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

• Published in: Molecular systems biology Vol. 14; no. 7; pp. e8071 - n/a
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.07.2018; John Wiley and Sons Inc; Springer Nature
• Subjects: Animals; Bioluminescence Resonance Energy Transfer Techniques; Chemical Precipitation; CSPα; EMBO17; EMBO22; EMBO26; Gene Regulatory Networks; HEK293 Cells; HSP40 Heat-Shock Proteins - chemistry; HSP40 Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins - metabolism; Humans; Luminescent Measurements; LuTHy; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Method; Methods; Mice; missense mutations; Mutation, Missense; Neuronal Ceroid-Lipofuscinoses - genetics; Protein Binding; protein–protein interactions; quantitative; Two-Hybrid System Techniques; protein–protein interactions; LuTHy; missense mutations; quantitative; CSPα
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.20178071

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. Synopsis A new double‐readout bioluminescence‐based two‐hybrid method deepens the coverage of protein interaction maps. It provides two quantitative interaction scores, recovers transient associations and monitors the effects of missense mutations and small molecules on interactions. LuTHy is a double‐readout technology that provides two quantitative scores for protein‐protein interactions in mammalian cells. Low‐ and high‐affinity interactions can be detected with high sensitivity. Nearly 50% of CSPα interactions with synaptic proteins are affected by ANCL‐disease mutations. Graphical Abstract A new double‐readout bioluminescence‐based two‐hybrid method deepens the coverage of protein interaction maps. It provides two quantitative interaction scores, recovers transient associations and monitors the effects of missense mutations and small molecules on interactions.