A novel miniTurbo knock-in mouse reveals a protein interaction network of USP46 in the brain

Abstract

Uncovering protein interaction networks in vivo is essential for understanding physiological and pathological processes. Here, we report the generation of a novel knock-in mouse model expressing miniTurbo, a highly active biotin ligase, fused to the endogenous Usp46 gene. This model enables proximity-dependent biotin labeling (BioID) of USP46-associated proteins in the brain. In adult mice, biotinylation was induced by feeding a 0.1% biotin diet. We further evaluated whether the combination of miniTurbo and dietary biotin supplementation is effective for BioID in the developing brain. Biotinylation was successfully induced in embryonic and neonatal brains via maternal biotin intake, demonstrating the transfer of biotin to the offspring through the placenta during pregnancy and through milk during lactation. This strategy enables proximity labeling under physiological conditions without invasive procedures, such as repetitive subcutaneous injections, during developmental stages. Using mass spectrometry, we identified USP46-proximal proteins, including known cofactors WDR48 and WDR20, in the adult brain. Gene Ontology analysis revealed enrichment in postsynaptic pathways, consistent with known localization of USP46. Among the identified proteins, PLPP3, a phospholipid phosphatase, was significantly downregulated in the hippocampus of Usp46-knockout mice. These findings establish the USP46-miniTurbo knock-in mouse as a powerful tool for in vivo interactome analysis and provide new insights into the molecular functions of USP46 in the brain.