Pregnane X receptor (PXR) belongs to the nuclear receptor superfamily that plays a crucial role in hepatic physiologic and pathologic conditions. Phase separation is a process in which biomacromolecules aggregate and condense into a dense phase as liquid condensates and coexist with a dilute phase, contributing to various cellular and biologic functions. Until now, whether PXR could undergo phase separation remains unclear. This study aimed to investigate whether PXR undergoes phase separation. Analysis of the intrinsically disordered regions (IDRs) using algorithm tools indicated a low propensity of PXR to undergo phase separation. Experimental assays such as hyperosmotic stress, agonist treatment, and optoDroplets assay demonstrated the absence of phase separation for PXR. OptoDroplets assay revealed the inability of the fusion protein of Cry2 with PXR to form condensates upon blue light stimulation. Moreover, phase separation of PXR did not occur even though the mRNA and protein expression levels of PXR target, cytochrome P450 3A4, changed after sorbitol treatment. In conclusion, for the first time, these findings suggested that exogenous PXR does not undergo phase separation following activation or under hyperosmotic stress in nucleus of cells.

SIGNIFICANCE STATEMENT PXR plays a critical role in hepatic physiological and pathological processes. The present study clearly demonstrated that exogenous PXR does not undergo phase separation after activation by agonist or under hyperosmotic stress in nucleus. These findings may help understand PXR biology.

This work was supported by the National Key R&D Program of China (2022YFA1104900), the Natural Science Foundation of China (Grants 82025034, U23A20535, 82304603), the Shenzhen Science and Technology Program (KQTD20190929174023858, China), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (Grant 2017BT01Y093, China), the National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province, Grant 2017B090903004, China), 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), the Guangdong Medical Research Foundation (Grant A2023109).

The authors have no financial disclosures, and no conflicts of interest with the contents of this article.

↵1P.Z. and Y.G. contributed equally to this work.

dx.doi.org/10.1124/dmd.123.001570.

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