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Time-gated detection of protein-protein interactions with transcriptional readout

By Min Woo Kim, Wenjing Wang, Mateo I Sanchez, Robert Coukos, Mark Von Zastrow, Alice Y Ting

Posted 21 Jul 2017
bioRxiv DOI: 10.1101/166462 (published DOI: 10.7554/eLife.30233)

Transcriptional assays such as yeast two hybrid, split ubiquitin, and Tango that convert transient protein-protein interactions (PPIs) in cells into stable expression of transgenes are powerful tools for PPI discovery, high-throughput screens, and analysis of large cell populations. However, these assays frequently suffer from high background and they lose all information about PPI dynamics. To address these limitations, we developed a light-gated transcriptional assay for PPI detection called PPI-FLARE (PPI-Fast Light- and Activity-Regulated Expression). PPI-FLARE requires both a PPI to deliver TEV protease proximal to its cleavage peptide, and externally-applied blue light to uncage the cleavage peptide, in order to release a membrane-tethered transcription factor (TF) for translocation to the nucleus. We used PPI-FLARE to detect the ligand-induced association of 12 different PPIs in living mammalian cells, with a temporal resolution of 5 minutes and a +/-ligand signal ratio up to 37. By systematically shifting the light irradiation window, we could reconstruct PPI time-courses, distinguishing between GPCRs that engage in transient versus sustained interactions with the cytosolic effector arrestin. When combined with FACS, PPI-FLARE enabled >100-fold enrichment of cells experiencing a specific GPCR-arrestin PPI during a short 10-minute light window over cells missing that PPI during the same time window. Due to its high specificity, sensitivity, and generality, PPI-FLARE should be a broadly useful tool for PPI analysis and discovery.

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