References of "De Pauw, Edwin"
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See detailMyoferlin is a novel exosomal protein and functional regulator of cancer-derived exosomes
Blomme, Arnaud; Fahmy, Karim; Peulen, Olivier ULg et al

in Oncotarget (2016)

Exosomes are communication mediators participating in the intercellular exchange of proteins, metabolites and nucleic acids. Recent studies have demonstrated that exosomes are characterized by a unique ... [more ▼]

Exosomes are communication mediators participating in the intercellular exchange of proteins, metabolites and nucleic acids. Recent studies have demonstrated that exosomes are characterized by a unique proteomic composition that is distinct from the cellular one. The mechanisms responsible for determining the proteome content of the exosomes remain however obscure. In the current study we employ ultrastructural approach to validate a novel exosomal protein myoferlin. This is a multiple C2-domain containing protein, known for its conserved physiological function in endocytosis and vesicle fusion biology. Emerging studies demonstrate that myoferlin is frequently overexpressed in cancer, where it promotes cancer cell migration and invasion. Our data expand these ndings by showing that myoferlin is a general component of cancer cell derived exosomes from different breast and pancreatic cancer cell lines. Using proteomic analysis, we demonstrate for the rst time that myoferlin depletion in cancer cells leads to a signi cantly modulated exosomal protein load. Such myoferlin-depleted exosomes were also functionally de cient as shown by their reduced capacity to transfer nucleic acids to human endothelial cells (HUVEC). Beyond this, myoferlin-depleted cancer exosomes also had a signi cantly reduced ability to induce migration and proliferation of HUVEC. The present study highlights myoferlin as a new functional player in exosome biology, calling for novel strategies to target this emerging oncogene in human cancer. [less ▲]

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See detailMyoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer
Blomme, Arnaud; Costanza, Brunella ULg; De Tullio, Pascal ULg et al

in Oncogene (2016)

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as ... [more ▼]

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients. [less ▲]

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See detailContribution of Cross-linking and Ion-mobility for the study of protein and complex structures
Baumans, France ULg; Grifnée, Elodie ULg; Hage, Christoph et al

Poster (2016, September 05)

The tridimensional structures of proteins and the mapping of protein-protein interactions are precious sources of information for the understanding of their function. Different techniques such as X-ray ... [more ▼]

The tridimensional structures of proteins and the mapping of protein-protein interactions are precious sources of information for the understanding of their function. Different techniques such as X-ray cristallography or nuclear magnetic resonance are usually used to achieve this goal. In the field of mass spectrometry, several tools were also developped. The one presented here is the chemical cross-linking in which two reactive residue side chains, spatially close, are linked thanks to a bifunctional chemical, called crosslinker. Ion-mobility coupled to mass spectrometry has also been investigated for the study of cross-linked products. The first results tend to show that cross-linkers allow to fix the shape of the protein in solution, leaving it intact when analysed in the gas phase. [less ▲]

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See detailMALDI-imaging guided microproteomics workflow for biomarker discovery of intra-tumor heterogeneity
Alberts, Deborah ULg; Longuespée, Rémi ULg; Smargiasso, Nicolas ULg et al

Poster (2016, June 09)

Introduction A single tumoral tissue can bear phenotypically different cell populations. This phenomenon called intra-tumor heterogeneity can lead to differential behaviors regarding metastasis seeding ... [more ▼]

