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.

This work was supported by the National Key R&D Program of China (2022YFA1104900), the Natural Science Foundation of China (Grants U23A20535, 82025034, and 82304603), the Shenzhen Science and Technology Program (Grant KQTD20190929174023858), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (Grant 2017BT01Y093), the National Engineering and Technology Research Center for New Drug Druggability Evaluation (Seed Program of Guangdong Province, Grant 2017B090903004m), the Science and Technology Innovation Project of Guangdong Medical Products Administration (Grant 2023ZDZ06), China National Postdoctoral Program for Innovative Talents (Grant BX20230151), the China Postdoctoral Science Foundation (Grant 2023M731570), the Guangdong Basic and Applied Basic Research Foundation (Grant 2023A1515012859), and the Guangdong Medical Research Foundation (Grant A2023109).

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

↵1 P.Z. and S.F. contributed equally to this work.

dx.doi.org/10.1124/jpet.124.002174.

↵This article has supplemental material available at jpet.aspetjournals.org.