A Novel Function of Novobiocin: Disrupting the Interaction of HIF 1α and p300/CBP through Direct Binding to the HIF1α C-Terminal Activation Domain
Figure 2
Novobiocin directly disrupts the HIF1αCTAD/p300 CH1 complex.
A. Novobiocin inhibits His-HIF1α FL protein pulling down p300 CH1. Insect cell expressed and purified His-HIF1α FL (∼5 µg), Flag-p300 CH1, 400 µM of antibiotic novobiocin or DMSO (control), and 15 µl of Ni2+-agarose were mixed in binding buffer containing 40 mM Hepes-NaOH (pH 7.9), 250 mM NaCl, 10% glycerol, and 40 mM imidazol, and incubated at 4°C for 2 hours. Bound proteins were eluted with 20 µl of elution buffer containing 300 mM imidazol and were then confirmed by Western blotting with anti-HIF1α and anti-Flag antibodies (upper panel). Western blot images were quantified using Quantity One software (BioRad) (lower panel). B. Novobiocin inhibited GST-p300 CH1 protein pulling down the HIF1α FL. GST-p300 CH1 protein (∼10 µg GST only as a negative control) was mixed with His-HIF1α FL, 15 µl of glutathione sepharose 4 Fast Flow beads, and novobiocin (100–400 µM). Bound proteins were then detected by Western blotting with anti-HIF1α antibody. Quantified HIF1α is depicted in the graph (lower panel). Percentages given above each bar represent bound HIF1α protein compared to lane 2. C. Novobiocin directly disrupts the HIF1α CTAD/p300 CH1 complex. The GST pull-down assay was performed by mixing (as indicated in the figure) ∼10 µg GST fusion proteins, 5 µg Flag-p300 CH1, and 100 or 400 µM novobiocin. Pulled-down p300 CH1 was detected by Western blotting with anti-Flag antibody. D. Novobiocin inhibited the interaction between overexpressed HIF1α FL and endogenous p300 in the presence or absence of CoCl2. HPC4-HIF1α FL was overexpressed in HeLa cells in the presence or absence of novobiocin (100 or 400 µM). Hypoxic conditions were induced by the addition of CoCl2 (150 µM) for 8 hours. Then, 50 µM MG132 was added 3 hours before harvest. At 72 hours after HPC4-HIF1α transfection, cells were harvested and an HPC4 immunoprecipitation assay was performed by mixing 250 µl whole-cell lysate, 2 µg HPC4 antibody, and 15 µl of glutathione sepharose. Bound p300 was then detected by Western blotting with anti-p300 antibody.