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1. Glycan Determinants of Heparin-Tau Interaction

Author

Isabelle Huvent, Peter M. Tessier, Guy Lippens, Robert J. Linhardt, Fuming Zhang, David Eliezer, Jing Zhao, Anqiang Zhang, Quanhong Li, Chunyu Wang, College of Food Science and Nutritional Engineering, China Agricultural University (CAU), Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute (RPI), Université de Lille, Sciences et Technologies, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry, Program in Structural Biology, Cornell University [New York], Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Department of Biology and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, The National Institutes of Health (AG019391 and AG025440 to D.E., and R01GM104130 to P.M.T.), China Agricultural University, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Cornell University, and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, 0301 basic medicine, Glycan, Protein Conformation, Biophysics, tau Proteins, macromolecular substances, Plasma protein binding, nmr spectrometry, héparine, Sodium Chloride, Dermatan sulfate, heparinic acid, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, medicine, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Binding site, Surface plasmon resonance, Dose-Response Relationship, Drug, 030102 biochemistry & molecular biology, biology, spectrométrie rmn, Heparin, Proteins, résonance plasmonique de surface, Nuclear magnetic resonance spectroscopy, Surface Plasmon Resonance, Peptide Fragments, 3. Good health, maladie d'alzheimer, 030104 developmental biology, chemistry, Biochemistry, biology.protein, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Protein Binding, medicine.drug
Abstract

Tau aggregates into paired helical filaments within neurons, a pathological hallmark of Alzheimer's disease. Heparin promotes tau aggregation and recently has been shown to be involved in the cellular uptake of tau aggregates. Although the tau-heparin interaction has been extensively studied, little is known about the glycan determinants of this interaction. Here, we used surface plasmon resonance (SPR) and NMR spectroscopy to characterize the interaction between two tau fragments, K18 and K19, and several polysaccharides, including heparin, heparin oligosaccharides, chemically modified heparin, and related glycans. Using a heparin-immobilized chip, SPR revealed that tau K18 and K19 bind heparin with a K-D of 0.2 and 70 mu M, respectively. In SPR competition experiments, N-desulfation and 2-O-desulfation had no effect on heparin binding to K18, whereas 6-O-desulfation severely reduced binding, suggesting a critical role for 6-O-sulfation in the tau-heparin interaction. The tau-heparin interaction became stronger with longer-chain heparin oligosaccharides. As expected for an electrostatics-driven interaction, a moderate amount of salt (0.3 M NaCl) abolished binding. NMR showed the largest chemical-shift perturbation (CSP) in R2 in tau K18, which was absent in K19, revealing differential binding sites in K18 and K19 to heparin. Dermatan sulfate binding produced minimal CSP, whereas dermatan disulfate, with the additional 6-O-sulfo group, induced much larger CSP. 2-O-desulfated heparin induced much larger CSP in K18 than 6-O-desulfated heparin. Our data demonstrate a crucial role for the 6-O-sulfo group in the tau-heparin interaction, which to our knowledge has not been reported before.

Published
2017

2. Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication

Author

François-Xavier Cantrelle, Xavier Hanoulle, Ralf Bartenschlager, Marie Dujardin, Guy Lippens, Vanesa Madan, Isabelle Huvent, Hélène Launay, Neha S. Gandhi, Université de Lille, Universität Heidelberg [Heidelberg] = Heidelberg University, School of Mathematical Sciences, Queensland University of Technology, School of Mathematical Sciences [Brisbane], Queensland University of Technology [Brisbane] (QUT)-Queensland University of Technology [Brisbane] (QUT), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Department of Molecular Virology (DMV), Toulouse Biotechnology Institute (TBI), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), ANRS French National Research Agency (ANR) A02014-2, Lille University, German Research Foundation (DFG) 112927078-TRR 83, Research Federation 'structural and functional biochemistry of BIOmolecular Assemblies' (CNRS) FR-3688, ANR-11-JSV8-0005,StruFunc5A5B,Etude des relations structurales et fonctionnelles entre les protéines NS5A et NS5B du virus de l'hépatite C et la Cyclophiline A humaine.(2011), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Universität Heidelberg [Heidelberg], Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Synthèse, structure et fonction des biomolécules (SSFB), Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), CNRS, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576, Université de Lille-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, nuclear magnetic resonance (NMR), [SDV]Life Sciences [q-bio], Allosteric regulation, prolyl isomerase, Cypa, nonstructural protein 5A (NS5A), Molecular Dynamics Simulation, Viral Nonstructural Proteins, Virus Replication, Intrinsically disordered proteins, Biochemistry, 03 medical and health sciences, Cyclophilin A, conformer ensemble, Prolyl isomerase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Structural motif, NS5A, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Cyclophilin, intrinsically disordered protein, hepatitis C virus (HCV), cyclophilin, allosteric regulation, RNA replication, 030102 biochemistry & molecular biology, biology, Chemistry, 030402 Biomolecular Modelling and Design, Cell Biology, biology.organism_classification, 3. Good health, Cell biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 030104 developmental biology, 030406 Proteins and Peptides, Protein Structure and Folding, RNA, Viral, 060112 Structural Biology (incl. Macromolecular Modelling)
Abstract

International audience; Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence , whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosteri-cally modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.