Introduction A single tumoral tissue can bear phenotypically different cell populations. This phenomenon called intra-tumor heterogeneity can lead to differential behaviors regarding metastasis seeding and therapy resistance [Zardavas et al., Nature Rev. Clin. Onc. 2015]. MALDI imaging has proven its efficiency for revealing hidden molecular features offering an insight into distinct cellular regions based on their molecular content. Further, proteomics applied to these regions could allow depicting the molecular context associated to particular cells groups and enable the collection of qualitative, quantitative and spatial information for each protein. Methods Breast cancer Formalin Fixed and Paraffin Embedded tissues, from patients whose outcome had been recorded over a period of 10 years, were provided by the department of Pathology of University of Liège. After Citric Acid Antigen Retrieval and trypsin digestion, images were obtained by MALDI-TOF/TOF-MS (Bruker, Germany). From the obtained datasets, segmentation and analytical data analysis were applied using SCiLS (Bruker, Germany) and the cloud software Multimaging (ImaBiotech, France). Small tissue areas were obtained by laser microdissection (LEICA LMD 700, Germany), upon which a combination of chemical processes was applied to ensure optimal protein antigen retrieval, extraction and digestion. Finally, the tissue pieces obtained were analyzed by LC-MS/MS using UPLC Waters Nanoacquity and Thermo Q-Exactive instruments. Preliminary data Based on mathematical calculations for the MALDI imaging datasets of the breast cancer FFPE tissues, Regions Of Interest (ROIs) were detected in a single tumor, revealing intra-tumoral heterogeneity, which can be correlated to the level of aggressiveness of the affliction and to the final prognosis of the patient. We aimed to compare the proteomic profiles of each of the small ROIs. Until today, proteomics applied to tissues composed by a restricted number of cells is quite tedious due to possible tissue losses during their handling. Recently, Longuespée [Longuespée et al., Methods 2015] published a method in order to retrieve the identification of 1400 proteins from microdissected tissue pieces containing only 2700 cells. This whole procedure allowed us to identify a panel of protein that characterizes tissue heterogeneity within a single tumor. This proves the applicability of the combination of MALDI imaging for the discovery of intra-tumoral heterogeneity without a priori, on a mathematical basis, and classical proteomics applied on laser-microdissected tissue samples of very restricted areas. This method will now be applied to several MALDI datasets in order to retrieve commune ROIs and to associate their presence with the information of each patient, such as their prognosis. Those ROIs will then be microdissected and subjected to microproteomic methods that will allow us to retrieve the extensive molecular context associated to bad patient prognosis and/or therapy resistance. The possibility to identify protein/peptide markers will have the power to predict the outcome of the breast cancer patient at the beginning of their treatment, and thus, improve the clinical care for the benefit of the patients. Novel aspect The workflow combines the unique advantages of MALDI imaging for de novo molecular features characterization and LMD-based microproteomics. [less ▲]

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See detailMethodology to fish peptide ligands of nAChRs from Cone snail venoms by MALDI-TOF mass spectrometry
Echterbille, Julien ULg; Gilles, Nicolas; Araoz, Romulo et al

Poster (2016, June)

More than 50,000 of venomous species are currently indexed in the world. Each of their venom is composed of hundreds of toxins which potentially exhibit a high selectivity for membrane receptors such as ... [more ▼]

More than 50,000 of venomous species are currently indexed in the world. Each of their venom is composed of hundreds of toxins which potentially exhibit a high selectivity for membrane receptors such as GPCRs or ion channels. Among them, nAChRs are a target for drug discovery, primarily for treating central nervous system troubles. Therefore, the discovery of pharmacological tools and innovative drugs targeting nAChRs from animal venoms appears as an evidence. This study proposes the use a mass-spectrometry based methodology1 to discover new nAChRs ligands from cone snails venoms, and particularly -conotoxins (a-CTXs), known as potential antagonists of nAChRs2. in few words, Torpedo membranes, containing a high concentration of nAChRs, are incubated with BSA tryptic digests (>100 peptides) doped by small amounts of known a-CTXs. After two hours incubation, free (i.e. containing molecules remaining in solution) and bound (i.e. peptides bound to the membranes) fractions were analyzed with a MALDI-TOF/TOF mass spectrometer. The POC (positive and negative controls) as well as a real screening of Conus ermineus venom are presented. [less ▲]

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See detailPeptidoglycan fragments separation by CE/LC-MS
Boulanger, Madeleine ULg; Delvaux, Cédric ULg; Far, Johann ULg et al

Poster (2016, May 24)

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from ... [more ▼]

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from the peptidoglycan (PG) degradation via an “AND Gate” regulation. This regulation involves the cellular stress induced by the beta-lactam, the membrane receptor BlaR1 and the PG turnover. Briefly, the induction occurs when the extracellular domain of BlaR1 is acylated by the antibiotic which leads to a reorganization of the transmembrane segments and the receptor autocleavage. Simultaneously, the beta-lactam partially inhibits the penicillin-binding protein 1 (PBP1), triggering increased PG turnover and accumulation of PG fragments. Some of these fragments could enter in the cytoplasm and undergo enzymatic degradations which lead to the formation of the pro co-activator (tripeptide L-Ala-D-Glu-m-A2pm). This pro co-activator generates the co-activator, the dipeptide D-Glu-m-A2pm. Nowadays the nature and the concentration of PG fragments inside the cytoplasm are unknown. Therefore, the development of different analytical methods is required in order to identify those cytoplasmic fragments. In this poster, three different ways to separate PG fragments are discussed. [less ▲]

