Proximity-dependent biotinylation reveals an interaction between ubiquitin-specific peptidase 46 and centrosome-related proteins.

Protein ubiquitination extensively modulates protein functions and controls various biological processes, such as protein degradation, signal transduction, transcription, and DNA repair. Ubiquitination is a reversible post-translational modification, and deubiquitinating enzymes cleave ubiquitin from proteins. Ubiquitin-specific peptidase 46 (USP46), a deubiquitinase, is highly expressed in the brain and regulates neural functions. Deleting lysine 92 (ΔK92) in USP46 reduces murine depression-like behavior in the tail suspension test. However, the molecular basis for USP46's role in regulating neural function has not yet been fully understood. Here we employed a proximity-dependent biotinylation approach to characterize the USP46 protein interaction partners. Using homology-independent targeted integration (HITI), a genome editing technique, we established knockin cell lines that stably express USP46 wildtype- or ΔK92-biotin ligase fusion protein. We identified 286 candidate interaction partners, including well-known binding partners of USP46. Although there were no obvious differences in the interactome of USP46 between wildtype and ΔK92, a gene ontology analysis revealed that centrosome-related proteins were significantly enriched in the proximal proteins of USP46. Several centrosome-related proteins were bound to USP46 in Neuro2a cells, but their protein expression levels were not affected in the brains of USP46-deficient mice. These results uncover a potential relationship between USP46 and centrosome regulation independently of protein stabilization.