95 results on '"Damblon C"'
Search Results
2. Erratum to: Identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica
- Author
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Carly, F., Gamboa-Melendez, H., Vandermies, M., Damblon, C., Nicaud, J. M., and Fickers, P.
- Published
- 2017
- Full Text
- View/download PDF
3. Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies
- Author
-
Mollard, C., Moali, C., cyril papamicael, Damblon, C., Vessilier, S., Amicosante, G., Cj Schofield, Galleni, M., Jm Frere, Gc Roberts, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Deleage, Gilbert
- Subjects
Models, Molecular ,Binding Sites ,Magnetic Resonance Spectroscopy ,Arginine ,Kinetics ,Structure-Activity Relationship ,Zinc ,Models, Chemical ,Spectrophotometry ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Mandelic Acids ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Sulfhydryl Compounds ,Enzyme Inhibitors ,beta-Lactamase Inhibitors ,Protein Binding - Abstract
International audience; Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.
- Published
- 2016
4. Interaction of Avibactam with Class B Metallo-β-Lactamases
- Author
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Abboud, M, Damblon, C, Brem, J, Smargiasso, N, Mercuri, P, Gilbert, B, Rydzik, A, Claridge, T, Schofield, C, and Frère, J
- Subjects
Magnetic Resonance Spectroscopy ,Mechanisms of Resistance ,Hydrolysis ,Spectrophotometry, Ultraviolet ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,Ceftazidime ,beta-Lactamases - Abstract
β-Lactamases are the most important mechanisms of resistance to the β-lactam antibacterials. There are two mechanistic classes of β-lactamases: the serine β-lactamases (SBLs) and the zinc-dependent metallo-β-lactamases (MBLs). Avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.
- Published
- 2016
5. Reinforced poly(hydroxyurethane) thermosets as high performance adhesives for aluminum substrates
- Author
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Panchireddy, S., primary, Thomassin, J.-M., additional, Grignard, B., additional, Damblon, C., additional, Tatton, A., additional, Jerome, C., additional, and Detrembleur, C., additional
- Published
- 2017
- Full Text
- View/download PDF
6. Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins
- Author
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ADAM, M, DAMBLON, C, JAMIN, M, ZORZI, W, DUSART, [No Value], GALLENI, M, ELKHARROUBI, A, PIRAS, G, SPRATT, BG, Keck, W, COYETTE, J, GHUYSEN, JM, NGUYENDISTECHE, M, FRERE, JM, and Stratingh Institute of Chemistry
- Subjects
Penicillin binding proteins ,BETA-LACTAMASES ,Stereochemistry ,Muramoylpentapeptide Carboxypeptidase ,Biochemistry ,Catalysis ,Substrate Specificity ,Cell membrane ,Hydrolysis ,Bacterial Proteins ,DD-PEPTIDASES ,Escherichia coli ,medicine ,polycyclic compounds ,Penicillin-Binding Proteins ,Moiety ,Sulfhydryl Compounds ,Molecular Biology ,chemistry.chemical_classification ,DIVISION ,Chemistry ,Aminobutyrates ,Hippurates ,organic chemicals ,Cell Membrane ,Esters ,Penicillin G ,Cell Biology ,Streptomyces ,Molecular Weight ,Kinetics ,medicine.anatomical_structure ,Enzyme ,Membrane ,Hexosyltransferases ,Acyltransferases ,ESCHERICHIA-COLI ,Acyltransferase ,Peptidyl Transferases ,SHAPE ,Carrier Proteins ,Enterococcus ,Research Article - Abstract
The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.
- Published
- 1991
7. A variety of roles for versatile zinc in metallo-β-lactamases
- Author
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Karsisiotis, A. I., primary, Damblon, C. F., additional, and Roberts, G. C. K., additional
- Published
- 2014
- Full Text
- View/download PDF
8. CG7054 solution structure
- Author
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Rautureau, G., primary, Jouvensal, L., additional, Vovelle, F., additional, Schoentgen, F., additional, Locker, D., additional, Decoville, M., additional, and Damblon, C., additional
- Published
- 2008
- Full Text
- View/download PDF
9. STRUCTURAL STUDIES OF HISTIDINES IN ZINC β-LACTAMASES AND THEIR INTERACTIONS WITH INHIBITORS
- Author
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Damblon, C., primary, Lian, C. LY, additional, Soto, RP., additional, Villadares, MH, additional, Galleni, M., additional, Frere, JM, additional, and Roberts, GCK, additional
