We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket., Conclusion: Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.
- Published
- 2013
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31. Structural and functional role of INI1 and LEDGF in the HIV-1 preintegration complex.
- Author
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Maillot B, Lévy N, Eiler S, Crucifix C, Granger F, Richert L, Didier P, Godet J, Pradeau-Aubreton K, Emiliani S, Nazabal A, Lesbats P, Parissi V, Mely Y, Moras D, Schultz P, and Ruff M
- Subjects
- Cryoelectron Microscopy, Fluorescence Polarization, HIV-1 enzymology, Humans, Mass Spectrometry, Protein Conformation, SMARCB1 Protein, Spectrometry, Fluorescence, Adaptor Proteins, Signal Transducing metabolism, Chromosomal Proteins, Non-Histone metabolism, DNA-Binding Proteins metabolism, HIV Integrase metabolism, HIV-1 physiology, Models, Molecular, Multiprotein Complexes metabolism, Transcription Factors metabolism, Virus Integration physiology
- Abstract
Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3' processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.
- Published
- 2013
- Full Text
- View/download PDF
32. Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors.
- Author
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Osz J, Brélivet Y, Peluso-Iltis C, Cura V, Eiler S, Ruff M, Bourguet W, Rochel N, and Moras D
- Subjects
- Allosteric Site, Biophysics methods, Crystallography, X-Ray methods, Dimerization, Humans, Kinetics, Ligands, Models, Biological, Models, Molecular, Molecular Conformation, Nuclear Receptor Coactivator 1 chemistry, Peptides chemistry, Protein Binding, Receptors, Calcitriol chemistry, Receptors, Retinoic Acid chemistry, Cell Nucleus metabolism
- Abstract
Transcription regulation by steroid hormones, vitamin derivatives, and metabolites is mediated by nuclear receptors (NRs), which play an important role in ligand-dependent gene expression and human health. NRs function as homodimers or heterodimers and are involved in a combinatorial, coordinated and sequentially orchestrated exchange between coregulators (corepressors, coactivators). The architecture of DNA-bound functional dimers positions the coregulators proteins. We previously demonstrated that retinoic acid (RAR-RXR) and vitamin D3 receptors (VDR-RXR) heterodimers recruit only one coactivator molecule asymmetrically without steric hindrance for the binding of a second cofactor. We now address the problem of homodimers for which the presence of two identical targets enhances the functional importance of the mode of binding. Using structural and biophysical methods and RAR as a model, we could dissect the molecular mechanism of coactivator recruitment to homodimers. Our study reveals an allosteric mechanism whereby binding of a coactivator promotes formation of nonsymmetrical RAR homodimers with a 21 stoichiometry. Ligand conformation and the cofactor binding site of the unbound receptor are affected through the dimer interface. A similar control mechanism is observed with estrogen receptor (ER) thus validating the negative cooperativity model for an established functional homodimer. Correlation with published data on other NRs confirms the general character of this regulatory pathway.
- Published
- 2012
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33. Towards high-throughput identification of endocrine disrupting compounds with mass spectrometry.
- Author
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Bovet C, Plet B, Ruff M, Eiler S, Granger F, Panagiotidis A, Wenzel R, Nazabal A, Moras D, and Zenobi R
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- Binding Sites, Dimerization, Estrogen Receptor alpha metabolism, Humans, Ligands, Endocrine Disruptors pharmacology, Estrogen Receptor alpha chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
High-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking has the ability to monitor the ligand-dependent dimerization of the human estrogen receptor alpha ligand binding domain (hERalpha LBD) in solution. Because only ER ligands enhance the homodimer abundance, we evaluated the ability of this label-free approach for identifying endocrine disrupting compounds (EDCs) in a high-throughput manner. This was achieved by combining an automated liquid handler with an automated MS acquisition procedure, which allowed a five-fold gain in operator time compared to a fully manual approach. To detect ligand binding with enough confidence, the receptor has to be incubated with at least a 10 microM concentration of the test compound. Based on the increase of the measured homodimer intensity, eight compounds with a relative binding affinity (RBA, relative to the natural hormone estradiol) >7% were identified as ER ligands among the 28 chemicals tested. Two other compounds, quercetin and 4-tert-amylphenol, were also identified as ER ligands, although their RBAs have been reported to be only 0.01% and 0.000055%, respectively. This suggests that these two ligands have a higher affinity for hERalpha LBD than reported in the literature. The high-mass MALDI approach thus allows identifying high affinity EDCs in an efficient way.
- Published
- 2009
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34. Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor.
- Author
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Michel F, Crucifix C, Granger F, Eiler S, Mouscadet JF, Korolev S, Agapkina J, Ziganshin R, Gottikh M, Nazabal A, Emiliani S, Benarous R, Moras D, Schultz P, and Ruff M
- Subjects
- Cryoelectron Microscopy, DNA, Viral genetics, DNA, Viral metabolism, HIV Integrase chemistry, HIV Integrase metabolism, Humans, Mass Spectrometry, Models, Molecular, Protein Conformation, Virus Replication, DNA, Viral chemistry, Genome, Human, Intercellular Signaling Peptides and Proteins chemistry, Intercellular Signaling Peptides and Proteins metabolism, Virus Integration
- Abstract
Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3' processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.
- Published
- 2009
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35. Monitoring ligand modulation of protein-protein interactions by mass spectrometry: estrogen receptor alpha-SRC1.
