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88 results on '"Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²)"'

1. Compared Binding Properties between Resveratrol and Other Polyphenols to Plasmatic Albumin: Consequences for the Health Protecting Effect of Dietary Plant Microcomponents

Author

Allan Lançon, René Buchet, Norbert Latruffe, Matthias Menzel, Dominique Delmas, Lipides - Nutrition - Cancer (U866) (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA), Université Bourgogne Franche-Comté [COMUE] (UBFC), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Centre National de la Recherche Scientifique (CNRS)-École Supérieure Chimie Physique Électronique de Lyon-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)-École Supérieure Chimie Physique Électronique de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon, Laboratoire Bio-PeroxIL. Biochimie du Peroxysome, Inflammation et Métabolisme Lipidique (Bio-PeroxIL), Université de Bourgogne (UB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Bio-PeroxIL. Biochimie du Peroxysome, Inflammation et Métabolisme Lipidique ( Bio-PeroxIL ), Université de Bourgogne ( UB ), Lipides - Nutrition - Cancer (U866) ( LNC ), Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ), Métabolisme, Enzymes et Mécanismes Moléculaires ( MEM² ), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires ( ICBMS ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon ( INSA Lyon ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS ) -Université Claude Bernard Lyon 1 ( UCBL ), and Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS )

Subjects
structural changes, [SDV]Life Sciences [q-bio], Pharmaceutical Science, Review, Resveratrol, resveratrol, 01 natural sciences, Analytical Chemistry, quercetin, chemistry.chemical_compound, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Resveratrol binding, Drug Discovery, Stilbenes, Bovine serum albumin, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, biology, food and beverages, Blood proteins, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 3. Good health, Biochemistry, Chemistry (miscellaneous), Health, Molecular Medicine, fluorescence, Plants, Edible, Quercetin, Serum albumin, lcsh:QD241-441, 03 medical and health sciences, lcsh:Organic chemistry, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Physical and Theoretical Chemistry, Binding site, Serum Albumin, 030304 developmental biology, Flavonoids, Binding Sites, 010405 organic chemistry, Organic Chemistry, Albumin, Polyphenols, 0104 chemical sciences, Diet, chemistry, Food, biology.protein, affinity
Abstract

International audience; Phytophenols are considered to have beneficial effects towards human physiology. They are food microcomponents with potent chemopreventive properties towards the most three frequent contemporary human diseases, e.g., cardiovascular alterations, cancer and neurodegenerative pathologies. Related to this, the plasmatic form and plasmatic level of plant polyphenols in the body circulation are crucial for their efficiency. Thus, determinations of the binding process of resveratrol and of common flavonoids produced by major edible plants, berries and fruits to plasma proteins are essential. The interactions between resveratrol and albumin, a major plasma protein, were compared with those already published, involving curcumin, genistein, quercetin and other well-known food-containing polyphenols. The approaches used are usually intrinsic fluorescence intensity changes, quenching of protein intrinsic fluorescence and infrared spectroscopy. It appears that: (1) all of the studied polyphenols interact with albumin; (2) while most of the studied polyphenols interact at one albumin binding site, there are two different types of resveratrol binding sites for bovine serum albumin, one with the highest affinity (apparent KD of 4 µM) with a stoichiometry of one per monomer and a second with a lower affinity (apparent KD of 20 µM) with also a stoichiometry of one per monomer; (3) at least one binding site is in the vicinity of one tryptophanyl residue of bovine serum albumin; and (4) resveratrol binding to bovine serum albumin produces a very small structural conformation change of the polypeptide chain. These results support a role played by polyphenols-albumin interactions in the plasma for the bio-activities of these food microcomponents in the body.

Published
2014

2. Nano/micro implant debris affect osteogenesis by chondrocytes: Comparison between ceramic and UHMWPE from hip walking simulator

Author

Jean Geringer, Marwa Ben Braham, Leyre Brizuela, Ana-Maria Trunfio-Sfarghiu, Ines Essefi, Saida Mebarek, Samir Hamza, Yves Berthier, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Mines Saint-Etienne, Univ Lyon, Univ Jean Monnet, INSERM, U 1059 Sainbiose, Centre CIS, F - 42023 Saint-Etienne France, Laboratoire de Mécanique des Contacts et des Structures [Villeurbanne] (LaMCoS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Modélisation et Simulation Multi Echelle (MSME), and Université Paris-Est Marne-la-Vallée (UPEM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Ceramics, Materials science, Osteolysis, Biocompatibility, Biomedical Engineering, Nanoparticle, Walking, Biomaterials, [SPI]Engineering Sciences [physics], chemistry.chemical_compound, Chondrocytes, Osteogenesis, Materials Testing, Nano, medicine, Humans, Ceramic, Lubricant, Simulation, Polyethylene, Intergranular corrosion, medicine.disease, Prosthesis Failure, chemistry, visual_art, visual_art.visual_art_medium, Hip Prosthesis, Polyethylenes
Abstract

A new generation of ceramic on ceramic (BIOLOX ®delta) bearings has emerged more than 10 years ago proving a high resistance to wear and good clinical results. However, biological reactions to wear debris, particularly the nanoparticles, need to be evaluated. The first originality of this study is to start from real wear particles obtained by the hip walking simulator (CERsim). These particles were compared with particles obtained by usual methods to assess the biocompatibility of materials: press machine (CERpress). Two ranges of ceramic particles were thus observed: ceramic particles with micron (intergranular fractures) and nano sizes (intragranular fractures), and characterized compared to ultra-high molecular weight polyethylene (UHMWPE). The second originality of this work is to assess the cellular reaction using the primary joint chondrocyte cultures simulating the osteogenesis process and not the cell lines, which are used to simulate the biological reaction of osteolysis. The first results showed a significant difference in cell viability between the cells in contact with particles from the walking simulator and those obtained with the press machine. On the other hand, it was found that the way of extraction of the particles from the lubricant could significantly affect the biological reaction. More interestingly, nano-sized ceramic particles showed a significant impact on the secretion of functional inflammatory mediators, agreeing with recent results in vivo. These novel methods of characterizing the osteogenic impact of UHMWPE and ceramic wear debris can complement the conventional expertise method focusing previously on the osteolysis aspect.

Published
2021

3. Phospholipase D inhibitors screening: Probing and evaluation of ancient and novel molecules

Author

Karim Bessaa, Meryem Aydin, Abdelkarim Abousalham, Alexandre Noiriel, Leyre Brizuela, Mickaël V. Cherrier, Renaud Rahier, Houda Abla, Yani Arhab, Florent Perret, Saida Mebarek, Arnaud Comte, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Université Grenoble Alpes (UGA), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Inhibitor, Cell, Drug Evaluation, Preclinical, 02 engineering and technology, Biochemistry, Chemical library, 03 medical and health sciences, chemistry.chemical_compound, X-Ray Diffraction, Structural Biology, Scattering, Small Angle, Phosphatidic acid, medicine, Phospholipase D, Humans, Vanadate, Enzyme Inhibitors, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Hydrolysis, Vigna, General Medicine, 021001 nanoscience & nanotechnology, High-Throughput Screening Assays, Enzyme, medicine.anatomical_structure, chemistry, Alcohols, Phosphodiester bond, Second messenger system, Salts, lipids (amino acids, peptides, and proteins), Vanadates, 0210 nano-technology
Abstract

International audience; Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 μM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.

Published
2021

4. Prostate cancer-derived exosomes promote osteoblast differentiation and activity through phospholipase D2

Author

Leyre Brizuela, Mathieu Borel, David Magne, Saida Mebarek, René Buchet, Giovanna Lollo, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'automatique, de génie des procédés et de génie pharmaceutique (LAGEPP), Université de Lyon-Université de Lyon-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Matériaux d'intérêt biologique, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0301 basic medicine, Male, [SDV]Life Sciences [q-bio], [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Exosomes, Exosome, Metastasis, 03 medical and health sciences, Mice, 0302 clinical medicine, medicine, Phospholipase D, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Cells, Cultured, Osteoblasts, Chemistry, PLD2, Bone metastasis, Prostatic Neoplasms, Osteoblast, Cell Differentiation, 3T3 Cells, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, medicine.disease, Microvesicles, 3. Good health, 030104 developmental biology, medicine.anatomical_structure, Tumor progression, 030220 oncology & carcinogenesis, Cancer research, Molecular Medicine
Abstract

Prostate cancer (PCa) is the most frequent cancer in men aged 65 and over. PCa mainly metastasizes in the bone, forming osteosclerotic lesions, inducing pain, fractures, and nerve compression. Cancer cell-derived exosomes participate in the metastatic spread, ranging from oncogenic reprogramming to the formation of pre-metastatic niches. Moreover, exosomes were recently involved in the dialog between PCa cells and the bone metastasis microenvironment. Phospholipase D (PLD) isoforms PLD1/2 catalyze the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), regulating tumor progression and metastasis. PLD is suspected to play a role in exosomes biogenesis. We aimed to determine whether PCa-derived exosomes, through PLD, interact with the bone microenvironment, especially osteoblasts, during the metastatic process. Here we demonstrate for the first time that PLD2 is present in exosomes of C4-2B and PC-3 cells. C4-2B-derived exosomes activate proliferation and differentiation of osteoblasts models, by stimulating ERK 1/2 phosphorylation, by increasing the tissue-nonspecific alkaline phosphatase activity and the expression of osteogenic differentiation markers. Contrariwise, when C4-2B exosomes are generated in the presence of halopemide, a PLD pan-inhibitor, they lose their ability to stimulate osteoblasts. Furthermore, the number of released exosomes diminishes significantly (-40%). When the PLD product PA is combined with halopemide, exosome secretion is fully restored. Taken together, our results indicate that PLD2 stimulates exosome secretion in PCa cell models as well as their ability to increase osteoblast activity. Thus, PLD2 could be considered as a potent player in the establishment of PCa bone metastasis acting through tumor cell derived-exosomes.

Published
2020

5. Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase

Author

E.L. Matera, J. Como, René Buchet, C. Mansouri, Caroline Fonta, Saida Mebarek, Anne Briolay, Marie Gleizes, A. El Jamal, José Luis Millán, Laurence Bessueille, Etienne Mornet, Charles Dumontet, David Magne, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Sanford Burnham Prebys Medical Discovery Institute, Centre de recherche cerveau et cognition (CERCO), Institut des sciences du cerveau de Toulouse. (ISCT), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, medicine.medical_specialty, Histology, Physiology, [SDV]Life Sciences [q-bio], Endocrinology, Diabetes and Metabolism, medicine.medical_treatment, Anti-Inflammatory Agents, 030209 endocrinology & metabolism, Article, 03 medical and health sciences, Mice, 0302 clinical medicine, Calcification, Physiologic, ATP hydrolysis, Nucleotidases, Nucleotidase, Internal medicine, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Osteoblasts, Chemistry, Mesenchymal stem cell, Hypophosphatasia, ALPL, medicine.disease, Alkaline Phosphatase, Adenosine, 030104 developmental biology, Endocrinology, Cytokine, Alkaline phosphatase, medicine.drug
Abstract

International audience; Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/− mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/− mice had significantly increased levels of Il-1β and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 μM of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1β, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Published
2020

6. NMR metabolomics for understanding the role of alkaline phosphatase in neurotransmission and inflammation processes

Author

BALAYSSAC, Stéphane, Cruz, Thomas, Gleizes, Marie, Lutin, Gary, Como, Juana, Briolay, Anne, Nowak, Lionel G, Malet-Martino, Myriam, Magne, David, Fonta, Caroline, Gilard, Véronique, Fonta, Caroline, Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique (SPCMIB), Institut de Chimie de Toulouse (ICT-FR 2599), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche cerveau et cognition (CERCO), Institut des sciences du cerveau de Toulouse. (ISCT), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Institut de Chimie de Toulouse (ICT), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
[CHIM] Chemical Sciences, [CHIM]Chemical Sciences
Abstract

International audience; Introduction Tissue Non-specific Alkaline Phosphatase (TNAP) is a ubiquitous ectoenzyme located in different body tissues. Mutations of the TNAP gene are responsible for hypophosphatasia, a rare disease characterized mainly by defective bone mineralization caused by pyrophosphate accumulation in bone tissue and accompanied by neurological symptoms (pain, seizure…) [1]. We aimed in identifying metabolic pathways associated with TNAP activity in brain and in exploring its role in the inflammatory process.Technological and methodological innovation Untargeted 1H NMR-based metabolomics was used on tissue extracts from two original models: (i) Akp2 mice, which is the rodent model of hypophosphatasia by invalidation of the TNAP gene and (ii) a model of postprandial inflammation based on food intake [2]. Results and impactEight metabolites differentiated TNAP-deficient mouse brain (in 7 day-old animals), among which GABA, adenosine and cystathionine were the most discriminant [3]. Metabolic changes were also observed in both liver and brain in the postprandial inflammation model. These metabolic changes suggest that TNAP is (i) involved in the production of anti-inflammatory agents (adenosine) or their precursor (cystathionine, precursor of H2S), (ii) regulated by pro-inflammatory cytokines

