Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 1 Pro-BMP9 and pro-BMP10 are equivalent ALK1-ligands. a Dose-dependent signalling assays in PAECs. Serum-starved PAECs were treated with different ligands at 2.48 pM (white bars), 8.27 pM (light grey bars) and 27.3 pM (dark grey bars) (using monomer molecular weight, equivalent to 0.03, 0.1 and 0.33 ng ml−1 BMP9 GF-domain concentration) for 1 h. Changes in the ID1 gene expression were monitored using RT-qPCR. Data were presented as fold change relative to untreated cells, and means ± SEM of three independent experiments are shown. Source data are provided as a Source Data file. b–d Volcano plots comparing changes in global gene expression in PAECs after pro-BMP9 or pro-BMP10 treatment. Serum-starved PAECs were treated with 25 pM of pro-BMP9 or pro-BMP10 (purity can be found on SDS-PAGE with silver staining in Supplementary Fig. 8a, lanes 1 and 4) for 1.5 h before RNA was extracted for microarray analysis. Four different primary PAEC lines were used. Red dots above the dashed line represent the changes in target genes with adjusted p values of less than 0.05. Several representative target genes are highlighted in c and d. Full list of genes can be found in Supplementary Data 1 and 2. e Affinity measurements of BMP9 and BMP10 for ALK1 using Biacore. A CM5 Biacore chip was immobilised with ALK1 dimer (ALK1-Fc) or monomer (in-house purified ALK1 ECD, purity can be seen in Supplementary Fig. 8a, lane 7). The sensorgrams of BMP9, pro-BMP9, BMP10 and pro- BMP10 binding raw data (in black lines) were overlaid with a global fit to a 1:1 model with mass transport limitations (red lines). f A summary of kinetic parameters for ligand-receptor interactions derived from the Biacore measurements in e.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Molecular Weight, Concentration Assay, Gene Expression, Quantitative RT-PCR, SDS Page, Silver Staining, Microarray, Binding Assay, Derivative Assay

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 2 Crystal structure of human BMP10:ALK1 complex at 2.3 Å. a Crystal structure of BMP10 (cyan) in complex with ALK1 (magenta), overlaid with the structure of BMP9:ALK1:ActRIIb complex (PDB:4FAO, in grey and semi-transparent). Mol A and Mol B are the two BMP monomers whose interfaces with ALK1 (Mol C) were analysed in b. b Comparison of the buried interface upon complex formation between BMP10:ALK1 and BMP9:ALK1 (from 4FAO). Total buried surface area and the contributing residues were calculated using PDBePISA server.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Comparison

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 3 Specificity determinants in the BMP9 and BMP10 subfamily. a Sequence alignment of representative ALK-binding BMPs. GF-domain sequences of ALK1-binding BMP9 and BMP10, ALK6-binding BMP14, ALK3-binding BMP2 and BMP4, as well as ALK2-binding BMP6 and BMP7 are aligned. Lines over and below the sequences highlight the residues at the type I and type II receptor-binding surface based on BMP10:ALK1 and BMP9:ALK1:ActRIIb structures, respectively. Asterisk (*) marks the residues that are conserved among at least 6 out of 7 aligned BMPs. Residues preferentially conserved between BMP9 and BMP10 are highlighted, in cyan for those at the type I site (conserved region 1), in blue for those at the type II site (conserved region 2) and in yellow for those outside receptor binding surface (conserved region 3). BMP10 D338 and P366 are also highlighted in cyan because they make conserved interactions with ALK1 in the crystal structure (Fig. 4). b Residues from conserved regions 1–3 plotted on BMP10 structure and labelled with full length proBMP10 residue numbers. Fifteen residues from conserved regions 1–3 are shown in spheres, coloured accordingly. The first Gly from conserved region 3 is not modelled in the crystal structure, and hence not plotted. c An overlay of BMP10 (grey) onto the structures of BMP9 (gold, from 4FAO)9, BMP2 (green, from 2GOO)31 and BMP7 (cyan, from 1M4U)61 is shown from the side view (left) and the top view (right). The red arrows indicate the unique insertion in BMP9 and BMP10.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Sequencing, Binding Assay, Residue

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 4 Conserved region 1 and ALK1-specificity determinants. a ALK1-binding residues are mapped onto the BMP10 surface (grey), with those conserved across all BMPs in Fig. 3a coloured in red, those from the BMP9 and BMP10 conserved region 1 in cyan, and other variable residues in yellow. b ALK1 (magenta cartoon) binding to BMP10 (surface), with residues interacting with BMP10 shown in sticks. c–f Detailed interactions between BMP10 and ALK1. g Sequence alignment of four BMP-binding type I receptors, ALK1, ALK2, ALK3 and ALK6, with the four specificity-determining residues in ALK1 highlighted in yellow. Loop 4 and loop 5 are the two loops surrounding the 310 helix (Supplementary Fig. 4). h Overlaid structures of BMP type I receptors. The structure of ALK1 in the BMP10:ALK1 complex (magenta) is overlaid onto ALK1 in BMP9:ALK1 complex (PBD:4FAO, orange), ALK3 (PDB:2GOO, light green) and ALK6 (PDB:3EVS, light grey). ALK1 residues highlighted in g are shown in sticks.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Binding Assay, Sequencing

