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

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

doi:10.7554/elife.30233

Abstract

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.

Highlights

Protein-protein interactions (PPIs) are central to cellular signal transduction
It should be facile to couple SPARK to the expression of other reporter genes as well, including APEX for EM (Martell et al, 2012; Lam et al, 2015) and proteomics (Rhee et al, 2013), antibiotic resistance genes, and various actuators such as opsins and toxins
The resulting tool is highly modular and generalizable; 12 of the 13 PPIs we cloned into SPARK worked on the first try, without any optimization of geometries or linkers

Summary

Introduction

Protein-protein interactions (PPIs) are central to cellular signal transduction. many assays have been developed to detect and study them, in the context of living cells, where native PPIs are unperturbed by cell lysis, detergents, fixatives, or dilution. Examples include the yeast two hybrid assay (Miller and Stagljar, 2004), the split ubiquitin assay (Petschnigg et al, 2014), and TANGO (Barnea et al, 2008; Inagaki et al, 2012; Kroeze et al, 2015) Benefits of these assays include scalability (because real-time microscopy is not needed), versatility of read out (transcription of a fluorescent protein or an antibiotic resistance gene, for example), and high sensitivity due to signal amplification. These properties have led to the widespread use of integrative PPI assays for PPI discovery and drug screening. This lays the foundation for the eventual use of SPARK for genetic screens and PPI discovery on a genome-wide scale

Design and optimization of SPARK

Discussion

Findings

Materials and methods