Published
2019

3. The Neuronal tau protein blocks in vitro fibrillation of the amyloid-beta (A beta) peptide at the oligomeric stage

Author

Guy Lippens, Cecilia Wallin, Astrid Gräslund, Yoshitaka Hiruma, Jüri Jarvet, Sebastian K.T.S. Wärmländer, Isabelle Huvent, Jan Pieter Abrahams, Jinghui Luo, Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, Divisions of Biochemistry, Netherlands Cancer Institute, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Biozentrum [Basel, Suisse], University of Basel (Unibas), Laboratory of Biomolecular Research, Institut Paul Scherrer (IPS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Region Midi-Pyrenees, ERDF, SICOVAL, Alzheimer Foundation [AF-642731], Swedish Research Council [2014-05867], department of Biology and Chemistry, Paul Scherrer Institute, UMR8576, Unité de glycobiologie structurale et fonctionnelle, Université de Lille, Biozentrum, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), and Université de Lille-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Circular dichroism, Surface Properties, alpha-synuclein, mouse model, [SDV]Life Sciences [q-bio], Tau protein, Peptide, tau Proteins, Microscopy, Atomic Force, Biochemistry, Catalysis, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Colloid and Surface Chemistry, mental disorders, medicine, Humans, chaperone, neurodegenerative diseases, atomic-resolution, Particle Size, alzheimers-disease, Nuclear Magnetic Resonance, Biomolecular, chemistry.chemical_classification, Fibrillation, Alpha-synuclein, Amyloid beta-Peptides, biology, Circular Dichroism, aggregation, transmission, General Chemistry, In vitro, Random coil, 030104 developmental biology, catalytic cycle, chemistry, Chaperone (protein), biology.protein, Biophysics, pathology, medicine.symptom, 030217 neurology & neurosurgery
Abstract

In Alzheimer's disease, amyloid-beta (A beta) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of A beta and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the A beta(40) peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the A beta(40) peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated A beta(40) peptides. The A beta monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on A beta fibrillation.

Published
2018

4. F2-06-01: MAJOR DIFFERENCES BETWEEN THE SELF-ASSEMBLY, SEEDING BEHAVIOR, AND INTERACTION WITH MODULATORS OF HEPARIN-INDUCED VERSUS IN-VITRO PHOSPHORYLATED TAU

Author

Chunyu Wang, Fuming Zhang, Guy Lippens, Isabelle Huvent, Thomas Schrader, Shiguo Chen, Zizheng Li, Jing Zhao, François-Xavier Cantrelle, Isabelle Landrieu, Robert J. Linhardt, Jianle Chen, Gal Bitan, Jing Di, Caroline Smet-Nocca, Béatrice Chambraud, Frank-Gerrit Klärner, and Clément Despres

Subjects
Epidemiology, Chemistry, Health Policy, Heparin, In vitro, Psychiatry and Mental health, Cellular and Molecular Neuroscience, Developmental Neuroscience, Tau phosphorylation, medicine, Biophysics, Seeding, Neurology (clinical), Geriatrics and Gerontology, medicine.drug
Published
2019

5. Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

Author

Joelle Rosseels, Raphaëlle Caillierez, Joris Winderickx, Sebastiaan Engelborghs, Guy Lippens, Marina Caldara, Eugeen Vanmechelen, Luc Buée, Valérie Buée-Scherrer, Marie-Luce Violet, Morvane Colin, Anthony Papegaey, Erwin Swinnen, Pierre Grognet, Juan José Marín López, Dirk Jacobs, Isabelle Huvent, Marie-Christine Galas, and Jeff Van den Brande

Subjects
biology, medicine.drug_class, Chemistry, Cell Biology, medicine.disease, Monoclonal antibody, Biochemistry, Molecular biology, Epitope, 3. Good health, Epitope mapping, mental disorders, medicine, biology.protein, Phosphorylation, Senile plaques, Alzheimer's disease, Binding site, Antibody, Molecular Biology
Abstract

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr18. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.

Published
2015

6. Identification of the Tau phosphorylation pattern that drives its aggregation

Author

François-Xavier Cantrelle, Haoling Qi, Yves Jacquot, Guy Lippens, Isabelle Huvent, Isabelle Landrieu, Cillian Byrne, Clément Despres, Béatrice Chambraud, Etienne-Emile Baulieu, Caroline Smet-Nocca, CNRS, Université de Lille, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF], Laboratoire des biomolécules [LBM UMR 7203], Université Pierre et Marie Curie - Paris 6 [UPMC], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Petites Molécules de neuroprotection, neurorégénération et remyélinisation, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés [LISBP], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC), Neuroprotection et neurogénération (UMR788), Neuroprotection et Neurodégénération (UMR788), TGE RMN THC (Tres Grands Equipements Resonance Magnetique Nucleaire Tres Hauts Champs, France) [FR-3050], FRABio (Federation de Recherche 'Biochimie Structurale & Fonctionnelle des Assemblages Biomoleculaires', France [FR 3688], MetaToul [MetaboHUB-AR-11-INBS-0010], Université de Lille-Centre National de la Recherche Scientifique (CNRS), Landrieu, Isabelle, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), and Lippens, Guy

Subjects
0301 basic medicine, Models, Molecular, Threonine, inorganic chemicals, Phosphorylation sites, Magnetic Resonance Spectroscopy, Tau, phosphorylation, aggregation, Alzheimer's disease, NMR, tau Proteins, Biotechnologies, macromolecular substances, Protein Aggregation, Pathological, environment and public health, Rats, Sprague-Dawley, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Microscopy, Electron, Transmission, Protein Domains, Alzheimer’s disease, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Serine, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, Binding Sites, Kinase, Nuclear magnetic resonance spectroscopy, Biological Sciences, Fluorescence, In vitro, Peptide Fragments, 3. Good health, enzymes and coenzymes (carbohydrates), 030104 developmental biology, chemistry, Biochemistry, Tau phosphorylation, Phosphorylation, bacteria, Thioflavin, 030217 neurology & neurosurgery
Abstract

International audience; Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.

