Citation
- Authors: Diao X. et al.
- Year: 2022
- Journal: Nat Commun 13 2529
- Applications: in vitro / DNA / jetPRIME
- Cell type: HEK-293
Description: Human embryonic kidney Fibroblast
Known as: HEK293, 293
Method
HEK293 cells were cultured in DMEM medium with 10% FBS in 6-well plates at 37 °C in 5% CO2, then 1 μg pCMV-Tag4, 1 μg pCMV-Tag4-HIF-3α1, or 1 μg pCMV-Tag4-HIF-3α1 (P490A) plasmids were transfected into HEK293 cells using the jetPRIME reagent when the cell density reached 70–80%. After 4 h of transfection, the medium was refreshed with or without OEA (25 μM). Another 24 h later, cells were harvested and lysed in 150 μl lysis buffer (1× TBS with 1 mM EDTA, 1% TRITON X-100 and 1× protease inhibitor cocktail) before quick spinning.
Abstract
Hypoxia-inducible factors (HIFs) are α/β heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/β heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.