LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells
| Autor: | Kirstin Rau, Sabrina Kruse, Craig C. Garner, Sabrina Golusik, Martina Zenkner, Simona Kostova, Erich E. Wanker, Sheila Hoffmann, Aline Schulz, Christopher Secker, Anne Tempelmeier, Konrad Klockmeier, Philipp Trepte, Alexander Buntru, Sigrid Schnoegl, Lisa Diez |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Bioluminescence Resonance Energy Transfer Techniques Gene regulatory network Method Plasma protein binding Network Biology metabolism [HSP40 Heat-Shock Proteins] Mice 0302 clinical medicine chemistry [HSP40 Heat-Shock Proteins] Methods Chemical Precipitation Gene Regulatory Networks genetics [Neuronal Ceroid-Lipofuscinoses] biology Applied Mathematics CSPα Small molecule cysteine string protein genetics [Membrane Proteins] Computational Theory and Mathematics Genome-Scale & Integrative Biology General Agricultural and Biological Sciences Function and Dysfunction of the Nervous System genetics [HSP40 Heat-Shock Proteins] Information Systems Protein Binding LuTHy Mutation Missense Computational biology protein–protein interactions Methods & Resources General Biochemistry Genetics and Molecular Biology Protein–protein interaction 03 medical and health sciences missense mutations quantitative ddc:570 Two-Hybrid System Techniques medicine Bioluminescence Animals Humans chemistry [Membrane Proteins] General Immunology and Microbiology HEK 293 cells Membrane Proteins HSP40 Heat-Shock Proteins medicine.disease 030104 developmental biology HEK293 Cells Chaperone (protein) Luminescent Measurements biology.protein Neuronal ceroid lipofuscinosis 030217 neurology & neurosurgery metabolism [Membrane Proteins] |
| Zdroj: | Molecular systems biology 14(7), e8071 (2018). doi:10.15252/msb.20178071 Molecular Systems Biology |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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