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5 results on '"Anne-Sophie Dabert-Gay"'

1. Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance

Author

Emilie Jaune, Hella Amdouni, Anne-Sophie Dabert-Gay, Jean-Sébastien Annicotte, Magali Plaisant, Solange Moréra, Delphine Debayle, Corine Bertolotto, Patricia Abbe, Thierry Passeron, Abdelali Lehraiki, Laurent Héliot, Robert Ballotti, Philippe Gual, Baharia Mograbi, Cyril Ronco, Mariano Gonzalez-Pisfil, Thomas Botton, Damien Alcor, Caroline Robert, Michael Cerezo, Stéphane Rocchi, Nabil Rabhi, Véronique Hofman, Armelle Vigouroux, Antoine Millet, Rachid Benhida, Maruf M.U. Ali, Paul Hofman, Florian Rouaud, Université Nice Sophia Antipolis - Faculté de Médecine (UNS UFR Médecine), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Centre méditerranéen de médecine moléculaire (C3M), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institutions et Dynamiques Historiques de l'Économie et de la Société (IDHES), École normale supérieure - Cachan (ENS Cachan)-Université Panthéon-Sorbonne (UP1)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Institut de Chimie de Nice (ICN), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Service de Dermatologie [Nice], Hôpital Archet 2 [Nice] (CHU), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre d'Etudes Lasers Intenses et Applications (CELIA), Université de Bordeaux (UB)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Génomique Intégrative et Modélisation des Maladies Métaboliques (EGID), Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Français de Recherche pour l'Exploitation de la Mer - Brest (IFREMER), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Microbiologie et enzymologie structurale (MESB3S), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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-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, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS), University of Stavanger, Laboratoire Chrono-environnement - UFC (UMR 6249) (LCE), Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Centre méditérannéen de médecine moléculaire ( C3M ), Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Nice Sophia Antipolis - Faculté de Médecine ( UNS UFR Médecine ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ), Institut de Chimie de Nice ( ICN ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Centre National de la Recherche Scientifique ( CNRS ), Hôpital Archet 2 [Nice] ( CHU ), Institut de Recherche sur le Cancer et le Vieillissement ( IRCAN ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Pathologie Clinique et Expérimentale, Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Hôpital pasteur [Colmar]-CHU Nice, Institut de pharmacologie moléculaire et cellulaire ( IPMC ), Génomique Intégrative et Modélisation des Maladies Métaboliques ( EGID ), Institut Pasteur de Lille, Réseau International des Instituts Pasteur ( RIIP ) -Réseau International des Instituts Pasteur ( RIIP ) -Université de Lille-Centre Hospitalier Régional Universitaire [Lille] ( CHRU Lille ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 ( PhLAM ), Université de Lille-Centre National de la Recherche Scientifique ( CNRS ), Département de médecine oncologique [Gustave Roussy], Institut Gustave Roussy ( IGR ), Microbiologie et enzymologie structurale ( MESB3S ), 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 ), Department of Life Sciences, Imperial College London, Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Université Paris 1 Panthéon-Sorbonne (UP1)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), 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 Côte d'Azur (UCA), Metabolic functional (epi)genomics and molecular mechanisms involved in type 2 diabetes and related diseases - UMR 8199 - UMR 1283 (EGENODIA (GI3M)), 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 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)-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), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Hôpital pasteur [Colmar]-Centre Hospitalier Universitaire de Nice (CHU Nice), Institut Gustave Roussy (IGR), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-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 Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Metabolic functional (epi)genomics and molecular mechanisms involved in type 2 diabetes and related diseases - UMR 8199 - UMR 1283 (GI3M), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Bordeaux (UB), Institut Français de Recherche pour l'Exploitation de la Mer - Brest (IFREMER Centre de Bretagne), Institutions et Dynamiques Historiques de l'Économie et de la Société ( IDHES ), École normale supérieure - Cachan ( ENS Cachan ) -Université Panthéon-Sorbonne ( UP1 ) -Université Paris 8 Vincennes-Saint-Denis ( UP8 ) -Université Paris Nanterre ( UPN ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Centre National de la Recherche Scientifique ( CNRS ), Centre d'Etudes Lasers Intenses et Applications ( CELIA ), Université de Bordeaux ( UB ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ), Institut Français de Recherche pour l'Exploitation de la Mer - Brest ( IFREMER ), Institut Français de Recherche pour l'Exploitation de la Mer ( IFREMER ), Centre National de la Recherche Scientifique ( CNRS ), Laboratoire Chrono-environnement ( LCE ), Université Bourgogne Franche-Comté ( UBFC ) -Centre National de la Recherche Scientifique ( CNRS ) -Université de Franche-Comté ( UFC ), and Houel, Ludivine

