title: Bioluminescence Resonance Energy Transfer (BRET)-Based Synthetic Sensor Platform for Drug Discovery.
abstract: Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein-protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine substrate. BRET is superior to FRET where issues such as autofluorescence, photobleaching, and light scattering can occur. Recently, BRET has been applied to design synthetic biosensors for monitoring autophagy in vivo and in vitro. Here, we report the methods for constructing a biosensor of human HsLC3a as a probe for autophagy biogenesis and the optimization of the intramolecular BRET assay that allows for high-throughput screening of chemical modulators of autophagy. User-friendly working interface with the BRET-based synthetic sensor of HsLC3a makes drug discovery easy and amenable for high-throughput. The BRET protocol described here could be easily applicable to generate other biosensors for monitoring PPIs by measurement of intermolecular BRET. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Department of Plant Biology and the Genome Center, College of Biological Sciences, University of California, Davis, California.
authors.forename: Jongchan
authors.initials: J
authors.lastname: Woo
authors.affiliation: Department of Plant Biology and the Genome Center, College of Biological Sciences, University of California, Davis, California.
authors.forename: Jason
authors.initials: J
authors.lastname: Hong
authors.affiliation: Department of Plant Biology and the Genome Center, College of Biological Sciences, University of California, Davis, California.
authors.forename: Savithramma P
authors.initials: SP
authors.lastname: Dinesh-Kumar
mesh_terms: D000818:Animals; D000906:Antibodies; D001343:Autophagy; D000071183:Autophagy-Related Proteins / Q000378:metabolism; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D049410:Luciferases, Renilla / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011719:Pyrazines / Q000737:chemistry
chemical_list: D000906:Antibodies; D000071183:Autophagy-Related Proteins; D007093:Imidazoles; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011719:Pyrazines; C017144:coelenterazine; D049410:Luciferases, Renilla; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases
keywords: HsATG4; HsLC3a; and luciferase; autophagy; bioluminescence resonance energy transfer (BRET); drug discovery; protein-protein interactions (PPIs)