OR33-03 Circadian Analysis Of Nuclear Receptor Protein Expression In Mouse Liver Reveals Peak COUP-TFII, EAR2, LXRa, LXRb And TR4 Expression At The End Of The Mouse Active Cycle ZT0

Abstract

Abstract Disclosure: M.F. Saikali: None. J. Li: None. C.L. Cummins: None. Nuclear Receptors (NRs) are a family of ligand-mediated transcription factors that control the expression of genes involved in a wide range of physiological processes. The ability to study NRs quantitatively at the protein level has been hindered by the poor quality of available antibodies, as well as the lack of available high throughput methods to study more than a few receptors at a time. To address this need, we have developed a mass spectrometry based targeted proteomic assay to quantify the absolute amounts of NR protein in mouse livers over a circadian period. C57Bl6 mice were sacrificed at 12 weeks every 4 hours from zeitgeber time ZT0 to ZT20. Their livers were processed using a transcription factor extraction protocol1. For each sample, 100 µg nuclear protein was processed using the NR assay described previously2. Briefly, the lysate is digested following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The sample is then spiked with 50 fmol of our internal standard mix. This mix is composed of 66 carefully selected peptides that account for 49 NRs, 11 histone peptides, and 6 circadian clock proteins. The resulting digest that contains the internal standards is desalted on C18 tips, followed by separation on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF mass spectrometer in parallel reaction monitoring mode. We used this assay to quantify the changes in NR protein expression in mouse livers for 9 receptors; RORα, CAR, COUP-TFII, EAR2, GR, LXRα, LXRβ, PPARα, and TR4. RORα showed peak nuclear expression at ZT0, with a peak-to-trough change of 2.2-fold. COUP-TFII, EAR2, LXRα, LXRβ, and TR4 also showed peak expression at ZT0 (the end of the active phase) with changes of 3.3, 2.4, 1.7, 2.0, and 1.6-fold respectively. GR showed peak nuclear expression at ZT12 with a 1.8-fold change. CAR showed 2.3-fold peak expression at ZT16. COUP-TFII, EAR2, and TR4 are considered orphan receptors with no known endogenous ligands. These data will be used to uncover endogenous tissue-specific ligands by analyzing extracts isolated at the zeitgeber time that the receptor is maximally expressed. Sources of Research Support: This project has been supported by the J. P. Bickell Foundation and the Canadian Institutes of Health Research New Frontiers Research Fund and International Development Research Fund. 1.Gillespie, M. A. et al. Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis. Mol Cell (2020) doi:10.1016/j.molcel.2020.03.031.2.Saikali, M. F. & Cummins, C. L. Quantifying the Protein Levels of All Nuclear Hormone Receptors by Mass Spectrometry. J Endocr Soc5, (2021). Presentation: Sunday, June 18, 2023