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

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 CSP?, 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 CSP? 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.

SUBMITTER: Trepte P

PROVIDER: S-EPMC6039870 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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

Trepte Philipp P Kruse Sabrina S Kostova Simona S Hoffmann Sheila S Buntru Alexander A Tempelmeier Anne A Secker Christopher C Diez Lisa L Schulz Aline A Klockmeier Konrad K Zenkner Martina M Golusik Sabrina S Rau Kirstin K Schnoegl Sigrid S Garner Craig C CC Wanker Erich E EE

Molecular systems biology 20180711 7

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 quantit ...[more]

PMID: 29997244

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Project description:BackgroundMost of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.ResultsIn this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented.ConclusionWe demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.

Dataset Information

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

Publications

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

Similar Datasets

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