Sirtuin 3-mediated delactylation of malic enzyme 2 disrupts redox balance and inhibits colorectal cancer growth.

PURPOSE: Post-translational modifications, such as lactylation, are emerging as critical regulators of metabolic enzymes in cancer progression. Mitochondrial malic enzyme 2 (ME2), a key enzyme in the TCA cycle, plays a pivotal role in maintaining redox homeostasis and supporting tumor metabolism. However, the functional significance of ME2 lactylation and its regulatory mechanisms remain unclear. This study investigates the role of ME2 K352 lactylation in modulating enzymatic activity, redox balance, and tumor progression. METHODS: Immunoprecipitation and western blotting were used to assess ME2 lactylation and its interaction with Sirtuin 3 (SIRT3). Mass spectrometry identified the lactylation site on ME2. Enzymatic activity was measured using NADH production assays. The functional effects of ME2 K352 lactylation were analyzed by measuring ROS levels, NADP⁺/NADPH ratios, metabolic intermediates, and mitochondrial respiration parameters. Cell proliferation was evaluated via CCK-8 and colony formation assays. Xenograft tumor models and Ki-67 immunohistochemical staining were used to assess tumor growth and proliferation in vivo. RESULTS: Mass spectrometry identified K352 as the primary lactylation site on ME2. Sodium lactate treatment enhanced ME2 lactylation and enzymatic activity, while SIRT3-mediated delactylation at K352 reduced ME2 activity, disrupting redox homeostasis. Cells expressing the K352R mutant exhibited elevated ROS levels, higher NADP⁺/NADPH ratios, and altered levels of metabolic intermediates, including increased malate and lactate with reduced pyruvate. Additionally, re-expression of ME2 K352R in HCT116 cells significantly impaired proliferation and colony formation. In vivo, xenograft models demonstrated that ME2 K352R expression suppressed tumor growth, as evidenced by reduced tumor volume, weight, and Ki-67 staining. CONCLUSIONS: This study reveals that ME2 K352 lactylation is a critical regulatory mechanism that modulates enzymatic activity, mitochondrial function, and tumor progression. SIRT3-mediated delactylation of ME2 K352 disrupts redox homeostasis and inhibits tumor growth. These findings highlight the potential of targeting ME2 lactylation as a therapeutic strategy in cancer treatment.