Detecting Protein‐Protein Interactions and Allosteric Regulation in the Corticosteroid Synthesis Pathway in Vertebrates

Abstract

The regulation of biochemical pathways is controlled at a variety of levels, from transcriptional timing to protein localization. Here we study the corticosteroid synthesis pathway in order to understand a transition from a biochemical pathway controlled by allosteric regulation to a phenotypically equivalent pathway controlled by differential expression of paralogs and enzyme specificity.The corticosteroids aldosterone and cortisol are produced in different layers of the adrenal cortex – a phenomenon known as zonation. Most mammals have a single enzyme, CYP11B, which produces aldosterone in the exterior layer of the adrenal cortex and cortisol in the interior layers. In mammals with a single‐CYP11B, the specific production of aldosterone and cortisol in different tissues is thought to be accomplished by allosteric regulation by another enzyme, CYP11A, which is only present in the interior layer of the cortex. It is thought that the presence of CYP11A changes the function of CYP11B so that it preferentially produces cortisol instead of aldosterone.However, throughout mammalian evolution CYP11B has duplicated independently in several lineages, giving rise to two CYP11B paralogs. These species still experience zonation and produce aldosterone and cortisol in different layers of the adrenal cortex. It has been demonstrated that this zonation in species with two CYP11B enzymes is accomplished by differential expression of the paralogs in the different tissues along with the evolution of enzymatic preference for their respective products. However, it is unknown whether the interaction between CYP11B and CYP11A has been lost and whether the zonation of hormones produced is regulated solely by the two differential expression and enzymatic activity of the CYP11B enzymes.Here we used Forster Resonance Energy Transfer (FRET) High Performance Liquid Chromatography (HPLC) to determine whether CYP11B and CYP11A physically interact and whether or not that interaction would contribute to the allosteric regulation of this pathway. Our preliminary analyses demonstrate a physical interaction between CYP11B and CYP11A in a single‐CYP11B species. Further experiments are aimed to test the interaction of duplicated CYP11Bs with CYP11A. We will monitor in vivo enzymatic assays by HPLC to determine what hormones are produced in the presence of different enzyme combinations. These results will shed light on the various ways metabolic pathways are regulated.Support or Funding InformationSD BRIN P20GM103443SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.