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

Molecular Systems Biology
Philipp TrepteErich E Wanker

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...Continue Reading

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Citations

Jul 20, 2018·Molecular Systems Biology·Mikko Taipale
Jul 27, 2018·Molecular Systems Biology·Maria Polychronidou
Aug 31, 2019·Nature Communications·Soon Gang ChoiYves Jacob
Feb 12, 2020·Progress in Neurobiology·Cameron B Gundersen
Mar 5, 2021·Biochimica Et Biophysica Acta. Molecular Cell Research·Hailong WangShiwen Luo
May 25, 2021·Disease Models & Mechanisms·Lauren G FalkenbergJoshua S Waxman
Aug 25, 2021·Current Opinion in Chemical Biology·Shivanthy PathmanathanIgor Stagljar
Sep 11, 2021·Proceedings of the National Academy of Sciences of the United States of America·Sri Murugan Poongkavithai VadevooByungheon Lee
Sep 7, 2021·Journal of Molecular Biology·Elena KudryashovaDmitri S Kudryashov

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Datasets Mentioned

BETA
IOH52832

Methods Mentioned

BETA
BRET
transfection
KISS
Y2H
FRET
fluorescence microscopy
proximity ligation
PCR
FCS
Light

Software Mentioned

pPA
IntAct
KISS
LuTHy
MAPPIT
GW
BACON
RL
DULIP
LUMIER

Related Concepts

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Batten Disease is a group of nervous system disorders known as neuronal ceroid lipofuscinosis. This feed focuses on neurobiological and neuropathological aspects of this disease.