Bioluminescence Resonance Energy Transfer (BRET)‐Based Synthetic Sensor Platform for Drug Discovery

Jongchan Woo1, Jason Hong1, Savithramma P. Dinesh‐Kumar1

1 Department of Plant Biology and the Genome Center, College of Biological Sciences, University of California, Davis, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.30
DOI:  10.1002/cpps.30
Online Posting Date:  April, 2017
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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.

Keywords: autophagy; bioluminescence resonance energy transfer (BRET); drug discovery; HsLC3a; HsATG4; and luciferase; protein‐protein interactions (PPIs)

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Table of Contents

  • Introduction
  • Basic Protocol 1: Chemical Library Screening for HsATG4b‐Mediated Processing of HsLC3a‐Sensor
  • Alternate Protocol 1: In Vitro Cleavage Assay by SDS‐PAGE and Immunoblotting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Chemical Library Screening for HsATG4b‐Mediated Processing of HsLC3a‐Sensor

  • Purified HsATG4b and HsLC3a‐sensor proteins (Seo et al., ; Woo et al., )
  • Ice
  • Coelenterazine (CLZ; see recipe)
  • Phosphate‐buffered saline (PBS) solution, pH 7.4 (e.g., VWR Life Science, cat no. E504‐500 ML)
  • Dimethyl sulfoxide (Sigma‐Aldrich, cat no. 472301‐500 ML)
  • Small‐molecule chemical library (user defined)
  • PCR 8‐tube strips (e.g., USA Scientific, cat no. 1402‐2700)
  • PCR tube rack
  • Aluminum foil
  • Multichannel pipette capable of handling 1 to 100 µl volumes
  • Multichannel pipette reservoir trough (e.g., VWR, cat no. 89094‐662)
  • 96‐well white, flat bottom, tissue culture treated, polystyrene plates (e.g., Corning, cat no. 3917)
  • Adhesive aluminum sealing film for 96‐well plates (e.g., ThermoFisher Scientific, cat no. AB0626)
  • Centrifuge
  • Plate reader (e.g., TECAN Infinite 200)
  • Excel software (Microsoft Office, Microsoft)

Alternate Protocol 1: In Vitro Cleavage Assay by SDS‐PAGE and Immunoblotting

  • Purified HsATG4b and HsLC3a‐sensor proteins (Seo et al., ; Woo et al., )
  • Dimethyl sulfoxide (DMSO; e.g., Sigma‐Aldrich, cat no. 472301‐500 ML)
  • Small‐molecule chemical library (user defined)
  • 2× Laemmli sample buffer (e.g., Bio‐rad cat no. 1610737)
  • SDS‐PAGE gel (e.g., Mini‐Protean TGX Precast Gels 10%; Bio‐rad, cat no. 4561034)
  • Protein marker (e.g., Spectra Multicolor Broad Range Protein Ladder, cat no. 26634)
  • 10× Tris/Glycine/SDS Buffer (see recipe)
  • Cathode buffer (see recipe)
  • Anode I and II buffers (see reciperecipes)
  • Methanol (e.g., Fisher Chemical, cat no. A412Sk‐4)
  • Distilled water
  • 5% and 3% nonfat milk (see recipes)
  • Primary antibody anti‐Renilla luciferase, clone 5B11.2 (e.g., EMD Millipore, cat no. MAB4400)
  • Phosphate‐buffered saline (PBS) solution, pH 7.4 (e.g., VWR Life Science, cat no. E504‐500 ML)
  • Tween‐20 (e.g., VWR Life Science, cat no. 0777‐1 liter)
  • Secondary antibody, anti‐Mouse IgG (whole molecule)‐peroxidase antibody produced in rabbit (e.g., Sigma‐Aldrich, cat no. A9044‐2 ML)
  • SuperSignal West Pico Chemiluminescent Substrate (e.g., ThermoFisher Scientific, cat no. 34077)
  • 50‐ml conical tubes (e.g., Corning, cat no. 430290)
  • Centrifuge
  • Water bath
  • Floating tube rack
  • Electrophoresis chamber (e.g., Mini‐PREOTEAN Tetra Vertical Electrophoresis Cell; cat no. 1658004)
  • Electrophoresis power supply (e.g., Bio‐Rad, PowerPac HC High‐Current Power Supply)
  • Orbital shaker (e.g., VWR Orbital Shaker, cat no. DS‐500E)
  • Plates (any plastic 4 in × 4 in container that can hold membrane and liquid solutions)
  • PVDF transfer membranes (e.g., Immobilon, cat no. IPVH00010)
  • Plate tray
  • Thick blot filter paper (e.g., Bio‐Rad, cat no. 1650921)
  • Transfer system (e.g., Trans‐Blot Turbo Transfer System; Bio‐Rad, cat no. 1704155)
  • Tweezers
  • Paper towels
  • Sheet protector non‐glare simple loading 8.5 in × 11 in (e.g., Office Depot, cat no. 535038)
  • Beakers (small and large)
  • 1.5‐ml microcentrifuge tubes
  • X‐ray film (e.g., Research Products International, cat no. 248300)
  • X‐ray film cassette
  • Film developer machine with dark room for film developing
  • Stir bars
  • Hot plate

