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LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells

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Item Type:Article
Title:LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells
Creators Name:Trepte, P. and Kruse, S. and Kostova, S. and Hoffmann, S. and Buntru, A. and Tempelmeier, A. and Secker, C. and Diez, L. and Schulz, A. and Klockmeier, K. and Zenkner, M. and Golusik, S. and Rau, K. and Schnoegl, S. and Garner, C.C. and Wanker, E.E.
Abstract:Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems.
Keywords:CSPα, LuTHy, Missense Mutations, Protein-Protein Interactions, Quantitative, Animals, Mice
Source:Molecular Systems Biology
ISSN:1744-4292
Publisher:Wiley (U.S.A.)
Volume:14
Number:7
Page Range:e8071
Date:11 July 2018
Official Publication:https://doi.org/10.15252/msb.20178071
PubMed:View item in PubMed

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