Optimization of an enzyme-linked immunosorbent assay to screen ligand of Peroxisome proliferator-activated receptor alpha

Abstract

Peroxisome proliferator-activated receptors (PPARs) are transcription factor which directly modulate gene expression by binding to specific agonists. It has been reported that PPARα controls lipid metabolism, inflammation, and atherosclerosis. PPARα activation by PPARα agonist can ultimately reduce the progression of atherosclerosis and decrease the incidence of coronary heart disease. In this study, we optimized enzyme-linked immunosorbent assay (ELISA) systems in order to screen putative PPARα agonists. These methods are based on the activation mechanism of PPARα where the ligand binding to PPARα induces the interaction of the receptor with transcriptional co-activators. Among co-activators such as SRC-1, TIF-2, and p300, although ligand-unbound PPARα had more strong binding with p300 at a lower concentrations of PPARα, ligand-bound PPARα had more specific and strong binding with SRC-1. We optimized and developed a novel and useful ELISA system to screen PPARα agonists. Wy14,643 and linoleic acid, the well-known PPARα ligands, increased the binding between PPARα and co-activators in a ligand dose-dependent manner. In this ELISA method to screen PPARα ligands, the use of specific anti-PPARα N-terminus antibody, full-length recombinant protein of human PPARα but not ligand-binding domain (LBD) of human PPARα, and his-tagged PPARα recombinant proteins but not GST-fused PPARα recombinant proteins is the critical factors. Development of this screening system may be useful in the discovery of PPARα ligands from various candidates such as chemical library and phytochemicals.

Keywords::

• Coactivator
• ELISA
• metabolic disorder
• PPARα
• PPARα
• ligand

Acknowledgments

This study was supported by the Korea Research Foundation grant funded by the South Korean Government (KRF-2006-311/312-E00119) and by a grant R01-2006-000-10145-0 from the Korea Science and Engineering Foundation. D.Y.Y. was partially supported by the Ministry for Health, Welfare and Family Affairs (KHIDI).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.