Published
2020

7. Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments

Author

Warren Madanamothoo, Michele Fiore, René Buchet, Dimitri Fayolle, Bernard Fenet, Emiliano Altamura, Pasquale Stano, Alice D'Onofrio, Fabio Mavelli, Peter Strazewski, Chimie Organique 2-Glycochimie (CO2GLYCO), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie, Catalyse, Polymères et Procédés, R 5265 (C2P2), Université de Lyon-Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre Commun de Résonance Magnétique Nucléaire, Université Lyon1 (CCRMN), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Roma Tre University, Fayolle, Dimitri, Altamura, Emiliano, D'Onofrio, Alice, Madanamothoo, Warren, Fenet, Bernard, Mavelli, Fabio, Buchet, René, Stano, Pasquale, Fiore, Michele, Strazewski, Peter, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Équipe Robotique et InteractionS (LAAS-RIS), Laboratoire d'analyse et d'architecture des systèmes [Toulouse] (LAAS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique [Toulouse] (INP)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UPS), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique [Toulouse] (INP)

Subjects
chemistry.chemical_classification, Phosphatidylethanolamine, Multidisciplinary, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Vesicle, Chemical structure, lcsh:R, Fatty acid, lcsh:Medicine, Phosphatidic acid, 010402 general chemistry, 01 natural sciences, Article, 0104 chemical sciences, chemistry.chemical_compound, Membrane, chemistry, Amphiphile, Biophysics, Glycerol, lcsh:Q, lcsh:Science
Abstract

It is an open question how the chemical structure of prebiotic vesicle-forming amphiphiles complexified to produce robust primitive compartments that could safely host foreign molecules. Previous work suggests that comparingly labile vesicles composed of plausibly prebiotic fatty acids were eventually chemically transformed with glycerol and a suitable phosphate source into phospholipids that would form robust vesicles. Here we show that phosphatidic acid (PA) and phosphatidylethanolamine (PE) lipids can be obtained from racemic dioleoyl glycerol under plausibly prebiotic phosphorylation conditions. Upon in situ hydration of the crude phosphorylation mixtures only those that contained rac-DOPA (not rac-DOPE) generated stable giant vesicles that were capable of encapsulating water-soluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction side-products (identified by IR and MS and quantified by 1H NMR) acted as co-surfactants and facilitated vesicle formation. To mimic the compositional variation of such primitive lipid mixtures, self-assembly of a combinatorial set of the above amphiphiles was tested, revealing that too high dioleoyl glycerol contents inhibited vesicle formation. We conclude that a decisive driving force for the gradual transition from unstable fatty acid vesicles to robust diacylglyceryl phosphate vesicles was to avoid the accumulation of unphosphorylated diacylglycerols in primitive vesicle membranes.

Published
2017

8. Overexpression of RANK and M-CSFR in Monocytes of G551D-Bearing Patients with Cystic Fibrosis

Author

Yvonne McCarthy, N.J. Ronan, Marie Laure Jourdain, Frédéric Velard, Dina Abdallah, Evelyn Flanagan, Barry J. Plant, Jacky Jacquot, Interfaces biomatériaux/tissus hôtes, Université de Reims Champagne-Ardenne (URCA)-IFR53-Institut National de la Santé et de la Recherche Médicale (INSERM), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Biomatériaux et inflammation en site osseux - EA 4691 (BIOS), Université de Reims Champagne-Ardenne (URCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), and Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)

Subjects
Male, 0301 basic medicine, Pulmonary and Respiratory Medicine, Oncology, medicine.medical_specialty, Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator, Receptor, Macrophage Colony-Stimulating Factor, Quinolones, Aminophenols, Critical Care and Intensive Care Medicine, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Cystic fibrosis, Monocytes, Cohort Studies, 03 medical and health sciences, 0302 clinical medicine, Internal medicine, medicine, Humans, Rank (graph theory), Chloride Channel Agonists, Receptor, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Regulation of gene expression, Receptor Activator of Nuclear Factor-kappa B, business.industry, Prognosis, medicine.disease, 030104 developmental biology, Gene Expression Regulation, 030228 respiratory system, Mutation, Female, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Cohort study
Abstract

International audience

Published
2018

9. Cooption of the pteridine biosynthesis pathway underlies the diversification of embryonic colors in water striders

Author

M. Emília Santos, Augustin Le Bouquin, Alexandra Berlioz-Barbier, François Bonneton, Abderrahman Khila, Carla Fernanda Burguez Floriano, Felipe Ferraz Figueiredo Moreira, Arnaud Salvador, Séverine Viala, Mathilde Bouchet, Marie Bernard, Aïdamalia Vargas-Lowman, David Armisén, Isabelle da Rocha Silva Cordeiro, Génétique Diversité et Ecophysiologie des Céréales (GDEC), Institut National de la Recherche Agronomique (INRA)-Université Blaise Pascal - Clermont-Ferrand 2 (UBP), Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), University of Basel (Unibas), ANABIO-MS - Analyse biomoléculaire par spectrométrie de masse - Biological Analysis by Mass Spectrometry, Institut des Sciences Analytiques (ISA), Centre National de la Recherche Scientifique (CNRS)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École normale supérieure - Lyon (ENS Lyon), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil., Physiopathologie, diagnostic et traitements des maladies osseuses / Pathophysiology, Diagnosis & Treatments of Bone Diseases (LYOS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut national de recherches archéologiques préventives (Inrap), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0106 biological sciences, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Embryo, Nonmammalian, diversification, genetic structures, Evolution, [SDV]Life Sciences [q-bio], Color, cooption, water striders, [SDV.BID]Life Sciences [q-bio]/Biodiversity, Biology, Eye, 010603 evolutionary biology, 01 natural sciences, Heteroptera, 03 medical and health sciences, Pigment, pteridine pathway, [CHIM]Chemical Sciences, Animals, [SDV.BDD]Life Sciences [q-bio]/Development Biology, ComputingMilieux_MISCELLANEOUS, Phylogeny, 030304 developmental biology, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Multidisciplinary, Pteridine biosynthesis, Phylogenetic tree, Pigmentation, Pteridines, Embryogenesis, Embryo, Pigments, Biological, Biological Sciences, biology.organism_classification, Embryonic stem cell, Biological Evolution, Phenotype, Developmental genetics, PNAS Plus, Evolutionary biology, visual_art, visual_art.visual_art_medium, Gerromorpha, Signal Transduction
Abstract

Significance Understanding how existing genomic content can be reused to generate new phenotypes is important for understanding how species diversify. Here, we address this question by studying the origin of a phenotype consisting of bright coloration in the embryos of water striders. We found that the pteridine biosynthesis pathway, originally active in the eyes, has been coopted in the embryo to produce various colors in the antennae and legs. The coopted pathway remained stable for over 200 million years, yet resulted in a striking diversification of colors and color patterns during the evolution of water striders. This work demonstrates how the activation of a complete pathway in new developmental contexts can drive the evolution of novelty and fuel species diversification., Naturalists have been fascinated for centuries by animal colors and color patterns. While widely studied at the adult stage, we know little about color patterns in the embryo. Here, we study a trait consisting of coloration that is specific to the embryo and absent from postembryonic stages in water striders (Gerromorpha). By combining developmental genetics with chemical and phylogenetic analyses across a broad sample of species, we uncovered the mechanisms underlying the emergence and diversification of embryonic colors in this group of insects. We show that the pteridine biosynthesis pathway, which ancestrally produces red pigment in the eyes, has been recruited during embryogenesis in various extraocular tissues including antennae and legs. In addition, we discovered that this cooption is common to all water striders and initially resulted in the production of yellow extraocular color. Subsequently, 6 lineages evolved bright red color and 2 lineages lost the color independently. Despite the high diversity in colors and color patterns, we show that the underlying biosynthesis pathway remained stable throughout the 200 million years of Gerromorpha evolutionary time. Finally, we identified erythropterin and xanthopterin as the pigments responsible for these colors in the embryo of various species. These findings demonstrate how traits can emerge through the activation of a biosynthesis pathway in new developmental contexts.

Published
2019

10. Exploring the influence of phospholipid monolayer conformation and environmental conditions on the interfacial binding of Gibberella Zeae lipase

Author

Abdelkarim Abousalham, Wuchong Chen, Fanghua Wang, Yonghua Wang, Bo Yang, Southern University of Science and Technology [Shenzhen] (SUSTech), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Circular dichroism, Gibberella, Molecular Conformation, Phospholipid, 02 engineering and technology, Sodium Chloride, Monolayer technology, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Maximum insertion pressure, Structural Biology, Phosphatidylcholine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Lipase, Molecular Biology, Phospholipids, 030304 developmental biology, Phosphocholine, 0303 health sciences, Degree of unsaturation, Dose-Response Relationship, Drug, biology, Gibberella zeae, Substrate (chemistry), General Medicine, Hydrogen-Ion Concentration, 021001 nanoscience & nanotechnology, Interfacial binding kinetics, chemistry, Biophysics, biology.protein, lipids (amino acids, peptides, and proteins), 0210 nano-technology, Protein Binding, Protein adsorption
Abstract

International audience; The involvement of different parameters on Gibberella zeae lipase (GZEL) membrane binding were characterized by using monomolecular film technology and circular dichroism spectroscopy. Among four kinds of phospholipid monolayers, 1,2‑dimyristoyl‑sn‑glycero‑3‑phosphoethanolamine have the highest maximum insertion pressure (MIP) value. Comparing the GZEL adsorption to phosphatidylcholine monolayers with different acyl chains in sn-1 and sn-2 positions, the higher MIP values were found for 1,2‑dilauroyl‑sn‑glycero‑3‑phosphocholine. Significantly improvement between 1,2‑dioleoyl‑sn‑glycero‑3‑phosphocholine and 1,2‑distearoyl‑sn‑glycero‑3‑phosphocholine suggested that the presence of fatty acid unsaturation may affect protein adsorption by changing the chemical structure in each phospholipid. The MIP value was shown higher (48.6 mN m−1) at pH 5 and pH 6 (47.5 ± 1.9 mN m−1) but decreased significantly (34.2 mN m−1) at pH 9. This may indicate that the proportion of helices in the protein decreases with the alteration of the catalytic center, thus affecting the binding of the protein to its substrate. The MIP values obviously decreased with increasing salt ion concentration, suggesting that excessive salt ion concentration may destabilize the secondary and tertiary structures of the protein, thereby affecting the characteristics of its adsorption at the interfaces. Present studies improve our understanding on the protein-membrane interaction of this enzyme.

Published
2019

11. Tribocorrosion of Polyethylene/Cobalt Contact Combined with Real-Time Fluorescence Assays on Living Macrophages: Development of A Multidisciplinary Biotribocorrosion Device

Author

Bernard Normand, C. Der-Loughian, Aline Bel-Brunon, Benoît Ter-Ovanessian, Saida Mebarek, L. Brizuela, Ana-Maria Trunfio-Sfarghiu, Yves Berthier, A. Impergre, Laboratoire de Mécanique des Contacts et des Structures [Villeurbanne] (LaMCoS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Matériaux, ingénierie et science [Villeurbanne] (MATEIS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Fluorescence microscopy, Materials science, Macrophage, Tribocorrosion, Metal ions in aqueous solution, chemistry.chemical_element, Ionic bonding, 02 engineering and technology, Polyethylene, 021001 nanoscience & nanotechnology, CoCrMo, Surfaces, Coatings and Films, Corrosion, Biomaterials, chemistry.chemical_compound, Chromium, 020303 mechanical engineering & transports, 0203 mechanical engineering, chemistry, Chemical engineering, Fluorescence microscope, Biotribocorrosion, [PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], 0210 nano-technology, Cobalt
Abstract

International audience; he test conditions currently used in biotribocorrosion devices often differ greatly from the physiological conditions of joint replacements, contributing to discrepancies between the simulated and actual life span of joint replacements. In this study, a multidisciplinary biotribocorrosion device was developed based on the limitations of existing tribocorrosimeters. The set-up enables corrosion measurements to be simultaneously performed with real-time visualization of living cells using fluorescence microscopy under dynamic loads and movements. The device was configured to simulate the joint contact of ankle prostheses, and the wear of ultra-high-molecular-weight polyethylene/cobalt alloy (CoCrMo) implants surrounded by murine macrophages was tested. Various characterization techniques (non-contact optical profilometry, scanning and fluorescence electron microscopy and quantitative analyses of metal ions and pro-inflammatory cytokines) were combined in-depth multidisciplinary study. Two experimental conditions were used to promote the production of either polyethylene wear particles or metal ions. The first results indicated two distinct tribocorrosion mechanisms: 1) adhesive wear coupled with slow ionic depassivation of the cobalt alloy. The main degradation products were micrometric spherical polyethylene particles that seem to have little impact effect on the metabolic activity of the macrophages. 2) Ionic wear with the production of small, fibrillar polyethylene particles was observed. The production of metal ions, mainly chromium, was the predominant degradation process. The cytotoxicity of the chromium ions was evaluated based on the secretion of pro-inflammatory cytokines (prostaglandin E2). Our findings indicate that simulated conditions that result in low mechanical wear but high ions release appear to be more harmful to cells.