Published
2017

7. A functional fragment of Tau forms fibers without the need for an intermolecular cysteine bridge

Author

Amina Kamah, Isabelle Landrieu, Isabelle Huvent, Guy Lippens, Christian Slomianny, François-Xavier Cantrelle, Nicolas Barois, Caroline Smet-Nocca, Université Lille Nord de France (COMUE), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Plateforme BioImaging Center Lille (BICeL), Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie Cellulaire : Canaux ioniques, inflammation et cancer - U 1003 (PHYCELL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université de Lille, Plate-forme BICeL (IFR142), Physiologie Cellulaire (PHYCEL) - U1003 (PHYCEL), Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Lille Nord de France (COMUE), Université de Lille-Centre National de la Recherche Scientifique (CNRS), and Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS)

Subjects
Magnetic Resonance Spectroscopy, Biophysics, tau Proteins, macromolecular substances, Fibril, Biochemistry, law.invention, Aggregation, 03 medical and health sciences, chemistry.chemical_compound, NMR spectroscopy, 0302 clinical medicine, law, Microtubule, mental disorders, Electron microscopy, Humans, Cysteine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding site, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Heparin, Cell Biology, Nuclear magnetic resonance spectroscopy, Fluorescence, Recombinant Proteins, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Crystallography, nervous system, chemistry, Nucleation, Thioflavin, Tau, Electron microscope, 030217 neurology & neurosurgery
Abstract

International audience; We study the aggregation of a fragment of the neuronal protein Tau that contains part of the proline rich domain and of the microtubule binding repeats. When incubated at 37 °C with heparin, the fragment readily forms fibers as witnessed by Thioflavin T fluorescence. Electron microscopy and NMR spectroscopy show bundled ribbon like structures with most residues rigidly incorporated in the fibril. Without its cysteines, this fragment still forms fibers of a similar morphology, but with lesser Thioflavin T binding sites and more mobility for the C-terminal residues.

Published
2014

8. The Neuronal Tau Protein Blocks In Vitro Fibrillation of the Amyloid-β (Aβ) Peptide

Author

Jüri Jarvet, Sebastian K.T.S. Wärmländer, Guy Lippens, Jinghui Luo, Cecilia Wallin, Jan Pieter Abrahams, Isabelle Huvent, Astrid Gräslund, and Yoshitaka Hiruma

Subjects
Fibrillation, biology, Amyloid β, Chemistry, Aβ peptide, Tau protein, Biophysics, biology.protein, medicine, medicine.symptom, In vitro, Cell biology
Published
2019

9. NMR Meets Tau: Insights into Its Function and Pathology

Author

Clément Despres, Isabelle Landrieu, Haoling Qi, Isabelle Huvent, Guy Lippens, Neha S. Gandhi, Benoît Gigant, Caroline Smet, Juan José Marín López, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), School of Mathematical Sciences, Queensland University of Technology, School of Mathematical Sciences [Brisbane], Queensland University of Technology [Brisbane] (QUT)-Queensland University of Technology [Brisbane] (QUT), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 ( UGSF ), Institut National de la Recherche Agronomique ( INRA ) -Université de Lille-Centre National de la Recherche Scientifique ( CNRS ), School of Mathematical Sciences, Queensland University of Technology [Brisbane] ( QUT ), Biochimie Structurale des Microtubules, des Kinésines et de leurs Cargos ( MIKICA ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Sciences Department-Chemistry, Pontificia Universidad Católica del Perú ( PUCP ), Queensland University of Technology [Brisbane] (QUT), Biochimie Structurale des Microtubules, des Kinésines et de leurs Cargos (MIKICA), Département Biochimie, Biophysique et Biologie Structurale (B3S), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Pontificia Universidad Católica del Perú (PUCP), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Queensland University of Technology [Brisbane] ( QUT ) -Queensland University of Technology [Brisbane] ( QUT ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), CNRS, Université de Lille, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés [LISBP], Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Queensland University of Technology [Brisbane] [QUT], Institut de Biologie Intégrative de la Cellule [I2BC], Pontificia Universidad Católica del Perú = Pontifical Catholic University of Peru [PUCP], and Lippens, Guy

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Tau protein, lcsh:QR1-502, tau Proteins, Review, Intrinsically disordered proteins, Biochemistry, protein interactions, lcsh:Microbiology, Protein–protein interaction, 03 medical and health sciences, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 0302 clinical medicine, NMR spectroscopy, mental disorders, Tau, intrinsically disordered protein, tubulin, aggregation, phosphorylation, protein/protein interactions, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, biology, [ SDV ] Life Sciences [q-bio], Chemistry, Kinase, protein, Nuclear magnetic resonance spectroscopy, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Intrinsically Disordered Proteins, 030104 developmental biology, Tubulin, biology.protein, Biophysics, Phosphorylation, Protein Processing, Post-Translational, 030217 neurology & neurosurgery, Function (biology)
Abstract

International audience; In this review, we focus on what we have learned from Nuclear Magnetic Resonance (NMR) studies on the neuronal microtubule-associated protein Tau. We consider both the mechanistic details of Tau: the tubulin relationship and its aggregation process. Phosphorylation of Tau is intimately linked to both aspects. NMR spectroscopy has depicted accurate phosphorylation patterns by different kinases, and its non-destructive character has allowed functional assays with the same samples. Finally, we will discuss other post-translational modifications of Tau and its interaction with other cellular factors in relationship to its (dys)function.

