The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα

Abstract

In this study, we investigated the effects of eccentric cleavage products of β-carotene, i.e. β-apocarotenoids (BACs), on retinoid X receptor alpha (RXRα) signaling. Transactivation assays were performed to test whether BACs activate or antagonize RXRα. Reporter gene constructs (RXRE-Luc, pRL-tk) and RXRα were transfected into Cos-1 cells and used to perform these assays. None of the BACs tested activated RXRα. Among the compounds tested, β-apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis-retinoic acid and was effective at concentrations as low as 1 nM. Molecular modeling studies revealed that β-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling.

Figure 1. The cleavage products of β-carotene

β-carotene can be cleaved either symmetrically (denoted as “e” cleavage) by β,β-carotene-15,15′-oxygenase (BCO1) yielding all-trans retinaldehyde that can be further oxidized to all-trans retinoic acid (ATRA) by retinal dehydrogenases. The second pathway of β-carotene cleavage is called the eccentric cleavage (denoted as “a”, “b”, “c”, “d” cleavages) and it occurs at double bonds other than the central 15,15’ double bond of the polyene chain of β-carotene to produce β-carotenals and β-apo-carotenones with different chain lengths.

Figure 2. The antagonistic effect of β-apo-13-carotenone on RXRα, dose response curve of 9-cis-RA alone (♦), and increasing concentrations of β-apo-13-carotenone: 10⁻⁹ M (●), 10⁻⁸ M (✖), 10⁻⁷ M (▲), and 10⁻⁶ M (■)

The fold induction of 10⁻⁵ M 9-cis-RA (the absolute fold induction of this point was 2.29 over the vehicle treated cells) was set to 100% and the other experimental points were calculated relative to this.

Figure 3. Docking of β-apo-13-carotenone with the RXRα tetramer

Shown is the structure of the ligand-binding domain of the tetrameric RXRα (in green) from PDB entry 1G5Y with: (a) the bound crystallized all-trans retinoic acid (ATRA) isomer (in white) and the Surfle-Dock docked structure of β-apo-13-carotenone (yellow) when built in a conformation resembling that of the bound ATRA; (b) same as in (a) with the protein helix 3 removed to facilitate visualization of the quality of docking in the binding site; (c) as in (a) with the RXR protein removed to show details of the similarity of the crystallized ATRA ligand and the docked pose of β-apo-13-carotenone.

Figure 4. Docking of β-apo-13-carotenone with the RXRα dimer

Shown is the structure of 9-cis-retinoic acid (9-cis-RA) (in white) crystallized in the binding site of the RXRα from PDB entry 1FBY, along with the docked structure of β-apo-13-carotenone (in yellow) in the ligand binding site after its construction in a conformation that resembles that of the bound 9-cis-RA (the protein structure has been removed to facilitate appreciation of the significant difference between the bound and docked structures).