Published
2019

12. Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization

Author

Meriem Chadli, Lukasz Bozycki, Maytê Bolean, José Luis Millán, Pietro Ciancaglini, Agnieszka Strzelecka-Kiliszek, Slawomir Pikula, Saida Mebarek, Nicola Rosato, Ana Maria Simao, René Buchet, Andrea Magrini, David Magne, Agnès Girard-Egrot, Massimo Bottini, Colin Farquharson, Justin S. Plaut, Sadik C. Esener, Nencki Institute of Experimental Biology, Polska Akademia Nauk = Polish Academy of Sciences (PAN), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Génie Enzymatique, Membrane Biomimétique et Assemblages Supramoléculaires (GEMBAS), and Centro Nacional de Microelectronica [Spain] (CNM)

Subjects
0301 basic medicine, Biomineralization, Mineralization, Biophysics, Nucleation, Chick Embryo, Microscopy, Atomic Force, Biochemistry, Mineralization (biology), Article, 03 medical and health sciences, Crystallinity, Atomic force microscopy, Extracellular Vesicles, Dynamic light scattering, Microscopy, Electron, Transmission, Spectroscopy, Fourier Transform Infrared, Zeta potential, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Settore BIO/10, Molecular Biology, Elastic modulus, Matrix vesicles, ComputingMilieux_MISCELLANEOUS, Nucleation core, 030102 biochemistry & molecular biology, Chemistry, Vesicle, Biomechanical Phenomena, 030104 developmental biology, Cartilage, Transmission electron microscopy, MICROSCOPIA
Abstract

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca(2+)) and inorganic phosphate (P(i)) within their lumen and forming a nucleation core (NC). After further sequestration of Ca(2+) and P(i), the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca(2+)) and non-mineralizing (–Ca(2+)) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca(2+). This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

Published
2019

13. Deletion the C-terminal peptides of Vibrio harveyi phospholipase D significantly improved its enzymatic properties

Author

Yonghua Wang, Wu Zongze, Bo Yang, Fanghua Wang, Abdelkarim Abousalham, Southern University of Science and Technology [Shenzhen] (SUSTech), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Tribologie et Dynamique des Systèmes (LTDS), École Centrale de Lyon (ECL), and Université de Lyon-Université de Lyon-École Nationale des Travaux Publics de l'État (ENTPE)-Ecole Nationale d'Ingénieurs de Saint Etienne-Centre National de la Recherche Scientifique (CNRS)

Subjects
Stereochemistry, Mutant, Phospholipid, 02 engineering and technology, Biochemistry, law.invention, 03 medical and health sciences, chemistry.chemical_compound, Surface-Active Agents, Structural Biology, law, Phospholipase D, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Enzyme kinetics, Amino Acid Sequence, Molecular Biology, Phospholipids, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Sequence Deletion, Vibrio, chemistry.chemical_classification, 0303 health sciences, biology, Vibrio harveyi, Temperature, General Medicine, Hydrogen-Ion Concentration, 021001 nanoscience & nanotechnology, biology.organism_classification, Peptide Fragments, Amino acid, Chemistry, Bioinorganic, Enzyme, chemistry, Recombinant DNA, Biocatalysis, 0210 nano-technology
Abstract

A novel phospholipase D that originate from marine Vibrio harveyi (VhPLD) was recombinant expressed and biochemically characterized. Moreover, effects of C-terminal peptides on catalytic and interfacial binding properties of VhPLD were investigated by constructing two truncated mutants (VhPLD-Δ(472–483) and VhPLD-Δ(437–483)). Optimal reaction temperature and pH value for wild-type VhPLD (VhPLD-WT) was 45 °C and pH 8.0. However, optimal reaction temperature of VhPLD-Δ(437–483) increased to 50 °C. Meanwhile, catalytic efficiency (kcat/KM) of VhPLD-Δ(472–483) and VhPLD-Δ(437–483) to the 1,2-Dioctanoyl-sn-glycero-3-phosphatidyl-p-nitrophenol (PpNP) was 12.9 and 14.2 times higher than that of VhPLD-WT. However, when compare the catalytic efficiency between VhPLD-Δ(472–483) and VhPLD-Δ(437–483), no significant change can be found between the two mutants. These results strongly indicated that the C-terminal 12 amino acids (472–483) have important role on the activity of VhPLD. Effects of C-terminal peptides on the interfacial binding properties of VhPLD to different phospholipid monolayers were also investigated by using monolayer film technology. Results of the maximum insertion pressure (MIP) indicated that deletion the C-terminal segment of VhPLD improved its interfacial binding properties to different phospholipid monolayers.

Published
2019

14. Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes

Author

Yuqing Wu, Saida Mebarek, Xiao-Yu Yuan, René Buchet, Carole Bougault, Leyre Brizuela, Zhongyuan Ren, David Magne, Chimie Supramoléculaire Appliquée (CSAP), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), State Key Laboratory of Supramolecular Structure and Materials, Jilin University, and Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²)

Subjects
Proteases, Cathepsin K, Clinical Biochemistry, Pharmaceutical Science, Matrix metalloproteinase, 01 natural sciences, Biochemistry, Cathepsin B, Chondrocyte, Cell Line, Mice, chemistry.chemical_compound, Chondrocytes, Nitriles, Drug Discovery, medicine, Animals, Humans, Protease Inhibitors, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Aza Compounds, 010405 organic chemistry, Organic Chemistry, Cell Dedifferentiation, Cysteine protease, 3. Good health, 0104 chemical sciences, Cell biology, 010404 medicinal & biomolecular chemistry, medicine.anatomical_structure, Arthritis therapy, chemistry, Drug Design, Molecular Medicine, Odanacatib
Abstract

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Published
2019

15. Identification of a new natural gastric lipase inhibitor from star anise

Author

Michele Fiore, Imed Koubaa, Ahmed Aloulou, Karine Alvarez, Mohamed Sellami, Renaud Rahier, Jannet Kamoun, Pascal Mansuelle, Régine Lebrun, Abdelkarim Abousalham, Frédéric Carrière, Alexandra Berlioz-Barbier, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Faculté des Sciences de Sfax, Université de Sfax - University of Sfax, Institut de Microbiologie de la Méditerranée (IMM), Plateforme Protéomique [Marseille], Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Plateforme de Spectrométrie de Masse Protéomique - Mass Spectrometry Proteomics Platform (MSPP), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA), Chimie Organique 2-Glycochimie (CO2GLYCO), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Enzymologie interfaciale et de physiologie de la lipolyse (EIPL), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-École Supérieure Chimie Physique Électronique de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-École Supérieure Chimie Physique Électronique de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon, Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Algorithmes, Composants, Modèles Et Services pour l'informatique répartie (ACMES-SAMOVAR), Services répartis, Architectures, MOdélisation, Validation, Administration des Réseaux (SAMOVAR), Institut Mines-Télécom [Paris] (IMT)-Télécom SudParis (TSP)-Institut Mines-Télécom [Paris] (IMT)-Télécom SudParis (TSP), Département Informatique (INF), Institut Mines-Télécom [Paris] (IMT)-Télécom SudParis (TSP), Institut Polytechnique de Paris (IP Paris), Centre National de la Recherche Scientifique (CNRS), Centre Commun de Spectrometrie de Masse (CCSM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Département Informatique (TSP - INF), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Star anise, [SDV]Life Sciences [q-bio], Flavonoid, Future application, Ether, Illicium, Mass Spectrometry, Serine, 03 medical and health sciences, chemistry.chemical_compound, Peptide mass fingerprinting, Gastric lipase, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Enzyme Inhibitors, Inhibition, chemistry.chemical_classification, 030109 nutrition & dietetics, Binding Sites, Chemistry, Plant Extracts, Stomach, General Medicine, Lipase, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Molecular Docking Simulation, Kinetics, 030104 developmental biology, Biochemistry, Molecular docking, Lipase inhibitors, Drug delivery, [SDV.AEN]Life Sciences [q-bio]/Food and Nutrition, Guided fractionation, Food Science
Abstract

International audience; The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

Published
2019

16. Pyrocarbon versus cobalt-chromium in the context of spherical interposition implants: an in vitro study on cultured chondrocytes

Author

Yves Berthier, Saida Mebarek, G. Ouenzerfi, Leyre Brizuela, A. Hannoun, M. Hassler, Carole Bougault, Ana-Maria Trunfio-Sfarghiu, Laboratoire de Mécanique des Contacts et des Structures [Villeurbanne] (LaMCoS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Wright Medical/Tornier, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
lcsh:Diseases of the musculoskeletal system, Cell Survival, Type II collagen, lcsh:Surgery, Biocompatible Materials, Context (language use), Matrix (biology), bone, Bone and Bones, Chondrocyte, Extracellular matrix, Mice, 03 medical and health sciences, Calcification, Physiologic, Chondrocytes, 0302 clinical medicine, medicine, Animals, [PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], rheology, cartilage, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Cell Proliferation, 030304 developmental biology, 030222 orthopedics, 0303 health sciences, Chemistry, Cartilage, Cell Membrane, Reproducibility of Results, Biomaterial, cultured chondrocytes, Implant, Prostheses and Implants, lcsh:RD1-811, cobalt-chromium, Carbon, Extracellular Matrix, medicine.anatomical_structure, Biophysics, pyrocarbon, Chromium Alloys, lcsh:RC925-935
Abstract

In the context of shoulder surgical replacement, a new generation of spherical interposition implants has been developed, with the implant being a mobile spacer rubbing against the glenoid cartilage and humeral bone cavity. The aim of the present study was to compare pyrocarbon (PyC) versus cobalt-chromium (CoCr) implants, regarding preservation and regeneration of the surrounding tissues. The effect of the biomaterials on chondrocytes was analysed in vitro. Murine primary chondrocytes were grown on discs made of PyC or CoCr using two culture media to mimic either cartilage-like or bone-like conditions (CLC or BLC). Chondrocytes did grow on PyC and CoCr without alteration in cell viability or manifestation of cytotoxicity. The tissue-like cell membranes grown under BLC were examined for the chondrocyte's ability to mineralise (by alizarin red matrix staining, calcium deposit and alkaline phosphatase activity) and for their mechanical properties (by rheological tests). For the chondrocytes grown under CLC and BLC, extracellular matrix components were analysed by histological staining and immunolabelling. Under CLC, PyC promoted type II collagen expression in chondrocytes, suggesting that they may generate a more cartilage-like matrix than samples grown on both CoCr and plastic control. In BLC, the tissue-like cell membranes grown on PyC were more mineralised and homogenous. The mechanical results corroborated the biological data, since the elastic modulus of the tissue-like cell membranes developed on the PyC surface was higher, indicating more stiffness. Overall, the results suggested that PyC might be a suitable biomaterial for spherical interposition implants.

Published
2019

17. Involvement of phospholipase D in mineralization process in cultured osteoblasts

Author

Abdallah, D., Skafi, N., Hamade, E., Reibel, S., Vitale, N., El Jamal, A., Bougault, C., Laroche, B., Vico, L., Badran, B., Hussein, N., Magne, D., Buchet, R., Brizuela, L, Mebarek, S., ICBMS, UMR5246, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

18. Phospholipase D: A new mediator during high phosphate-induced vascular calcification associated with chronic kidney disease

Author

Nader Hussein, Leyre Brizuela, Nicolas Vitale, Christophe O. Soulage, Najwa Skafi, Wissam H. Faour, Dina Abdallah, David Magne, Sophie Reibel, Bassam Badran, Eva Hamade, Saida Mebarek, René Buchet, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Physiologie intégrative, cellulaire et moléculaire (PICM), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Genomic and Health Laboratory, Genomic and Health Laboratory/PRASE-EDST, Plateforme d'hébergement et d'exploration fonctionnelle (Chronobiotron), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Cellulaires et Intégratives (INCI), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National de la Recherche Agronomique (INRA), Lebanese University [Beirut] (LU), Lebanese University (LU), Lebanese National Council for Scientific Research (CNRS-L), and Universite Claude Bernard Lyon 1

Subjects
Male, 0301 basic medicine, medicine.medical_specialty, Vascular smooth muscle, Phosphodiesterase Inhibitors, Physiology, [SDV]Life Sciences [q-bio], Myocytes, Smooth Muscle, Clinical Biochemistry, chronic kidney disease (CKD), Muscle, Smooth, Vascular, Cell Line, Rats, Sprague-Dawley, Tissue Culture Techniques, Mice, 03 medical and health sciences, 0302 clinical medicine, Internal medicine, Phospholipase D, medicine, Animals, Myocyte, Renal Insufficiency, Chronic, Vascular Calcification, vascular calcification (VC), Protein kinase C, Mice, Knockout, phospholipase D (PLD), Chemistry, PLD2, Cell Biology, Ascorbic acid, medicine.disease, 3. Good health, Calcium, Dietary, Disease Models, Animal, 030104 developmental biology, Endocrinology, 030220 oncology & carcinogenesis, Cell Transdifferentiation, Phosphorus, Dietary, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Ex vivo, Signal Transduction, Calcification
Abstract

International audience; Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and beta-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.