- Published
- 1999
- Full Text
- View/download PDF
10. The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.
- Author
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Damblon, C, primary, Raquet, X, additional, Lian, L Y, additional, Lamotte-Brasseur, J, additional, Fonze, E, additional, Charlier, P, additional, Roberts, G C, additional, and Frère, J M, additional
- Published
- 1996
- Full Text
- View/download PDF
11. Breakdown of the stereospecificity of dd-peptidases and β-lactamases with thiolester substrates
- Author
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Damblon, C, primary, Zhao, G H, additional, Jamin, M, additional, Ledent, P, additional, Dubus, A, additional, Vanhove, M, additional, Raquet, X, additional, Christiaens, L, additional, and Frère, J M, additional
- Published
- 1995
- Full Text
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12. Streptomyces K15 active-site serine dd-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions
- Author
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Grandchamps, J, primary, Nguyen-Distèche, M, additional, Damblon, C, additional, Frère, J M, additional, and Ghuysen, J M, additional
- Published
- 1995
- Full Text
- View/download PDF
13. Direct n.m.r. evidence for substrate-induced conformational changes in a β-lactamase
- Author
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Jamin, M, primary, Damblon, C, additional, Bauduin-Misselyn, A M, additional, Durant, F, additional, Roberts, G C K, additional, Charlier, P, additional, Llabres, G, additional, and Frère, J M, additional
- Published
- 1994
- Full Text
- View/download PDF
14. Synthesis, purification and kinetic properties of fluorescein-labelled penicillins
- Author
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Lakaye, B, primary, Damblon, C, additional, Jamin, M, additional, Galleni, M, additional, Lepage, S, additional, Joris, B, additional, Marchand-Brynaert, J, additional, Frydrych, C, additional, and Frere, J M, additional
- Published
- 1994
- Full Text
- View/download PDF
15. Penicillin-binding protein 2x of Streptococcus pneumoniae: enzymic activities and interactions with β-lactams
- Author
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Jamin, M, primary, Damblon, C, additional, Millier, S, additional, Hakenbeck, R, additional, and Frère, J M, additional
- Published
- 1993
- Full Text
- View/download PDF
16. The mechanism of action of dd-peptidases: the role of tyrosine-159 in the Streptomyces R61 dd-peptidase
- Author
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Wilkin, J M, primary, Jamin, M, additional, Damblon, C, additional, Zhao, G H, additional, Joris, B, additional, Duez, C, additional, and Frère, J M, additional
- Published
- 1993
- Full Text
- View/download PDF
17. Accumulation of acyl-enzyme in dd-peptidase-catalysed reactions with analogues of peptide substrates
- Author
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Jamin, M, primary, Adam, M, additional, Damblon, C, additional, Christiaens, L, additional, and Frère, J M, additional
- Published
- 1991
- Full Text
- View/download PDF
18. The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence
- Author
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Brannigan, J, primary, Matagne, A, additional, Jacob, F, additional, Damblon, C, additional, Joris, B, additional, Klein, D, additional, Spratt, B G, additional, and Frère, J M, additional
- Published
- 1991
- Full Text
- View/download PDF
19. Chromogenic depsipeptide substrates for β-lactamases and penicillin-sensitive dd-peptidases
- Author
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Adam, M, primary, Damblon, C, additional, Plaitin, B, additional, Christiaens, L, additional, and Frère, J M, additional
- Published
- 1990
- Full Text
- View/download PDF
20. Synthesis of a Simplified Bryostatin C-Ring Analogue That Binds to the CRD2 of Human PKC-α and Construction of a Novel BC-Analogue by an Unusual Julia Olefination Process
- Author
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Hale, K. J., Frigerio, M., Manaviazar, S., Hummersone, M. G., Fillingham, I. J., Barsukov, I. G., Damblon, C. F., Gescher, A., and Roberts, G. C. K.
- Abstract
The synthesis of two truncated bryostatin analogues 2 and3 is described. High-field NMR measurements on the C-ring analogue3 in C2H3 CN containing 25% 2H2 O have shown that it binds to the CRD2 of human PKC-α at virtually the same position as phorbol-13-acetate (PA) and bryostatin 1 (1 ). NMR titration studies have also revealed that3 binds to the CRD2 with a potency similar in magnitude to PA but much less potently than1 .- Published
- 2003
21. Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies.
- Author
-
Mollard, C, Moali, C, Papamicael, C, Damblon, C, Vessilier, S, Amicosante, G, Schofield, C J, Galleni, M, Frere, J M, and Roberts, G C
- Abstract
Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.
- Published
- 2001
- Full Text
- View/download PDF
22. DNA-binding mechanism of the Escherichia coli Ada O(6)-alkylguanine-DNA alkyltransferase.
- Author
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Verdemato, P E, Brannigan, J A, Damblon, C, Zuccotto, F, Moody, P C, and Lian, L Y
- Abstract
The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O:(6)-methylguanine (O:(6)meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.
- Published
- 2000
- Full Text
- View/download PDF
23. DNA-binding mechanism of the Escherichia coli Ada O<SUP>6</SUP>-alkylguanine-DNA alkyltransferase
- Author
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Brannigan, J.A., Zuccotto, F., Verdemato, P.E., Damblon, C., Moody, P.C.E., and Lian, L-Y.
