English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/65549
logo share SHARE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:


Combined biophysical and cell-based approaches for the assessment of ligand binding to PPARγ

AuthorsZorrilla, Silvia ; Pérez-Sala, Dolores
KeywordsPPAR activity assay
PPAR ligand
Fluorescence anisotropy assay
Issue Date2013
PublisherHumana Press
CitationMethods in Molecular Biology, 952, Chapter 18 : 237-252 (2013)
AbstractTranscription 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
Description16 páginas, 4 figuras -- PAGS nros. 237-252
Publisher version (URL)https://dx.doi.org/10.1007/978-1-62703-155-4_18
Appears in Collections:(IQFR) Libros y partes de libros
(CIB) Libros y partes de libros
Files in This Item:
File Description SizeFormat 
restringido.pdf21,67 kBAdobe PDFThumbnail
Show full item record
Review this work

Related articles:

WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.