Published
2019

19. Multi-physical characterization of chondrocyte cultures in contact with different orthopaedic implants biomaterials

Author

Hannoun, A., Ouenzer, G., Mebarek, S., Bougault, C., Hassler, M., Berthier, Y., Sfarghi-Trunfio, A.M., Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), and ICBMS, UMR5246

Subjects
[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

20. Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators

Author

René Buchet, Camila B. Tovani, Ekeveliny Amabile Veschi, Ana Maria Simao, Bruno Z. Favarin, Maytê Bolean, José Luis Millán, Massimo Bottini, Ana Paula Ramos, Pietro Ciancaglini, Universidade de São Paulo (USP), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Sanford Burnham Medical Research Institute, and La Jolla

Subjects
Biomineralization, 0301 basic medicine, Proteolipids, Endocrinology, Diabetes and Metabolism, 030209 endocrinology & metabolism, Mineralization (biology), Article, Proteoliposome, Alkaline phosphatase, Matrix vesicles, Nucleational core, 03 medical and health sciences, chemistry.chemical_compound, Adenosine Triphosphate, Calcification, Physiologic, 0302 clinical medicine, Endocrinology, Spectroscopy, Fourier Transform Infrared, Animals, Humans, Orthopedics and Sports Medicine, Amorphous calcium phosphate, Settore BIO/10, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, Hydrolysis, Vesicle, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, Phosphatidylserine, Alkaline Phosphatase, Lipids, Dynamic Light Scattering, Kinetics, Membrane, Cellular Microenvironment, chemistry, Biophysics, lipids (amino acids, peptides, and proteins), METABOLISMO, 030101 anatomy & morphology, Sphingomyelin
Abstract

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators. We also found that the addition of cholesterol or sphingomyelin to TNAP-proteoliposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine reduced the ability of TNAP to induce biomineralization. Our results suggest that the lipid microenvironment is essential for the induction and propagation of minerals mediated by TNAP.

Published
2018

21. From electrochemical behaviour to biotribocorrosion of metal alloys dedicated to joint prostheses

Author

Impergre, A., Trunfio-Sfarghiu, Ana-Maria, Der Loughian, Christelle, Ter-Ovanessian, Benoît, Bel-Brunon, Aline, Youjil, Saida, Brizuela, Leyre, Berthier, Yves, Normand, Bernard, Laboratoire de Mécanique des Contacts et des Structures [Villeurbanne] (LaMCoS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Matériaux, ingénierie et science [Villeurbanne] (MATEIS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Tribologie et Mécanique des Interfaces (TMI), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Bel-Brunon, Aline

Subjects
[SPI]Engineering Sciences [physics], [SPI] Engineering Sciences [physics], [SPI.MAT] Engineering Sciences [physics]/Materials, ComputingMilieux_MISCELLANEOUS, [SPI.MAT]Engineering Sciences [physics]/Materials
Abstract

International audience

Published
2018

22. Plant phospholipase D mining unravels new conserved residues important for catalytic activity

Author

Abdelkarim Abousalham, Yani Arhab, Alexandre Noiriel, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, 0301 basic medicine, Gene isoform, Arabidopsis thaliana, Arabidopsis, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, transphosphatidylation, Catalytic Domain, Consensus sequence, Phospholipase D, Data Mining, Protein Isoforms, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Molecular Biology, C2 domain, Conserved Sequence, Phylogeny, chemistry.chemical_classification, Chemistry, Arabidopsis Proteins, HKD motif, Cell Biology, Phosphatidic acid, Plants, Small molecule, 030104 developmental biology, Enzyme, post-translational modification, Biochemistry, Mutation, Posttranslational modification, lipids (amino acids, peptides, and proteins), 010606 plant biology & botany
Abstract

International audience; Phospholipase D (PLD) is a key enzyme involved in numerous processes in all living organisms. Hydrolysis of phospholipids by PLD allows the release of phosphatidic acid which is a crucial intermediate of multiple pathways and signaling reactions, including tumorigenesis in mammals and defense responses in plants. One common feature found in the plant alpha isoform (PLDα), in some PLD from microbes and in all PLD from eukaryotes, is a duplicated motif named HKD involved in the catalysis. However, other residues are strictly conserved among these organisms and their role remains obscure. To gain further insights into PLD structure and the role of these conserved residues, we first looked for all the plant PLDα sequences available in public databases. With >200 sequences retrieved, a generic sequence was constructed showing that 138 residues are strictly conserved among plant PLDα, with some of them identical to residues found in mammalian PLDs. Using site-directed mutagenesis of the PLDα from Arabidopsis thaliana, we demonstrated that mutation of some of these residues abolished the PLD activity. Moreover, mutation of the residues around both HKD motifs enabled us to re-define the consensus sequence of these motifs. By sequential deletions of the N-terminal extremity, the minimum length of the domain required for catalytic activity was determined. Overall, this work furthers our understanding of the structure of eukaryotic PLDs and it may lead to the discovery of new regions involved in the catalytic reaction that could be targeted by small molecule modulators of PLDs.

Published
2018

23. Production, purification and biochemical characterization of a thermoactive, alkaline lipase from a newly isolated Serratia sp. W3 Tunisian strain

Author

Fatma Aloui, Abdelkarim Abousalham, Youssef Gargouri, Alexandre Noiriel, Nabil Smichi, Ahlem Eddehech, Zied Zarai, Université de Sfax - University of Sfax, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Serratia, Tributyrin, [SDV]Life Sciences [q-bio], Lipolysis, Detergents, 02 engineering and technology, Alkalies, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Hydrolysis, Structural Biology, Enzyme Stability, Extracellular, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Response surface methodology, Amino Acid Sequence, Lipase, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Ions, 0303 health sciences, Analysis of Variance, Chromatography, biology, Molecular mass, Chemistry, Temperature, General Medicine, Hydrogen-Ion Concentration, Models, Theoretical, 021001 nanoscience & nanotechnology, biology.organism_classification, 6. Clean water, Metals, biology.protein, Solvents, Specific activity, 0210 nano-technology
Abstract

A newly isolated Serratia sp. W3 strain was shown to secrete a non-induced lipase in the culture medium. Lipolytic activity was optimized using the response surface methodology (RSM) and the extracellular lipase from Serratia sp. W3 (SmL) was purified to homogeneity with a total yield of 10% and its molecular mass was estimated of about 67 kDa by SDS-PAGE. The amino acid sequence of the first 7 N-terminal residues of SmL revealed a high degree of homology with other Serratia lipase sequences. The purified SmL can be considered as thermoactive lipase, its maximal specific activity measured at pH 9 and 55 °C was shown to be 625 U/mg and 300 U/mg using tributyrin and olive oil emulsion as substrate, respectively. In contrast to other described Serratia lipases, SmL was found to be stable at a large scale of pH between pH 5 and pH 12. SmL was also able to hydrolyze its substrate in presence of various oxidizing agents as well as in presence of surfactants and some commercial detergents. Then, considering the overall biochemical properties of SmL, it can be considered as a potential candidate for industrial and biotechnological applications, such as synthesis of biodiesel and in the detergent industry.

Published
2018

24. Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies

Author

Arhab, Yani, Rahier, Renaud, Noiriel, Alexandre, Cherrier, Mickael V, Abousalham, Abdelkarim, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Chromatography, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Vigna, Gene Expression, food and beverages, Octyl-Sepharose, Pichia, Recombinant Proteins, Enzyme Activation, Transformation, Genetic, Pichia pastoris, pGAPZB vector, Phospholipase D, Cloning, Molecular, Vigna unguiculata, Plasmids
Abstract

International audience; The production of pure enzymes in high quantities is a proven strategy to study the catalytic mechanism as well as the solving of structure at the atomic scale for therapeutic or industrial purposes. Phospholipase D (PLD, EC 3.1.4.4) is found in a wide majority of living organisms and has been shown to be involved in signal transduction, vesicle trafficking, and membrane metabolism processes. Located at the membrane-cytoplasm interface, plant PLDs are soluble but also bear an evident hydrophobic aspect making challenging its expression and its purification in large quantity. So far there is no high-resolution three-dimensional structure for a eukaryotic PLD. The protocols herein describe the cloning of the eukaryotic recombinant PLDα of Vigna unguiculata (cowpea) into the yeast expression system Pichia pastoris and its two-step purification process. This allowed us to purify to homogeneity hundreds of micrograms of highly pure protein to conduct in fine structural studies.

Published
2018

25. Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus

Author

Wuchong Chen, Yonghua Wang, Ruixia Wei, Fanghua Wang, Bo Yang, Abdelkarim Abousalham, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Tribologie et Dynamique des Systèmes (LTDS), École Centrale de Lyon (ECL), and Université de Lyon-Université de Lyon-École Nationale des Travaux Publics de l'État (ENTPE)-Ecole Nationale d'Ingénieurs de Saint Etienne-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Phospholipid, Peptide, Catalysis, lcsh:Chemistry, Inorganic Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Monolayer, Phospholipase D, interfacial properties, Physical and Theoretical Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:QH301-705.5, Molecular Biology, Protein secondary structure, Vibrio parahaemolyticus, Spectroscopy, Binding selectivity, ComputingMilieux_MISCELLANEOUS, binding parameters, chemistry.chemical_classification, Organic Chemistry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, 3. Good health, Computer Science Applications, Amino acid, 030104 developmental biology, Enzyme, lcsh:Biology (General), lcsh:QD1-999, chemistry, Biophysics, lipids (amino acids, peptides, and proteins), monomolecular film
Abstract

The effects of N-terminal (1&ndash, 34 amino acids) and C-terminal (434&ndash, 487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor &ldquo, a&rdquo, indicated that the loop structure (1&ndash, 25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26&ndash, 34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434&ndash, 487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434&ndash, 469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

Published
2018

26. Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Author

Saida Mebarek, Julian A. Tanner, Lina Li, Mei Li Khong, Eric Lik Hang Lui, René Buchet, David Magne, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Indoles, Light, 02 engineering and technology, Chick Embryo, Biochemistry, law.invention, Extracellular matrix, chemistry.chemical_compound, law, Osteogenesis, Polyphosphates, Scattering, Radiation, Polyacrylamide gel electrophoresis, ComputingMilieux_MISCELLANEOUS, Microscopy, Confocal, Chemistry, Vesicle, Hydrolysis, Osteoblast, pathological conditions, signs and symptoms, 021001 nanoscience & nanotechnology, Extracellular Matrix, surgical procedures, operative, medicine.anatomical_structure, 0210 nano-technology, Biophysics, Bone and Bones, 03 medical and health sciences, Calcification, Physiologic, Microscopy, Electron, Transmission, Confocal microscopy, Cell Line, Tumor, otorhinolaryngologic diseases, medicine, Extracellular, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, DAPI, neoplasms, Molecular Biology, Osteoblasts, Cartilage, Alkaline Phosphatase, digestive system diseases, 030104 developmental biology, Durapatite, Type C Phospholipases, Calcium
Abstract

Background Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation. Methods PolyP was quantified by 4′,6-diamidino-2-phenylindole (DAPI) fluorescence and characterized by enzymatic methods coupled to urea polyacrylamide gel electrophoresis. Transmission electron microscopy and confocal microscopy were used to investigate polyP localization. A chicken embryo cartilage model was used to investigate the effect of polyP on mineralization. Results PolyP increased in concentration as SaOS-2 cells matured and mineralized. Particularly high levels of polyP were observed in MVs. The average length of MV polyP was determined to be longer than 196 Pi residues by gel chromatography. Electron micrographs of MVs, stained by two polyP-specific staining approaches, revealed polyP localization in the vicinity of the MV membrane. Additional extracellular polyP binds to MVs and inhibits MV-induced hydroxyapatite formation. Conclusion PolyP is highly enriched in matrix vesicles and can inhibit apatite formation. PolyP may be hydrolysed to phosphate for further mineralization in the extracellular matrix. General significance PolyP is a unique yet underappreciated macromolecule which plays a critical role in extracellular mineralization in matrix vesicles.

Published
2018

27. Molecular mechanisms of vascular smooth muscle cell trans-differentiation and calcification in atherosclerosis

Author

Roszkowska, Monika, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Université de Lyon, Instytut biologii doświadczalnej im. M. Nenckiego (Pologne), David Magne, Slawomir Pikula, and STAR, ABES

Subjects
Cellule musculaire lisse, Vascular smooth muscle cell, Tissue-nonspecific alkaline phosphatase, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphatase alcaline, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Calcification vasculaire, Chondrocyte, Vascular calcification
Abstract

