
Proximity labeling coupled to mass spectrometry can be used to comprehensively map proteasome interactions and substrates in cultured cells and mouse models.


coremsg

Proteasomes 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. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and 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.

### Competing Interest Statement

A patent application covering part of the data presented in this manuscript has been filed at the European Patent Office with application number PCT/EP2023/069680. 

Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [61][1] partner repository and they are accessible with the identifier:

PXD032833 following login credentials Username: reviewer_pxd032833@ebi.ac.uk password: UeTUvffr, for the HEK293T ProteasomeID dataset for PSMA4 and PSMC2;

PXD034874 following login credentials Username:reviewer_pxd034874@ebi.ac.uk password: JSKJIvmX, for the HEK293T size exclusion chromatography dataset;

PXD033008 following login credentials Username: reviewer_pxd033008@ebi.ac.uk password: eRoFg8LW, for the MG132 ProteasomeID dataset;

PXD032976 following login credentials Username: reviewer_pxd032976@ebi.ac.uk password: kcQb7byl for the PROTAC ProteasomeID dataset;

PXD034965 following login credentials Username: reviewer_pxd034965@ebi.ac.uk password:

DHtFE3zX, for the MG132 whole cell proteome dataset;

Mass spectrometry proteomics data have been deposited to ProteomeXchange Consortium via the MassIVE partner repository and they are accessible with the identifier:

MSV000092396 following login credential Username: MSV000092396_reviewer password: a, for the mouse ProteasomeID dataset

MSV000092407 following login credential Username: MSV000092407_reviewer password: proteasomeID, for the HEK293T ProteasomeID for PSMD3

 [1]: #ref-61

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