Development of a Homogeneous, Fluorescence Resonance Energy Transfer-Based in Vitro Recruitment Assay for Peroxisome Proliferator-Activated Receptor δ via Selection of Active LXXLL Coactivator Peptides
✍ Scribed by Katherine A. Drake; Ji-Hu Zhang; Richard K. Harrison; Gerald M. McGeehan
- Publisher
- Elsevier Science
- Year
- 2002
- Tongue
- English
- Weight
- 88 KB
- Volume
- 304
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors activated by fatty acids and their metabolites. The PPAR␦ subtype is believed to be involved in lipoprotein regulation and may have a role in reverse cholesterol transport. While the range of biological roles of PPAR␦ still remains unclear, it is of therapeutic interest in cardiovascular diseases. Here we report a homogeneous in vitro assay for studying ligand activation of PPAR␦. We surveyed a panel of peptides containing the LXXLL motifs derived from coactivator protein sequences. Peptides with the best response were used to develop a sensitive and homogeneous recruitment assay for PPAR␦. The optimized assay has a signalto-background ratio of about 8:1 and an assay quality parameter Z-factor value of 0.8. The assay signal generated is stable for hours to even overnight. This simple recruitment assay can provide agonist and/or antagonist information that cannot be assessed by receptorbinding assay, and can be used for characterization and screening of ligands that modulate the activation of PPAR␦.