Vascular calcification (VC) is a hallmark of atherosclerosis plaques. Calcification (formation of apatite) of advanced lesions share common features with endochondral ossification of long bones and appears to stabilize plaques. This process is associated with trans-differentiation of vascular smooth muscle cells (VSMCs) into chondrocyte-like cells. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. The two proteins necessary for physiological mineralization are tissue-nonspecific alkaline phosphatase (TNAP) and collagen. Under pathological conditions, TNAP is activated by inflammatory cytokines in VSMCs, whereas collagen is produced constantly. The activation of TNAP appears to induce calcification of these cells. Therefore, the objective of this PhD thesis was to study the role of TNAP and generated apatite crystals in the VSMC trans-differentiation and determine underlying molecular mechanisms. Based on the obtained results, we propose that activation of BMP-2, a strong inducer of ectopic calcification, and formation of apatite crystals generated by TNAP represents a likely mechanism responsible for stimulation of VSMC trans-differentiation. Moreover, we were interested in localization and function of mineralization markers such as TNAP and annexins in mineralization process mediated by trans-differentiated VSMCs and VSMC-derived matrix vesicles (MVs). We observed that, similarly as in the case of typical mineralizing cells, increased TNAP activity in VSMC-derived MVs and association with collagen were important for their ability to mineralize, Chez les patients atteints d'athérosclérose, les calcifications vasculaires sont une caracteristique des plaques d'athérome. Elles résultent de la trans-différenciation des cellules musculaires lisses (CMLs) en cellules de type ostéoblastique et/ou chondrocytaire, notamment en réponse à des cytokines inflammatoires. Les CMLs forment alors des cristaux par l'activité de la phosphatase alcaline non-spécifique du tissu (TNAP). A la lumière de résultats récents, nous avons émis l'hypothèse que la TNAP module la trans-différenciation des CMLs. Nos objectifs étaient donc de déterminer l'effet de la TNAP dans la trans-différenciation des CMLs, et d'étudier les mécanismes impliqués dans son induction. Nous avons observé que l'ajout de phosphatase alcaline purifiée ou la surexpression de TNAP stimule l'expression de marqueurs chondrocytaires en culture de CMLs et de cellules souches mésenchymateuses. De plus, l'inhibition de la TNAP bloque la maturation de chondrocytes primaires. Nous avons observé un rôle des cristaux formés par la TNAP, puisque l'ajout de cristaux seuls ou associés à une matrice collagénique a reproduit les effets de la TNAP. Nous suspectons que la TNAP agit en hydrolysant le PPi et en générant des cristaux. Ces cristaux ensuite induisent l'expression du facteur ostéogénique BMP-2 et l'inhibition des effets de la BMP-2 annule les effets de la TNAP. De plus, nous étions intéressés par les la localisation et la fonction de marqueurs de minéralisation comme les annexines en parallèle de la TNAP. Nous avons observé que l'activité TNAP des CMLs induit la minéralisation en grande partie quand la TNAP est associée aux vésicules matricielles et au fibres de collagène

Published
2018

28. Mécanismes moléculaires de la transdifférenciation des cellules musculaires lisses et calcification dans l'athérosclérose

Author

Roszkowska, Monika, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Université de Lyon, Instytut biologii doświadczalnej im. M. Nenckiego (Pologne), David Magne, and Slawomir Pikula

Subjects
Cellule musculaire lisse, Vascular smooth muscle cell, Tissue-nonspecific alkaline phosphatase, Phosphatase alcaline, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Calcification vasculaire, Chondrocyte, Vascular calcification
Abstract

Vascular calcification (VC) is a hallmark of atherosclerosis plaques. Calcification (formation of apatite) of advanced lesions share common features with endochondral ossification of long bones and appears to stabilize plaques. This process is associated with trans-differentiation of vascular smooth muscle cells (VSMCs) into chondrocyte-like cells. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. The two proteins necessary for physiological mineralization are tissue-nonspecific alkaline phosphatase (TNAP) and collagen. Under pathological conditions, TNAP is activated by inflammatory cytokines in VSMCs, whereas collagen is produced constantly. The activation of TNAP appears to induce calcification of these cells. Therefore, the objective of this PhD thesis was to study the role of TNAP and generated apatite crystals in the VSMC trans-differentiation and determine underlying molecular mechanisms. Based on the obtained results, we propose that activation of BMP-2, a strong inducer of ectopic calcification, and formation of apatite crystals generated by TNAP represents a likely mechanism responsible for stimulation of VSMC trans-differentiation. Moreover, we were interested in localization and function of mineralization markers such as TNAP and annexins in mineralization process mediated by trans-differentiated VSMCs and VSMC-derived matrix vesicles (MVs). We observed that, similarly as in the case of typical mineralizing cells, increased TNAP activity in VSMC-derived MVs and association with collagen were important for their ability to mineralize; Chez les patients atteints d'athérosclérose, les calcifications vasculaires sont une caracteristique des plaques d'athérome. Elles résultent de la trans-différenciation des cellules musculaires lisses (CMLs) en cellules de type ostéoblastique et/ou chondrocytaire, notamment en réponse à des cytokines inflammatoires. Les CMLs forment alors des cristaux par l'activité de la phosphatase alcaline non-spécifique du tissu (TNAP). A la lumière de résultats récents, nous avons émis l'hypothèse que la TNAP module la trans-différenciation des CMLs. Nos objectifs étaient donc de déterminer l'effet de la TNAP dans la trans-différenciation des CMLs, et d'étudier les mécanismes impliqués dans son induction. Nous avons observé que l'ajout de phosphatase alcaline purifiée ou la surexpression de TNAP stimule l'expression de marqueurs chondrocytaires en culture de CMLs et de cellules souches mésenchymateuses. De plus, l'inhibition de la TNAP bloque la maturation de chondrocytes primaires. Nous avons observé un rôle des cristaux formés par la TNAP, puisque l'ajout de cristaux seuls ou associés à une matrice collagénique a reproduit les effets de la TNAP. Nous suspectons que la TNAP agit en hydrolysant le PPi et en générant des cristaux. Ces cristaux ensuite induisent l'expression du facteur ostéogénique BMP-2 et l'inhibition des effets de la BMP-2 annule les effets de la TNAP. De plus, nous étions intéressés par les la localisation et la fonction de marqueurs de minéralisation comme les annexines en parallèle de la TNAP. Nous avons observé que l'activité TNAP des CMLs induit la minéralisation en grande partie quand la TNAP est associée aux vésicules matricielles et au fibres de collagène

Published
2018

29. An Optimized Method to Generate Human Active Osteoclasts From Peripheral Blood Monocytes

Author

Abdallah, Dina, Jourdain, Marie-Laure, Braux, Julien, Guillaume, Christine, Gangloff, Sophie C., Jacquot, Jacky, Velard, Frédéric, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Biomatériaux et inflammation en site osseux - EA 4691 (BIOS), Université de Reims Champagne-Ardenne (URCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV), Interfaces biomatériaux/tissus hôtes, Université de Reims Champagne-Ardenne (URCA)-IFR53-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université de Reims Champagne-Ardenne (URCA)

Subjects
human peripheral blood mononuclear cells, vitamin D3, lcsh:Immunologic diseases. Allergy, Macrophage Colony-Stimulating Factor, RANK Ligand, Immunology, Cell Culture Techniques, Cell Differentiation, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], dentin matrix resorption, osteoclasts, Blood Circulation, Dentin, CD14+ monocytes, Methods, Leukocytes, Mononuclear, Animals, Humans, Immunology and Allergy, Cattle, Bone Resorption, lcsh:RC581-607, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Cholecalciferol
Abstract

Osteoclasts (OCs), the bone-resorbing cells, play a key role in skeletal development and adult bone remodeling. They also participate in the pathogenesis of various bone disorders. One of the major technical difficulties in the generation of OCs, when working on human material, is the ability to achieve large differentiation of mature OCs from human peripheral blood mononuclear cells (PBMCs). Access to a standardized source of active OCs is needed to better analyze the roles of human OCs. The aim of this study was to develop a procedure yielding active and mature OCs from fresh human PBMCs. We therefore examined the differentiation of PBMCs to OCs in different cell culture media, using non-stripped and charcoal-stripped sera in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). We also studied the effects of vitamin D3 in the differentiation level of PBMCs to OCs. Phalloidin-AlexaFluor®488/DAPI fluorescent stainings and dentin resorption analyses by scanning electron microscopy were used to identify the number and size of differentiated OCs, number of nuclei per cell and resorption activities of OCs for a 7–14–21-day culture period. This study reports an optimized method for an efficient production of human active OCs from a low seeding density of PBMCs, after a 14-day culture period by using a medium containing fetal bovine charcoal-stripped serum in the presence of M-CSF and RANKL, and in the absence of vitamin D3.

Published
2018

30. Breast Cancer Targeting through Inhibition of the Endoplasmic Reticulum-Based Apoptosis Regulator Nrh/BCL2L10

Author

Adrien Nougarède, Nikolay Popgeorgiev, Stephane Borel, Olivier Marcillat, Loay Kassem, Soleilmane Omarjee, Rudy Gadet, Ruth Rimokh, Isabelle Treilleux, Jonathan Lopez, Bruno O. Villoutreix, Germain Gillet, Ivan Mikaelian, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Clinical Oncology Department, Cairo University - Faculty of Medicine, Aptanomics, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Département d'anatomopathologie, biopathologie, Centre Léon Bérard [Lyon], Molécules Thérapeutiques in silico (MTI), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie et chimie des protéines [Lyon] (IBCP), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Cancer Research, [SDV]Life Sciences [q-bio], Apoptosis, Breast Neoplasms, [SDV.CAN]Life Sciences [q-bio]/Cancer, Drug resistance, Mice, SCID, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Endoplasmic Reticulum, 03 medical and health sciences, 0302 clinical medicine, Breast cancer, In vivo, Cell Line, Tumor, Proto-Oncogene Proteins, Biomarkers, Tumor, Medicine, Neoplasm, Animals, Humans, Inositol 1,4,5-Trisphosphate Receptors, Molecular Targeted Therapy, Receptor, Binding Sites, Apoptosis Regulator, business.industry, Endoplasmic reticulum, Cancer, medicine.disease, Prognosis, Xenograft Model Antitumor Assays, Peptide Fragments, 3. Good health, 030104 developmental biology, Oncology, Proto-Oncogene Proteins c-bcl-2, Drug Resistance, Neoplasm, 030220 oncology & carcinogenesis, Cancer research, Calcium, Female, business, Peptides
Abstract

Drug resistance and metastatic relapse remain a top challenge in breast cancer treatment. In this study, we present preclinical evidence for a strategy to eradicate advanced breast cancers by targeting the BCL-2 homolog Nrh/BCL2L10, which we discovered to be overexpressed in >45% of a large cohort of breast invasive carcinomas. Nrh expression in these tumors correlated with reduced metastasis-free survival, and we determined it to be an independent marker of poor prognosis. Nrh protein localized to the endoplasmic reticulum. Mechanistic investigations showed that Nrh made BH4 domain–dependent interactions with the ligand-binding domain of the inositol-1,4,5-triphosphate receptor (IP3R), a type 1/3 Ca2+ channel, allowing Nrh to negatively regulate ER-Ca2+ release and to mediate antiapoptosis. Notably, disrupting Nrh/IP3R complexes by BH4 mimetic peptides was sufficient to inhibit the growth of breast cancer cells in vitro and in vivo. Taken together, our results highlighted Nrh as a novel prognostic marker and a candidate therapeutic target for late stage breast cancers that may be addicted to Nrh. Significance: These findings offer a comprehensive molecular model for the activity of Nrh/BCL2L10, a little studied antiapoptotic molecule, prognostic marker, and candidate drug target in breast cancer. Cancer Res; 78(6); 1404–17. ©2018 AACR.

Published
2018

31. Collagen promotes matrix vesicle-mediated mineralization by vascular smooth muscle cells

Author

David Magne, Laurence Bessueille, Agnieszka Strzelecka-Kiliszek, Monika Roszkowska, René Buchet, Slawomir Pikula, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Nencki Institute of Experimental Biology, and Polska Akademia Nauk = Polish Academy of Sciences (PAN)

Subjects
0301 basic medicine, Vascular smooth muscle, Myocytes, Smooth Muscle, 030204 cardiovascular system & hematology, Biochemistry, Mineralization (biology), Muscle, Smooth, Vascular, Inorganic Chemistry, 03 medical and health sciences, Mice, 0302 clinical medicine, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Vascular Calcification, Endochondral ossification, ComputingMilieux_MISCELLANEOUS, Chemistry, Vesicle, medicine.disease, Ascorbic acid, Alkaline Phosphatase, Atherosclerosis, 3. Good health, Cell biology, 030104 developmental biology, Collagenase, Alkaline phosphatase, Collagen, Calcification, medicine.drug
Abstract

Vascular calcification (VC) is a hallmark of atherosclerotic plaques. Calcification of advanced plaques shares common features with endochondral ossification of long bones and appears to be protective. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. Tissue-nonspecific alkaline phosphatase (TNAP) and collagen are the two proteins necessary for physiological mineralization. Here, we demonstrate the presence of membrane-bound TNAP, detected by immunofluorescence, that seems to form clusters on the plasma membrane of vascular smooth muscle cells (VSMCs) cultured in mineralizing conditions. We observed that TNAP activity and mineralization were increased when VSMCs were cultured in the presence of ascorbic acid (AA) and β-glycerophosphate (β-GP). Increased TNAP activity was observed in whole cell lysates, total membrane fractions and, more particularly, in matrix vesicles (MVs). We have shown that TNAP-enriched MVs released from VSMCs subjected to collagenase contained more apatite-like mineral than the less TNAP-rich/TNAP-enriched vesicles isolated without collagenase treatment. These results suggest a role for collagen in promoting calcification induced by TNAP in atherosclerotic plaques.

Published
2018

32. Direct and Continuous Measurement of Phospholipase D Activities Using the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline

Author

Abdelkarim Abousalham, Renaud Rahier, Yani Arhab, Houda Abla, Alexandre Noiriel, ICBMS, UMR5246, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, chemistry.chemical_classification, 030102 biochemistry & molecular biology, Phospholipase D, Substrate (chemistry), Phosphatidic acid, Fluorescence, 3. Good health, enzymes and coenzymes (carbohydrates), 03 medical and health sciences, Hydrolysis, chemistry.chemical_compound, Enzyme, chemistry, Phosphatidylcholine, Biophysics, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, lipids (amino acids, peptides, and proteins), Chelation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

Phospholipase D (PLD) hydrolyzes phospholipids to form phosphatidic acid (PA) and the corresponding headgroup. To date, PLD has been linked to several pathologies, such as cancer, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released upon phosphatidylcholine (PC) hydrolysis. Therefore, we designed a PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline. This assay exhibits a strong fluorescence signal upon Ca2+ complexation with the PLD-generated PA and is not limited to PC as the substrate but allows the use of natural phospholipids with various headgroups. Besides, this easy-to-handle assay allows to monitor prokaryotic and eukaryotic PLD activities in a continuous way and on a microplate scale.