Subjects
0301 basic medicine, Proto-Oncogene Proteins B-raf, Cancer Research, Programmed cell death, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Pharmacology, Biology, Transcriptome, 03 medical and health sciences, Mice, 0302 clinical medicine, Cell Line, Tumor, medicine, Animals, Humans, Endoplasmic Reticulum Chaperone BiP, Melanoma, Protein Kinase Inhibitors, neoplasms, Heat-Shock Proteins, 030304 developmental biology, Cell Proliferation, Regulation of gene expression, 0303 health sciences, Sulfonamides, [ SDV ] Life Sciences [q-bio], Autophagy, Cell Biology, medicine.disease, Endoplasmic Reticulum Stress, Xenograft Model Antitumor Assays, [SDV] Life Sciences [q-bio], Gene Expression Regulation, Neoplastic, 030104 developmental biology, Oncology, Cell culture, Apoptosis, Drug Resistance, Neoplasm, 030220 oncology & carcinogenesis, Unfolded protein response, Cancer research
Abstract

International audience; Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal epsilon, epsilon-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt. To analyse the importance of primary sequence in these regions, we have combined selection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3'-part of the bulge are important. Such a bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of (-)-strand DNA in HBV reverse transcription.

Published
2016
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2. Evidence that the Amyloid-β Protein Precursor Intracellular Domain, AICD, Derives From β-Secretase-Generated C-Terminal Fragment

Author

Raphaëlle Pardossi-Piquard, Jean Sevalle, Brice Flammang, Delphine Debayle, Frédéric Checler, Anne-Sophie Dabert-Gay, Inger Lauritzen, Aurelie Thevenet, Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)

Subjects
Intracellular Fluid, Time Factors, Proteolysis, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, amyloid-β, Transfection, Substrate Specificity, Amyloid beta-Protein Precursor, Neuroblastoma, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Cell Line, Tumor, Extracellular, medicine, Humans, Immunoprecipitation, Senile plaques, Protein precursor, Chromatography, High Pressure Liquid, C99, Cell Line, Transformed, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, AICD, medicine.diagnostic_test, biology, secretases, General Neuroscience, General Medicine, Alzheimer's disease, Peptide Fragments, Protein Structure, Tertiary, Amino acid, C83, Psychiatry and Mental health, Clinical Psychology, amyloidogenic pathway, chemistry, Biochemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, biology.protein, Amyloid Precursor Protein Secretases, Geriatrics and Gerontology, Amyloid precursor protein secretase, 030217 neurology & neurosurgery
Abstract

International audience; One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is the senile plaque, an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39-43 amino acids) referred to as amyloid-β (Aβ) peptides. Aβ peptides are derived from combined proteolytic cleavages undergone on the amyloid-β protein precursor (AβPP) by a set of enzymes called secretases. Several lines of anatomical and biological evidence suggest that Aβ peptides would not account for all pathological stigmata and molecular dysfunctions taking place in AD. In amyloidogenic and non-amyloidogenic pathways, AβPP first undergoes β- or α-secretases-mediated cleavages yielding C99 and C83, respectively. These two membrane-embedded C-terminal fragments are both potential targets of subsequent γ-secretase-mediated proteolysis. The latter cleavage not only generates either p3 or Aβ peptides but similarly gives rise to an AβPP IntraCellular Domain (AICD fragment) that could modulate the transcription of several genes linked to AD pathology. It is therefore striking that AICD theoretically derives from both amyloidogenic and non-amyloidogenic AβPP processing pathways. Here we show that AICD predominantly derives from C99 by means of recombinant substrates and transiently transfected cells expressing C99. Our data suggest a preferred pathogenic pathway for AICD production and suggests that this fragment, in addition to C99 and Aβ peptides, could contribute to AD pathology.

Published
2012
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3. Group X secreted phospholipase A2 proenzyme is matured by a furin-like proprotein convertase and releases arachidonic acid inside of human HEK293 cells

Author

Christine Payré, Khaoula Chargui, Rob C. Oslund, Sabine Scarzello, Michael H. Gelb, Hiromi, Gérard Lambeau, Anne Sophie Dabert-Gay, Dominique Douguet, Ikram Jemel, Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Department of Chemistry, University of Washington [Seattle], and Department of Biochemistry [Washington ]

Subjects
MESH: Group X Phospholipases A2, MESH: Mutation, medicine.medical_treatment, Amino Acid Motifs, Biochemistry, 03 medical and health sciences, Mice, MESH: Amino Acid Motifs, MESH: Enzyme Precursors, 0302 clinical medicine, Phospholipase A2, medicine, Animals, Group X Phospholipases A2, Humans, Secretion, Protease Inhibitors, MESH: Animals, Protein precursor, Molecular Biology, Furin, MESH: Mice, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Enzyme Precursors, Protease, Arachidonic Acid, MESH: Protease Inhibitors, MESH: Humans, biology, Cell Biology, Proprotein convertase, Lipids, Amino acid, MESH: Arachidonic Acid, HEK293 Cells, chemistry, 030220 oncology & carcinogenesis, MESH: HEK293 Cells, Mutation, biology.protein, MESH: Proprotein Convertases, [SDV.IMM]Life Sciences [q-bio]/Immunology, Proprotein Convertases
Abstract

International audience; Among mammalian secreted phospholipases A(2) (sPLA(2)s), group X sPLA(2) has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA(2) is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA(2) in HEK293 cells, which have been extensively used to analyze sPLA(2)-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA(2) inhibitors and protease inhibitors, we demonstrate that group X sPLA(2) is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA(2) inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA(2) maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.

