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251. 2-Amino diphenylsulfides as new inhibitors of trypanothione reductase

Author

Ali Ouaissi, Rodolfo Fernandez-Gomez, André Tartar, Christian Sergheraert, Gilles Bethegnies, Mireille Moutiez, Marc Aumercier, Michel Luyckx, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de biologie de Lille - IBL (IBLI), Université de Lille, Sciences et Technologies-Institut Pasteur de Lille, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS)

Subjects
Microbiology (medical), chemistry.chemical_classification, biology, Glutathione reductase, Trypanothione, Kinetoplastida, Biological activity, General Medicine, Glutathione, biology.organism_classification, In vitro, chemistry.chemical_compound, Infectious Diseases, Enzyme, chemistry, Biochemistry, Enzyme inhibitor, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, biology.protein, Pharmacology (medical), [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS
Abstract

Trypanothione reductase (TR) is the primary enzyme responsible for the reduction of trypanothione, the analog of glutathione found in trypanosomatidae. We have discovered a series of diphenylsulfides which are potent inhibitors of TR and have no activity on mammalian glutathione reductase. These compounds are also active in vitro on various stages of the parasite. Although structurally related to phenothiazines, which are known to be TR inhibitors, these compounds are devoided of any neuroleptic activity, making them attractive leads to develop specific and non toxic anti-chagasic drugs.

Published
1995
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252. Compared recognition of di- and trisulfide substrates by glutathione and trypanothione reductases

Author

André Tartar, Christian Sergheraert, Mireille Moutiez, Bernard Parmentier, Marc Aumercier, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Stereochemistry, Glutathione reductase, Kinetics, Biophysics, Trypanothione, Biochemistry, Substrate Specificity, chemistry.chemical_compound, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, NADH, NADPH Oxidoreductases, Disulfides, Hydrogen Sulfide, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Chromatography, High Pressure Liquid, chemistry.chemical_classification, Glutathione Disulfide, Substrate (chemistry), Glutathione, Enzyme, Glutathione Reductase, chemistry, Glutathione disulfide, Trypanothione reductase, Oxidation-Reduction, NADP
Abstract

Trypanothione trisulfide was synthesized according to two strategies. It was found to be recognized and reduced by trypanothione reductase as the natural disulfide substrate. At the difference with the mechanism observed for the reduction of glutathione trisulfide by glutathione reductase, the intermediate trypanothione persulfide was rapidly reduced. The enzymatic reduction of another trisulfide derived from an alternative substrate of trypanothione reductase was also studied. The structure of the trisulfide bridge of the substrate (intra- or intermolecular) appeared to be a determining factor in the enzymatic reduction pattern. Moreover, in the case of the alternative substrate of trypanothione reductase, differences of kinetics appeared for the first time between a di- and a trisulfide species. All kinetic parameters are given.

Published
1995
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253. A particularly labile Asp-Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage

Author

I, Ségalas, R, Thai, R, Ménez, C, Vita, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Models, Molecular, Magnetic Resonance Spectroscopy, Protein Conformation, Reptilian Proteins, medicine.disease_cause, Biochemistry, Protein structure, Structural Biology, Muscarinic acetylcholine receptor, ComputingMilieux_MISCELLANEOUS, Chromatography, High Pressure Liquid, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Structure, Chemistry, Circular Dichroism, 030302 biochemistry & molecular biology, Mamba, Hydrogen-Ion Concentration, Muscarinic toxin, Electrophoresis, Peptide bond cleavage, Proline, Stereochemistry, Molecular Sequence Data, Neurotoxins, Biophysics, Protein degradation, Cleavage (embryo), 03 medical and health sciences, Structure-Activity Relationship, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Genetics, medicine, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Asp-Pro bond, Molecular Biology, Bond cleavage, 030304 developmental biology, Elapid Venoms, Aspartic Acid, Toxin, Structural integrity, Peptide fragment, Cell Biology, biology.organism_classification, Peptide Fragments
Abstract

The single Asp53-Pro54 bond of the MTX2 toxin from the mamba snake Dendroaspis angusticeps is rapidly and efficiently cleaved in acidic solution (pH 1.5–2.5) at 45°C. Unfolding of the toxin slows down the cleavage reaction by several times. Modelling studies indicate that the native toxin conformation can catalyse the Asp53-Pro54 bond cleavage. The implications of this study are: (i) cleavage of Asp-Pro bond for sequence determination may occur better in absence than in presence of denaturant, (ii) mild acid conditions, commonly used in NMR structure determinations, may irreversibly affect the structural integrity of Asp--Pro containing peptides and proteins.

