Showing papers by "Sylvie Ricard-Blum published in 2017"

Pro-angiogenic capacities of microvesicles produced by skin wound myofibroblasts.

Abstract: Wound healing is a very highly organized process where numerous cell types are tightly regulated to restore injured tissue. Myofibroblasts are cells that produce new extracellular matrix and contract wound edges. We previously reported that the human myofibroblasts isolated from normal wound (WMyos) produced microvesicles (MVs) in the presence of the serum. In this study, MVs were further characterized using a proteomic strategy and potential functions of the MVs were determined. MV proteins isolated from six WMyo populations were separated using two-dimensional differential gel electrophoresis. Highly conserved spots were selected and analyzed using mass spectrometry resulting in the identification of 381 different human proteins. Using the DAVID database, clusters of proteins involved in cell motion, apoptosis and adhesion, but also in extracellular matrix production (21 proteins, enrichment score: 3.32) and in blood vessel development/angiogenesis (19 proteins, enrichment score: 2.66) were identified. Another analysis using the functional enrichment analysis tool FunRich was consistent with these results. While the action of the myofibroblasts on extracellular matrix formation is well known, their angiogenic potential is less studied. To further characterize the angiogenic activity of the MVs, they were added to cultured microvascular endothelial cells to evaluate their influence on cell growth and migration using scratch test and capillary-like structure formation in Matrigel®. The addition of a MV-enriched preparation significantly increased endothelial cell growth, migration and capillary formation compared with controls. The release of microvesicles by the wound myofibroblasts brings new perspectives to the field of communication between cells during the normal healing process.

Glycosaminoglycanomics: where we are.

Abstract: Glycosaminoglycans regulate numerous physiopathological processes such as development, angiogenesis, innate immunity, cancer and neurodegenerative diseases. Cell surface GAGs are involved in cell-cell and cell-matrix interactions, cell adhesion and signaling, and host-pathogen interactions. GAGs contribute to the assembly of the extracellular matrix and heparan sulfate chains are able to sequester growth factors in the ECM. Their biological activities are regulated by their interactions with proteins. The structural heterogeneity of GAGs, mostly due to chemical modifications occurring during and after their synthesis, makes the development of analytical techniques for their profiling in cells, tissues, and biological fluids, and of computational tools for mining GAG-protein interaction data very challenging. We give here an overview of the experimental approaches used in glycosaminoglycomics, of the major GAG-protein interactomes characterized so far, and of the computational tools and databases available to analyze and store GAG structures and interactions.

Overall Structural Model of NS5A Protein from Hepatitis C Virus and Modulation by Mutations Confering Resistance of Virus Replication to Cyclosporin A.

Abstract: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding phosphoprotein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3) The knowledge of the functional architecture of this multifunctional protein remains limited We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes We also investigated the eff

The Multimerization State of the Amyloid-β42 Amyloid Peptide Governs its Interaction Network with the Extracellular Matrix.

Abstract: The goals of this work were i) to identify the interactions of amyloid-β (Aβ)42 under monomeric, oligomeric, and fibrillar forms with the extracellular matrix (ECM) and receptors, ii) to determine the influence of Aβ42 supramolecular organization on these interactions, and iii) to identify the molecular functions, biological processes, and pathways targeted by Aβ42 in the ECM. The ECM and cell surface partners of Aβ42 and its supramolecular forms were identified with protein and glycosaminoglycan (GAG) arrays (81 molecules in triplicate) probed by surface plasmon resonance imaging. The number of partners of Aβ42 increased upon its multimerization, ranging from 4 for the peptide up to 53 for the fibrillar aggregates. The peptide interacted only with ECM proteins but their percentage among Aβ42 partners decreased upon multimerization. Aβ42 and its supramolecular forms recognized different molecular features on their partners, and the partners of Aβ42 fibrillar forms were enriched in laminin IV-A, N-terminal, and EGF-like domains. Aβ42 oligomerization triggered interactions with receptors, whereas Aβ42 fibrillogenesis promoted binding to GAGs, proteoglycans, enzymes, and growth factors and the ability to interact with perineuronal nets. Fibril aggregation bind to further membrane proteins including tumor endothelial marker-8, syndecan-4, and discoidin-domain receptor-2. The partners of the Aβ42 supramolecular forms are enriched in proteins contributing to cell growth and/or maintenance, involved in integrin cell surface interactions and expressed in kidney cancer, preadipocytes, and dentin. In conclusion, the supramolecular assembly of Aβ42 governs its ability to interact in vitro with ECM proteins, remodeling and crosslinking ECM enzymes, proteoglycans, and receptors.

Protein–glycosaminoglycan interaction networks: Focus on heparan sulfate☆

Abstract: Summary Sulfated glycosaminoglycans (GAGs) are complex polysaccharides, which are covalently bound to protein cores to form proteoglycans. They are mostly located at the cell surface and in the extracellular matrix (ECM) where they regulate numerous biological processes. The aim of our work is (i) to identify and characterize protein–GAG interactions occurring at the cell surface and in the ECM, (ii) to study the assembly of multimolecular complexes formed at the cell surface via protein–heparan sulfate interactions, (iii) to determine the roles of these complexes in the ECM maturation and assembly, which are initiated in the pericellular matrix, and in pathological situations such as angiogenesis and host–pathogen interactions, (iv) to build, contextualize and analyze the corresponding protein–heparan sulfate interaction networks to identify molecular connections between the physio-pathological processes mentioned above and to select protein–GAG complexes specifically formed in a pathological situation and which might be therapeutic targets.

