Helix 12 stabilization contributes to basal transcriptional activity of PXR.

Content Provider	Europe PMC
Author	Shizu, Ryota Nishiguchi, Hikaru Tashiro, Sarii Sato, Takumi Sugawara, Ayaka Kanno, Yuichiro Hosaka, Takuomi Sasaki, Takamitsu Yoshinari, Kouichi
Copyright Year	2021
Abstract	Pregnane X receptor (PXR) plays an important role in xenobiotic metabolism. While ligand binding induces PXR-dependent gene transcription, PXR shows constitutive transcriptional activity in the absence of ligands when expressed in cultured cells. This constitutive activity sometimes hampers investigation of PXR activation by compounds of interest. In this study, we investigated the molecular mechanism of PXR activation. In the reported crystal structures of unliganded PXR, helix 12 (H12), including a coactivator binding motif, was stabilized, while it is destabilized in the unliganded structures of other nuclear receptors, suggesting a role for H12 stabilization in the basal activity of PXR. Since Phe420, located in the loop between H11 and H12, is thought to interact with Leu411 and Ile414 to stabilize H12, we substituted alanine at Phe420 (PXR-F420A) and separately inserted three alanine residues directly after Phe420 (PXR-3A) and investigated their influence on PXR-mediated transcription. Reporter gene assays demonstrated that the mutants showed drastically reduced basal activity and enhanced responses to various ligands, which was further enhanced by coexpression of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1α. Mutations of both Leu411 and Ile414 to alanine also suppressed basal activity. Mammalian two-hybrid assays showed that PXR-F420A and PXR-3A bound to corepressors and coactivators in the absence and presence of ligands, respectively. We conclude that the intramolecular interactions of Phe420 with Leu411 and Ile414 stabilize H12 to recruit coactivators even in the absence of ligands, contributing to the basal transcriptional activity of PXR. We propose that the generated mutants might be useful for PXR ligand screening.
ISSN	00219258
Volume Number	297
PubMed Central reference number	PMC8390552
Issue Number	3
PubMed reference number	34284062
Journal	The Journal of Biological Chemistry [J. Biol. Chem]
e-ISSN	1083351X
DOI	10.1016/j.jbc.2021.100978
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2021-07-17
Access Restriction	Open
Rights License	This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). © 2021 The Authors
Subject Keyword	coactivator nuclear receptor pregnane X receptor transactivation domain AF2, activation function 2 CAR, constitutive active/androstane receptor DBD, DNA-binding domain DMEM, Dulbecco's modified Eagle medium DMSO, dimethyl sulfoxide LBD, ligand-binding domain PGC1α, peroxisome proliferator-activated receptor gamma coactivator-1α PPAR, peroxisome proliferator-activated receptor PXR, pregnane X receptor RXR, retinoid X receptor SRC, steroid receptor coactivator VDR, vitamin D receptor WT, wild-type
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry