biostudies-literatureHaowei ZShenzhen Science and Technology Innovation CommissionScience and Technology Department of Henan Province (Henan Provincial Department of Science and Technology)National Natural Science Foundation of China (National Science Foundation of China)225https://www.ebi.ac.uk/biostudies/studies/S-EPMC12921045biostudies-literatureUnknown17(1)The mitochondria-localized deubiquitinase USP30 regulates mitophagy and mitochondrial homeostasis, playing a significant role in the progression of neurodegenerative diseases. However, its role in cancer remains poorly understood. Through metabolism-related gene set enrichment analysis, we identified USP30 as a key modulator of glucose metabolism in cancer cells. Utilizing quantitative proteomic and ubiquitinomic approaches coupled with co-immunoprecipitation assays, we elucidated that USP30 regulates glycolysis by interacting with hexokinase HK1 and HK2, a process dependent on its enzymatic activity. USP30 modulates the ubiquitination profile of HK1 and HK2 by preferentially removing atypical ubiquitin chains, thereby enhancing their stability, mitochondrial localization, VDAC1 binding anCell death & diseaseUSP30-mediated Deubiquitination of Hexokinase 2 controls the metabolic fate of glucose and tumor progression.PMC1292104532200438, 32301053252102310403JCYJ20240813142003005Jiang YYang HLi XLi YXie WXu NZhang YLiao WHaowei ZQin XZeng HfalseUSP30-mediated Deubiquitination of Hexokinase 2 controls the metabolic fate of glucose and tumor progression.The mitochondria-localized deubiquitinase USP30 regulates mitophagy and mitochondrial homeostasis, playing a significant role in the progression of neurodegenerative diseases. However, its role in cancer remains poorly understood. Through metabolism-related gene set enrichment analysis, we identified USP30 as a key modulator of glucose metabolism in cancer cells. Utilizing quantitative proteomic and ubiquitinomic approaches coupled with co-immunoprecipitation assays, we elucidated that USP30 regulates glycolysis by interacting with hexokinase HK1 and HK2, a process dependent on its enzymatic activity. USP30 modulates the ubiquitination profile of HK1 and HK2 by preferentially removing atypical ubiquitin chains, thereby enhancing their stability, mitochondrial localization, VDAC1 binding an2026-01-01T00:00:00Z2026 Feb2026-07-16T12:33:35.994Z2026-07-09T10:55:29.764ZS-EPMC129210454168844310.1038/s41419-026-08459-w