Ta respectively in Fig 3A. Amino acids within both of theseFig

Ta respectively in Fig 3A. Amino acids within both of theseFig 3. MAGE-A2 interacts with the p53-binding hydrophobic cleft in the N-terminus of MDM2. (A) The structure of the N-terminus (p53-binding domain) of MDM2 (1YCR) showing the positions of MAGE-A2 binding peptides 51?5 (red) and 91?05 (magenta). The backbone is shown in green. The locations of the amino acids at the flanks of fpsyg.2017.00209 each of these peptides are shown. (B) Schematic showing the structure of the GST-MDM2 mini-protein, MP1, used in this experiment. A version in which GST was substituted by GFP and was used for cultured cell expression and immunoprecipitation analysis. (C) Coimmunoprecipitation analysis was carried out following expression of GFP-MP1 (or GFP alone as control) in H1299 cells together with MAGE-A2 or p53 (as positive control). (D) GST pull-down assays were performed in which 35S-radiolabelled MAGE-A2, or 35S-radiolabelled p53 as control, were captured on glutathione sepharose 4B beads using GST linked to the MDM2 mini-protein, MP1, which encompasses amino acids 1?10 including the hydrophobic p53-binding cleft. The association of MAGE-A2 or p53 was measured in the presence of increasing concentrations of Nutlin-3a. The co-precipitating MAGE-A2 and p53 proteins were detected by SDS-PAGE followed by fluorography. Note that in vitro transcription/translation of p53 gives rise to two polypeptides. (E) Quantification of the pull-down experiment following densitometry. In all cases the data are order Z-DEVD-FMK representative of at least three independent experiments. doi:10.1371/CPI-455 web journal.pone.0127713.gPLOS ONE | DOI:10.1371/journal.pone.0127713 May 22,9 /MAGE-A Inhibits MDM2 and Increases MDM4 Levelsregions are required for p53 binding [44] suggesting either that MAGE-A2 may interact with MDM2 in a manner similar to that of p53 or that it may influence the p53/MDM2 interaction. To determine whether MAGE-A2 binds to the N-terminus of MDM2 in a cellular context, the “MP1” MDM2 mini-protein (comprising amino acids 1?10 of MDM2) was expressed as a fusion with green fluorescent protein (GFP; Fig 3B) together with MAGE-A2 in H1299 cells. As a positive control, p53 was expressed in place of GFP-MP1. Co-immunoprecipitation analysis (Fig 3C) confirmed that both MAGE-A2 and the p53 control were able to associate with the GFP-MP1(MDM2) wcs.1183 in the cultured cells. No association was observed when GFP lacking the MP1 extension was used. To test the idea that p53 and MAGE-A2 bind to MDM2 in a similar manner, GST pulldown experiments were carried out in vitro to determine whether the competitive inhibitor, Nutlin-3a, which mimics p53 and binds tightly within the hydrophobic groove in MDM2 that constitutes the p53 binding site, could block the interaction of MAGE-A2 with MDM2 [45]. The data show that increasing concentrations of Nutlin-3a do indeed prevent MAGE-A2 from binding to the N-terminus of MDM2 (Fig 3D and 3E). The interaction of p53 with MDM2, which was measured in the same experiment as a control, was also progressively inhibited by increasing concentrations of Nutlin-3a (Fig 3D and 3E). The IC50 values for dissociation of p53 and MAGE-A2 respectively from MDM2 were 18 M and 10 M indicating that p53 binds to this region comparatively better than MAGE-A2. Curiously, higher levels of Nutlin-3a actually reversed inhibition and stimulated binding of MAGE-A2 to MDM2, an effect that did not occur with p53. One possible explanation of this effect is that prior binding of p53 (or a p53 analogue) may inf.Ta respectively in Fig 3A. Amino acids within both of theseFig 3. MAGE-A2 interacts with the p53-binding hydrophobic cleft in the N-terminus of MDM2. (A) The structure of the N-terminus (p53-binding domain) of MDM2 (1YCR) showing the positions of MAGE-A2 binding peptides 51?5 (red) and 91?05 (magenta). The backbone is shown in green. The locations of the amino acids at the flanks of fpsyg.2017.00209 each of these peptides are shown. (B) Schematic showing the structure of the GST-MDM2 mini-protein, MP1, used in this experiment. A version in which GST was substituted by GFP and was used for cultured cell expression and immunoprecipitation analysis. (C) Coimmunoprecipitation analysis was carried out following expression of GFP-MP1 (or GFP alone as control) in H1299 cells together with MAGE-A2 or p53 (as positive control). (D) GST pull-down assays were performed in which 35S-radiolabelled MAGE-A2, or 35S-radiolabelled p53 as control, were captured on glutathione sepharose 4B beads using GST linked to the MDM2 mini-protein, MP1, which encompasses amino acids 1?10 including the hydrophobic p53-binding cleft. The association of MAGE-A2 or p53 was measured in the presence of increasing concentrations of Nutlin-3a. The co-precipitating MAGE-A2 and p53 proteins were detected by SDS-PAGE followed by fluorography. Note that in vitro transcription/translation of p53 gives rise to two polypeptides. (E) Quantification of the pull-down experiment following densitometry. In all cases the data are representative of at least three independent experiments. doi:10.1371/journal.pone.0127713.gPLOS ONE | DOI:10.1371/journal.pone.0127713 May 22,9 /MAGE-A Inhibits MDM2 and Increases MDM4 Levelsregions are required for p53 binding [44] suggesting either that MAGE-A2 may interact with MDM2 in a manner similar to that of p53 or that it may influence the p53/MDM2 interaction. To determine whether MAGE-A2 binds to the N-terminus of MDM2 in a cellular context, the “MP1” MDM2 mini-protein (comprising amino acids 1?10 of MDM2) was expressed as a fusion with green fluorescent protein (GFP; Fig 3B) together with MAGE-A2 in H1299 cells. As a positive control, p53 was expressed in place of GFP-MP1. Co-immunoprecipitation analysis (Fig 3C) confirmed that both MAGE-A2 and the p53 control were able to associate with the GFP-MP1(MDM2) wcs.1183 in the cultured cells. No association was observed when GFP lacking the MP1 extension was used. To test the idea that p53 and MAGE-A2 bind to MDM2 in a similar manner, GST pulldown experiments were carried out in vitro to determine whether the competitive inhibitor, Nutlin-3a, which mimics p53 and binds tightly within the hydrophobic groove in MDM2 that constitutes the p53 binding site, could block the interaction of MAGE-A2 with MDM2 [45]. The data show that increasing concentrations of Nutlin-3a do indeed prevent MAGE-A2 from binding to the N-terminus of MDM2 (Fig 3D and 3E). The interaction of p53 with MDM2, which was measured in the same experiment as a control, was also progressively inhibited by increasing concentrations of Nutlin-3a (Fig 3D and 3E). The IC50 values for dissociation of p53 and MAGE-A2 respectively from MDM2 were 18 M and 10 M indicating that p53 binds to this region comparatively better than MAGE-A2. Curiously, higher levels of Nutlin-3a actually reversed inhibition and stimulated binding of MAGE-A2 to MDM2, an effect that did not occur with p53. One possible explanation of this effect is that prior binding of p53 (or a p53 analogue) may inf.