Constitutive Androstane Receptor and Peroxisome Proliferator-Activated Receptor α Do Not Perform Liquid-Liquid Phase Separation in Cells.

Abstract

Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are members of the nuclear receptor superfamily, which regulates various physiologic and pathologic processes. Phase separation is a dynamic biophysical process in which biomacromolecules form liquid-like condensates, which have been identified as contributors to many cellular functions, such as signal transduction and transcription regulation. However, the possibility of phase separation for CAR and PPARα remains unknown. This study explored the potential phase separation of CAR and PPARα The computational analysis utilizing algorithm tools examining the intrinsically disordered regions of CAR and PPARα suggested a limited likelihood of undergoing phase separation. Experimental assays under varying conditions of hyperosmotic stress and agonist treatments confirmed the absence of phase separation for these receptors. Additionally, the optoDroplets assay, which utilizes blue light stimulation to induce condensate formation, showed that there was no condensate formation of the fusion protein of Cry2 with CAR or PPARα Furthermore, phase separation of CAR or PPARα did not occur despite reduced target expression under hyperosmotic stress. In conclusion, these findings revealed that neither the activation of CAR and PPARα nor hyperosmotic stress induces phase separation of CAR and PPARα in cells. SIGNIFICANCE STATEMENT: Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are key regulators of various functions in the body. This study showed that CAR and PPARα do not exhibit phase separation under hyperosmotic stress or after agonist-induced activation. These findings provide new insights into the CAR and PPARα biology and physiology.