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 .

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF‐β signaling as primary targets

“…In agreement with the specificity of the antibody used in Western blotting, the peptides observed by mass spectrometry cover the N‐terminal assembly domain of fibronectin. The precise cleavage site was then determined using a new approach called ATOMS (22, 23) coupled to TMT labeling and to a multienzymatic digestion. The 5 pep tides with the highest P:C ratios resulted from a unique and unambiguous cleavage site: V‐R‐AA 292 ↓ 293 Y‐Y‐Q,‐P (Fig.…”

“…Proteins were separated using SDS‐PAGE and stained with Coomassie blue. The cleavage site was determined by iTRAQ‐ATOMS according to the reported procedure (23). Investigation of testis morphology of WT or Adamts2 –/– males was performed by hematoxylin and eosin staining of paraffin‐embedded transversal sections.…”

BMP-1/tolloid-like proteinases synchronize matrix assembly with growth factor activation to promote morphogenesis and tissue remodeling

“…These functions seem particularly coherent in the contexts of development, growth and tissue repair. However, recent proteomic studies aimed at discovering new BTP substrates [43] suggest that we are just beginning to uncover the full spectrum of their activities and that future studies will reveal new and unexpected roles. In this regard, the BTP regulatory proteins described above may represent interesting alternatives to target specific functions without affecting other activities of BTPs.…”

“…2; [42]), other proteinases such as MMPs and plasmin can perform the subsequent steps which consist in the degradation of LAP and in the release of active TGF-β1. More recently, we have shown that BTPs could also contribute to maintaining high levels of active TGF-β1 in tissues by promoting the degradation of two TGF-β antagonists (soluble betaglycan and CD109; [43]). …”

BMP1-like proteinases are essential to the structure and wound healing of skin

“…Evidence has previously been presented that BTPs may be involved in modulating TGFβ signalling via cleavage of the extracellular antagonist LTBP (Latent TGFβ Binding Protein) [17], and by cleavage of TGFβ co-receptors betaglycan and CD109 [55]. TGFβ can enhance ECM formation via recruitment of ECM-producing myofibroblasts and by up-regulation of many ECM-related genes, such as those encoding collagens I and III [22, 56].…”