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See detailNew methodology to detect toxin-nAChRs binding by MALDI-TOF mass spectrometry
Echterbille, Julien ULg; Gilles, Nicolas; Araoz, Romulo et al

Conference (2016, April 18)

More than 50 thousands of venomous species are currently indexed in the world. Each of their venoms is composed of 200 to 1000 different toxins which potentially exhibit a high selectivity for membrane ... [more ▼]

More than 50 thousands of venomous species are currently indexed in the world. Each of their venoms is composed of 200 to 1000 different toxins which potentially exhibit a high selectivity for membrane receptors such as G-protein coupled receptors or ion channels such nicotinic acetylcholine receptors (nAChRs). The latter have been a target for drug discovery efforts, primarily for central nervous system indications. Therefore, it appears of prime interest to design new pharmacological tools and potentially discover future drugs targeting this kind of ion channels. In 2015, our group published a new mass spectrometry-based methodology to screen peptide ligands for G protein coupled receptors1. The proof of concept of this methodology was built by studying the binding of [Arg8]-vasopressin (AVP) on type 2-vasopressin receptor (V2). We extended this methodology to another system ligand-receptor. As all Conus species venoms investigated so far contain at least one toxin antagonizing nAChRs: the alpha-conotoxins. Therefore, the ligand-receptor model couple that has been chosen is nAChRs-alpha-conotoxins. Experimentally, fragments of cellular membranes over-expressing nAChRs were incubated with Bovine Serum Albumine (BSA) tryptic digest (~100 peptide toxins) doped by a small amount of Alpha-conotoxins. After 2 hours incubation, free and bound fractions were purified with a combination of centrifugation and micro column purifications. Samples were finally analyzed with a MALDI-TOF/TOF mass spectrometer. By comparison of the intensity of Alpha-conotoxins in the free and in the bound fractions, we clearly detect an enrichment of nAChRs ligand in the latter. In order to transpose the methodology to natural mixture, we applied the workflow to crude conus venoms. We incubated membranes over-expressing nAChRs with Conus textile venom which is known to possess at least 5 different alpha-conotoxins. Thanks to our approach, we were able to detect an enrichment of these known ligands in the bound fraction. In order to validate the potential of our approach, the next step of this work will be the incubation of a Conus venom for which no alpha-conotoxins have been described. [less ▲]

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See detailFrom Valeriana officinalis to cancer therapy: the success of a bio-sourced compound
Hamaïdia, Malik ULg; Barez, Pierre-Yves ULg; Carpentier, Alexandre ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment (2016), 20

Over the centuries, bio-sourced compounds isolated from plants, insects and microorganisms have been a potent source of drugs for the treatment of human diseases. In this review, we recapitulate the story ... [more ▼]

Over the centuries, bio-sourced compounds isolated from plants, insects and microorganisms have been a potent source of drugs for the treatment of human diseases. In this review, we recapitulate the story of one of these compounds, 2-propylpentanoic acid, derived from the Valeriana officinalis flowering plant and its path to validation as a cancer treatment. [less ▲]

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See detailContribution of Capillary Electrophoresis and Ion Mobility Spectrometry to probe conformational change during desolvation
Far, Johann ULg; Delvaux, Cédric ULg; Kune, Christopher ULg et al

Conference (2016, January 28)

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium ... [more ▼]

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium conformation in the gas phase. The pathway to reach the thermodynamically stable conformation involves crossing potential barriers of different heights. When these barriers are too high compared to the internal energy of the ions, it will result in “partial memories” (as structural preservation) of the conformation in solution. In order to evaluate the effect of the solvent evaporation and of the various collision processes encountered by the ions in the mass spectrometer, we developed two strategies: The first strategy consists in comparing in a single experiment the shape of the ions in solution and in the gas phase. Data are obtained by coupling Capillary Electrophoresis (CE) with Ion Mobility Mass Spectrometry (IMS). Drift times in solution and in the gas phase are directly compared. Deviations from their correlation points out changes in folding upon desolvatation. Preliminary results show that some of peptides issued from tryptic digest of BSA clearly change their conformation during desolvatation. The second strategy consists to monitor the shape of the different conformers observed for the intact cytochrome C during native runs in capillary electrophoresis and “native” ion mobility mass spectrometry, both using a physiological TRIS acetic pH7 buffer compared to a “MS friendly” ammonium acetate pH7 buffer. Finally as preliminary study, we proposed to use homopolymers for better understanding about the folding behavior in gas phase and the resulting shape of these ions using ion mobility spectrometry. [less ▲]

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