Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation

J Biol Chem. 2021 Oct;297(4):101142. doi: 10.1016/j.jbc.2021.101142. Epub 2021 Sep 1.

Abstract

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid-binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the kcat/Km values either decreased 5-fold or were equal to the respective free retinoid values. The kcat/Km value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in kcat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.

Keywords: MS; cytochrome P450; dehydroretinoid; enzyme kinetics; protein–protein interaction; retinoid; retinoid-binding protein; vitamin A.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cytochrome P450 Family 27 / chemistry*
  • Cytochrome P450 Family 27 / metabolism
  • Humans
  • Receptors, Retinoic Acid / chemistry*
  • Receptors, Retinoic Acid / metabolism
  • Retinoids / chemistry*
  • Retinoids / metabolism
  • Retinol-Binding Proteins, Cellular / chemistry*
  • Retinol-Binding Proteins, Cellular / metabolism

Substances

  • RBP1 protein, human
  • Receptors, Retinoic Acid
  • Retinoids
  • Retinol-Binding Proteins, Cellular
  • retinoic acid binding protein I, cellular
  • retinoic acid binding protein II, cellular
  • CYP27C1 protein, human
  • Cytochrome P450 Family 27