Published
1995
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254. Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Author

Elodie Marcon, Gilles Mourier, Nicolas Gilles, Laura Vera, Enrico A. Stura, Carole Fruchart-Gaillard, Renée Ménez, Guillaume Blanchet, Denis Servent, Laboratoire de Toxinologie Moléculaire et Biotechnologies (LTMB), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université Pierre et Marie Curie - Paris 6 (UPMC)

Subjects
Models, Molecular, Nicotinic Acetylcholine Receptors, Protein Structure, Adrenergic receptor, Science, Molecular Sequence Data, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Protein Synthesis, Computational biology, Biology, Toxicology, Ligands, Protein Engineering, Biochemistry, Muscarinic acetylcholine receptor, Toxin Binding, Amino Acid Sequence, Biomacromolecule-Ligand Interactions, Protein Interactions, Receptor, Peptide sequence, Toxins, Biological, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Sequence Homology, Amino Acid, Rational design, Proteins, Protein engineering, Directed evolution, Receptors, Muscarinic, Fusion protein, Receptors, Adrenergic, Transmembrane Proteins, Acetylcholine Receptors, Medicine, Muscarinic Acetylcholine Receptors, Crystallization, Research Article
Abstract

International audience; Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test ‘‘loop grafting,’’ a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the $\alpha_{1A}$-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and $\alpha_{1A}$-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used todesign new three-finger proteins with original pharmacological profiles

Published
2012
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255. Identification of two-histidines one-carboxylate binding motifs in proteins amenable to facial coordination to metals

Author

Maurus Schmid, Philippe Cuniasse, Florian P. Seebeck, Thomas R. Ward, François Gilardoni, Beat Amrein, Guillaume Collet, Laboratoire Chimie pour le Vivant, ingénierie moléculaire pour la santé (LCV), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Models, Molecular, Carboxypeptidases A, Protein Conformation, MESH: Metalloproteins, Amino Acid Motifs, Carboxylic Acids, Protein Data Bank (RCSB PDB), MESH: Amino Acid Sequence, 01 natural sciences, Biochemistry, MESH: Amino Acid Motifs, MESH: Protein Structure, Tertiary, MESH: Histidine, chemistry.chemical_compound, MESH: Protein Conformation, Databases, Protein, chemistry.chemical_classification, 0303 health sciences, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Metals and Alloys, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Amino acid, MESH: Carboxylic Acids, Metals, Chemistry (miscellaneous), Identification (biology), MESH: Models, Molecular, MESH: Computational Biology, MESH: Databases, Protein, Coordination sphere, Stereochemistry, MESH: Molecular Structure, Biophysics, 010402 general chemistry, Biomaterials, MESH: Software, 03 medical and health sciences, Metalloproteins, Histidine, Amino Acid Sequence, Carboxylate, 030304 developmental biology, Binding Sites, MESH: Metals, Computational Biology, Active site, MESH: Carboxypeptidases A, Protein Structure, Tertiary, 0104 chemical sciences, Sequence homology, MESH: Binding Sites, chemistry, biology.protein, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Software
Abstract

International audience; Among natural metalloenzymes, the facial two-histidines one-carboxylate binding motif (FTM) is a widely represented first coordination sphere motif present in the active site of a variety of metalloenzymes. A PDB search revealed a total of 1685 structures bearing such FTMs bound to a metal. Sixty statistically representative FTMs were selected and used as template for the identification of structurally characterized proteins bearing these three amino acids in a propitious environment for binding to a transition metal. This geometrical superposition search, carried out using the STAMPS software, returned 2320 hits. While most consisted of either apo-FTMs or bore strong sequence homology to known FTMs, seven such structures lying within a cavity were identified as novel and viable scaffolds for the creation of artificial metalloenzymes bearing an FTM.

