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Delolme, F; Anastasi, C; Alcaraz, LB; Mendoza, V; Vadon-Le Goff, S; Talantikite, M; Capomaccio, R; Mevaere, J; Fortin, L; Mazzocut, D; Damour, O; Zanella-Cléon, I; Hulmes, DJ; Overall, CM; Valcourt, U; Lopez-Casillas, F; Moali, C (2015)

PROTEOLYTIC CONTROL OF TGF-B CO-RECEPTOR ACTIVITY BY BMP-1/ TOLLOID-LIKE PROTEASES REVEALED BY QUANTITATIVE ITRAQ PROTEOMICS

Cellular and Molecular Life Sciences 72(5):1009-1027
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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 .