Support Protocol 1:

  • Film (see protocol 2Alternate Protocol)
  • Image scanner
  • ImageJ program (e.g., Fiji program, NIH)
  • Excel program
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Literature Cited

Literature Cited
  Bacart, J., Corbel, C., Jockers, R., Bach, S., & Couturier, C. (2008). The BRET technology and its application to screening assays. Biotechnology Journal, 3, 311–324. doi: 10.1002/biot.200700222
  Deane, C. M., Salwinski, L., Xenarios, I., & Eisenberg, D. (2002). Protein interactions: Two methods for assessment of the reliability of high throughput observations. Molecular & Cellular Proteomics, 1, 349–356. doi: 10.1074/mcp.M100037‐MCP200
  Dragulescu‐Andrasi, A., Chan, C. T., De, A., Massoud, T. F., & Gambhir, S. S. (2011). Bioluminescence resonance energy transfer (BRET) imaging of protein‐protein interactions within deep tissues of living subjects. Proceedings of the National Academy of Sciences of the United States of America, 108, 12060–12065. doi: 10.1073/pnas.1100923108
  Eidne, K. A., Kroeger, K. M., & Hanyaloglu, A. C. (2002). Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends in Endocrinology and Metabolism, 13, 415–421. doi: 10.1016/S1043‐2760(02)00669‐0
  Fields, S., & Song, O. (1989). A novel genetic system to detect protein‐protein interactions. Nature, 340, 245–246. doi: 10.1038/340245a0
  Hayward, A. P., & Dinesh‐Kumar, S. P. (2011). What can plant autophagy do for an innate immune response? Annual Review of Phytopathology, 49, 557–576. doi: 10.1146/annurev‐phyto‐072910‐095333
  He, C., & Klionsky, D. J. (2009). Regulation mechanisms and signaling pathways of autophagy. Annual Review of Genetics, 43, 67–93. doi: 10.1146/annurev‐genet‐102808‐114910
  Huynh, K., & Partch, C. L. (2015). Analysis of protein stability and ligand interactions by thermal shift assay. Current Protocols in Protein Science, 79, 28.9.1–29.9.14. doi: 10.1002/0471140864.ps2809s79
  Jin, M., Liu, X., & Klionsky, D. J. (2013). SnapShot: Selective autophagy. Cell, 152, 368–368 e362. doi: 10.1016/j.cell.2013.01.004
  Kaur, J., & Debnath, J. (2015). Autophagy at the crossroads of catabolism and anabolism. Nature Reviews. Molecular Cell Biology, 16, 461–472. doi: 10.1038/nrm4024
  Ohsumi, Y. (2001). Molecular dissection of autophagy: Two ubiquitin‐like systems. Nature Reviews. Molecular Cell Biology, 2, 211–216. doi: 10.1038/35056522
  Persani, L., Calebiro, D., & Bonomi, M. (2007). Technology Insight: Modern methods to monitor protein‐protein interactions reveal functional TSH receptor oligomerization. Nature Clinical Practice. Endocrinology & Metabolism, 3, 180–190. doi: 10.1038/ncpendmet0401
  Phizicky, E. M., & Fields, S. (1995). Protein‐protein interactions: Methods for detection and analysis. Microbiology Reviews, 59, 94–123.
  Rubinsztein, D. C., Codogno, P., & Levine, B. (2012). Autophagy modulation as a potential therapeutic target for diverse diseases. Nature Reviews. Drug Discovery, 11, 709–730. doi: 10.1038/nrd3802
