SUMOylation of proteins regulates cell behaviors and is reversibly removed by small ubiquitin-like modifier(SUMO)-specific proteases(SENPs).The SENP family member SENP3 is involved in SUMO2/3 deconjugation and has been reported to sense cell stress and accumulate in several human cancer cells and macrophages.We previously reported that Senp3-knockout heterozygous mice showed smaller liver,but the pertinent mechanisms of SENP3 and SUMOylated substrates remain unclear.Thus,in this study,we investigated the interacting proteins with SENP3 and the alteration in hepatocytes treated with the xenobiotic diethylnitrosamine(DEN),which is specifi-cally transformed in the liver and induces DNA double-strand breaks.Our data revealed that a certain amount of SENP3 was present in normal,untreated hepatocytes;however,DEN treatment promoted rapid SENP3 accumulation.SENP3 was mainly localized in the nuclei,and its level was significantly increased in the cytoplasm after 2 h of DEN treatment.The application of the recent proximity-dependent biotinylation(BioID)method led to the identification of 310 SENP3-interacting proteins that were involved in not only gene transcription but also RNA splicing,protein fold-ing,and metabolism.Furthermore,after DEN exposure for a short duration,ribosomal proteins as well as proteins associated with mitochondrial ATP synthesis,membrane transport,and bile acid synthesis,rather than DNA repair proteins,were identified.This study provides insights into the diverse regulatory roles of SENP3,and the BiolD method seems to be efficient for identifying physiologically relevant insoluble proteins.