Two protein/protein interaction assays in one go.

Mikko Taipale

doi:10.15252/msb.20188485

Abstract

Discovering and characterizing protein–protein interactions (PPIs) that contribute to cellular homeostasis, development, and disease is a key priority in proteomics. Numerous assays for protein–protein interactions have been developed, but each one comes with its own strengths, weaknesses, and false‐positive/false‐negative rates. Therefore, it seems rather intuitive that combining multiple assays is beneficial for robust and reliable discovery of interactions. Along those lines, in their recent study, Wanker and colleagues (Trepte et al , 2018) combined two complementary and quantitative interaction assays in one pot. One assay is luminescence‐based and depends on protein proximity in living cells, while the other relies on formation of more stable complexes detected by co‐precipitation with a luminescence‐based readout, which facilitates confident identification and quantitation of interactions in high throughput.

Highlights

Discovering and characterizing protein– protein interactions (PPIs) that contribute to cellular homeostasis, development, and disease is a key priority in proteomics
The methods are as diverse as their acronyms; some are based on genetic selection (Y2H: yeast two-hybrid) or reporter genes (MaMTH: mammalian membrane two-hybrid; MAPPIT: mammalian protein/protein interaction trap; and KISS: kinase substrate sensor), others on protein complementation (BiFC: bimolecular fluorescence complementation; NanoBit: NanoLuc complementation assay; and DHFR-PCA: dihydrofolate reductase proteinfragment complementation assay) or co-purification (AP-MS: affinity purification–mass spectrometry; LUMIER: luminescence-based mammalian interactome assay; and NAPPA: nucleic-acid programmable protein array), yet others on protein proximity rather than direct interaction (BRET: bioluminescence resonance energy transfer; FRET: fluorescence/Foerster resonance energy transfer; and BioID: proximity-dependent biotin identification)
PPIs occur in all compartments, with their characteristic pH, redox environment, lipid constitution, and posttranslational modifications

Summary

Introduction

Discovering and characterizing protein– protein interactions (PPIs) that contribute to cellular homeostasis, development, and disease is a key priority in proteomics. They have developed LuTHy, a novel method which combines copurification and proximity methods in one system to assay binary PPIs (Fig 1). BRET can be measured in living cells but the assay is highly sensitive to tag location.

Results

Conclusion