title: Regulation of bone morphogenetic protein 9 (BMP9) by redox-dependent proteolysis.
abstract: BMP9, a member of the TGFβ superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.
authors.affiliation: From the Department of Medicine, University of Cambridge, School of Clinical Medicine, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Zhenquan
authors.initials: Z
authors.lastname: Wei
authors.affiliation: From the Department of Medicine, University of Cambridge, School of Clinical Medicine, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Salmon
authors.affiliation: From the Department of Medicine, University of Cambridge, School of Clinical Medicine, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Upton
authors.affiliation: From the Department of Medicine, University of Cambridge, School of Clinical Medicine, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, United Kingdom nwm23@cam.ac.uk.
authors.forename: Nicholas W
authors.initials: NW
authors.lastname: Morrell
authors.affiliation: From the Department of Medicine, University of Cambridge, School of Clinical Medicine, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, United Kingdom wl225@cam.ac.uk.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D042783:Endothelial Cells / Q000166:cytology; D005786:Gene Expression Regulation; D055427:Growth Differentiation Factor 2 / Q000378:metabolism; D055412:Growth Differentiation Factors / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D010084:Oxidation-Reduction; D010641:Phenotype; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018076:DNA, Complementary; D004220:Disulfides; C525887:GDF2 protein, human; C526108:Gdf2 protein, mouse; D055427:Growth Differentiation Factor 2; D055412:Growth Differentiation Factors; D008024:Ligands; D011994:Recombinant Proteins
keywords: Bone Morphogenetic Protein (BMP); Cell Signaling; Crystal Structure; Disulfide; Endothelial Cell; Redox Regulation