Abstract
Retinoic X receptor (RXR) is a promising target for drug discovery against cancer and metabolic syndromes. Here, we identified a specific RXRα antagonist, danthron, from the traditional Chinese medicine rhubarb. Danthron repressed all tested RXRα-involved response element transcription, including the RXRE, PPRE, FXRE, and LXRE. Results from native PAGE and isothermal titration calorimetry (ITC)-based assays indicated that danthron bound to the tetrameric RXRα-LBD in a specific stoichimetric ratio, and such a binding could influence the corepressor SMRT affinity to the receptor. Additionally, a unique tetrameric structure of the apo-RXRα ligand-binding domain (LBD) was determined, which exhibited a larger tetramer interface and different ligand-binding pocket size compared with the one previously reported. Together with the biochemical and biophysical results, the determined crystal structure of danthron-soaked RXRα-LBD suggested a new mechanism for danthron antagonism to tetrameric RXRα. Moreover, the in vivo efficient improvement of insulin sensitivity by danthron was observed in diet-induced obese (DIO) mice. Thus, our findings were expected to supply new insights into the structural basis of RXRα antagonist for its further potential therapeutic application.
Highlights
Retinoic X receptor (RXR),4 as a member of nuclear receptors (NRs), plays a central role in NR-regulated signaling pathways, for its obligate heterodimer partnership with almost one
The tetramer is formed with two dimers packed in a bottom-to-bottom manner (11). Binding of agonist such as RXR natural ligand 9-cis retinoic acid (9cRA) induces notable conformational changes in which the activation function-2 (AF-2) domain rotates and moves to its active position to seal off the ligand-binding pocket (LBP), recruiting coactivators to initiate transcription (12–14)
This natural product repressed all of the tested RXR␣-involved response elements transcription, including RXRE, PPRE, FXRE, and LXRE, without binding to the corresponding nuclear receptors, except RXR␣
Summary
Materials—All the cell culture reagents were purchased from Invitrogen. The UAS-TK-Luc reporter was generously donated by Dr Daniel P. Edwards (The Medicine Institute at University of California, Los Angeles), and pGL3-pro-FXRE-Luc vector was kindly provided by Dr Majlis Hermansson (AstraZeneca, Molndal, Sweden). Protein Purification—Preparation of RXR␣-LBD was performed according to the previously published approach (14). The coding sequence of human RXR␣-LBD (residues 221– 458) was cloned to the vector pET15b, and Escherichia coli strain BL21 (DE3) was used for protein expression. SPR Technology-based Assay—Binding affinity of danthron toward RXR␣-LBD was assayed with a BIACORE 3000 instrument (BIACORE) based on our previous report (20). Native PAGE Assay—The separating gel was prepared by mixing 4.8 ml of water, 2.7 ml of 30% acrylamide/0.8% bisacrylamide, 2.5 ml 5 M Tris-Cl, pH 8.8, 0.05 ml of 10% (w/v) ammonium persulfate, and 0.005 ml of N,N,NЈ,NЈ-tetramethylethylenediamine.
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