Published
2012
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256. A microplate assay for trypanothione reductase inhibitors

Author

Christian Sergheraert, Mireille Moutiez, André Tartar, Marc Aumercier, Djalal Meziane-Cherif, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, Chemistry, Spermidine, Biophysics, Cell Biology, Biochemistry, Glutathione, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, NADH, NADPH Oxidoreductases, Trypanothione reductase, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
1994
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257. Reduction of a Trisulfide Derivative of Glutathione by Glutathione Reductase

Author

André Tartar, Mireille Moutiez, Elisabeth Teissier, Christian Sergheraert, B. Parmentier, Marc Aumercier, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
GPX1, GPX3, Hydrogen sulfide, Glutathione reductase, Biophysics, Sulfides, GPX4, Biochemistry, Medicinal chemistry, Mass Spectrometry, chemistry.chemical_compound, Glutaredoxin, Electrochemistry, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Chromatography, High Pressure Liquid, ComputingMilieux_MISCELLANEOUS, Chemistry, Cell Biology, Glutathione, Glutathione Reductase, Glutathione disulfide, Cattle, Oxidation-Reduction
Abstract

Glutathione trisulfide was synthesized from glutathione disulfide and its reduction by glutathione reductase was studied. A two-step reaction was observed. In a first step, the rate of reduction was similar to that observed with glutathione disulfide. In addition to glutathione, a persulfide intermediate was detected by an electrochemical method and was carboxymethylated by iodoacetate to be identified by Plasma Desorption Mass Spectrometry. During the second step the reduction of this intermediate led to the formation of hydrogen sulfide and a second equivalent of glutathione.

Published
1994
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258. Preparation of trisulfide derivatives of cystine and their formation as by-products during peptide synthesis

Author

Mireille Moutiez, Christian Sergheraert, B. Parmentier, André Tartar, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
chemistry.chemical_compound, chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Reagent, Organic Chemistry, Drug Discovery, Cystine, Side reaction, Peptide synthesis, Organic chemistry, Biochemistry, ComputingMilieux_MISCELLANEOUS
Abstract

The formation of a trisulfide derivative is observed when di-NH 2 -protected cystine, is used during peptide synthesis. This side reaction occurs during the coupling step with several coupling reagents. A convenient method for the preparation of these derivatives in mild conditions is described.

Published
1994
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259. Compared Reactivities of Trypanothione and Glutathione in Conjugation Reactions

Author

Christian Sergheraert, Djalal Meziane-Cherif, Marc Aumercier, André Tartar, Mireille Moutiez, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
chemistry.chemical_classification, Stereochemistry, Trypanothione, General Chemistry, General Medicine, Glutathione, chemistry.chemical_compound, chemistry, Nucleophile, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Reagent, Drug Discovery, Electrophile, Thiol, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Xenobiotic, ComputingMilieux_MISCELLANEOUS, Conjugate
Abstract

In order to compare the non-enzymatic capacities of the xenobiotic conjugation of trypanothione (a spermidine-glutathione conjugate unique to kinetoplastidae) and glutathione, the reactivity of their respective thiols was investigated. The acido-basic properties of both compounds and their nucleophilicity toward Elliman's reagent and 1-chloro-2, 4-dinitrobenzene were studied. Our results show that although glutathione is a better nucleophile than trypanothione, the latter is more reactive because it is more ionized in a large pH range. This pH range likely includes the pH to which such conjugation reactions are expected to happen in vivo. Thus, the better conjugation capacity of trypanothione could make it the cornerstone for the xenobiotic detoxication of trypanosomatidae.