Glycosaminoglycans: major biological players

Abstract: Glycosaminoglycans are complex polysaccharides, which play major biological roles in health and disease, intracellularly, at the cell surface and in the extracellular matrix. The sulfated glycosaminoglycans, namely chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate are covalently attached to various protein cores to form proteoglycans [1]. In contrast hyaluronan, a non-sulfated glycosaminoglycan, does not form proteoglycans but mediates the formation of highmolecular-weight proteoglycan aggregates. In this special issue, various aspects of glycosaminoglycans and their biological roles are discussed. Glycosaminoglycans with unique sulfation patterns are present in primitive chordates. Their structure, biological functions, intracellular and extracellular distribution in different species and tissues of ascidians are reviewed by Karamanou et al. The variability in heparin sequence and in structure between different cells and tissues in the same species are investigated by Mulloy et al., who focus on the GAGs carried by the proteoglycan serglycin in mast cells and show that mast cell populations differ not only in the proportions of different GAGs, but also in their fine structure. Sulfation patterns contribute indeed to the structural diversity and biological activities of GAGs. The role of 6O-sulfation in the structure/function relationships of heparan sulfate, its synthesis by 6-O-sulfotransferases, and its removal by 6-O-endosulfatases are detailed by ElMasri et al. A complex interplay of heparan sulfate, chondroitin/dermatan sulfate and hyaluronan biosynthesis regulates cancer cell viability, motility and adhesion to the extracellular matrix (Viola et al.). These findings are important for the development of GAGtargeted therapeutic approaches in cancer. The role of heparan sulfate proteoglycans in breast cancer and the use of heparin and nano-heparin formulations with anti-metastatic and antiangiogenic activities as therapeutic tools in breast cancer are reviewed by Afratis et al. Polysaccharides mimicking heparan sulfate, called ReGeneraTing Agents (RGTAs), have been chemically engineered to replace heparan sulfate in injured tissues and facilitate tissue repair, which opens new perspectives in regenerative medicine (Barritault et al.). Sulfated glycosaminoglycans are attached to core proteins. Heparan sulfate, proteoglycans such as syndecans, glypicans, perlecan, and agrin, which bind to growth factors, chemokines and cytokines, are key regulators of the mesenchymal niche of hematopoietic stem cells and play a role in the interaction of hematopoietic stem cells with their endosteal niche in bone marrow (Papy-Garcia and Albanese). Frey et al. report a novel biological function of the soluble proteoglycan biglycan, which is mediated by its interaction with Toll-like receptor-2. This small leucine-rich proteoglycan promotes the synthesis of erythropoietin, which leads to secondary polycythemia disease characterized by a selective increase in circulating mature erythrocytes. GAGs are able to bind a variety of proteins and these interactions are mediated and regulated among other things by their sulfation pattern and epimerization. The characterization at the molecular level of GAG-protein interactions is crucial to decipher the molecular mechanisms supporting GAG biological activities and to design inhibitors targeting these interactions. The interactions established by chondroitin sulfate, the most abundant GAG in the central nervous system extracellular matrix, with various protein families involved in physiological and cognitive mechanisms are reviewed by Djerbal et al. Babik et al. characterize the interactions of Fibroblast Growth Factor-1 with heparin and heparin derivatives by docking techniques and * Sylvie Ricard-Blum sylvie.ricard-blum@univ-lyon1.fr

Chapter 11:Strategies for Building Protein–Glycosaminoglycan Interaction Networks Combining SPRi, SPR, and BLI

Abstract: Sulfated glycosaminoglycans (GAGs) are complex, linear polysaccharides that are covalently linked to proteins to form proteoglycans They are located in the extracellular matrix and at the cell surface and interact with many proteins More than 400 interactions have been reported for heparin/heparan sulfate and these interactions are involved in numerous biological processes such as development, angiogenesis, tumor growth, host–pathogen interactions and inflammation, extracellular matrix (ECM) assembly, cell–matrix interactions and signaling The building of GAG–protein interaction networks is required to determine how these individual interactions influence each other in vivo, are coordinated in biological processes, and are altered in diseases This chapter reports the roadmap designed to build and analyze these interaction networks New interactions were identified by surface plasmon resonance imaging (SPRi) using a Biacore Flexchip system and were combined with data manually curated from the literature to build a GAG–protein network The values of equilibrium dissociation constants and of association and dissociation rates, calculated by SPR and biolayer interferometry (BLI), were integrated into the network The network was then analyzed in silico to determine the biological processes and pathways associated with GAG partners

Interaction of Complement Defence Collagens C1q and Mannose-Binding Lectin with BMP-1/Tolloid-like Proteinases.

Abstract: The defence collagens C1q and mannose-binding lectin (MBL) are immune recognition proteins that associate with the serine proteinases C1r/C1s and MBL-associated serine proteases (MASPs) to trigger activation of complement, a major innate immune system. Bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (BTPs) are metalloproteinases with major roles in extracellular matrix assembly and growth factor signalling. Despite their different functions, C1r/C1s/MASPs and BTPs share structural similarities, including a specific CUB-EGF-CUB domain arrangement found only in these enzymes that mediates interactions with collagen-like proteins, suggesting a possible functional relationship. Here we investigated the potential interactions between the defence collagens C1q and MBL and the BTPs BMP-1 and mammalian tolloid-like-1 (mTLL-1). C1q and MBL bound to immobilized BMP-1 and mTLL-1 with nanomolar affinities. These interactions involved the collagen-like regions of the defence collagens and were inhibited by pre-incubation of C1q or MBL with their cognate complement proteinases. Soluble BMP-1 and mTLL-1 did not inhibit complement activation and the defence collagens were neither substrates nor inhibitors of BMP-1. Finally, C1q co-localized with BMP-1 in skin biopsies following melanoma excision and from patients with recessive dystrophic epidermolysis bullosa. The observed interactions provide support for a functional link between complement and BTPs during inflammation and tissue repair.