- Abstract
The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O6-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O6-methylguanine (O6meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.
- Published
- 2000
24. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes
- Author
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Rowe J, Teulade-Fichou Marie-Paule, De Pauw Edwin, Rosu Frédéric, Damblon Christian, Gabelica Valérie, Smargiasso Nicolas, and Claessens Antoine
- Subjects
Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.
- Published
- 2009
- Full Text
- View/download PDF
25. Biosynthesis of the antioxidant γ-glutamyl-cysteine with engineered Yarrowia lipolytica.
- Author
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Do D, Guruk M, Kus-Liśkiewicz M, Damblon C, Arguelles-Arias A, Erten H, and Fickers P
- Subjects
- Cysteine metabolism, Glutathione, Glutamates metabolism, Antioxidants metabolism, Yarrowia genetics, Yarrowia metabolism
- Abstract
The dipeptide γ-glutamylcysteine (γ-GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age-related disorders such as Alzheimer and Parkinson diseases. The unusual γ-linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non-conventional yeast Yarrowia lipolytica for efficient γ-GC synthesis. The yeast was first converted into a γ-GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4, CYSE, CYSF, and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ-GC titer of 464 nmol mg
-1 protein (93 mg gDCW-1 ) was obtained within 24 h of cell growth., (© 2024 Wiley-VCH GmbH.)- Published
- 2024
- Full Text
- View/download PDF
26. Human Pancreatic Islets React to Glucolipotoxicity by Secreting Pyruvate and Citrate.
- Author
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Perrier J, Nawrot M, Madec AM, Chikh K, Chauvin MA, Damblon C, Sabatier J, Thivolet CH, Rieusset J, Rautureau GJP, and Panthu B
- Subjects
- Rats, Animals, Humans, Pyruvic Acid metabolism, Citric Acid pharmacology, Citric Acid metabolism, Glucose pharmacology, Glucose metabolism, Insulin metabolism, Diabetes Mellitus, Type 2 metabolism, Islets of Langerhans, Insulin-Secreting Cells metabolism
- Abstract
Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.
- Published
- 2023
- Full Text
- View/download PDF
27. Variability in the substitution pattern of hydroxypropyl cellulose affects its physico-chemical properties.
- Author
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Cremer G, Danthine S, Van Hoed V, Dombree A, Laveaux AS, Damblon C, Karoui R, and Blecker C
- Abstract
Hydroxypropyl cellulose (HPC) is a water-soluble polymer with many applications in food, pharmaceutical, medical, or paints industries. Past studies have reported that differences in functionality can occur between products of similar pharmaceutical grades. Understanding the origin of these differences is a major challenge for the industry. In this work, the structure and physico-chemical properties of several HPC samples of the same commercial grade were studied. Structural analysis by NMR and enzymatic hydrolysis were performed to study molar substitution and distribution of substituents along the polymer chain respectively. Water-polymer interactions, surface properties as well as rheological and thermal behavior were characterized to tentatively correlate them with the structure, and gain new insights into the structure-function relationship of this polymer. The differences in structure revealed between the samples affect their properties. The unexpected behavior of one sample was attributed to a more heterogeneous substitution pattern, with the coexistence of highly and weakly substituted regions along the same polymer chain. The more block-like distribution of substituents has a great effect on the clouding behavior and surface tension reduction ability of the polymer., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Published by Elsevier Ltd.)
- Published
- 2023
- Full Text
- View/download PDF
28. Encapsulation of Vitamin C by Glycerol-Derived Dendrimers, Their Interaction with Biomimetic Models of Stratum corneum and Their Cytotoxicity.
- Author
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Bacha K, Chemotti C, Monboisse JC, Robert A, Furlan AL, Smeralda W, Damblon C, Estager J, Brassart-Pasco S, Mbakidi JP, Pršić J, Bouquillon S, and Deleu M
- Subjects
- Humans, Ascorbic Acid pharmacology, Glycerol, Biomimetics, Liposomes, Vitamins, Lipids, Dendrimers pharmacology, Dendrimers chemistry
- Abstract
Vitamin C is one of the most sensitive cosmetic active ingredients. To avoid its degradation, its encapsulation into biobased carriers such as dendrimers is one alternative of interest. In this work, we wanted to evaluate the potential of two biobased glycerodendrimer families (GlyceroDendrimers-Poly(AmidoAmine) (GD-PAMAMs) or GlyceroDendrimers-Poly(Propylene Imine) (GD-PPIs)) as a vitamin C carrier for topical application. The higher encapsulation capacity of GD-PAMAM-3 compared to commercial PAMAM-3 and different GD-PPIs, and its absence of cytotoxicity towards dermal cells, make it a good candidate. Investigation of its mechanism of action was done by using two kinds of biomimetic models of stratum corneum (SC), lipid monolayers and liposomes. GD-PAMAM-3 and VitC@GD-PAMAM-3 (GD-PAMAM-3 with encapsulated vitamin C) can both interact with the lipid representatives of the SC lipid matrix, whichever pH is considered. However, only pH 5.0 is suggested to be favorable to release vitamin C into the SC matrix. Their binding to SC-biomimetic liposomes revealed only a slight effect on membrane permeability in accordance with the absence of cytotoxicity but an increase in membrane rigidity, suggesting a reinforcement of the SC barrier property. Globally, our results suggest that the dendrimer GD-PAMAM-3 could be an efficient carrier for cosmetic applications.