Published
2016

10. Isomerisation and oligomerization of truncated and mutated tau forms by FKBP52 are independent processes

Author

Julien Giustiniani, Yves Jacquot, Isabelle Landrieu, Béatrice Chambraud, Guy Lippens, François-Xavier Cantrelle, Caroline Smet, Etienne-Emile Baulieu, Isabelle Huvent, Kevin Guillemeau, Amina Kamah, Cillian Byrne, Université Lille Nord de France (COMUE), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé-Université de Lille, Sciences et Technologies, Neuroprotection et neurogénération (UMR788), Neuroprotection et Neurodégénération (UMR788), Peptides, glycoconjugués et métaux en biologie (LBM-E1), Laboratoire des biomolécules (LBM UMR 7203), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC), ANR (program MALZ-TAF), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Lille-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Isomerase activity, Tau protein, Mutant, tau Proteins, Peptide, Protein Aggregation, Pathological, Tacrolimus Binding Proteins, 03 medical and health sciences, 0302 clinical medicine, Isomerism, Structural Biology, mental disorders, Humans, Molecular Biology, FK506 binding, chemistry.chemical_classification, biology, Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, aggregation, Alzheimer's disease, FKBP52, prolyl cis/trans isomerase activity, 030104 developmental biology, FKBP, Biochemistry, Biophysics, biology.protein, Protein Multimerization, Tau, 030217 neurology & neurosurgery, Heteronuclear single quantum coherence spectroscopy
Abstract

International audience; The aggregation of the neuronal Tau protein is one molecular hallmark of Alzheimer's disease and other related tauopathies, but the precise molecular mechanisms of the aggregation process remain unclear. The FK506 binding protein FKBP52 is able to induce oligomers in the pathogenic Tau P301L mutant and in a truncated form of the wild-type human Tau protein. Here, we investigate whether FKBP52's capacity to induce Tau oligomers depends on its prolyl cis/trans isomerase activity. We find that FKBP52 indeed can isomerize selected prolyl bonds in the different Tau proteins, and that this activity is carried solely by its first FK506 binding domain. Its capacity to oligomerize Tau is however not linked to this PPIase activity. In addition, we identified a novel molecular interaction implying the PHF6 peptide of Tau and the FK1/FK2 domains of FKBP52 independent of FK506 binding; these data point towards a non-catalytic molecular interaction that might govern the effect of FKBP52 on Tau.

Published
2016

11. Proline Conformation in a Functional Tau Fragment

Author

Puneet Ahuja, Guy Lippens, Caroline Smet, Jean-Michel Wieruszeski, Juan José Marín López, Xavier Hanoulle, Isabelle Landrieu, François-Xavier Cantrelle, Isabelle Huvent, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université de Lille, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, Proline, Stereochemistry, Protein Conformation, Tau protein, tau Proteins, Isomerase, 03 medical and health sciences, Protein structure, Structural Biology, Humans, Phosphorylation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Conformational isomerism, ComputingMilieux_MISCELLANEOUS, biology, Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Folding (chemistry), 030104 developmental biology, biology.protein, Two-dimensional nuclear magnetic resonance spectroscopy, Protein Processing, Post-Translational, Heteronuclear single quantum coherence spectroscopy
Abstract

The conformational state of distinct prolines can determine the folding of a protein but equally other biological processes when coupled to a conformation-sensitive secondary reaction. For the neuronal tau protein, the importance of proline conformation is underscored by its interaction with different prolyl cis/trans isomerases. The proline conformation would gain even further importance after phosphorylation of the preceding residue by various proline-directed kinases. A number of molecular diseases including Alzheimer's disease and traumatic brain injury were thereby recently qualified as "cistauosis", as they would imply a cis conformation for the pThr231-Pro232 prolyl bond. We here investigate by NMR spectroscopy the conformation of all prolines in a functional Tau fragment, Tau[208-324]. Although we can detect and identify some minor conformers in the cis form, we show that all prolines are for over 90% in the trans conformation. Phosphorylation by CDK2/CycA3, which notably leads to complete modification of the Thr231 residue, does not change this conclusion. Our data hence disagree with the notion that specific prolyl bonds in tau would adopt preferentially the cis conformation.

Published
2016

12. Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Author

Haoling Qi, Robert Schneider, Xavier Hanoulle, Hamida Merzougui, Isabelle Landrieu, François-Xavier Cantrelle, Clément Despres, Clément Danis, Caroline Smet-Nocca, Luiza M. Bessa, Idir Malki, Guy Lippens, Isabelle Huvent, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer (JPArc - U837 Inserm), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université Lille 2 - Faculté de Médecine, CNRS, Université de Lille, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U837 [JPArc], Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U1172 Inserm - U837 (JPArc), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Lille Nord de France (COMUE)-Université de Lille, Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U837 (JPArc), and Université Lille Nord de France (COMUE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, General Chemical Engineering, Tau protein, tau Proteins, Intrinsically disordered proteins, Biochemistry, General Biochemistry, Genetics and Molecular Biology, Isotopic labeling, 03 medical and health sciences, Alzheimer Disease, Protein purification, mental disorders, Triple-resonance nuclear magnetic resonance spectroscopy, Humans, Protein phosphorylation, Phosphorylation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, biology, General Immunology and Microbiology, Chemistry, General Neuroscience, Nuclear magnetic resonance spectroscopy, Recombinant Proteins, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Intrinsically Disordered Proteins, 030104 developmental biology, biology.protein, Issue 118, nuclear magnetic resonance spectroscopy, isotopic labeling, intrinsically disordered protein, recombinantprotein, protein purification, protein phosphorylation, NMR data acquisition, resonance assignment
Abstract