Published
2018

33. Matrix vesicles from chondrocytes and osteoblasts: their biogenesis, properties, functions and biomimetic models

Author

Joanna Bandorowicz Pikula, Karen L. Anderson, Massimo Bottini, Pietro Ciancaglini, David Magne, Ana Maria Simao, Dorit Hanein, Agnieszka Strzelecka-Kiliszek, Lukasz Bozycki, René Buchet, Saida Mebarek, Maytê Bolean, José Luis Millán, Niels Volkmann, Slawomir Pikula, Régulations métaboliques, nutrition et diabètes - UM55 (RMND UM55), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Recherche Agronomique (INRA), Mid Sweden University, Nencki Institute of Experimental Biology, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Sanford Burnham Prebys Medical Discovery Institute, Sanford Burnham Medical Research Institute, La Jolla, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Proteoliposomes, [SDV]Life Sciences [q-bio], Proteomics, Biochemistry, Mineralization (biology), Atomic force microscopy, 0302 clinical medicine, Biomimetic Materials, Models, Apatites, Matrix vesicles, Lipid raft, ComputingMilieux_MISCELLANEOUS, Minerals, Organelle Biogenesis, Chemistry, Vesicle, Calcinosis, Extracellular Matrix, Cell biology, 030220 oncology & carcinogenesis, Intramembranous ossification, Collagen, Mineralization, Proteolipids, Biophysics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Models, Biological, Article, Calcification, Specimen Handling, Extracellular Vesicles, 03 medical and health sciences, Calcification, Physiologic, Chondrocytes, Membrane Microdomains, COLÁGENO, Lipidomics, Electron microscopy, Animals, Humans, Settore BIO/10, Physiologic, Vascular Calcification, Molecular Biology, Endochondral ossification, Hypertrophy, Osteoblasts, Biological, 030104 developmental biology, Biogenesis
Abstract

BACKGROUND: Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100–300 nm diameter harboring the biochemical machinery needed to induce mineralization. SCOPE OF THE REVIEW: The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs. MAJOR CONCLUSIONS: MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies. GRAPHICAL ABSTRACT LEGEND: Matrix vesicles are extracellular vesicles that bind to collagen and can induce formation of apatitic mineral during physiological and ectopic mineralization. Lipid and protein compositions in matrix vesicles resemble those of lipid rafts. Mechanisms of the biogenesis of matrix vesicles and processes leading to mineral/apatite formation are still unclear. Proteoliposomes can serve as biomimetic models to understand matrix vesicle-mediated mineralization.

Published
2018

34. A Continuous and Sensitive Spectrophotometric Assay for Lipase and Phospholipase A Activities Using α-Eleostearic Acid-Containing Substrates

Author

Yves Queneau, Meddy El Alaoui, Laurent Soulère, Florence Popowycz, Abdelkarim Abousalham, Lucie Grand, Jorge Alberto Rodríguez-González, Alexandre Noiriel, Priscila Sutto-Ortiz, Chimie Organique et Bioorganique (COB), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), and ICBMS, UMR5246

Subjects
chemistry.chemical_classification, Phospholipase A, Chromatography, biology, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Chemistry, 010401 analytical chemistry, Substrate (chemistry), Fatty acid, 030204 cardiovascular system & hematology, [CHIM.ORGA] Chemical Sciences/Organic chemistry, 01 natural sciences, 0104 chemical sciences, Absorbance, 03 medical and health sciences, 0302 clinical medicine, Phospholipase A2, Phospholipase A1, biology.protein, Moiety, Lipase, ComputingMilieux_MISCELLANEOUS
Abstract

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272 nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA1 and PLA2 inhibitors.

Published
2018

35. Phospholipases: An Overview

Author

Ahmed Aloulou, Renaud Rahier, Yani Arhab, Alexandre Noiriel, Abdelkarim Abousalham, ICBMS, UMR5246, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, 030220 oncology & carcinogenesis, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2018

36. Multi-scale mechanical characterization of prostate cancer cell lines : relevant biological markers to evaluate the cell metastatic potential

Author

Agnès Piednoir, Agnès Girard-Egrot, Saida Mebarek, J. Zouaoui, L. Brizuela, Ofelia Maniti, Thierry Granjon, B. Munteanu, Ahmed Landoulsi, Ana-Maria Trunfio-Sfarghiu, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Faculté des Sciences de Bizerte [Université de Carthage], Université de Carthage - University of Carthage, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Mécanique des Contacts et des Structures [Villeurbanne] (LaMCoS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut Lumière Matière [Villeurbanne] (ILM), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de recherches sur la catalyse (IRC), Centre National de la Recherche Scientifique (CNRS), Génie Enzymatique, Membrane Biomimétique et Assemblages Supramoléculaires (GEMBAS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Male, Cell viscoelasticity, Cell, Biophysics, Microscopy, Atomic Force, Biochemistry, Viscoelasticity, Cell membrane, Diffusion, 03 medical and health sciences, Atomic force microscopy, 0302 clinical medicine, Cell Line, Tumor, Monolayer, medicine, Humans, [CHIM]Chemical Sciences, Neoplasm Metastasis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Cytoskeleton, Molecular Biology, Prostate cancer, Chemistry, Viscosity, Cancer, Prostatic Neoplasms, Middle Aged, medicine.disease, Elasticity, 3. Good health, Cell biology, 030104 developmental biology, medicine.anatomical_structure, Membrane, 030220 oncology & carcinogenesis, Cancer cell, FRAP, Stress, Mechanical, Rheology, Biomarkers, Fluorescence Recovery After Photobleaching
Abstract

International audience; BackgroundConsidering the importance of cellular mechanics in the birth and evolution of cancer towards increasingly aggressive stages, we compared nano-mechanical properties of non-tumoral (WPMY-1) and highly aggressive metastatic (PC-3) prostate cell lines both on cell aggregates, single cells, and membrane lipids.MethodsCell aggregate rheological properties were analyzed during dynamic compression stress performed on a homemade rheometer. Single cell visco-elasticity measurements were performed by Atomic Force Microscopy using a cantilever with round tip on surface-attached cells. At a molecular level, the lateral diffusion coefficient of total extracted lipids deposited as a Langmuir monolayer on an air-water interface was measured by the FRAP technique.ResultsAt cellular pellet scale, and at single cell scale, PC-3 cells were less stiff, less viscous, and thus more prone to deformation than the WPMY-1 control. Interestingly, stress-relaxation curves indicated a two-step response, which we attributed to a differential response coming from two cell elements, successively stressed. Both responses are faster for PC-3 cells. At a molecular scale, the dynamics of the PC-3 lipid extracts are also faster than that of WPMY-1 lipid extracts.ConclusionsAs the evolution of cancer towards increasingly aggressive stages is accompanied by alterations both in membrane composition and in cytoskeleton dynamical properties, we attribute differences in viscoelasticity between PC-3 and WPMY-1 cells to modifications of both elements.General significanceA decrease in stiffness and a less viscous behavior may be one of the diverse mechanisms that cancer cells adopt to cope with the various physiological conditions that they encounter.

Published
2017

37. Inflammation: a culprit for vascular calcification in atherosclerosis and diabetes

Author

Laurence Bessueille, David Magne, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Pathology, medicine.medical_specialty, Vascular smooth muscle, Inflammasomes, Osteoporosis, Inflammation, Disease, Bioinformatics, Models, Biological, Muscle, Smooth, Vascular, Proinflammatory cytokine, Cellular and Molecular Neuroscience, Chondrocytes, Diabetes mellitus, medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Vascular Calcification, Molecular Biology, Pharmacology, Osteoblasts, Tumor Necrosis Factor-alpha, [CHIM.ORGA]Chemical Sciences/Organic chemistry, business.industry, Type 2 Diabetes Mellitus, Cell Differentiation, Inflammasome, Cell Biology, Atherosclerosis, medicine.disease, 3. Good health, Diabetes Mellitus, Type 2, Cytokines, Molecular Medicine, medicine.symptom, business, Interleukin-1, medicine.drug
Abstract

International audience; It is today acknowledged that aging is associated with a low-grade chronic inflammatory status, and that inflammation exacerbates age-related diseases such as osteoporosis, Alzheimer's disease, atherosclerosis and type 2 diabetes mellitus (T2DM). Vascular calcification is a complication that also occurs during aging, in particular in association with atherosclerosis and T2DM. Recent studies provided compelling evidence that vascular calcification is associated with inflammatory status and is enhanced by inflammatory cytokines. In the present review, we propose on one hand to highlight the most important and recent findings on the cellular and molecular mechanisms of vascular inflammation in atherosclerosis and T2DM. On the other hand, we will present the effects of inflammatory mediators on the trans-differentiation of vascular smooth muscle cell and on the deposition of crystals. Since vascular calcification significantly impacts morbidity and mortality in affected individuals, a better understanding of its induction and development will pave the way to develop new therapeutic strategies.

Published
2015

38. Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2

Author

Youssef Gargouri, Nabil Miled, Vincent Arondel, Neila Achouri, Ahmed Fendri, Houcemeddine Othman, Nabil Smichi, Soumaya Triki, Alexandre Noiriel, Abdelkarim Abousalham, Najet Srairi-Abid, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Enzymologie interfaciale et de physiologie de la lipolyse (EIPL), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université de Tunis El Manar (UTM), Laboratoire des Venins et Biomolécules Thérapeutiques - Laboratory of Venoms and Therapeutic Biomolecules (LR11IPT08), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Biotechnologie de Sfax (CBS), Laboratoire de biogenèse membranaire (LBM), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Ministry of higher education and scientific research of Tunisia., We thank Dr. Pascal MANSUELLE for technical assistance with MALDI-TOF spectrometers., Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Sparidae, Molecular model, Biochemistry, law.invention, MESH: Models, Molecular, 03 medical and health sciences, law, Sparidae phospholipase A(2) cloning, MESH: Animals, MESH: Sea Bream/metabolism, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Monomolecular film technique, Molecular Biology, Phylogeny, chemistry.chemical_classification, Gel electrophoresis, 030102 biochemistry & molecular biology, Molecular mass, biology, Organic Chemistry, MESH: Phospholipases A2/metabolism, Cell Biology, biology.organism_classification, Structural characterization, Amino acid, 030104 developmental biology, Enzyme, chemistry, Expression and purification, Recombinant DNA, MESH: Phospholipases A2/chemistry, lipids (amino acids, peptides, and proteins), Heterologous expression
Abstract

International audience; Here we report the cDNA cloning of a phospholipase A2 (PLA2) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA2. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA2 are closer to avian PLA2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA2 (AsPLA2). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg(-1) measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA2. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA2 at high temperatures was explained. Using the monomolecular film technique, AsPLA2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm(-1). Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA2, provides new insights on structure-activity relationships of fish PLA2.

Published
2017

39. Functional and Structural Characterization of a Thermostable Phospholipase A(2) from a Sparidae Fish (Diplodus annularis)

Author

Alexandre Noiriel, Neila Achouri, Youssef Gargouri, Nabil Miled, Houcemeddine Othman, Najet Srairi-Abid, Abdelkarim Abousalham, Ahmed Fendri, Vincent Arondel, Nabil Smichi, École Nationale d'Ingénieurs de Sfax | National School of Engineers of Sfax (ENIS), Université de Tunis El Manar (UTM), Laboratoire des Venins et Biomolécules Thérapeutiques - Laboratory of Venoms and Therapeutic Biomolecules (LR11IPT08), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biogenèse membranaire (LBM), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), and Ministry of Higher Education, Scientific Research, in Tunisia

Subjects
0301 basic medicine, phospholipase A(2), Sparidae, refolding and purification, Phospholipase, Biology, 03 medical and health sciences, Phospholipase A2, Thermostability, chemistry.chemical_classification, Phospholipase A, 030102 biochemistry & molecular biology, Molecular mass, monolayer properties, E. coli expression, General Chemistry, Diplodus, biology.organism_classification, Molecular biology, thermostability, structural characterization, 030104 developmental biology, Enzyme, chemistry, Biochemistry, biology.protein, General Agricultural and Biological Sciences, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Novel phospholipase (PLA(2)) genes from the Sparidae family were cloned. The sequenced PLA(2) revealed an identity with pancreatic PLA(2) group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA(2) (DaPLA(2)) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPIA(2) was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 31) structure of DaPLA(2) was constructed to gain insights into the functional properties of sparidae PLA(2). Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA(2). Using the monolayer technique, the purified DaPLA(2) was found to be active on various phospholipids ranging from 10 to 20 mN-m(-1), which explained the absence of the hemolytic activity for DaPLA(2).