- Published
- 2022
- Full Text
- View/download PDF
29. Phytochemical Investigation and Biological Activities of Lantana rhodesiensis .
- Author
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Nea F, Bitchi MB, Genva M, Ledoux A, Tchinda AT, Damblon C, Frederich M, Tonzibo ZF, and Fauconnier ML
- Subjects
- Antimalarials chemistry, Antioxidants chemistry, Phytochemicals chemistry, Plant Leaves chemistry, Polyphenols analysis, Antimalarials pharmacology, Antioxidants pharmacology, Lantana chemistry, Phytochemicals pharmacology
- Abstract
Lantana rhodesiensis Moldenke is a plant widely used to treat diseases, such as rheumatism, diabetes, and malaria in traditional medicine. To better understand the traditional uses of this plant, a phytochemical study was undertaken, revealing a higher proportion of polyphenols, including flavonoids in L. rhodesiensis leaf extract and moderate proportion in stem and root extracts. The antioxidant activity of the extracts was also determined using three different assays: the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, the FRAP method (Ferric-reducing antioxidant power) and the β-carotene bleaching test. The anti-malarial activity of each extract was also evaluated using asexual erythrocyte stages of Plasmodium falciparum , chloroquine-sensitive strain 3D7. The results showed that the leaf extract exhibited higher antioxidant and anti-malarial activities in comparison with the stem and root extracts, probably due to the presence of higher quantities of polyphenols including flavonoids in the leaves. A positive linear correlation was established between the phenolic compound content (total polyphenols including flavonoids and tannins; and total flavonoids) and the antioxidant activity of all extracts. Furthermore, four flavones were isolated from leaf dichloromethane and ethyl acetate fractions: a new flavone named rhodescine (5,6,3',5'-tetrahydroxy-7,4'-dimethoxyflavone) ( 1 ), 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone ( 2 ), 5-hydroxy-6,7,3',4'-tetramethoxyflavone ( 3 ), and 5,6,3'-trihydroxy-7,4'-dimethoxyflavone ( 4 ). Their structures were elucidated by
1 H,13 CNMR, COSY, HSQC, HMBC, and MS-EI spectral methods. Aside from compound 2 , all other molecules were described for the first time in this plant species.- Published
- 2021
- Full Text
- View/download PDF
30. Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures.
- Author
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Balcerczyk A, Damblon C, Elena-Herrmann B, Panthu B, and Rautureau GJP
- Subjects
- Animals, Cell Culture Techniques, Culture Media, Humans, Metabolome, Xenobiotics metabolism, Xenobiotics pharmacology, Magnetic Resonance Spectroscopy methods, Mass Spectrometry methods, Metabolomics methods
- Abstract
Biological organisms are constantly exposed to an immense repertoire of molecules that cover environmental or food-derived molecules and drugs, triggering a continuous flow of stimuli-dependent adaptations. The diversity of these chemicals as well as their concentrations contribute to the multiplicity of induced effects, including activation, stimulation, or inhibition of physiological processes and toxicity. Metabolism, as the foremost phenotype and manifestation of life, has proven to be immensely sensitive and highly adaptive to chemical stimuli. Therefore, studying the effect of endo- or xenobiotics over cellular metabolism delivers valuable knowledge to apprehend potential cellular activity of individual molecules and evaluate their acute or chronic benefits and toxicity. The development of modern metabolomics technologies such as mass spectrometry or nuclear magnetic resonance spectroscopy now offers unprecedented solutions for the rapid and efficient determination of metabolic profiles of cells and more complex biological systems. Combined with the availability of well-established cell culture techniques, these analytical methods appear perfectly suited to determine the biological activity and estimate the positive and negative effects of chemicals in a variety of cell types and models, even at hardly detectable concentrations. Metabolic phenotypes can be estimated from studying intracellular metabolites at homeostasis in vivo, while in vitro cell cultures provide additional access to metabolites exchanged with growth media. This article discusses analytical solutions available for metabolic phenotyping of cell culture metabolism as well as the general metabolomics workflow suitable for testing the biological activity of molecular compounds. We emphasize how metabolic profiling of cell supernatants and intracellular extracts can deliver valuable and complementary insights for evaluating the effects of xenobiotics on cellular metabolism. We note that the concepts and methods discussed primarily for xenobiotics exposure are widely applicable to drug testing in general, including endobiotics that cover active metabolites, nutrients, peptides and proteins, cytokines, hormones, vitamins, etc.
