title: Development of a homogeneous, fluorescence resonance energy transfer-based in vitro recruitment assay for peroxisome proliferator-activated receptor delta via selection of active LXXLL coactivator peptides.
abstract: The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors activated by fatty acids and their metabolites. The PPARdelta 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 PPARdelta still remains unclear, it is of therapeutic interest in cardiovascular diseases. Here we report a homogeneous in vitro assay for studying ligand activation of PPARdelta. 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 PPARdelta. The optimized assay has a signal-to-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 receptor-binding assay, and can be used for characterization and screening of ligands that modulate the activation of PPARdelta.
authors.affiliation: Dupont Pharmaceutical Research Laboratories, Leads Discovery, Experimental Station, Wilmington, Delaware 19880, USA.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Drake
authors.forename: Ji-Hu
authors.initials: JH
authors.lastname: Zhang
authors.forename: Richard K
authors.initials: RK
authors.lastname: Harrison
authors.forename: Gerald M
authors.initials: GM
authors.lastname: McGeehan
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004735:Energy Transfer; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism*
chemical_list: D008024:Ligands; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors