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

Constitutive androstane receptor (CAR) and peroxisome proliferatoractivated receptor a (PPARa) 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 PPARa remains unknown. This study explored the potential phase separation of CAR and PPARa. The computational analysis utilizing algorithm tools examining the intrinsically disordered regions of CAR induce condensate formation, showed that there was no condensate formation of the fusion protein of Cry2 with CAR or PPARa. Furthermore, phase separation of CAR or PPARa did not occur despite reduced target expression under hyperosmotic stress. In conclusion, these findings revealed that neither the activation of CAR and PPARa nor hyperosmotic stress induces phase separation of CAR and PPARa in cells. SIGNIFICANCE STATEMENT Constitutive androstane receptor (CAR) and peroxisome proliferatoractivated receptor a (PPARa) are key regulators of various functions in the body. This study showed that CAR and PPARa do not exhibit phase separation under hyperosmotic stress or after agonistinduced activation. These findings provide new insights into the CAR and PPARa biology and physiology. and PPARa 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