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 5 Conserved region 2 and type II site analysis. a ActRIIb-binding residues (based on PDB:4FAO, ActRIIb in semi-transparent cartoon) are mapped onto BMP9 surface (grey), with those conserved across all BMPs in Fig. 3a coloured in red, those from conserved region 2 in blue, and other variable residues in yellow. b Type II binding surface of BMP9 (left) and BMP10 (right), showing as electrostatic surface (generated in PyMOL, red representing negatively charged and blue positively charged surface). ActRIIb is shown in orange, semi-transparent cartoon. c Residues from BMP9 conserved region 2 make three backbone β-sheet and one sidechain H-bond interactions with ENG (PDB:5HZW, ENG in green, BMP9 in cyan). BMP10 is overlaid onto BMP9 and shown in grey, with four conserved region 2 residues shown in blue spheres. Sidechains of other residues are omitted for clarity. d Sequence alignment of human BMP10 prodomain (hBMP10_pro) with mouse BMP9 prodomain (mBMP9_pro) and human BMP9 prodomain (hBMP9_pro). Residues at the BMP9-binding surface are highlighted in yellow and those that make direct interactions with BMP9 GF-domain are marked with *. Residues that make main chain interactions are also marked with ^. Only the prodomain regions that interact with BMP9 GF-domain are shown, and full-length alignment of hBMP9_pro and hBMP10_pro can be found in Supplementary Fig. 5. e Residues in conserved region 2 of BMP9 make four backbone H-bond β-sheet interactions with prodomain (PDB:4YCG; prodomain in orange, BMP9 in cyan. BMP10 is overlaid on BMP9 and shown in grey. Four conserved BMP10 residues are in blue spheres).

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Binding Assay, Generated, Sequencing

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 7 CV2 does not inhibit BMP9 signalling. a Structural analysis. BMP10 (grey, with conserved region 2 residues in blue spheres) was overlaid onto the BMP2:CV2 structure (PDB:3BK3, CV2 in magenta and BMP2 in green). Four mainchain H-bonds that stabilise the BMP2:CV2 β-sheet interaction are shown. BMP9 has the same conformation as BMP10 in this region. b, c CV2 does not inhibit BMP9 signalling in PAECs. Serum-starved PAECs were treated with BMP9 or pro-BMP9 (at 1 ng ml−1 GF-domain concentration) without or with CV2 at 10-fold, 20-fold, 50-fold or 250-fold molar excess for 15 min to assess Smad1/5 phosphorylation using immunoblots (b) or for 1 h to assess ID1 gene expression using qPCR (c). One representative of three independent experiments is shown in b. Band intensity was quantified using Image J (version 1.51s). d CV2 inhibits BMP4 signalling in PASMCs. Serum-starved PASMCs were treated with BMP4 (25 ng ml−1) without or with CV2 at indicated molar excess for 15 min. Immunoblots and quantification were carried out as above. N = 3 independent experiments and one representative blot is shown. e CV2 inhibits BMP2 but not BMP9 signalling in C2C12 cells. Serum-starved C2C12 cells were treated with BMP2 (130 ng ml−1) or BMP9 (25 ng ml−1) without or with CV2 at the indicated molar excess for 68 h. ALP activity in the cell lysate were analysed (see Methods section). N = 7 independent experiments. For all panels, means ± SEM are shown. d, e One-way ANOVA for each BMP treatment group, Dunnett’s post hoc analysis against BMP alone-treated controls. Source data are provided as a Source Data file.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Gene Expression, Activity Assay

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 8 Modifying BMP9 signalling specificity by mutagenesis. A panel of BMP9 mutants were generated, and tested in vitro and in vivo as described in the Methods. a Mutant proteins were subject to endothelial cell signalling assays (at 0.3 ng ml−1 GF-domain concentration) using induction of ID1 gene expression in hPAECs as a readout, and osteogenic signalling assays (at 10 ng ml−1 GF-domain concentration) using ALP induction in C2C12 cells as a readout. Data were normalised to WT BMP9 and shown as fold change relative to WT upon mutation. Each treatment condition was repeated in 3–7 independent experiments alongside untreated and WT controls. The exact N number for each condition is given under the column. Means ± SEM are shown. b Recombinant WT pro-BMP9, pro-BMP9 D366E, pro-BMP10, as well as BMP2 GF-domain were subject to in vivo heterotopic bone-forming assays in the presence and absence of cardiotoxin. Each data point represents the HO result from an independent injection in one leg. N number for each treatment condition is given under each column. Data are presented as % ossification volume relative to the average of BMP2-treated controls. Means ± SEM are shown. c Representative CT images (left) and histological staining (right) of in vivo formed heterotopic bones after stimulation of indicated BMP molecules in the presence and absence of cardiotoxin. B: osteoid matrix; M: muscle cells. Scale bar = 500 µm.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Mutagenesis, Generated, In Vitro, In Vivo, Concentration Assay, Gene Expression, Recombinant, Injection, Staining

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

Article Snippet: BMP9 (GDF-2) and BMP10 were purchased from PeproTech (Cat.no 120–07 and 120–40, respectively).

Techniques: Expressing, Transfection, Control, Isolation, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: Graphical representation of the role of GATA6 in coordinating cross-talk between BMP10 and oxidative stress axis in PAH. GATA6 is an activator of anti-oxidant enzymes and its deficiency in PAEC and PASMC induces oxidative stress and mitochondrial dysfunction. BMP10 induces expression of GATA6 through the ALK1, BMPRII, ENG and ERK pathway. GATA6, in turn, transcriptionally activates BMP receptors in PAEC. Endothelial GATA6 regulates PASMC function via paracrine factors. TGFβ2 secreted by GATA6 deficient PAEC induces PASMC proliferation. Administration of dimethyl fumarate (DMF) to mice with endothelial Gata6 loss restores expression of BMP receptors, resolves oxidative stress, and reverses PH.

Article Snippet: BMP9 (GDF-2) and BMP10 were purchased from PeproTech (Cat.no 120–07 and 120–40, respectively).

Techniques: Expressing