title: Proteolytic control of TGF-β co-receptor activity by BMP-1/tolloid-like proteases revealed by quantitative iTRAQ proteomics.
abstract: The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-β superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-β co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-β was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-β co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi: 10.6019/PXD000786 .
authors.affiliation: UMR 5305, Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, CNRS/Université de Lyon, 69367, Lyon, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Delolme
authors.forename: Cyril
authors.initials: C
authors.lastname: Anastasi
authors.forename: Lindsay B
authors.initials: LB
authors.lastname: Alcaraz
authors.forename: Valentin
authors.initials: V
authors.lastname: Mendoza
authors.forename: Sandrine
authors.initials: S
authors.lastname: Vadon-Le Goff
authors.forename: Maya
authors.initials: M
authors.lastname: Talantikite
authors.forename: Robin
authors.initials: R
authors.lastname: Capomaccio
authors.forename: Jimmy
authors.initials: J
authors.lastname: Mevaere
authors.forename: Laëtitia
authors.initials: L
authors.lastname: Fortin
authors.forename: Dominique
authors.initials: D
authors.lastname: Mazzocut
authors.forename: Odile
authors.initials: O
authors.lastname: Damour
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zanella-Cléon
authors.forename: David J S
authors.initials: DJ
authors.lastname: Hulmes
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.forename: Ulrich
authors.initials: U
authors.lastname: Valcourt
authors.forename: Fernando
authors.initials: F
authors.lastname: Lopez-Casillas
authors.forename: Catherine
authors.initials: C
authors.lastname: Moali
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D055395:Bone Morphogenetic Protein 1 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D005109:Extracellular Matrix / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000378:metabolism; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism; D039942:Neuropilin-1 / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C120523:CD109 protein, human; D058851:GPI-Linked Proteins; D009363:Neoplasm Proteins; D010455:Peptides; D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D039942:Neuropilin-1; C071736:betaglycan; D055395:Bone Morphogenetic Protein 1