  Salahpour, A., Espinoza, S., Masri, B., Lam, V., Barak, L. S., & Gainetdinov, R. R. (2012). BRET biosensors to study GPCR biology, pharmacology, and signal transduction. Frontiers in Endocrinology, 3, 105. doi: 10.3389/fendo.2012.00105
  Schindelin, J., Arganda‐Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: An open‐source platform for biological‐image analysis. Nature Methods, 9, 676–682. doi: 10.1038/nmeth.2019
  Sekar, R. B., & Periasamy, A. (2003). Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. The Journal of Cell Biology, 160, 629–633. doi: 10.1083/jcb.200210140
  Seo, E., Woo, J., Park, E., Bertolani, S. J., Siegel, J. B., Choi, D., & Dinesh‐Kumar, S. P. (2016). Comparative analyses of ubiquitin‐like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross‐kingdom processing of ATG8 by ATG4. Autophagy, 12, 2054–2068. doi: 10.1080/15548627.2016.1217373
  Shimomura, O., Johnson, F. H., & Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 59, 223–239. doi: 10.1002/jcp.1030590302
  Siddiqui, S., Cong, W. N., Daimon, C. M., Martin, B., & Maudsley, S. (2013). BRET biosensor analysis of receptor tyrosine kinase functionality. Frontiers in Endocrinology (Lausanne), 4, 46. doi: 10.3389/fendo.2013.00046
  Subramanian, C., Woo, J., Cai, X., Xu, X., Servick, S., Johnson, C. H., … von Arnim, A. G. (2006). A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET). The Plant Journal, 48, 138–152. doi: 10.1111/j.1365‐313X.2006.02851.x
  Villalobos, V., Naik, S., & Piwnica‐Worms, D. (2007). Current state of imaging protein‐protein interactions in vivo with genetically encoded reporters. Annual Review of Biomedical Engineering, 9, 321–349. doi: 10.1146/annurev.bioeng.9.060906.152044
  Wen, X., & Klionsky, D. J. (2016). An overview of macroautophagy in yeast. Journal of Molecular Biology, 428, 1681–1699. doi: 10.1016/j.jmb.2016.02.021
  Woo, J. (2008). Application and optimization of bioluminescence resonance energy transfer (BRET) for real time detection of protein‐protein interactions in transgenic Arabidopsis as well as structure‐based functional studies on the active site of coelenterazine‐dependent luciferase from Renilla and its improvement by protein engineering. Knoxville: University of Tennessee.
  Woo, J., & von Arnim, A. G. 2008. Mutational optimization of the coelenterazine‐dependent luciferase from Renilla. Plant Methods, 4, 23. doi: 10.1186/1746‐4811‐4‐23
  Woo, J., Park, E., & Dinesh‐Kumar, S. P. (2014). Differential processing of Arabidopsis ubiquitin‐like Atg8 autophagy proteins by Atg4 cysteine proteases. Proceedings of the National Academy of Sciences of the United States of America, 111, 863–868. doi: 10.1073/pnas.1318207111
  Xu, Y., Piston, D. W., & Johnson, C. H. (1999). A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences of the United States of America, 96, 151–156. doi: 10.1073/pnas.96.1.151
  Zhang, L., Li, J., Ouyang, L., Liu, B., & Cheng, Y. (2016). Unraveling the roles of Atg4 proteases from autophagy modulation to targeted cancer therapy. Cancer Letters, 373, 19–26. doi: 10.1016/j.canlet.2016.01.022
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