ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Abstract

SUMMARYProteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Proteasomes have been exploited for the development of small molecule degraders for otherwise undruggable proteins. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small molecule-induced proteasome substrates.