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed. 118

Published
2016

13. Studying Intrinsically disordered proteins under true in vivo conditions by combined cross-polarization and carbonyl-detection NMR spectroscopy

Author

Guy Lippens, Juan José Marín López, Robert Schneider, Isabelle Huvent, François-Xavier Cantrelle, Departamento de Ciencias-Quimica, Pontificia Universidad Católica del Perú (PUCP), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique, LABEX DISTALZ grant (Development of Innovative Strategies for a Transdisciplinary Approach to Alzheimer's Disease), CNRS Large Scale Facility NMR THC [Fr3050], European Community, CNRS, Region Nord-Pas de Calais (France), University of Lille 1, Institut Pasteur de Lille, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Pontificia Universidad Católica del Perú = Pontifical Catholic University of Peru (PUCP), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, Proton, Nitrogen, alpha-synuclein, [SDV]Life Sciences [q-bio], 010402 general chemistry, Intrinsically disordered proteins, 01 natural sciences, Catalysis, Protein Carbonylation, 03 medical and health sciences, Nuclear magnetic resonance, In vivo, cross-polarization, Bacteria, Chemistry, Cross polarization, Pulse sequence, General Medicine, General Chemistry, Nuclear magnetic resonance spectroscopy, Correlation spectrum, 0104 chemical sciences, NMR spectra database, 030104 developmental biology, carbon detection, in-cell NMR spectroscopy, intrinsically disordered proteins
Abstract

Under physiological conditions, studies of intrinsically disordered proteins (IDPs) by conventional NMR methods based on proton detection are severely limited by fast amide-proton exchange with water. C-13 detection has been proposed as a solution to the exchange problem, but is hampered by low sensitivity. We propose a new pulse sequence combining proton-nitrogen cross-polarization and carbonyl detection to record high-resolution, high-sensitivity NMR spectra of IDPs under physiological conditions. To demonstrate the efficacy of this approach, we recorded a high-quality N-CO correlation spectrum of alpha-synuclein in bacterial cells at 37 degrees C.

Published
2016

14. Structural characterization by nuclear magnetic resonance of the impact of phosphorylation in the proline-rich region of the disordered Tau protein

Author

Nathalie Sibille, Dries Verdegem, Laziza Amniai, Jean-Michel Wieruszeski, Guy Lippens, Caroline Fauquant, Isabelle Landrieu, Isabelle Huvent, and Arnaud Leroy

Subjects
0303 health sciences, biology, Chemistry, Kinase, Cyclin-dependent kinase 2, Tau protein, Nuclear magnetic resonance spectroscopy, 010402 general chemistry, 01 natural sciences, Biochemistry, 0104 chemical sciences, 03 medical and health sciences, Tubulin, Nuclear magnetic resonance, Structural Biology, Microtubule, biology.protein, Phosphorylation, Molecular Biology, Protein secondary structure, 030304 developmental biology
Abstract

Phosphorylation of the neuronal Tau protein is implicated in both the regulation of its physiological function of microtubule stabilization and its pathological propensity to aggregate into the fibers that characterize Alzheimer's diseased neurons. However, how specific phosphorylation events influence both aspects of Tau biology remains largely unknown. In this study, we address the structural impact of phosphorylation of the Tau protein by Nuclear Magnetic Resonance (NMR) spectroscopy on a functional fragment of Tau (Tau[Ser208-Ser324] = TauF4). TauF4 was phosphorylated by the proline-directed CDK2/CycA3 kinase on Thr231 (generating the AT180 epitope), Ser235, and equally on Thr212 and Thr217 in the Proline-rich region (Tau[Ser208-Gln244] or PRR). These modifications strongly decrease the capacity of TauF4 to polymerize tubulin into microtubules. While all the NMR parameters are consistent with a globally disordered Tau protein fragment, local clusters of structuration can be defined. The most salient result of our NMR analysis is that phosphorylation in the PRR stabilizes a short α-helix that runs from pSer235 till the very beginning of the microtubule-binding region (Tau[Thr245-Ser324] or MTBR of TauF4). Phosphorylation of Thr231/Ser235 creates a N-cap with helix stabilizing role while phosphorylation of Thr212/Thr217 does not induce modification of the local transient secondary structure, showing that the stabilizing effect is sequence specific. Using paramagnetic relaxation experiments, we additionally show a transient interaction between the PRR and the MTBR, observed in both TauF4 and phospho-TauF4. Proteins 2011;. © 2011 Wiley Periodicals, Inc.

Published
2011

15. Structure–based Mechanism of Ligand Binding for Periplasmic Solute-binding Protein of the Bug Family

Author

Arnaud Leroy, Françoise Jacob-Dubuisson, Camille Locht, Julien Herrou, David Hot, Rudy Antoine, Isabelle Huvent, Vincent Villeret, Coralie Bompard, Prakash Rucktooa, Mécanismes moléculaires de la pathogenèse bactérienne, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de Lille - IBL (IBLI), Université de Lille, Sciences et Technologies-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé, Laboratoire de Biochimie Appliquée, and Université Paris-Sud - Paris 11 (UP11)

Subjects
Models, Molecular, Circular dichroism, Stereochemistry, Amino Acid Motifs, Molecular Sequence Data, Plasma protein binding, Crystallography, X-Ray, Ligands, Niacin, Bordetella pertussis, Protein Structure, Secondary, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Carboxylate, Molecular Biology, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Hydrogen bond, Circular Dichroism, Binding protein, Periplasmic space, Ligand (biochemistry), Protein Structure, Tertiary, Crystallography, chemistry, Periplasmic Binding Proteins, Protein Binding
Abstract