- Published
- 2020
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31. Exploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design.
- Author
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De Franco S, Vandenameele J, Brans A, Verlaine O, Bendak K, Damblon C, Matagne A, Segal DJ, Galleni M, Mackay JP, and Vandevenne M
- Subjects
- Base Sequence, Binding Sites, Drug Design, Humans, Models, Molecular, Protein Binding, Protein Conformation, Protein Engineering, RNA genetics, RNA-Binding Proteins chemistry, RNA-Binding Proteins metabolism, Structure-Activity Relationship, Zinc Fingers, RNA-Binding Proteins genetics, SELEX Aptamer Technique methods
- Abstract
Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.
- Published
- 2019
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32. "NOTA-PRGD 2 and NODAGA-PRGD 2 : Bioconjugation, characterization, radiolabelling, and design space".
- Author
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Salvé M, Avohou HT, Monbaliu JM, Lebrun P, Lemaire C, Damblon C, de Tullio P, Hubert P, Hustinx R, and Luxen A
- Subjects
- Amides chemistry, Automation instrumentation, Automation methods, Chemistry Techniques, Synthetic instrumentation, Chemistry Techniques, Synthetic methods, Microfluidics instrumentation, Microfluidics methods, Polycyclic Compounds chemistry, Amides chemical synthesis, Gallium Radioisotopes chemistry, Oligopeptides chemistry, Organometallic Compounds chemistry, Polycyclic Compounds chemical synthesis, Radiopharmaceuticals chemical synthesis
- Abstract
This work reports on the development of amide bond bioconjugation for the production of -NOTA and -NODAGA PRGD
2 using batch strategy and microfluidic reactor technology. The final radiolabelling step was fully optimized using Design of Experiments and Design Space approaches, hence targeting robust labelling yields in routine. Optimal labelling conditions were defined in sodium acetate buffer as 168 μg/mL peptide concentration, 4.9 pH, 47.5°C temperature, and 12.5-minute reaction time. Upon optimization, the Gallium-68 radiolabelling was fully automated. All the work was designed to be compliant to the GMP environment and to support the pharmaceutical scale-up., (Copyright © 2018 John Wiley & Sons, Ltd.)- Published
- 2018
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33. 1 H, 13 C and 15 N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants.
- Author
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Thorn D, Kay J, Rhazi N, Dumoulin M, Corazza A, and Damblon C
- Subjects
- Mutant Proteins genetics, beta-Lactamases genetics, Bacillus licheniformis enzymology, Mutant Proteins chemistry, Mutation, beta-Lactamases chemistry
- Abstract
Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used the β-lactamase BlaP from Bacillus licheniformis 749/C (BlaP) as a protein scaffold to create model polyglutamine (polyQ) proteins in order to better understand the mechanism(s) by which an expanded polyQ sequence triggers the formation of amyloid fibrils. The model chimeras were designed by inserting a polyQ sequence of various lengths at two different locations within BlaP (i.e. position 197 or position 216) allowing a detailed comparison of the effects of subtle differences in the environment of the polyQ sequence on its ability to trigger protein aggregation. In order to investigate the effects of the polyQ insertion at both positions on the structure, stability and dynamics of BlaP, a series of NMR experiments including H/D exchange are foreseen. Accordingly, as necessitated by these studies, here we report the NMR assignment of the wild-type BlaP (BlaP-WT) and of the two reference proteins, BlaP197Q0 and BlaP216Q0, wherein a Pro-Gly dipeptide has been introduced at position 197 and 216, respectively; this dipeptide originates from the addition of the Sma1 restriction site at the genetic level to allow further polyQ sequence insertion.
- Published
- 2018
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34. Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH, and on Substrate Relative Concentration.
- Author
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Bafort F, Damblon C, Smargiasso N, De Pauw E, Perraudin JP, and Jijakli MH
- Subjects
- Antifungal Agents chemistry, Antifungal Agents metabolism, Dose-Response Relationship, Drug, Hydrogen-Ion Concentration, Iodine chemistry, Iodine metabolism, Microbial Sensitivity Tests, Osmolar Concentration, Oxidation-Reduction, Structure-Activity Relationship, Thiocyanates chemistry, Thiocyanates metabolism, Antifungal Agents pharmacology, Iodine pharmacology, Lactoperoxidase metabolism, Penicillium drug effects, Thiocyanates pharmacology
- Abstract
The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I
- and SCN- in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3-fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength.13 C-labelled SCN- and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I2 SCN- ), giving an intense signal at 49.7 ppm in13 C-NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN- : I- ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN- >I- . We demonstrated that the formation of the signal required the interaction between I2 and SCN- , and MS showed the presence of I2 SCN- ., (© 2018 Wiley-VHCA AG, Zurich, Switzerland.)- Published
- 2018
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35. G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets.
