Structural basis for the high all-trans-retinaldehyde reductase activity of the tumor marker AKR1B10

Abstract

AKR1B10 is a human aldo-keto reductase (AKR) found to be elevated in several cancer types and in precancerous lesions. In vitro, AKR1B10 exhibits a much higher retinaldehyde reductase activity than any other human AKR, including AKR1B1 (aldose reductase). We here demonstrate that AKR1B10 also acts as a retinaldehyde reductase in vivo. This activity may be relevant in controlling the first step of retinoic acid synthesis. Up-regulation of AKR1B10, resulting in retinoic acid depletion, may lead to cellular proliferation. Both in vitro and in vivo activities of AKR1B10 were inhibited by tolrestat, an AKR1B1 inhibitor developed for diabetes treatment. The crystal structure of the ternary complex AKR1B10-NADP(+)-tolrestat was determined at 1.25-A resolution. Molecular dynamics models of AKR1B10 and AKR1B1 with retinaldehyde isomers and site-directed mutagenesis show that subtle differences at the entrance of the retinoid-binding site, especially at position 125, are determinant for the all-trans-retinaldehyde specificity of AKR1B10. Substitutions in the retinaldehyde cyclohexene ring also influence the specificity. These structural features should facilitate the design of specific inhibitors, with potential use in cancer and diabetes treatments.

Fig. 1.

Retinaldehyde reductase activity of AKR1B10 and effect of tolrestat in vitro and in vivo. (A) Retinoid metabolism in COS-1 cells transiently expressing AKR1B10. Cellular retinoid content was measured by HPLC after incubating cells for 30 min with 10 μM retinaldehyde or 10 μM retinol. (B) Determination of tolrestat IC₅₀ for retinaldehyde reductase activity of AKR1B10 using 0.5 μM retinaldehyde as a substrate. (C) Tolrestat inhibition of cellular AKR1B10 activity. COS-1 cells transfected with pCMV-HA-AKR1B10 were incubated with 10 μM all-trans-retinaldehyde and different concentrations of tolrestat. Data are expressed as the percentage of conversion of the retinoid taken up by cells (reduced retinaldehyde or oxidized retinol). Conversion for COS-1 cells transfected with empty vector (pCMV-HA) is shown as a control. Results are expressed as the mean ± SEM of at least three determinations.

Fig. 2.

Crystal structure of AKR1B10 complexed with NADP⁺ and tolrestat. (A) View from the top of the (α/β)₈ barrel. The catalytic site is located in the center of the barrel. (B) View of the (α/β)₈ barrel after rotating 90°. Cofactor approaches the catalytic site from one side of the barrel, and tolrestat enters the barrel from the upper face. (C) LIGPLOT (32) describing interactions of the tolrestat molecule in the AKR1B10–NADP⁺–tolrestat complex. (D) LIGPLOT of tolrestat in the AKR1B1–NADP⁺–tolrestat complex.

Fig. 3.

Models of all-trans-retinaldehyde docked into the AKR1B10 and AKR1B1 structures. (A) Tolrestat-binding pocket in the AKR1B10–NADP⁺–tolrestat crystal. (B) all-trans-retinaldehyde-binding pocket of AKR1B10 predicted by our model. (C) Tolrestat-binding pocket in the AKR1B1–NADP⁺–tolrestat crystal (PDB entry 2FZD). (D) all-trans-retinaldehyde-binding pocket of AKR1B1 predicted by our model. The molecular surface is colored according to the local electrostatic potential as calculated with the program PYMOL (www.pymol.org). Residues around the substrate define a highly hydrophobic and well adjusted pocket, protecting the retinaldehyde molecule from the polar solvent.

Fig. 4.

Retinaldehyde isomers docked into the AKR1B10 and AKR1B1 structures. (A) Superimposition of critical residues for 9-cis-retinaldehyde (blue) and all-trans-retinaldehyde (orange) binding predicted by the AKR1B10 model. (B) Superimposition for 9-cis- and all-trans-retinaldehyde in the AKR1B1 model.