Published
2017

40. Involvement of sphingosine kinase/sphingosine 1-phosphate metabolic pathway in spondyloarthritis

Author

Benoit Le Goff, Leyre Brizuela, Anne Briolay, T Garraud, David Magne, Marie-Astrid Boutet, Alaeddine El Jamal, Frédéric Blanchard, Carole Bougault, Saida Mebarek, maurice, sandrine, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Physiopathologie des Adaptations Nutritionnelles (PhAN), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN), Sarcomes osseux et remodelage des tissus calcifiés - Phy-Os [Nantes - INSERM U1238] (Phy-Os), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Bretagne Loire (UBL)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), This work was supported by grant from 'Société Française de Rhumatologie (SFR)' [2818, 2014–2015]., Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université Bretagne Loire (UBL)

Subjects
0301 basic medicine, Adult, Male, Histology, Mineralization, Adolescent, Physiology, Endocrinology, Diabetes and Metabolism, Sphingosine kinase, Biology, Chondrocyte, 03 medical and health sciences, chemistry.chemical_compound, Mice, Young Adult, Calcification, Physiologic, Sphingosine, Spondylarthritis, Spondyloarthritis, medicine, Animals, Humans, Sphingosine-1-phosphate, Aged, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Kinase, Osteoblast, Cell cycle, Middle Aged, 3. Good health, Phosphotransferases (Alcohol Group Acceptor), 030104 developmental biology, medicine.anatomical_structure, chemistry, Sphingosine 1-phosphate, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Immunology, Cancer research, Alkaline phosphatase, Female, Lysophospholipids
Abstract

International audience; Spondyloarthritis (SpA) is a relatively common chronic inflammatory joint disorder, with a prevalence of about 0.2–0.5% worldwide. The primary target of the pathological process is the enthesis, where tendons and ligaments attach to underlying bone. These insertion sites are hotspots of bone formation (enthesophytes), which can lead to ankylosis. Unfortunately, the mechanisms causing the onset and progression of entheseal ossification remain largely unknown. Sphingosine 1-phosphate (S1P), a lipid generated after sphingosine phosphorylation by sphin-gosine kinases 1 and 2 (SK1/2), plays important roles in cell proliferation, differentiation and survival. S1P regulates fundamental biological processes such as cell cycle, inflammatory response or bone homeostasis. Indeed, S1P has been involved in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. On the other hand, the implication of S1P in SpA has not been explored yet. In the present work, we observed by ELISA that S1P content was significantly increased in the serum of SpA patients (6.1 ± 4.2 μM, n = 21) compared to healthy donors (1.6 ± 0.9 μM, n = 12). In vitro, gene expression of SK1 and SK2 as well as their activity were increased during differentiation of primary murine chondrocytes and osteoblasts into mineralizing cells. In addition, mRNA of the S1P-specific transporter Spns2 and S1P secretion were augmented. Using the pharmacological drugs SKi (SK pan-inhibitor), PF-543 (SK1 specific inhibitor) or K-145 (SK2 specific inhibitor), we showed that the inhibition of SK1 and/or SK2 decreased matrix mineralization, alkaline phosphatase activity and the mRNA expression of Runx2 and Bglap in chondrocytes and osteoblasts. To our knowledge, this is the first study indicating that S1P levels are significantly increased in serum from SpA patients. Moreover, we showed in vitro that SK activity was involved in the mineralization capacity of osteoblasts and chondrocytes. S1P metabolic pathway may represent an ingenious therapeutic target for SpA in the future.

Published
2017

41. Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions

Author

Michele Fiore, Alexandra Berlioz-Barbier, Agnès Girard-Egrot, Peter Strazewski, René Buchet, Warren Madanamoothoo, Ofelia Maniti, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Génie Enzymatique, Membrane Biomimétique et Assemblages Supramoléculaires (GEMBAS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Chimie Organique 2-Glycochimie (CO2GLYCO), Université de Lyon, Laboratoire des biomolécules (LBM UMR 7203), Institut National de la Santé et de la Recherche Médicale (INSERM)-Chimie Moléculaire de Paris Centre (FR 2769), 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)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École Supérieure Chimie Physique Électronique de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-École Supérieure Chimie Physique Électronique de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
medicine.medical_treatment, 010402 general chemistry, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Amphiphile, medicine, Urea, Organic chemistry, Physical and Theoretical Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Unilamellar Liposomes, Alkyl, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Prebiotic, Organic Chemistry, Membrane structure, Small molecule, 0104 chemical sciences, Prebiotics, Membrane, chemistry, Chemical engineering, Cyanamide, Hydrophobic and Hydrophilic Interactions, Macromolecule
Abstract

International audience; Giant lipid vesicles resemble compartments of biological cells, mimicking them in their dimension, membrane structure and partly in their membrane composition. The spontanenous appearance of closed membranes composed of bilayers of self-assembling amphiphiles was likely a prerequisite for Darwinian competitive behavior to set in at the molecular level. Such compartments should be dynamic in their membrane composition (evolvable), and sufficiently stable to harbor macromolecules (leak-free), yet semi-permeable for reactive small molecules to get across the membrane (stay away from chemical equilibrium). Here we describe bottom-up experiments simulating prebiotic environments that support the formation of simple amphiphilic molecules capable of self-assembling into vesicular objects on the micrometer scale. Long-chain alkyl phosphates, together with related amphiphilic compounds, were formed under simulated prebiotic phosphorylation conditions by using cyanamide, a recognized prebiotic chemical activator and a precursor for several compound classes. Crude dry material of the thus obtained prebiotic mixtures formed multilamellar giant vesicles once rehydrated at the appropriate pH and in the presence of plausibly prebiotic co-surfactants, as observed by optical microscopy. The size and the shape of lipid aggregates tentatively suggest that prebiotic lipid assemblies could encapsulate peptides or nucleic acids that could be formed under similar chemical prebiotic conditions. The formation of prebiotic amphiphiles was monitored by using TLC, IR, NMR and ESI-MS and UPLC-HRMS. In addition we provide a spectroscopic analysis of cyanamide under simulated prebiotic conditions in the presence of phosphate sources and spectroscopic analysis of O-phosphorylethanolamine as a plausible precursor for phosphoethanolamine lipids.

Published
2017

42. Carrier-inside-carrier: polyelectrolyte microcapsules as reservoir for drug-loaded liposomes

Author

Jie Zhao, Olivier Marcillat, Thierry Granjon, Yi Jia, Loïc J. Blum, Joe Sarkis, Agnès Girard-Egrot, Junbai Li, Ofelia Maniti, Samuel Rebaud, Génie Enzymatique, Membrane Biomimétique et Assemblages Supramoléculaires (GEMBAS), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), and Depierre, Frédérique

Subjects
Drug, Materials science, Polymers, Surface Properties, media_common.quotation_subject, Pharmaceutical Science, Capsules, Nanotechnology, 02 engineering and technology, Polyethylene glycol, 010402 general chemistry, 01 natural sciences, Calcium Carbonate, Electrolytes, chemistry.chemical_compound, Particle Size, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], media_common, chemistry.chemical_classification, Drug Carriers, Liposome, Polymer, 021001 nanoscience & nanotechnology, Polyelectrolyte, 0104 chemical sciences, Calcium carbonate, chemistry, Liposomes, Drug delivery, Carbonate, 0210 nano-technology, Porosity
Abstract

International audience; Conventional liposomes have a short life-time in blood, unless they are protected by a polymer envelope, most often polyethylene glycol. However, these stabilizing polymers frequently interfere with cellular uptake, impede liposome-membrane fusion and inhibit escape of liposome content from endosomes. To overcome such drawbacks, polymer-based systems as carriers for liposomes are currently developed. Conforming to this approach, we propose a new and convenient method for embedding small size liposomes, 30–100 nm, inside porous calcium carbonate microparticles. These microparticles served as templates for deposition of various polyelectrolytes to form a protective shell. The carbonate particles were then dissolved to yield hollow polyelectrolyte microcapsules. The main advantage of using this method for liposome encapsulation is that carbonate particles can serve as a sacrificial template for deposition of virtually any polyelectrolyte. By carefully choosing the shell composition, bioavailability of the liposomes and of the encapsulated drug can be modulated to respond to biological requirements and to improve drug delivery to the cytoplasm and avoid endosomal escape.

Published
2014

43. Fatty acid composition in matrix vesicles and in microvilli from femurs of chicken embryos revealed selective recruitment of fatty acids

Author

Raghida Abou Merhi, Dina Abdallah, Saida Mebarek, Eva Hamade, Badran Bassam, René Buchet, Depierre, Frédérique, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Université Américaine de Beyrouth, and Université libre de Bruxelles (ULB)

Subjects
Biophysics, Chick Embryo, Biology, Biochemistry, Mineralization (biology), 03 medical and health sciences, chemistry.chemical_compound, Calcification, Physiologic, Chondrocytes, Eicosadienoic Acid, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Femur, Molecular Biology, Cells, Cultured, 030304 developmental biology, 0303 health sciences, Microvilli, Vesicle, Fatty Acids, 030302 biochemistry & molecular biology, Embryo, Cell Biology, Phosphate, Oleic acid, Membrane, chemistry, Arachidonic acid, Chickens
Abstract

International audience; Hypertrophic chondrocytes participate in matrix mineralization by releasing matrix vesicles (MVs). These MVs, by accumulating Ca2+ and phosphate initiate the formation of hydroxyapatite. To determine the types of lipids essential for mineralization, we analyzed fatty acids (FAs) in MVs, microvilli and in membrane fractions of chondrocytes isolated from femurs of chicken embryos. The FA composition in the MVs was almost identical to that in microvilli, indicating that the MVs originated from microvilli. These fractions contained more monounsaturated FAs especially oleic acid than in membrane homogenates of chondrocytes. They were enriched in 5,8,11-eicosatrienoic acid (20:3n−9), in eicosadienoic acid (20:2n−6), and in arachidonic acid (20:4n−6). In contrast, membrane homogenates from chondrocytes were enriched in 20:1n−9, 18:3n−3, 22:5n−3 and 22:5n−6. Due to their relatively high content in MVs and to their selective recruitment within microvilli from where MV originate, we concluded that 20:2n−6 and 20:3n−9 (pooled values), 18:1n−9 and 20:4n−6 are essential for the biogenesis of MVs and for bone mineralization.

Published
2014

44. Crosstalk between tyrosine kinase receptors, GSK3 and BMP2 signaling during osteoblastic differentiation of human mesenchymal stem cells

Author

Cyril Thouverey, David Magne, Joseph Caverzasio, Emmanuel Biver, Depierre, Frédérique, Service de rhumatologie[Lille], Hôpital Roger Salengro [Lille]-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Division of bone diseases, University Hospital, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
Male, Fibroblast growth factor, Bone Morphogenetic Protein 2, Smad Proteins, Biochemistry, Receptor tyrosine kinase, Glycogen Synthase Kinase 3, Phosphatidylinositol 3-Kinases, Endocrinology, Receptors, Platelet-Derived Growth Factor, Extracellular Signal-Regulated MAP Kinases, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Platelet-Derived Growth Factor, biology, Chemistry, Cell Differentiation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Cell biology, Osteoblastic differentiation, Signal transduction, Platelet-derived growth factor receptor, Signal Transduction, medicine.medical_specialty, Cell signaling, animal structures, Models, Biological, Bone morphogenetic protein 2, Young Adult, Wnt/ß-catenin, Bone morphogenic proteins, Internal medicine, medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein Kinase Inhibitors, Molecular Biology, PI3K/AKT/mTOR pathway, Cell Proliferation, Osteoblasts, Platelet derived growth factor, fungi, JNK Mitogen-Activated Protein Kinases, Receptor Protein-Tyrosine Kinases, Receptor Cross-Talk, Transforming growth factor beta, Receptors, Fibroblast Growth Factor, Enzyme Activation, Fibroblast Growth Factors, ddc:618.97, biology.protein, Mesenchymal stem cells
Abstract

International audience; Bone morphogenic proteins (BMPs) promote mesenchymal stem cell (MSC) osteogenic differentiation, whereas platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) activate their proliferation through receptors tyrosine kinase (RTK). The effects of PDGF or FGF receptor signaling pathway on BMP2-induced osteoblastic differentiation was investigated in human MSC (HMSC). Inhibition of PDGF or/and FGF receptors enhanced BMP2-induced alkaline phosphatase (ALP) activity, expression of Osterix, ALP and Bone sialoprotein, and matrix calcification. These effects were associated with increased Smad-1 activity, indicating that mitogenic factors interfere with Smad signaling in HMSC differentiation. RTK activate MAPK and inhibit GSK3 through the PI3K/Akt pathway. Biochemical analysis indicated that MAPK JNK and GSK3 especially are potential signaling molecules regulating BMP-induced osteoblastic HMSC differentiation. These observations highlight that the osteogenic effects of BMP2 are modulated by mitogenic factors acting through RTK.

Published
2014

45. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities

Author

El Alaoui, Meddy, Soulere, L., Noiriel, A., Popowycz, F., Khatib, A., Queneau, Y., Abousalham, A., Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Chimie Organique et Bioorganique (COB)

Subjects
[CHIM.ORGA]Chemical Sciences/Organic chemistry, lipids (amino acids, peptides, and proteins), [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
Abstract

International audience; A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.