- Author
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Harris LM, Monsell KR, Noulin F, Famodimu MT, Smargiasso N, Damblon C, Horrocks P, and Merrick CJ
- Subjects
- Humans, Malaria, Falciparum microbiology, Antimalarials pharmacology, G-Quadruplexes drug effects, Plasmodium falciparum drug effects, Plasmodium falciparum genetics
- Abstract
G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs., (Copyright © 2018 American Society for Microbiology.)
- Published
- 2018
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36. In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.
- Author
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Degueldre M, Echterbille J, Smargiasso N, Damblon C, Gouin C, Mourier G, Gilles N, De Pauw E, and Quinton L
- Subjects
- Amino Acid Sequence, Animals, Chromatography, Liquid, Dendroaspis genetics, Elapid Venoms analysis, Elapid Venoms chemistry, Elapid Venoms genetics, Glycopeptides genetics, Molecular Structure, Protein Processing, Post-Translational, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem Mass Spectrometry, Dendroaspis metabolism, Glycopeptides analysis, Glycopeptides chemistry, Proteomics methods
- Abstract
Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S -cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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37. The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase.
- Author
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Montagner C, Nigen M, Jacquin O, Willet N, Dumoulin M, Karsisiotis AI, Roberts GC, Damblon C, Redfield C, and Matagne A
- Subjects
- Catalytic Domain, Hydrophobic and Hydrophilic Interactions, Protein Structure, Secondary, Bacillus cereus enzymology, Protein Unfolding, Zinc chemistry, beta-Lactamases chemistry
- Abstract
Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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38. The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.
- Author
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Figueroa M, Sleutel M, Vandevenne M, Parvizi G, Attout S, Jacquin O, Vandenameele J, Fischer AW, Damblon C, Goormaghtigh E, Valerio-Lepiniec M, Urvoas A, Durand D, Pardon E, Steyaert J, Minard P, Maes D, Meiler J, Matagne A, Martial JA, and Van de Weerdt C
- Subjects
- Crystallography, X-Ray, Humans, Protein Folding, Protein Structure, Tertiary, Computer Simulation standards, Directed Molecular Evolution methods, Proteins chemistry, Recombinant Proteins chemistry
- Abstract
Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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39. Investigation of a suitable in vitro dissolution test for itraconazole-based solid dispersions.
- Author
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Thiry J, Broze G, Pestieau A, Tatton AS, Baumans F, Damblon C, Krier F, and Evrard B
- Subjects
- Bile Acids and Salts chemistry, Chemistry, Pharmaceutical methods, Dynamic Light Scattering methods, Excipients chemistry, Magnetic Resonance Spectroscopy methods, Polymers chemistry, Sodium Dodecyl Sulfate chemistry, Solubility, Surface-Active Agents chemistry, Water chemistry, Itraconazole chemistry
- Abstract
The difficulty to find a relevant in vitro dissolution test to evaluate poorly soluble drugs is a well-known issue. One way to enhance their aqueous solubility is to formulate them as amorphous solid dispersions. In this study, three formulations containing itraconazole (ITZ), a model drug, were tested in seven different conditions (different USP apparatuses and different media). Two of the formulations were amorphous solid dispersions namely Sporanox®, the marketed product, and extrudates composed of Soluplus® and ITZ produced by hot melt extrusion; and the last one was pure crystalline ITZ capsules. After each test, a ranking of the formulations was established. Surprisingly, the two amorphous solid dispersions exhibited very different behavior depending primarily on the dissolution media. Indeed, the extrudates showed a better release profile than Sporanox® in non-sink and in biphasic conditions, whilst Sporanox® showed a higher release profile than the extrudates in sink and fasted simulated gastric conditions. The disintegration, dynamic light scattering and nuclear magnetic resonance results highlighted the presence of interaction between the surfactants and Soluplus®, which slowed down the erosion of the polymer matrix. Indeed, the negative charge of sodium dodecyl sulfate (SDS) and bile salts interacted with the surface of the extrudates that formed a barrier through which the water hardly diffused. Moreover, Soluplus® and SDS formed mixed micelles in solution in which ITZ interacts with SDS, but no longer with Soluplus®. Regarding the biphasic dissolution test, the interactions between the octanol dissolved in the aqueous media disrupted the polymer--ITZ system leading to a reduced release of ITZ from Sporanox®, whilst it had no influence on the extrudates. All together these results pointed out the difficulty of finding a suitable in vitro dissolution test due to interactions between the excipients that complicates the prediction of the behavior of these solid dispersions in vivo., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
40. Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.
