Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics

Simple Summary O-GlcNAcylation is a rapid and reversible posttranslational modification involved in the adaptation of cells to nutrient availability. The interplay of O-GlcNAcylation and phosphorylation controls signal transduction pathways to maintain cellular homeostasis. To date, studies have concentrated on the O-GlcNAcylation of proteins by the nuclear-cytoplasmic isoform of O-GclNAc transferase (ncOGT) whereas the function of mitochondrial OGT (mOGT) is poorly understood. The goal of our research was to investigate how mOGT affects the phosphorylation of mitochondrial proteins and the synthesis of ATP. Based on our results, we suggested that the deregulation of mOGT might be sufficient for changes in electron transport chain activity and ATP synthesis. Inhibition of the energy metabolism of cancer cells is important for potential cancer therapies; therefore, mOGT could be considered as a therapeutic target.Abstract O-linked beta-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol.