Fig. 2: PADI4 promotes HIF-1α citrullination at R698.
From: Citrullination modulation stabilizes HIF-1α to promote tumour progression
a Diagram showing the in vitro citrullination assay with His-PADI4 protein and GST-HIF-1α proteins in catalytic buffer at 37 °C for 1.5 h. Modified GST-HIF-1α proteins were separated by SDS-PAGE and subsequently subjected to liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS) analysis. b The citrulline modification sites in HIF-1α were determined by LC-MS/MS. They were R17, R273, R463, R665, R671, and R698. c LC-MS/MS spectrum of the citrullinated peptide containing the R698 site in HIF-1α. d Molecular docking model for the interaction of PADI4 (RCSB PDB: 1WDA) with HIF-1α. A close-up view of the peptide binding site of PADI4 is shown in the cartoon, in which green represents the surface and orange represents the pocket structure. The HIF-1α 693VALSQRTTVP702 peptide is shown as a magenta ribbon and the stick represents side chains (coloured by atom type: oxygen, red; nitrogen, blue; C, grey). The interfacial regions between PADI4 (blue) and HIF-1α (magenta) indicate an interaction between R698 on HIF-1α and the pocket structure of PADI4. W347, D350, V469, H471, H640 and C645 of PADI4 come in close contact. e The surface electrostatic potential of the docking model. Positively charged R698 of the HIF-1α 693VALSQRTTVP702 peptide with the negatively charged pocket structure of PADI4. The electrostatic potential is colour-coded as −69.540 kcal/mol (red) to +69.540 kcal/mol (blue), thus displaying negative or positive charges, respectively. f HEK293T cells expressing WT Flag-HIF-1α or Flag-HIF-1α R698A together with HA-PADI4 were used for immunoprecipitation analysis. g An in vitro citrullination assay was performed by incubating purified WT GST-HIF-1α or GST-HIF-1αR698A with His-PADI4 at 37 °C for 1.5 h, followed by western blotting analysis. h IF analysis of the colocalization of citrullinated HIF-1αR698 with HIF-1α or PADI4 in Hep3B cells under hypoxic conditions. Scale bars: 10 μm (left panel). Intensity profiles of each line were quantified by ImageJ software and drawn with GraphPad Prism 7.0 (right panel). Immunoblots and immunofluorescence are representative of three independent experiments (f–h). Figure 2a, created with BioRender.com, is released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.