- Author
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Laurent C, Lekeux G, Ukuwela AA, Xiao Z, Charlier JB, Bosman B, Carnol M, Motte P, Damblon C, Galleni M, and Hanikenne M
- Subjects
- Adenosine Triphosphatases genetics, Amino Acid Motifs, Arabidopsis genetics, Arabidopsis Proteins genetics, Biological Transport, Cadmium metabolism, Cell Membrane, Cloning, Molecular, Magnetic Resonance Spectroscopy, Models, Molecular, Mutation, Protein Binding, Protein Conformation, Protein Transport, Zinc metabolism, Adenosine Triphosphatases metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Gene Expression Regulation, Plant physiology, Metals metabolism
- Abstract
PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.
- Published
- 2016
- Full Text
- View/download PDF
41. Complete ¹H, ¹⁵N, and ¹³C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid.
- Author
-
Karsisiotis AI, Damblon C, and Roberts GC
- Subjects
- beta-Lactamase Inhibitors metabolism, beta-Lactamase Inhibitors pharmacology, Bacillus cereus enzymology, Mandelic Acids metabolism, Mandelic Acids pharmacology, Nuclear Magnetic Resonance, Biomolecular, Sulfhydryl Compounds metabolism, Sulfhydryl Compounds pharmacology, beta-Lactamases chemistry, beta-Lactamases metabolism
- Abstract
β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96%) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.
- Published
- 2014
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42. Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A.
- Author
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Gahungu M, Arguelles-Arias A, Fickers P, Zervosen A, Joris B, Damblon C, and Luxen A
- Subjects
- 2-Amino-5-phosphonovalerate analogs & derivatives, 2-Amino-5-phosphonovalerate chemical synthesis, 2-Amino-5-phosphonovalerate chemistry, Anti-Infective Agents chemistry, Anti-Infective Agents pharmacology, Bacillus subtilis metabolism, Carbon-Oxygen Lyases metabolism, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Fungi drug effects, Fungi enzymology, Gram-Negative Bacteria drug effects, Gram-Negative Bacteria enzymology, Gram-Positive Bacteria drug effects, Gram-Positive Bacteria enzymology, Oligopeptides chemistry, Oligopeptides pharmacology, Organophosphorus Compounds chemistry, Organophosphorus Compounds pharmacology, Stereoisomerism, Streptomyces metabolism, Anti-Infective Agents chemical synthesis, Carbon-Oxygen Lyases antagonists & inhibitors, Enzyme Inhibitors chemical synthesis, Oligopeptides chemical synthesis, Organophosphorus Compounds chemical synthesis
- Abstract
Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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43. Efficient determination of diffusion coefficients by monitoring transport during recovery delays in NMR.
- Author
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Augustyniak R, Ferrage F, Damblon C, Bodenhausen G, and Pelupessy P
- Subjects
- Diffusion, Enzyme Inhibitors chemistry, Humans, Proto-Oncogene Proteins c-raf antagonists & inhibitors, Proto-Oncogene Proteins c-raf metabolism, RNA metabolism, Ubiquitin chemistry, Ubiquitin metabolism, beta-Lactamase Inhibitors, beta-Lactamases metabolism, Magnetic Resonance Spectroscopy
- Abstract
A novel NMR approach allows one to efficiently determine translational diffusion coefficients of macromolecules in solution. This method for Signal Optimization with Recovery in Diffusion Delays (SORDID) monitors transport occurring during the recovery times between consecutive scans so that the duration of the measurements can be reduced approximately by a factor two., (This journal is © The Royal Society of Chemistry 2012)
- Published
- 2012
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44. Ligand binding study of human PEBP1/RKIP: interaction with nucleotides and Raf-1 peptides evidenced by NMR and mass spectrometry.
- Author
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Tavel L, Jaquillard L, Karsisiotis AI, Saab F, Jouvensal L, Brans A, Delmas AF, Schoentgen F, Cadene M, and Damblon C
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Flavin Mononucleotide metabolism, Guanosine Triphosphate metabolism, Humans, Hydrogen-Ion Concentration, Mice, Models, Molecular, Molecular Sequence Data, Peptide Fragments chemistry, Phosphatidylethanolamine Binding Protein chemistry, Phosphorylation, Protein Binding drug effects, Protein Conformation, Rats, Salts pharmacology, Temperature, Ligands, Mass Spectrometry, Nuclear Magnetic Resonance, Biomolecular, Nucleotides metabolism, Peptide Fragments metabolism, Phosphatidylethanolamine Binding Protein metabolism, Proto-Oncogene Proteins c-raf chemistry
- Abstract
Background: Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown., Methods/findings: In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K(D) for different ligands. Native mass spectrometry was used as an alternative method for measuring K(D) values., Conclusions/significance: Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.
