References of "De Pauw, Edwin"
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See detailStructural Determinants of Specificity and Catalytic Mechanism in mammalian 25-kDa Thiamine Triphosphatase
Delvaux, David; Kerff, Frédéric ULg; Murty, Mamidanna R.V.S. et al

in Biochimica et Biophysica Acta - General Subjects (2013), 1830

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine ... [more ▼]

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. Methods: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. Results: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. Conclusion: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. General significance: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins. [less ▲]

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See detailExpression of a protease of biotechnological interest cloned from C. d. collilineatus venom gland
Boldrini-Franca, Johaha; Rodrigues, RS; Santos-Silva, LK et al

Poster (2013)

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See detailPeptide backbone fragmentation initiated by side-chain loss at cysteine residue in matrixassisted laser desorption/ionization in-source decay mass spectrometry
Asakawa, Daiki; Smargiasso, Nicolas ULg; Quinton, Loïc ULg et al

in Journal of Mass Spectrometry [=JMS] (2013), 48

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical-induced ... [more ▼]

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical-induced cleavage leading to c0/z• fragments pair. MALDI-ISD is a very powerful method to obtain long sequence tags from proteins or to do de novo sequencing of peptides. Besides classical fragmentation, MALDI-ISD also shows specific fragments for which the mechanism of formation enlightened the MALDI-ISD process. In this study, the MALDI-ISD mechanism is reviewed, and a specific mechanism is studied in details: the N-terminal side of Cys residue (Xxx-Cys) is described to promote the generation of c0 and w fragments in MALDI-ISD. Our data suggest that for sequences containing Xxx-Cys motifs, the N–Ca bond cleavage occurs following the hydrogen attachment to the thiol group of Cys side-chain. The c•/w fragments pair is formed by side-chain loss of the Cys residue with subsequent radical-induced cleavage at the N–Ca bond located at the left side (N-terminal direction) of the Cys residue. This fragmentation pathway preferentially occurs at free Cys residue and is suppressedwhen the cysteines are involved in disulfide bonds. Hydrogen attachment to alkylated Cys residues using iodoacetamide gives free Cys residue by the loss of •CH2CONH2 radical. The presence of alkylated Cys residue also suppress the formation of c•/w fragments pair via the (Cb)-centered radical, whereas w fragment is still observed as intense signal. In this case, the z• fragment formed by hydrogen attachment of carbonyl oxygen followed side-chain loss at alkylated Cys leads to a w fragment. Hydrogen attachment on peptide backbone and side-chain of Cys residue occurs therefore competitively during MALDI-ISD process. [less ▲]

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See detailInfluence of lignin in Reticulitermes santonensis: symbiotic interations investigated through proteomics
Bauwens, Julien ULg; Tarayre, Cédric ULg; Brasseur, Catherine ULg et al

in Symbiosis (2013)

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting ... [more ▼]

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting together and with the insect host. Their likely ability to degrade ligno-cellulosic compounds could lead to improvements in second generation biofuels production. Lignin elimination represents a critical point as this polymer significantly interferes with industrial process of cellulose. Although host produces its own lignin-degrading enzymes, some symbionts may participate in digestion of lignin and its degradation products in termite gut. Here, we compared gut proteomes from R. santonensis after rearing on artificial diets composed of cellulose with and without lignin. The effect of lignin in artificial diets on different parts of the digestive tract was compared through liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) experiments. Enzymatic assays were performed to characterize activities present in R. santonensis digestive tract after feeding on artificial diets. Microscopic observations of microbial communities provided some information on population balances after feeding experiment. [less ▲]

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See detailFirst evidence of the possible implication of the 11-deoxycorticosterone (DOC) in immune activity of Eurasian perch (Perca fluviatilis, L.): Comparison with cortisol
Mathieu, Cédric; Mila, Sylvain; Mandiki, Robert et al

in Comparative Biochemistry & Physiology Part A : Molecular & Integrative Physiology (2013), 165(2), 149-158

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See detailIon-Mobility mass spectrometry as a potential tool to assign disulfide bonds arrangements in peptides with multiple disulfide bridges.
Echterbille, Julien ULg; Quinton, Loïc ULg; Gilles, Nicolas et al

in Analytical Chemistry (2013)

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually ... [more ▼]

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using MS/MS spectra of the totally reduced unfolded species but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of non-classical ions. MS/MS alone does not allow the cysteine pairing nor the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining Ion Mobility Spectrometry (IMS)and Collision Induced Dissociation(CID). It is assumed that the opening of one S-S bridges in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence, but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteines connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference. [less ▲]

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See detailSéminaire des chercheurs Télévie 2013
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, December 10)

Séminaire des chercheurs Télévie 2013

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See detailSecretion and maturation of conotoxins in the venom ducts of Conus textile
Dobson, Rowan ULg; Collodoro, Mike; Gilles, Nicolas et al

in Toxicon (2012), 60(8), 1370-1379

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See detailUtilisation des termites comme source de microorganismes dans la filière de production du bioéthanol de seconde génération
Tarayre, Cédric ULg; Bauwens, Julien ULg; Brasseur, Catherine ULg et al

Poster (2012, November 14)

