Lysine β-hydroxybutyrylation (Kbhb) is a post-translational modification induced by the ketogenic diet (KD), a diet showing therapeutic effects on multiple human diseases. Little is known how cellular processes are regulated by Kbhb. Here we show that protein Kbhb is strongly affected by the KD through a multi-omics analysis of mouse livers. Using a small training dataset with known functions, we developed a bioinformatics method for the prediction of functionally important lysine modification sites (pFunK), which revealed functionally relevant Kbhb sites on various proteins, including aldolase B (ALDOB) Lys108. KD consumption or β-hydroxybutyrate supplementation in hepatocellular carcinoma cells increases ALDOB Lys108bhb and inhibits the enzymatic activity of ALDOB. A Kbhb-mimicking mutation (p.Lys108Gln) attenuates ALDOB activity and its binding to substrate fructose-1,6-bisphosphate, inhibits mammalian target of rapamycin signalling and glycolysis, and markedly suppresses cancer cell proliferation. Our study reveals a critical role of Kbhb in regulating cancer cell metabolism and provides a generally applicable algorithm for predicting functionally important lysine modification sites.

赖氨酸β-羟基丁酰化（Kbhb）是生酮饮食（KD）诱导的翻译后修饰，生酮饮食对多种人类疾病具有治疗作用。人们对 Kbhb 如何调节细胞过程知之甚少。在这里，我们通过对小鼠肝脏的多组学分析表明蛋白质 Kbhb 受到 KD 的强烈影响。使用具有已知功能的小型训练数据集，我们开发了一种生物信息学方法来预测功能上重要的赖氨酸修饰位点 (pFunK)，该方法揭示了各种蛋白质上的功能相关 Kbhb 位点，包括醛缩酶 B (ALDOB) Lys108。肝细胞癌细胞中消耗 KD 或补充 β-羟基丁酸会增加 ALDOB Lys108bhb 并抑制 ALDOB 的酶活性。 Kbhb 模拟突变 (p.Lys108Gln) 会减弱 ALDOB 活性及其与底物果糖 1,6-二磷酸的结合，抑制哺乳动物雷帕霉素信号传导和糖酵解的靶标，并显着抑制癌细胞增殖。我们的研究揭示了 Kbhb 在调节癌细胞代谢中的关键作用，并提供了一种普遍适用的算法来预测功能上重要的赖氨酸修饰位点。