Non-histone proteins localized in membrane, cytosol and nucleus are lactylated to promote tumor progression. However, whether mitochondrial proteins undergo lactylation and contribute to tumor progression remains unexplored. Here, we identified multiple lactylated mitochondrial proteins in human renal cell carcinoma (RCC) cells using lactylome profiling. Among these, malate dehydrogenase 2 (MDH2)–the only lactylated protein in the tricarboxylic acid (TCA) cycle–emerged as a key target, with K239 as its lactylation site. MDH2 K239la levels are regulated by KAT8 and SIRT3. Under low-glucose, high-lactate conditions mimicking the tumor microenvironment, MDH2 K239la elevated the NADH/NAD + ratio to drive ATP production and boosted NADPH generation to reduce ROS. This enables cells to alleviate oxidative stress, sustain mitochondrial function, and promote RCC malignancy in vitro and in vivo . Mechanistically, MDH2 K239la enhances MDH2 enzymatic activity and strengthens its interaction with the citrate transporter SLC25A1. This facilitates citrate efflux, fueling IDH1-dependent NADPH production when lactate serves as an energy source. Collectively, we unveil a lactylation-dependent mechanism that reprograms mitochondrial metabolism to confer oxidative stress resistance and drive RCC progression. Targeting mitochondrial proteins lactylation, exemplified by MDH2 K239la , represents a promising therapeutic strategy for RCC.
Wenjuan Liu,Junjie Li,Sihan Xu,Yixu Wang,Jiawei Li,Shou Wang,Li Fu,Min Jiang,Gang Bai
ABSTRACT Background Fever is one of the main pathophysiological reactions that occurs during the acute phase of various diseases. Excessive body temperature can lead to various adverse consequences such as brain tissue damage and abnormal immune responses. Phillyrin (Phr) is the main active ingredient in Forsythia suspensa (Thunb.) Vahl (Lian Qiao) and has antipyretic effects; however, its antipyretic mechanism of action remains unclear. Purpose This study aimed to explore the antipyretic mechanisms of Phr and provide a new treatment plan for fever. Methods The antipyretic effects of Phr were evaluated using a mouse model of pneumonia fever. The main metabolites of Phr involved in its antipyretic function were identified using a mitochondrial temperature-sensitive probe. Further synthesis of the main metabolite, phillygenin (Phg), an alkynylated probe, was performed, and chemical proteomics was used to capture and analyze its direct target for antipyretic effects. The mechanism of action of Phg and its antipyretic targets was explored using metabolomics and various molecular biology methods. Results Phr showed significant antipyretic and anti-inflammatory effects in a mouse model of lipopolysaccharide-induced fever. Phg reversibly targeted the nicotinamide adenine dinucleotide (NAD + ) binding domain of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malate dehydrogenase 2 (MDH2), and isocitrate dehydrogenase 2 (IDH2) to inhibit their enzymatic activity. In-depth analysis of cellular metabolomics and mitochondrial stress testing indicated that inhibition of GAPDH, MDH2, and IDH2 enzyme activity by Phg led to a decrease in cellular energy supply and heat production regulated by glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation signaling pathways. Phg specifically targeted macrophages and inhibited LPS-induced macrophage activation by downregulating GAPDH enzyme activity, thereby exerting anti-inflammatory effects. In vivo experiments also confirmed that the antipyretic effect of Phr in LPS-induced fever model mice was related to its main metabolites, Phg and Phg-sulfonate (Phg-S), which directly targeted the NAD + binding domain of GAPDH, IDH2, and MDH2, inhibiting the activity of these enzymes, thereby reducing energy supply and regulating febrile-related inflammatory factors. Conclusion This study reported for the first time that the antipyretic effect of Phr is produced by targeting GAPDH, IDH2, and MDH2 to regulate energy supply and febrile-related inflammatory factors through its main metabolites Phg and Phg-S. This study not only provides potential drugs for fever treatment but also provides new ideas for improving clinical fever treatment plans.
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