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Studying OTUD6B-OTUB1 protein-protein interaction by low-throughput GFP-trap assays and high-throughput AlphaScreen assays.

In: Proteomic Profiling. Berlin [u.a.]: Springer, 2021. 381-394 (Methods Mol. Biol. ; 2261)
Postprint DOI
Open Access Green
Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors.
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Publication type Article: Edited volume or book chapter
Keywords Alphascreen ; Deubiquitinase ; Dub ; Gfp-trap ; Homogeneous Proximity Assay ; Immunoprecipitation ; Otub1 ; Otud6b ; Protein–protein Interactions ; Ubiquitin
ISSN (print) / ISBN 1064-3745
e-ISSN 1940-6029
Book Volume Title Proteomic Profiling
Quellenangaben Volume: 2261, Issue: , Pages: 381-394 Article Number: , Supplement: ,
Publisher Springer
Publishing Place Berlin [u.a.]
Reviewing status Peer reviewed