Abstract
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor regulating cellular responses to hypoxia and is composed of alpha and beta subunits. During normoxia, factor inhibiting HIF-1 (FIH-1) inhibits the activity of HIF-1 by preventing HIF-1alpha binding to p300/CBP via modification of the Asn(803) residue. However, it is not known whether FIH-1 activity can be regulated in an oxygen-independent manner. In this study, we survey possible binding proteins to FIH-1 and identify Mint3/APBA3, which has been reported to bind Alzheimer beta-amyloid precursor protein. Purified Mint3 binds FIH-1 and inhibits the ability of FIH-1 to modify HIF-1alpha in vitro. In a reporter assay, the activity of HIF-1alpha is suppressed because of endogenous FIH-1 in HEK293 cells, and expression of Mint3 antagonizes this suppression. Macrophages are known to depend on glycolysis for ATP production because of elevated HIF-1 activity. FIH-1 activity is suppressed in macrophages by Mint3 so as to maintain HIF-1 activity. FIH-1 forms a complex with Mint3, and these two factors co-localize within the perinuclear region. Knockdown of Mint3 expression in macrophages leads to redistribution of FIH-1 to the cytoplasm and decreases glycolysis and ATP production. Thus, Mint3 regulates the FIH-1-HIF-1 pathway, which controls ATP production in macrophages and therefore represents a potential new therapeutic target to regulate macrophage-mediated inflammation.
Highlights
We further show that Mint3 regulates Hypoxia-inducible factor-1 (HIF-1) activity by modulating factor inhibiting HIF-1 (FIH-1) and that constitutive suppression of FIH-1 by Mint3 plays a pivotal role in the activation of HIF-1, glycolytic activity, and ATP production in macrophages
Each candidate protein was expressed as a FLAG-tagged fusion protein, and we examined the ability of these fusions to bind Myc-tagged FIH-1 that was co-expressed in HEK293 cells with the FLAG-tagged fusions (Fig. 1B)
Myc-tagged FIH-1 in the precipitates (IP) or in the whole cell lysates was detected by immunoblot analysis, using anti-Myc antibody (IB, lower two panels)
Summary
Yeast Two-hybrid Assay—The yeast two-hybrid screens were performed by Hybrigenics (Paris, France) using FIH-1 as a bait to screen a human placenta random-primed cDNA library, and each protein interaction was assigned with a statistical confidence score as described previously [10]. The lysates were separated by SDS-PAGE, transferred to membrane filters, and subjected to immunoblot analysis using anti-lamin A/C mouse antibody (BD Biosciences), using anti-HIF-1␣ mouse antibody (EXALPHA, Shirley, MA), anti-Myc mouse antibody (Roche Applied Science), anti-V5 mouse antibody (Invitrogen), anti-Mint mouse antibody (BD Biosciences), anti-FIH-1 goat antibody (Santa Cruz Biotechnology), anti-actin mouse antibody (Millipore), or anti-FLAG epitope M2 antibody (Sigma). After blocking in phosphate-buffered saline containing 5% goat serum and 3% bovine serum albumin, the cells were incubated with mouse anti-FLAG M2 antibody (Sigma), mouse anti-Mint antibody (BD Biosciences), mouse anti-GM130 (BD Biosciences), rabbit anti-GM130 (Novus Biologicals), or rabbit anti-FIH-1 antibody (Novus Biologicals) for 1 h and washed three times and incubated for 1 h with anti-mouse IgG Alexa488 conjugate or anti-rabbit Alexa594 (Invitrogen). The unpaired Student’s t test was used for analyzing differences between experimental groups
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