Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

Abstract

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.