Published
1994
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260. Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation

Author

Françoise Bouet, Philippe Poujeol, André Ménez, Françoise Bontems, Alan L. Harvey, Claudio Vita, Flavio Toma, Hossein Vatanpour, Michel Tauc, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Service d'anatomie et cytologie pathologiques [Rennes] = Anatomy and Cytopathology [Rennes], CHU Pontchaillou [Rennes], Laboratoire CNRS 3093, Université de Nice-Sophia Antipolis, Centre National de la Recherche Scientifique (CNRS), Physiologie cellulaire et moléculaire des systèmes intégrés (PCMSI), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Service d'anatomie et cytologie pathologiques [Rennes], Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Hôpital Pontchaillou-CHU Pontchaillou [Rennes], and COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Protein Folding, Circular dichroism, Magnetic Resonance Spectroscopy, Potassium Channels, Charybdotoxin, Stereochemistry, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Scorpion Venoms, Peptide, Kidney, Biochemistry, Cell Line, 03 medical and health sciences, chemistry.chemical_compound, Dogs, Animals, Amino Acid Sequence, Disulfides, Peptide sequence, Protein secondary structure, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Circular Dichroism, 030302 biochemistry & molecular biology, Brain, Glutathione, Peptide Fragments, Rats, Amino acid, chemistry, Calcium, Spectrophotometry, Ultraviolet, Protein folding, Pyroglutamic acid, Synaptosomes
Abstract

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.

Published
1993
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261. P19-23. Putative prefusion mechanism of HIV-1 Env revealed by ligand-induced quaternary alterations in o-gp140 trimers

Author

Loïc Martin, R Cheng, Dj Green, Indresh K. Srivastava, S Poon, Frank I. Lin, Li Xing, Susan W. Barnett, Cg Moscoso, Elaine Kan, Yide Sun, Department of Chemistry - The Chinese University of Hong-Kong, The Chinese University of Hong Kong [Hong Kong], Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de Recherche en Acquisition et Traitement de l'Image pour la Santé (CREATIS), Université Jean Monnet [Saint-Étienne] (UJM)-Hospices Civils de Lyon (HCL)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
lcsh:Immunologic diseases. Allergy, Chemokine, viruses, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Gp41, Cleavage (embryo), Bioinformatics, 03 medical and health sciences, Protein structure, Mediator, Virology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Chemistry, virus diseases, Lipid bilayer fusion, Transmembrane protein, 3. Good health, Cell biology, Cytosol, Infectious Diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Poster Presentation, biology.protein, lcsh:RC581-607
Abstract

Background The human immunodeficiency virus envelope spike is the key mediator of membrane fusion with the host cell and is a promising research target for vaccine development. The surface spike gp120 and the transmembrane protein gp41 result from cleavage of immature cytosolic gp160 and are expressed as trimers. Conformational rearrangement upon binding of gp120 to host CD4 receptors and chemokine coreceptors mediates membrane fusion, exposes the fusion peptide of gp41 and enables viral genome insertion.

Published
2009
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262. Scorpion-Toxin Mimics of CD4 in Complex with Human Immunodeficiency Virus gp120 Crystal Structures,Molecular Mimicry, and Neutralization Breadth

Author

Huang, Chih-Chin, Stricher, François, Martin, Loïc, Decker, Julie, Majeed, Shahzad, Barthe, Philippe, Hendrickson, Wayne, Robinson, James, Roumestand, Christian, Sodroski, Joseph, Wyatt, Richard, Shaw, George, Vita, Claudio, Kwong, Peter, National Institutes of Health [Bethesda] (NIH), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Alabama at Birmingham [ Birmingham] (UAB), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Columbia University [New York], Tulane University, Harvard School of Public Health, Support was provided by the Agence Nationale de Recherches sur le SIDA (ANRS)., Support was provided by the 6th European Union Framework Program European Microbicides Project, the Howard Hughes Medical Institute, and the National Institutes of Health., and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, virus diseases, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
Abstract

International audience; The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.

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263. Pinnatoxins : an emergent family of marine phycotoxins targeting nicotinic acetylcholine receptors with high affinity

Author

Rómulo Aráoz, Denis Servent, Jordi Molgo, Bogdan Iorga, Fruchard-Gaillard, C., Evelyne BENOIT, Zhenhua Gu, Craig Stivala, Armen Zakarian, Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Neurobiologie et Développement (N&eD), Centre National de la Recherche Scientifique (CNRS), Institut de Neurobiologie Alfred Fessard (INAF), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry and Biochemistry [Santa Barbara], University of California [Santa Barbara] (UCSB), University of California-University of California, J. Barbier, E. Benoit, N. Gilles, D. Ladant, M.-F. Martin-Eauclaire, C. Mattéi, J. Molgó, M.R. Poppof & D. Servent, SFET, Brunet, Jocelyne, University of California [Santa Barbara] (UC Santa Barbara), and University of California (UC)-University of California (UC)