Bug proteins form a large family of periplasmic solute-binding proteins well represented in beta-proteobacteria. They adopt a characteristic Venus flytrap fold with two globular domains bisected by a ligand-binding cleft. The structures of two liganded Bug proteins have revealed that the family is specific for carboxylated solutes, with a characteristic mode of binding involving two highly conserved beta strand-beta turn-alpha helix motifs originating from each domain. These two motifs form hydrogen bonds with a carboxylate group of the ligand, both directly and via conserved water molecules, and have thus been termed the carboxylate pincers. In both crystallized Bug proteins, the ligands were found enclosed between the two domains and inaccessible to solvent, suggesting an inter-domain hinge-bending motion upon ligand binding. We report here the first structures of an open, unliganded Bug protein and of the same protein with a citrate ion bound in the open cavity. One of the ligand carboxylate groups is bound to one half of the carboxylate pincers by the beta strand-beta turn-alpha helix motif from domain 1, and the citrate ion forms several additional interactions with domain 1. The ligand is accessible to solvent and has very few contacts with domain 2. In this open, liganded structure, the second part of the carboxylate pincers originating from domain 2 is not stabilized by ligand binding, and a loop replaces the beta turn. In the unliganded structure, both motifs of the carboxylate pincers are highly mobile, and neither of the two beta turns is formed. Thus, ligand recognition is performed by domain 1, with the carboxylate group serving as an initial anchoring point. Stabilization of the closed conformation requires proper interactions to be established with domain 2, and thus domain 2 discriminates between productively and non-productively bound ligands.

Published
2007

16. Crystal Structures of two Bordetella pertussis Periplasmic Receptors Contribute to Defining a Novel Pyroglutamic Acid Binding DctP Subfamily

Author

Julien Herrou, Vincent Villeret, Camille Locht, Isabelle Huvent, Prakash Rucktooa, Coralie Bompard, Rudy Antoine, and Françoise Jacob-Dubuisson

Subjects
Models, Molecular, Stereochemistry, Molecular Sequence Data, Sequence alignment, Plasma protein binding, Biology, Crystallography, X-Ray, Bordetella pertussis, chemistry.chemical_compound, Protein structure, Bacterial Proteins, Structural Biology, Amino Acid Sequence, Molecular Biology, Peptide sequence, Permease, Membrane Transport Proteins, Biological Transport, Periplasmic space, Protein Structure, Tertiary, Pyrrolidonecarboxylic Acid, Transport protein, Biochemistry, chemistry, Pyroglutamic acid, Sequence Alignment, Protein Binding
Abstract

Gram-negative bacteria have developed several different transport systems for solute uptake. One of these, the tripartite ATP independent periplasmic transport system (TRAP-T), makes use of an extracytoplasmic solute receptor (ESR) which captures specific solutes with high affinity and transfers them to their partner permease complex located in the bacterial inner membrane. We hereby report the structures of DctP6 and DctP7, two such ESRs from Bordetella pertussis. These two proteins display a high degree of sequence and structural similarity and possess the "Venus flytrap" fold characteristic of ESRs, comprising two globular alpha/beta domains hinged together to form a ligand binding cleft. DctP6 and DctP7 both show a closed conformation due to the presence of one pyroglutamic acid molecule bound by highly conserved residues in their respective ligand binding sites. BLAST analyses have revealed that the DctP6 and DctP7 residues involved in ligand binding are strictly present in a number of predicted TRAP-T ESRs from other bacteria. In most cases, the genes encoding these TRAP-T systems are located in the vicinity of a gene coding for a pyroglutamic acid metabolising enzyme. Both the high degree of conservation of these ligand binding residues and the genomic context of these TRAP-T-coding operons in a number of bacterial species, suggest that DctP6 and DctP7 constitute the prototypes of a novel TRAP-T DctP subfamily involved in pyroglutamic acid transport.

Published
2007

17. The FK506-binding protein FKBP52 in vitro induces aggregation of truncated Tau forms with prion-like behavior

Author

Julien Giustiniani, Guy Lippens, Isabelle Landrieu, Alain Schmitt, Etienne-Emile Baulieu, Malika Hamdane, Luc Buée, Omar Dounane, Kevin Guillemeau, Elodie Sardin, Isabelle Huvent, Béatrice Chambraud, Neuroprotection et neurogénération (UMR788), Neuroprotection et Neurodégénération (UMR788), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre de recherche Jean-Pierre Aubert-Neurosciences et Cancer, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Droit et Santé, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U1172 Inserm - U837 (JPArc), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Lille Nord de France (COMUE)-Université de Lille, Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U837 (JPArc), and Université Lille Nord de France (COMUE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille

Subjects
Male, Prions, Tau protein, Mutant, tau Proteins, Microtubules, Biochemistry, law.invention, Rats, Sprague-Dawley, Tacrolimus Binding Proteins, Alzheimer Disease, Microtubule, law, Cell Line, Tumor, mental disorders, Genetics, medicine, Animals, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, biology, Chemistry, Brain, Human brain, FKBP52, In vitro, Rats, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], FKBP, medicine.anatomical_structure, Tauopathies, Biophysics, biology.protein, Recombinant DNA, Protein Binding, Biotechnology
Abstract