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Bovet C, Ruff M, Eiler S, Granger F, Wenzel R, Nazabal A, Moras D, and Zenobi R
- Subjects
- Amino Acid Sequence, Dimerization, Endocrine Disruptors chemistry, Estrogen Receptor alpha agonists, Estrogen Receptor alpha antagonists & inhibitors, Estrogen Receptor alpha chemistry, Humans, Ligands, Mass Spectrometry, Peptides chemistry, Peptides metabolism, Pharmacology, Protein Binding drug effects, Protein Structure, Quaternary, Protein Structure, Tertiary, Endocrine Disruptors metabolism, Endocrine Disruptors pharmacology, Estrogen Receptor alpha metabolism
- Abstract
Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.
- Published
- 2008
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36. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERalpha in complex with synthetic ligands.
- Author
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Cura V, Gangloff M, Eiler S, Moras D, and Ruff M
- Subjects
- Binding Sites, Crystallization, Crystallography, X-Ray, Estrogen Receptor alpha genetics, Estrogen Receptor alpha metabolism, Humans, Ligands, Polymorphism, Single Nucleotide, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Solubility, Thioredoxins genetics, Thioredoxins metabolism, Estrogen Receptor alpha chemistry, Estrogen Receptor alpha isolation & purification, Thioredoxins chemistry
- Abstract
The ligand-binding domain (LBD) of human oestrogen receptor alpha was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD-ligand-coactivator peptide complexes at 2.3 A resolution. This technique is likely to be applicable to other low-solubility proteins.
- Published
- 2008
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37. Estrogen receptor-ligand complexes measured by chip-based nanoelectrospray mass spectrometry: an approach for the screening of endocrine disruptors.
- Author
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Bovet C, Wortmann A, Eiler S, Granger F, Ruff M, Gerrits B, Moras D, and Zenobi R
- Subjects
- Endocrine Disruptors analysis, Endocrine Disruptors metabolism, Estrogen Receptor alpha metabolism, Humans, Nanotechnology methods, Protein Binding, Endocrine Disruptors chemistry, Estrogen Receptor alpha chemistry, Ligands, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.
- Published
- 2007
- Full Text
- View/download PDF
38. Overexpression, purification, and crystal structure of native ER alpha LBD.
- Author
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Eiler S, Gangloff M, Duclaud S, Moras D, and Ruff M
- Subjects
- Cloning, Molecular, Computer Simulation, Crystallization, Crystallography, X-Ray, Dimerization, Estrogen Receptor alpha, Humans, Ligands, Models, Molecular, Peptide Fragments genetics, Peptide Fragments metabolism, Protein Folding, Protein Structure, Secondary genetics, Protein Structure, Tertiary genetics, Receptors, Estrogen genetics, Receptors, Estrogen metabolism, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Receptors, Estrogen chemistry, Receptors, Estrogen isolation & purification
- Abstract
Several crystal structures of human estrogen receptor alpha ligand-binding domain (hERalpha LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturation problems. In this work, high-level protein expression and purification together with crystallization screening procedure yielded high amounts of soluble protein without renaturation or modifications steps. The native protein crystallizes in the space group P3(2) 21 with three molecules in the asymmetric unit. The overall structure is very similar to that previously reported for the hERalpha LBD with cysteine carboxymethylated residues thus validating the modification approach. The present strategy can be adapted to other cases where the solubility and the proper folding is a difficulty., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
39. Crystal structure of a mutant hERalpha ligand-binding domain reveals key structural features for the mechanism of partial agonism.
- Author
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Gangloff M, Ruff M, Eiler S, Duclaud S, Wurtz JM, and Moras D
- Subjects
- Cloning, Molecular, Crystallography, X-Ray, Estrogen Antagonists pharmacology, Estrogen Receptor alpha, Humans, Hydrogen Bonding, Ligands, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, Receptors, Estrogen agonists, Receptors, Estrogen genetics, Receptors, Estrogen chemistry, Receptors, Estrogen metabolism
- Abstract
The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.
- Published
- 2001
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40. An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase.
- Author
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Briand C, Poterszman A, Eiler S, Webster G, Thierry J, and Moras D
- Subjects
- Anticodon chemistry, Anticodon genetics, Anticodon metabolism, Aspartate-tRNA Ligase genetics, Base Sequence, Binding Sites, Catalytic Domain, Crystallography, X-Ray, Escherichia coli genetics, Hydrogen Bonding, Kinetics, Models, Molecular, Molecular Sequence Data, Protein Conformation, RNA, Bacterial chemistry, RNA, Bacterial genetics, RNA, Transfer, Asp chemistry, RNA, Transfer, Asp genetics, Structure-Activity Relationship, Temperature, Aspartate-tRNA Ligase chemistry, Aspartate-tRNA Ligase metabolism, RNA, Bacterial metabolism, RNA, Transfer, Asp metabolism, Thermus thermophilus enzymology, Thermus thermophilus genetics
- Abstract
The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed., (Copyright 2000 Academic Press.)
- Published
- 2000
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41. Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation.
- Author
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Martin F, Eriani G, Eiler S, Moras D, Dirheimer G, and Gangloff J
- Subjects
- Anticodon, Aspartate-tRNA Ligase metabolism, Base Composition, Base Sequence, Cloning, Molecular, Crystallography, X-Ray, Guanine chemistry, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Transfer, Asp chemistry, Saccharomyces cerevisiae metabolism, Structure-Activity Relationship, Escherichia coli metabolism, Guanine analogs & derivatives, RNA, Transfer, Asp metabolism, Transfer RNA Aminoacylation
- Abstract
Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.
- Published
- 1993
- Full Text
- View/download PDF
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