Subjects
[CHIM.ORGA]Chemical Sciences/Organic chemistry, [SDV]Life Sciences [q-bio], [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.TOX]Life Sciences [q-bio]/Toxicology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [CHIM.ORGA] Chemical Sciences/Organic chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

264. Glutathione-dependent activities of Trypanosoma cruzi p52 makes it a new member of the thiol:disulphide oxidoreductase family

Author

Valérie Lucas, Christian Sergheraert, Eric Quéméneur, André Tartar, Elisabeth Davioud-Charvet, Mireille Moutiez, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Service de Chimie Bio-Organique et de Marquage (SCBM), Département d'Ingenierie et d'Etudes des Protéines (DIEP), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Trypanosoma cruzi, Trypanothione, Protein Disulfide-Isomerases, Oxidative phosphorylation, Biology, Biochemistry, Catalysis, Substrate Specificity, Enzyme activator, chemistry.chemical_compound, Oxidoreductase, parasitic diseases, Animals, Insulin, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Disulfides, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein disulfide-isomerase, Isomerases, Molecular Biology, chemistry.chemical_classification, Oxidase test, Cell Biology, Glutathione, Enzyme Activation, Enzyme, chemistry, Oxidoreductases, Oxidation-Reduction, Research Article
Abstract

International audience; Trypanothione:glutathione disulphide thioltransferase of Trypanosoma cruzi (p52) is a key enzyme in the regulation of the intracellular thiol-disulphide redox balance by reducing glutathione disulphide. Here we show that p52, like other disulphide oxidoreductases possessing the CXXC active site motif, catalyses the reduction of low-molecular-mass disulphides (hydroxyethyldisulphide) as well as protein disulphides (insulin). However, p52 seems to be a poor oxidase under physiological conditions as evidenced by its very low rate for oxidative renaturation of reduced ribonuclease A. Like thioltransferase and protein disulphide isomerase, p52 was found to possess a glutathione-dependent dehydroascorbate reductase activity. The kinetic parameters were in the same range as those determined for mammalian dehydroascorbate reductases. A catalytic mechanism taking into account both trypanothione- and glutathione-dependent reduction reactions was proposed. This newly characterized enzyme is specific for the parasite and provides a new target for specific chemotherapy.

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265. High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

Author

Renaud Vincentelli, Philippe Cuniasse, Carole Fruchart-Gaillard, Muriel Gondry, Badreddine Douzi, Mireille Moutiez, Fabrice Beau, François Fenaille, Natalie J. Saez, Vincent Dive, Denis Servent, Hervé Nozach, Robert Thai, Oscar H. P. Ramos, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Service de Pharmacologie et Immunoanalyse (SPI), Laboratoire Chimie pour le Vivant, ingénierie moléculaire pour la santé (LCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Enzymologie et Biosynthèse Peptidique Non Ribosomale (BIOSYN), Département Microbiologie (Dpt Microbio), 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)-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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), This work was supported by the ANR PFTV, the GIS IBiSA and by INSTRUCT (ESFRI), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), European Project: 278346,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,VENOMICS(2011), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Médicaments et Technologies pour la Santé (MTS), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-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), and ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)

Subjects
Protein Folding, Cytoplasm, Recombinant Fusion Proteins, High-throughput screening, Protein Disulfide-Isomerases, Bioengineering, medicine.disease_cause, Applied Microbiology and Biotechnology, Matrix-assisted laser desorption-ionization, 03 medical and health sciences, Recombinant expression, High throughput screening, medicine, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Disulfides, Disulfide bonds, Protein disulfide-isomerase, 030304 developmental biology, 0303 health sciences, biology, Chemistry, Spectrometry, Research, 030302 biochemistry & molecular biology, Disulfide bond, Proteins, Mass, biology.organism_classification, Fusion protein, Biochemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Disulfide-rich, bacteria, Protein folding, Oxidation-Reduction, Bacteria, Biotechnology
Abstract

International audience; BackgroundDisulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.ResultsHere we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.ConclusionsOur results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB - /gor - strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

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