Tauopathies, including Alzheimer's disease (AD), are neurodegenerative diseases associated with the pathologic aggregation of human brain Tau protein. Neuronal Tau is involved in microtubule (MT) formation and stabilization. We showed previously that the immunophilin FK506-binding protein of MW ∼52 kDa (FKBP52) interferes with this function of full-length Tau and provokes aggregation of a disease-related mutant of Tau. To dissect the molecular interaction between recombinant human FKBP52 and Tau, here, we study the effect of FKBP52 on a functional Tau fragment (Tau-F4, Ser(208)-Ser(324)) containing part of the proline- rich region and MT-binding repeats. Therefore, we perform MT assembly and light-scattering assays, blue native PAGE analysis, electron microscopy, and Tau seeding experiments in SH-SY5Y human neuroblastoma cells. We show that FKBP52 (6 µM) prevents MT formation generated by Tau-F4 (5 µM) and induces Tau-F4 oligomerization and aggregation. Electron microscopy analyses show granular oligomers and filaments of Tau-F4 after short-time FKBP52 incubation. We demonstrate that the terminal parts of Tau interfere with the effects of FKBP52. Finally, we find that FKBP52-induced Tau-F4 oligomers cannot only generate in vitro, direct conformational changes in full-length Tau and that their uptake into neuronal cells can equally lead to aggregation of wild-type endogenous Tau. This suggests a potential prion-like property of these particular Tau-F4 aggregates. Collectively, our results strengthen the hypothesis of FKBP52 involvement in the Tau pathogenicity process.

Published
2015

18. Structural analysis ofBordetella pertussisBugE solute receptor in a bound conformation

Author

Rudy Antoine, Coralie Bompard, Françoise Jacob-Dubuisson, Hassan Belrhali, Vincent Villeret, Camille Locht, and Isabelle Huvent

Subjects
chemistry.chemical_classification, Bordetella pertussis, Ligand, Amino Acid Motifs, Glutamate binding, General Medicine, Periplasmic space, Biology, Crystallography, X-Ray, Ligands, biology.organism_classification, Protein Structure, Tertiary, Transport protein, Amino acid, Biochemistry, chemistry, Structural Biology, Periplasmic Binding Proteins, Periplasm, Aspartic acid
Abstract

The Bug proteins form a large family of periplasmic solute-binding receptors present in a number of bacterial species. Here, the crystal structure of Bordetella pertussis BugE, a member of the Bug family coded by the gene BP0250, is reported. It adopts the Venus flytrap architecture of periplasmic binding proteins, with two domains separated by a deep cleft. BugE has a bound ligand, identified as a glutamate. The structure of B. pertussis BugD, which is an aspartic acid transporter, has recently been reported. These structures reveal high conservation of the Bug architecture, despite limited sequence identity. They share a common carboxylate-binding motif defined by two strand-beta-turn-alpha-helix motifs, also involving two water molecules to bridge the carboxylate O atoms to the protein. The two water molecules are hydrogen bonded to a common main-chain carbonyl group. Although the features of the carboxylate-binding motif are totally conserved, the ligand in BugE is bound by its side-chain carboxylate group rather than by its alpha-carboxylate as in BugD. This specific ligand-binding motif is highly conserved in Bug proteins and the BugE structure suggests that the cavity of the Bug proteins might also accommodate carboxylated solutes other than amino acids. The vast expansion of the Bug family in several bacterial genera is likely to be explained by the possible diversity of ligands. No charged residues are involved in glutamate binding by BugE, unlike what has been described for all glutamate receptors reported so far.

Published
2006

19. H/D exchange of a 15 N labelled Tau fragment as measured by a simple Relax-EXSY experiment

Author

François-Xavier Cantrelle, Isabelle Landrieu, Isabelle Huvent, Guy Lippens, Juan José Marín López, Puneet Ahuja, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
H/D exchange, Nuclear and High Energy Physics, Intrinsically unfolded protein, Biophysics, 010402 general chemistry, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Nuclear magnetic resonance, Labelling, Amide, Moiety, Phosphorylation, Amide protons, 030304 developmental biology, 0303 health sciences, Isotope, Chemistry, Hydrogen bond, Fragment (computer graphics), Spin–lattice relaxation, Condensed Matter Physics, Phosphate, 3. Good health, 0104 chemical sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Crystallography
Abstract

We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T1 relaxation rates in solvents of different D2O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.

Published
2014

20. Mechanism of Tau-promoted microtubule assembly as probed by NMR spectroscopy

Author

Isabelle Huvent, Benoît Gigant, Caroline Fauquant, Isabelle Landrieu, Pascale Barbier, Jean-Michel Wieruszeski, Marcel Knossow, Guy Lippens, Laboratoire d’Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université Lille Nord de France (COMUE), Centre de Recherches en Oncologie biologique et Oncopharmacologie (CRO2), Institut National de la Santé et de la Recherche Médicale (INSERM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Aix Marseille Université (AMU), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Aix Marseille Université (AMU)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Tau protein, tau Proteins, Peptide, macromolecular substances, Microtubules, Biochemistry, Catalysis, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Colloid and Surface Chemistry, Tubulin, Microtubule, Nuclear Magnetic Resonance, Biomolecular, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Microtubule assembly, General Chemistry, Nuclear magnetic resonance spectroscopy, Affinities, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Monomer, chemistry, Biophysics, biology.protein, 030217 neurology & neurosurgery
Abstract

International audience; Determining the molecular mechanism of the neuronal Tau protein in the tubulin heterodimer assembly has been a challenge owing to the dynamic character of the complex and the large size of microtubules. We use here defined constructs comprising one or two tubulin heterodimers to characterize their association with a functional fragment of Tau, named TauF4. TauF4 binds with high affinities to the tubulin heterodimer complexes, but NMR spectroscopy shows that it remains highly dynamic, partly because of the interaction with the acidic C-terminal tails of the tubulin monomers. When bound to a single tubulin heterodimer, TauF4 is characterized by an overhanging peptide corresponding to the first of the four microtubule binding repeats of Tau. This peptide becomes immobilized in the complex with two longitudinally associated tubulin heterodimers. The longitudinal associations are favored by the fragment and contribute to Tau's functional role in microtubule assembly.

