A molecular toolbox for ADP-ribosyl binding proteins
Autor: | Heli I. Alanen, Sven T. Sowa, Albert Galera-Prat, Sarah Wazir, Lari Lehtiö, Mirko M. Maksimainen |
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Rok vydání: | 2021 |
Předmět: |
High-throughput screening
Gi alpha subunit Peptide macrodomain Biochemistry Genetics and Molecular Biology (miscellaneous) Biochemistry DNA-binding protein high-throughput screening Article protein labelling inhibitors Genetics Radiology Nuclear Medicine and imaging G alpha subunit chemistry.chemical_classification binding assay Drug discovery Chemistry Ligand binding assay Computer Science Applications Blot Förster resonance energy transfer post-translational modification Biotechnology ADP-ribosylation |
Zdroj: | Cell Reports Methods |
ISSN: | 2667-2375 |
Popis: | Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl-binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in E. coli and will help in ADP-ribosylation research and drug discovery. Graphical Abstract ADP-ribosyl binding proteins are integral actors in many cellular processes, however only a few inhibitors have been discovered thus far. Sowa et al. report a range of easy-to-produce in vitro tools for detection of ADP-ribosylation that also facilitate assay development allowing for screening of inhibitors in a high-throughput setup. |
Databáze: | OpenAIRE |
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