Open AccessLu TH y: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells
Author(s) -
Trepte Philipp,
Kruse Sabrina,
Kostova Simona,
Hoffmann Sheila,
Buntru Alexander,
Tempelmeier Anne,
Secker Christopher,
Diez Lisa,
Schulz Aline,
Klockmeier Konrad,
Zenkner Martina,
Golusik Sabrina,
Rau Kirstin,
Schnoegl Sigrid,
Garner Craig C,
Wanker Erich E
Publication year - 2018
Publication title -
molecular systems biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 8.523
H-Index - 148
ISSN - 1744-4292
DOI - 10.15252/msb.20178071
Subject(s) - bioluminescence , biology , computational biology , lysis , protein–protein interaction , luciferase , missense mutation , microbiology and biotechnology , mutation , cell culture , biochemistry , genetics , gene , transfection
Abstract Information on protein–protein interactions ( PPI s) 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 Lu TH y, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPI s 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 PPI s. Applying Lu TH y in a focused screen, we identified 42 interactions for the presynaptic chaperone CSP α, causative to adult‐onset neuronal ceroid lipofuscinosis ( ANCL ), a progressive neurodegenerative disease. Nearly 50% of PPI s were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ∆L116 in CSP α with Lu TH y. 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.