Published
2014

21. Nuclear Magnetic Resonance Analysis of the Acetylation Pattern of the Neuronal Tau Protein

Author

Isabelle Landrieu, François-Xavier Cantrelle, Guy Lippens, Amina Kamah, Isabelle Huvent, Caroline Smet-Nocca, Haoling Qi, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université de Lille, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université Lille Nord de France (COMUE), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
Lysine, Tau protein, tau Proteins, Biochemistry, 03 medical and health sciences, 0302 clinical medicine, Nuclear magnetic resonance, Microtubule, Humans, Post-translational regulation, p300-CBP Transcription Factors, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cysteine, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, Nitrogen Isotopes, Chemistry, Heparin, Acetylation, Nuclear magnetic resonance spectroscopy, In vitro, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Kinetics, Acetyltransferase, biology.protein, Protein Processing, Post-Translational, 030217 neurology & neurosurgery
Abstract

International audience; Lysine acetylation of the neuronal Tau protein was described as a novel mechanism of posttranslational regulation of Tau functions with important outcomes in microtubule binding and aggregation processes related to Alzheimer's disease. Here, we unravel at a per-residue resolution the acetylation pattern of full-length Tau by the Creb-binding protein (CBP) acetyltransferase using high-resolution nuclear magnetic resonance spectroscopy. Our study gives a quantitative overview of CBP-mediated acetylation and examines the catalytic proficiency because the nonenzymatic reaction with acetyl-coenzyme A occurs in vitro. Furthermore, we have investigated with this characterized acetylated Tau the effect of acetylation on Tau fibrillization in a heparin-induced aggregation assay and on heparin binding.

Published
2014

22. Spectroscopic studies of GSK3{beta} phosphorylation of the neuronal tau protein and its interaction with the N-terminal domain of apolipoprotein E

Author

Isabelle Landrieu, Isabelle Huvent, Guy Lippens, Dominique Legrand, Arnaud Leroy, Bernadette Codeville, Jean-Michel Wieruszeski, Laboratoire de Biochimie Appliquée, Université Paris-Sud - Paris 11 (UP11), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
Tau protein, Mutation, Missense, tau Proteins, Mitogen-activated protein kinase kinase, Biochemistry, MAP2K7, Glycogen Synthase Kinase 3, 03 medical and health sciences, Apolipoproteins E, 0302 clinical medicine, Alzheimer Disease, GSK-3, Humans, Protein phosphorylation, Phosphorylation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein kinase A, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, 030304 developmental biology, 0303 health sciences, Glycogen Synthase Kinase 3 beta, biology, Chemistry, Cyclin-dependent kinase 2, Cell Biology, Protein Structure, Tertiary, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Amino Acid Substitution, biology.protein, Molecular Biophysics, 030217 neurology & neurosurgery
Abstract

International audience; Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.

Published
2010

23. NMR spectroscopy of the neuronal tau protein: normal function and implication in Alzheimer's disease

Author

Arnaud Leroy, Isabelle Huvent, Luc Buée, Isabelle Landrieu, Malika Hamdane, Guy Lippens, Caroline Smet-Nocca, Laziza Amniai, Jean-Michel Wieruszeski, Nathalie Sibille, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U1172 Inserm - U837 (JPArc), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Lille Nord de France (COMUE)-Université de Lille, Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U837 (JPArc), Université Lille Nord de France (COMUE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer (JPArc - U837 Inserm), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université Lille 2 - Faculté de Médecine

Subjects
Models, Molecular, Tau protein, MESH: Neurons, tau Proteins, Isomerase, Proteomics, Biochemistry, 03 medical and health sciences, Alzheimer Disease, MESH: Nuclear Magnetic Resonance, Biomolecular, Animals, Humans, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Phosphorylation, MESH: Protein Kinases, Nuclear Magnetic Resonance, Biomolecular, 030304 developmental biology, chemistry.chemical_classification, Neurons, 0303 health sciences, MESH: Humans, MESH: Phosphorylation, biology, Kinase, 030302 biochemistry & molecular biology, Nuclear magnetic resonance spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: tau Proteins, Amino acid, chemistry, PIN1, biology.protein, Protein Kinases, MESH: Alzheimer Disease, MESH: Models, Molecular
Abstract

International audience; NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis-trans isomerase Pin1.

Published
2010

24. Crystal structure of Bordetella pertussis BugD solute receptor unveils the basis of ligand binding in a new family of periplasmic binding proteins

Author

Coralie Bompard, Hassan Belrhali, Isabelle Huvent, Rudy Antoine, Vincent Villeret, Camille Locht, and Françoise Jacob-Dubuisson

Subjects
Models, Molecular, Hydrogen bond, Stereochemistry, Chemistry, Binding protein, Molecular Sequence Data, Cooperative binding, Membrane Transport Proteins, Plasma protein binding, Ligand (biochemistry), Crystallography, X-Ray, Ligands, Bordetella pertussis, Protein Structure, Tertiary, Crystallography, Protein structure, Bacterial Proteins, Structural Biology, Periplasmic Binding Proteins, Peptide bond, Amino Acid Sequence, Molecular Biology, Sequence Alignment, Protein Binding
Abstract

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.

Published
2005

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