USP30-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 and hexokinase activity. Lysine 144 emerges as a critical regulatory site for USP30-mediated deubiquitination of HK2. Mutation of K144 enhances HK2 stability, increases its mitochondrial localization and binding to VDAC1, and significantly augments hexokinase activity. Furthermore, the HK2 K144 mutation markedly enhances tumor cell glycolysis, fostering increased proliferation and migration both in vitro and in vivo. These findings underscore USP30 as a novel regulator of glycolysis in cancer cells via modulation of HK2 ubiquitination dynamics, suggesting its potential as a therapeutic target in cancer metabolism.