Les termites abritent une microflore symbiotique qui intervient dans la dégradation des fibres constitutives du bois, synthétisant des enzymes capables d’hydrolyser ses composants. Les sucres ... [more ▼]

Les termites abritent une microflore symbiotique qui intervient dans la dégradation des fibres constitutives du bois, synthétisant des enzymes capables d’hydrolyser ses composants. Les sucres fermentescibles libérés suite à cette hydrolyse sont utilisables dans le cadre de la production du bioéthanol de seconde génération. [less ▲]

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See detailDetermination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, October 13)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and me<x>tastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by me<x>tastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

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See detailPotential of CE/MS for Small Carboxylic Acids Analysis as Alternative to GC/MS Reference Analytical Methods
Far, Johann ULg; Falmagne, Jean-Bernard, ANALIS R&D; de l'Escaille, François, ANALIS R&D et al

Poster (2012, October)

GC/MS (Gas Chromatography coupled to Mass Spectrometry) is the analytical method of choice for carboxylic acids analysis because of good sensitivity, low limit of detection and the possibility to compare ... [more ▼]

GC/MS (Gas Chromatography coupled to Mass Spectrometry) is the analytical method of choice for carboxylic acids analysis because of good sensitivity, low limit of detection and the possibility to compare the pattern of fragmentation with existing databases for identification. However GC requires that the analytes are volatile. If it is not the case, the use of chemicals in order to perform the derivatization is mandatory, this may induce analytical bias. CE (Capillary electrophoresis) is a technique of choice for ions separation without prior treatments. The method is fast and do not require highly technical skills. A UV detector is the most common detection method; however electrospray mass spectrometry detection is recently gaining interest, while it really helps for structural information of the detected compounds. In this poster, the preliminary results of CE/MS analysis of several carboxylic acids are presented. All carboxylic acids are analyzed without any sample pretreatment. These acids looked at are from the “Citric Acid Cycle” including pyruvate and some isotope labeled analogues but also glyoxylate, lactate, oxalate and tartrate. Moreover, the preliminary results of a sample preparation approach to remove phosphate salts are presented. Phosphate is a very common salt that is often used in biological buffers but prevents the derivatization of carboxylic acid for GC/MS analysis and reduces the reproducibility of results for both GC/MS and CE/MS analysis. [less ▲]

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See detailDevelopment of a quantitative approach to measure phospholipids in dried drops by Raman spectroscopy
Malherbe, Cédric ULg; Jadoul, Laure; Gilbert, Bernard ULg et al

Poster (2012, October)

We present here the results obtained during our tentative to analyse quantitatively dried drops of phospholipidic solutions by Raman spectroscopy. Drops of different solutions of phospholipid were deposed ... [more ▼]

We present here the results obtained during our tentative to analyse quantitatively dried drops of phospholipidic solutions by Raman spectroscopy. Drops of different solutions of phospholipid were deposed onto different material supports. The spots were then analyses by confocal Raman microspectroscopy. Experimental settings have been optimised and the analysis of the intensity profile of the Raman signal inside the spot allows the establishment of a calibration curve for the determination of the phospholipids amount within a 1 µL solution. [less ▲]

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See detailFuran formation upon degradation of carbohydrates in combination with proteins and lipids
Owczarek, Agnieszka; De Meulenaer, Bruno; Scholl, Georges ULg et al

Conference (2012, September 20)

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See detailIntraocular Lens Adsorbome: a Proteomic Study of Adsorbed Proteins onto Acrylic Materials and Its Implication in Secondary Cataract
Huang, Yi-Shiang ULg; Bertrand, Virginie ULg; Mazzucchelli, Gabriel ULg et al

Poster (2012, September 17)

The intraocular lens (IOL) is a polymer implant designed to replace the natural lens after cataract surgery. When the implant is introduced into the lens capsule, the polymer starts to interact with the ... [more ▼]

The intraocular lens (IOL) is a polymer implant designed to replace the natural lens after cataract surgery. When the implant is introduced into the lens capsule, the polymer starts to interact with the aqueous humour and the exchange of molecules between the solid and the liquid begins. The nature of exchange in water, ions, and biomolecules may result in several postoperative complications including glistening, calcification, and posterior capsular opacification. The posterior capsular opacification (PCO, also called “Secondary Cataract”) is raised from the over-growth of residual lens epithelial cells. The first step of the over-growth process of the cells is their adhesion to the deposited biomolecules, such as proteins involved in extra-cellular matrices. The purpose of this study is to identify the principal proteins adsorbed onto the acrylic polymers by mass spectrometry. The concept of adsorbome is to generate a list of adsorbed proteins to the hydrophilic and hydrophobic polymers, and then compare the difference to the original component of aqueous humour in order to see the affinity of individual protein to each material. Two kinds of hydrophilic and two kinds of hydrophobic acrylic polymers were tested for their adsorbomes by treating them with an aqueous humour analogue and the major adsorbed proteins were identified by mass spectrometry. Interestingly, the hydrophilic acrylic polymer shows a relative lower protein adsorption rate but shows a higher incidence of secondary cataract. This phenomenon implies the adsorbed proteins play a crucial role in progress of secondary cataract. [less ▲]

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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Ongena, Marc ULg et al

Conference (2012, September 05)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

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