Combined Biophysical and Cell-Based Approaches for the Assessment of Ligand Binding to PPAR
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Transcription factors of the peroxisome proliferator-activated receptor (PPAR) family are ligand-activated receptors that
play key roles in lipid metabolism and inflammation. The γ isoform (PPARγ) is involved in adipocyte differentiation, insulin
sensitization, and vascular pathophysiology, including inflammation and atherosclerosis, for which it is considered an important
drug target. PPARγ ligands display varied structures and include fatty acids, electrophilic lipids, and certain drugs. These
agonists promote conformational changes allowing interaction of PPARγ with coactivators and hence transcriptional regulation.
Here we present a panoply of methods to study PPARγ interactions with ligands and activation in vitro and in cells. The first
method is based on the competition of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) with PPARγ ligands for
the ligand binding pocket, allowing detection and quantification of ligand binding to PPARγ. This method is specific for PPARγ
while ANS displays negligible interaction with other nuclear receptors such as PPARα and retinoid X receptor α (RXRα). The
ANS competition assay has been validated through comparison of the affinities determined for well-known PPARγ ligands by this
method with those reported in the literature. We also describe here gel-based competition assays that show limited performance
with non-covalently bound ligands. In addition, we present a fluorescence anisotropy assay to analyze PPARγ activation by
ligands in vitro through their capacity of eliciting PPARγ interaction with a fluorescently labeled peptide derived from one
of its coactivators (SRC-1). Finally, we show cell-based assays to investigate PPARγ activation by interaction with its ligands.
We believe that combined approaches using ANS, fluorescent coactivator peptides, and in-cell assays to monitor PPARγ binding
and interactions may provide valuable strategies for the identification and characterization of PPARγ ligands.