Published
2011
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4. Detection of matrix metalloproteinase active forms in complex proteomes: evaluation of affinity versus photoaffinity capture

Author

Athanasios Yiotakis, Fabrice Beau, Céline Huillet, Laurent Devel, Anne-Sophie Dabert-Gay, Bertrand Czarny, Sarah Bregant, Robert Thai, Vincent Dive, 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), 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 National and Kapodistrian University of Athens (NKUA)

Subjects
Proteomics, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Proteome, Molecular Sequence Data, [SDV.CAN]Life Sciences [q-bio]/Cancer, Photoaffinity Labels, Matrix metalloproteinase, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Mice, Biotin, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Cell Line, Tumor, Matrix Metalloproteinase 12, Animals, Humans, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Peptide sequence, Polyacrylamide gel electrophoresis, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Mice, Inbred BALB C, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Structure, 010405 organic chemistry, Chemistry, Active site, Affinity Labels, General Chemistry, Matrix Metalloproteinases, 0104 chemical sciences, Matrix-assisted laser desorption/ionization, Matrix Metalloproteinase 8, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, biology.protein, Matrix Metalloproteinase 2, Electrophoresis, Polyacrylamide Gel, Linker
Abstract

Various attempts to detect matrix metalloproteinase (MMP) active forms from complex proteomes, based on the use of specific photoactivatable affinity probes, have up to now failed. To overcome this failure, an affinity approach has been evaluated as an alternative to the photoaffinity one. For this purpose, two probes were synthesized to interact specifically with the active site of MMPs and allow isolation of MMP/probe complexes on magnetic beads through a biotin linker. Using phosphinic peptide chemistry, we prepared an affinity probe displaying picomolar potency toward several MMPs, and a related photoaffinity probe incorporating a photoactivatable azido group exhibiting subnanomolar affinity toward these targets. By a combination of silver-staining detection and MALDI peptide mass fingerprints, a systematic comparison was made of both strategies in terms of hMMP-12 and hMMP-8 recovery and identification when present in mixtures of different complexity. The results obtained show that the affinity protocol is superior to the photoaffinity strategy in terms of quantity of captured MMPs and number of MMP tryptic fragments detected in MALDI-MS. The specificity and efficiency of the affinity capture protocol developed in this study allowed easy, fast, and unambiguous detection by MALDI-MS of three hMMPs (2, 8, and 12), from a single affinity capture experiment, when added (10-36 ng of MMPs) to a tumor extract (10 microg). Thus, the tools and approaches reported should enable us to progress in the detection of endogenous active forms of MMPs in complex proteomes, an important objective with many diagnostic applications.

Published
2009

5. Molecular Determinants of Matrix Metalloproteinase-12 Covalent Modification by a Photoaffinity Probe

Author

Athanasios Yiotakis, Bertrand Czarny, Fabrice Beau, Sarah Bregant, Robert Thai, Vincent Dive, Anne-Sophie Dabert-Gay, E. Lajeunesse, Laurent Devel, 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), and National and Kapodistrian University of Athens (NKUA)

Subjects
[SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, [SDV.CAN]Life Sciences [q-bio]/Cancer, Matrix metalloproteinase, Matrix (biology), Photoaffinity Labels, 01 natural sciences, Biochemistry, 03 medical and health sciences, Residue (chemistry), [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Side chain, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Chemistry, Mutagenesis, Active site, Cell Biology, 0104 chemical sciences, Covalent bond, Biophysics, biology.protein
Abstract

Mass spectroscopy, microsequencing, and site-directed mutagenesis studies have been performed to identify in human matrix metalloelastase (hMMP-12) residues covalently modified by a photoaffinity probe, previously shown to be able to covalently label specifically the active site of matrix metalloproteinases (MMPs). Results obtained led us to conclude that photoactivation of this probe in complex with hMMP-12 affects a single residue in human MMP-12, Lys241, through covalent modification of its side chain e NH2 group. Because x-ray and NMR studies of hMMP-12 indicate that Lys241 side chain is highly flexible, our data reveal the existence of particular Lys241 side-chain conformation in which the e NH2 group points toward the photolabile group of the probe, an event explaining the high levels of cross-linking yield between hMMP-12 and the probe. Lys241 is not conserved in MMPs, thus differences in cross-linking yields observed with this probe between MMP members may be linked to the residue variability observed at position 241 in this family.

Published
2008

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