Published
2016

46. Synthesis of benzofuran derivatives as selective inhibitors of tissue-nonspecific alkaline phosphatase : effects on cell toxicity and osteoblast-induced mineralization

Author

Florence Popowycz, Stéphanie Marquès, Saida Mebarek, René Buchet, Marc Lemaire, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Chimie Organique et Bioorganique (COB), Catalyse Synthèse et Environnement (CASYEN), and Depierre, Frédérique

Subjects
0301 basic medicine, Clinical Biochemistry, Pharmaceutical Science, Osteoclasts, 01 natural sciences, Biochemistry, Pyrophosphate, Substrate Specificity, Extracellular matrix, Ectopic calcification, chemistry.chemical_compound, Mice, Drug Discovery, Benzofuran derivatives, Benzofuran, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Structure, Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Osteoblast, Calcification disease, 3. Good health, medicine.anatomical_structure, Molecular Medicine, Alkaline phosphatase, Mineralization, Cell Survival, education, Cell Line, 03 medical and health sciences, Structure-Activity Relationship, Calcification, Physiologic, medicine, Animals, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Benzofurans, Dose-Response Relationship, Drug, Inhibitors, Organic Chemistry, [CHIM.ORGA] Chemical Sciences/Organic chemistry, medicine.disease, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, 030104 developmental biology, Cell culture, Calcification
Abstract

International audience; Tissue-nonspecific alkaline phosphatase (TNAP) by hydrolyzing pyrophosphate, an inhibitor of apatite formation, promotes extracellular matrix calcification during bone formation and growth, as well as during ectopic calcification under pathological conditions. TNAP is a target for the treatment of soft tissue pathological ossification. We synthesized a series of benzofuran derivatives. Among these, SMA14, displayed TNAP activity better than levamisole. SMA14 was found to be not toxic at doses of up to 40μM in osteoblast-like Saos-2 cells and primary osteoblasts. As probed by Alizarin Red staining, this compound inhibited mineral formation in murine primary osteoblast and in osteoblast-like Saos-2 cells.

Published
2016

47. A convenient and versatile synthesis of Laurdan-like fluorescent membrane probes: characterization of their fluorescence properties

Author

David Gueyrard, Mouhedine Cheniour, Peter G. Goekjian, Olivier Marcillat, Thierry Granjon, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Chimie Organique 2-Glycochimie (CO2GLYCO), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0301 basic medicine, Liposome, [CHIM.ORGA]Chemical Sciences/Organic chemistry, General Chemical Engineering, Natural phospholipids, Membrane lipids, technology, industry, and agriculture, General Chemistry, Fluorescence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Characterization (materials science), 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, Membrane, chemistry, Visible range, Biophysics, Organic chemistry, lipids (amino acids, peptides, and proteins), [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Laurdan
Abstract

International audience; It is now well established that biomembranes show a high degree of lateral organization, with the coexistence of lipid domains showing increased order within a fluid phase. Environment-sensitive fluorescent probes such as Laurdan have played an important role in the study of membrane lipids properties. However, it has been shown that, despite its interesting spectroscopic features, including efficient two-photon excitation in the visible range, Laurdan can be difficult to use for investigating cellular membranes and its capacity to accurately document membrane properties has been challenged. We present here a simple and versatile two-step synthetic scheme that readily leads to Laurdan analogs differing by their polar head. We prepared a small library in order to evaluate the influence of the polar headgroup on the probe's ability to monitor membrane properties. The spectroscopic properties of the probes dissolved in different organic solvents or inserted into liposomes made of synthetic as well as natural phospholipids at different temperatures are described. Comparison of the fluorescence properties of existing Laurdan and C-Laurdan with the newly synthesized fluorescent probes indicates that the structure of the electron-donating amino group is a key parameter for the development of better-performing probes, especially in membranes containing negatively charged phospholipids.

Published
2016

48. Biochemical characterization of phospholipases D and their functional domains : novel method for measuring phospholipase D activities

Author

Rahier-Corticchiato , Renaud, STAR, ABES, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Université de Lyon, Abdelkarim Abousalham, Alexandre Noiriel, Métabolisme, Enzymes et Mécanismes Moléculaires ( MEM² ), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires ( ICBMS ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon ( INSA Lyon ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS ) -Université Claude Bernard Lyon 1 ( UCBL ), and Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS )

Subjects
8-hydroxyquinoline, Pichia pastoris, Arabidopsis thaliana, Phospholipase D, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Domaine C2, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, C2 domains, Phospholipases D, 8-Hydroxyquinoléine, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract

Phospholipase D (PLD) hydrolyses membrane phospholipids, leading to the formation of free polar headgroup and phosphatidic acid releasing, involved in cell signaling. To understand the biochemical properties of PLDs, this work has been made around two axes. The one first concerns the recombinant expression and purification of the PLDa of Arabidopsis thaliana (AtPLDa) in the yeast Pichia pastoris. The N-terminal sequence of the recombinant AtPLDa has been determined and found to lack its first 35 amino acids, suggesting the involvement of a maturing mechanism. However, plant PLDs exhibit a C2-lipid binding domain at their N-terminal region, which is involved in their Ca2+-dependent membrane targeting. Thus, to assess the impact of such a cleavage, whole and mature-like C2 domains of AtPLDa, as well as of AtPLDß, for the sake of comparison were studied. Thus, the characterization of their affinity for phospholipids, combined with their three-dimensional modeling have demonstrated that the differences observed in their regulation by Ca2+, observed between whole and mature-like forms, originated from the presence of a N-terminus amphipathic a helix, removed during the maturation process. The second axis concerns the development of a novel PLD assay that measure PA in a direct, specific and continuous manner, using the chelation enhanced fluorescence property of 8-hydroxyquinoline in the presence of Ca2+. Thus, this assay appears suitable for monitoring both the inhibition of PLDs as well as their substrate specificity, using natural phospholipids with different polar headgroups, and at a microplate scale, La phospholipase D (PLD) hydrolyse les phospholipides membranaires en libérant leur tête polaire afin de générer l'acide phosphatidique (PA), impliqué dans la signalisation cellulaire. Pour comprendre les propriétés biochimiques des PLDs, les travaux présentés ont été réalisés autour de deux axes. Le premier axe concerne l'expression recombinante et la purification de la PLDa d'Arabidopsis thaliana (AtPLDa) dans la levure Pichia pastoris. La détermination de la séquence N-terminale a révélé que l'AtPLDa est amputée de ses 35 premiers résidus, suggérant ainsi la participation d'un mécanisme de maturation. Cependant, la région N-terminale des PLDs de plantes est homologue au domaine C2, impliqué dans leur interaction Ca2+-dépendante avec la membrane. Afin d'évaluer l'impact d'un tel clivage, les domaines C2 de l'AtPLDa mais également de l'AtPLDß, à titre de comparaison, ont été étudiés sous leur forme entière ou mature. Ainsi, la caractérisation de leur affinité pour les phospholipides, associée à leur modélisation tridimensionnelle, ont permis de démontrer que les différences de régulation par le Ca2+, observées entre les formes entières et mature, provenait de la présence d'une hélice a amphipathique, retirée lors du processus de maturation. Le second axe concerne le développement d'une nouvelle méthode de mesure des activités PLD via le dosage de manière direct, spécifique et continu du PA grâce à la propriété d'amplification de fluorescence par chélation de la 8-hydroxyquinoléine, en présence de Ca2+. Ainsi, ce test apparait adapté pour le suivi de l'inhibition des PLDs et pour l'étude de leur spécificité de substrat, en utilisant des phospholipides naturels avec différentes tête polaires, et à l'échelle d'une microplaque

Published
2016

49. Membranes and pathophysiological mineralization

Author

Roszkowska, M., Strzelecka-Kiliszek, A., Magne, D., Pikula, S., Bessueille, L.., Métabolisme, Enzymes et Mécanismes Moléculaires (MEM²), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratory of Biochemistry of Lipids, Nencki Institute of Experimental Biology of Polish, and Depierre, Frédérique

Subjects
lipid rafts, vascular calcification, [CHIM.ORGA]Chemical Sciences/Organic chemistry, cholesterol, mineralization, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [CHIM.ORGA] Chemical Sciences/Organic chemistry, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], fatty acids, matrix vesicles
Abstract

International audience; Vascular calcification accompanies the pathological process of atherosclerotic plaque formation. Artery calcification results from trans-differentiation of vascular smooth muscle cells (VSMCs) into cells resembling mineralization-competent cells such as osteoblasts and chondrocytes. The activity of tissue-nonspecific alkaline phosphatase (TNAP), a GPI-anchored enzyme necessary for physiological mineralization, is induced in VSMCs in response to inflammation. TNAP achieves its mineralizing function being anchored to plasma membrane of mineralizing cells and to the surface of their derived matrix vesicles (MVs), and numerous important reports indicate that membranes play a crucial role in initiating the crystal formation. In this review, we would like to highlight various functions of lipids and proteins associated to membranes at different stages of both physiological mineralization and vascular calcification, with an emphasis on the pathological process of atherosclerotic plaque formation.

Published
2016

50. Expression, purification and re-evaluation of the role of CGI-58 protein in lipids metabolism

Author

Khatib , Abdallah, STAR, ABES, Métabolisme, Enzymes et Mécanismes Moléculaires ( MEM² ), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires ( ICBMS ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon ( INSA Lyon ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Université de Lyon, Abdelkarim Abousalham, Alexandre Noiriel, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), 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)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
CGI-58, PGAT, PDAT, E. coli, humanities, NLSD, ATGL, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], health services administration, A. thaliana, mental disorders, TG, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
Abstract

Triglycerides (TG) hydrolysis in adipose tissue is initialized by the action of the adipose TG lipase (ATGL) and its cofactor, the CGI-58 protein. Mutations in the gene coding the CGI-58 or ATGL proteins are the cause of the syndrome of Chanarin-Dorfman and of neutral lipids storage disease, respectively. CGI-58 protein belongs to the a/ß-hydrolase family, harboring the catalytic triad Ser-Asp/Glu-His, characteristic of carboxylester hydrolases, as well as the HX4D motif, characteristic of acyltransferase activity. Nowadays, the role and the enzymatic activity of the CGI-58 protein, in mammalians and plants, are not quite clear. In order to better understand the function of CGI-58 protein in lipid metabolism, we developed a new strategy using a new set of plasmids that enabled us, with the use of pET28b(+) plasmid, to express and purify the CGI-58 protein of mice and plants as well as the murine ATGL. These proteins were expressed in different E. coli strains, to test in vitro their activity. In addition, we have set up, using the generated plasmids and different E. coli strains, a system that allowed us to test the in vivo acyltransferase activity (LPAAT and/or LPGAT) of the CGI-58 proteins expressed in E. coli. Using these techniques, we demonstrated in vivo and in vitro that both mice and plant CGI-58 proteins, are neither able to catalyze a LPAAT or a LPGAT reaction, and that the plant CGI-58 protein is devoid of TG lipase or phospholipase activity. However we have shown, by analyzing lipid extracts, by thin layer chromatography and by mass spectrometry of different E. coli strains expressing the CGI-58 protein, that the expression of the plant CGI-58 protein, but not the mice one, results in a decrease of phosphatidylglycerol (PG) content in the different strains tested. However the mutation of the serine or histidine residue of the putative catalytic triad restores the wild-typephenotype. These results allowed us to propose that plant CGI-58 protein is involved in the metabolism of PG, L’hydrolyse des triglycérides (TG) du tissu adipeux est initialisée par l’action de l’adipocyte TG lipase (ATGL), activée par son cofacteur la protéine CGI-58. Des mutations du gène codant les protéines CGI-58 ou ATGL sont respectivement à l’origine du syndrome de Chanarin-Dorfmann et de maladies de stockagede lipides neutres. La protéine CGI-58 appartient à la famille des a/ß-hydrolases, elle en possède la triade catalytique Ser-Asp/Glu-His caractéristique des carboxylester hydrolases, ainsi que le motif HX4D, caractéristique d’une activité acyltransférase. A ce jour, le rôle de la protéine CGI-58, chez les mammifères ou chez les plantes, n’est pas totalement éclairci, de même que son activité enzymatique. Dans le but de mieux comprendre le rôle de cette protéine CGI-58 dans le métabolisme lipidique, nous avons développé une nouvelle stratégie, en générant notamment de nouveaux plasmides qui nous ont permis d’exprimer et de purifier la protéine CGI-58 de souris et de plantes, ainsi que l’ATGL murine, dans différents souches d’E. coli, pour tester l’activité in vitro de ces protéines. De plus, nous avons mis en place, en utilisant ces plasmides générés et différentes souches E. coli, un système qui nous a permis de tester in vivo, dans E. coli, l’activité acyltransférase (LPAAT et/ou LPGAT) de la protéine CGI-58. En utilisant ces différentes techniques, nous avons pu montrer, aussi bien in vivo qu’in vitro, que la protéine CGI-58 de plante, ainsique celle de mammifère, ne possède ni activité LPAAT ni activité LPGAT, et que la protéine CGI-58 de plante est dépourvue d’activité TG lipase ou phospholipase. Cependant, nous avons montré, en analysant, par chromatographie sur couche mince et par spectrométrie de masse, des extraits lipidiques des différentes souches d’E. coli exprimant la protéine CGI-58, que l’expression de la protéine de plante, et non celle de mammifère, aboutit à une diminution du taux de phosphatidylglycérol (PG) dans les différentes souchestestées, et a contrario nous avons montré que la mutation de la sérine, ainsi que la mutation de l’histidine de la triade catalytique potentielle, restaure le phénotype sauvage. Ces résultats nous ont permis de proposer que la protéine CGI-58 de plante est impliquée dans le métabolisme du PG

Published
2016

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