- Published
- 2012
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45. An alternative flexible conformation of the E. coli HUβ₂ protein: structural, dynamics, and functional aspects.
- Author
-
Garnier N, Loth K, Coste F, Augustyniak R, Nadan V, Damblon C, and Castaing B
- Subjects
- Bacterial Proteins metabolism, DNA-Binding Proteins metabolism, Escherichia coli Proteins metabolism, Hot Temperature, Magnetic Resonance Spectroscopy, Protein Conformation, Time Factors, Bacterial Proteins chemistry, DNA-Binding Proteins chemistry, Escherichia coli Proteins chemistry, Molecular Dynamics Simulation, Protein Denaturation
- Abstract
The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα₂, EcHUβ₂, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N₂ ↔ I₂) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N₂ to I₂ thermal transition of the EcHUβ₂ homodimer. On the basis of these data, a realistic 3D model is proposed for the major I₂ conformation of EcHUβ₂. This model is in agreement with previous experimental data.
- Published
- 2011
- Full Text
- View/download PDF
46. 1H, 13C and 15N backbone resonance assignments for the BS3 class A β-lactamase from Bacillus licheniformis.
- Author
-
Vandenameele J, Matagne A, and Damblon C
- Subjects
- Carbon Isotopes, Hydrogen, Nitrogen Isotopes, Bacillus enzymology, Nuclear Magnetic Resonance, Biomolecular, beta-Lactamases chemistry
- Abstract
Class A β-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-α domain formed by five to eight helices and an α/β domain consisting of a five-stranded antiparallel β-sheet covered by three to four α-helices. The α domain (~150 residues) is made up of the central part of the polypeptide chain whereas the α/β domain (111-135 residues) is constituted by the N- and C-termini of the protein. Our goal is to determine in which order the different secondary structure elements are formed during the folding of BS3. With this aim, we will use pulse-labelling hydrogen/deuterium exchange experiments, in combination with 2D-NMR measurements, to monitor the time-course of formation and stabilization of secondary structure elements. Here we report the backbone resonance assignments as the requirement for further hydrogen/deuterium exchange studies.
- Published
- 2010
- Full Text
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47. NMR structure of a phosphatidyl-ethanolamine binding protein from Drosophila.
- Author
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Rautureau GJ, Vovelle F, Schoentgen F, Decoville M, Locker D, Damblon C, and Jouvensal L
- Subjects
- Amino Acid Sequence, Animals, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Protein Structure, Secondary, Solutions, Structural Homology, Protein, Drosophila melanogaster chemistry, Phosphatidylethanolamine Binding Protein chemistry
- Published
- 2010
- Full Text
- View/download PDF
48. Positively cooperative binding of zinc ions to Bacillus cereus 569/H/9 beta-lactamase II suggests that the binuclear enzyme is the only relevant form for catalysis.
- Author
-
Jacquin O, Balbeur D, Damblon C, Marchot P, De Pauw E, Roberts GC, Frère JM, and Matagne A
- Subjects
- Circular Dichroism, Kinetics, Magnetic Resonance Spectroscopy, Mass Spectrometry, Protein Binding, Spectrophotometry, Anti-Bacterial Agents metabolism, Bacillus cereus enzymology, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Zinc metabolism, beta-Lactamases chemistry, beta-Lactamases metabolism, beta-Lactams metabolism
- Abstract
Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K(1)/K(2)> or =5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K(2)<80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility.
- Published
- 2009
- Full Text
- View/download PDF
49. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes.
- Author
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Smargiasso N, Gabelica V, Damblon C, Rosu F, De Pauw E, Teulade-Fichou MP, Rowe JA, and Claessens A
- Subjects
- Amino Acid Sequence, Animals, Antigens, Protozoan genetics, Circular Dichroism, Computational Biology, DNA, Protozoan genetics, Gene Expression Regulation, Magnetic Resonance Imaging, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, DNA, G-Quadruplexes, Plasmodium falciparum genetics, Promoter Regions, Genetic
- Abstract
Background: Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro., Results: We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q., Conclusion: This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.
- Published
- 2009
- Full Text
- View/download PDF
50. High-level biosynthesis of the anteiso-C(17) isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity.
- Author
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Fickers P, Guez JS, Damblon C, Leclère V, Béchet M, Jacques P, and Joris B
- Subjects
- Bacillus subtilis genetics, Biosynthetic Pathways genetics, Genes, Bacterial, Genetic Engineering, Lipoproteins biosynthesis, Microbial Sensitivity Tests, Protein Isoforms metabolism, Antifungal Agents biosynthesis, Bacillus subtilis metabolism, Candida drug effects
- Abstract
High-level production (880 mg liter(-1)) and isolation of the anteiso-C(17) isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.
- Published
- 2009
- Full Text
- View/download PDF
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