Mitochondrial O-GlcNAc Transferase Interacts with and Modifies Many Proteins and Its Up-Regulation Affects Mitochondrial Function and Cellular Energy Homeostasis

Abstract

O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT’s role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.

Role and Function of O-GlcNAcylation in Cancer

“…Substrates are specific to the isoforms of OGT, and the three isoforms are expressed differently [20,21]. For instance, mOGT is expressed transiently and in much lower than ncOGT, due to its susceptibility to the cellular glucose level [22,23]. The exact mechanisms for substrate recognition by OGT are yet to be clarified; however, the adaptor protein hypothesis and non-specific O-GlcNAcylation are recognized as the plausible mechanisms [24][25][26][27].…”

Short O-GlcNAcase Is Targeted to the Mitochondria and Regulates Mitochondrial Reactive Oxygen Species Level

“…Increases in O-GlcNAcylation of mitochondrial proteins have been observed upon high-glucose conditions [ 8 , 9 ], and recent work pointed to perturbation in the localization of mOGT in cardiomyocytes mitochondria from diabetic mice [ 10 ]. Several lines of evidence indicate that alteration of O-GlcNAc cycle disrupts mitochondrial homoeostasis [ 7 , 11 , 12 , 13 , 14 , 15 , 16 ], including alteration in reactive oxygen species production [ 14 , 16 , 17 , 18 , 19 , 20 ].…”

“…Biochemistry and proteomic studies have revealed that hundreds of mitochondrial proteins are O-GlcNAcylated, including key proteins involved in mitochondria bioenergetics, such as components of the respiratory chain complexes, ATP synthase, pyruvate dehydrogenase, Krebs cycle, and lipid metabolism enzymes [ 8 , 13 , 16 , 20 , 21 , 22 , 23 , 24 ]. Although it cannot be excluded that some of these proteins are O-GlcNAcylated in the cytosol prior to their export into mitochondria, several proteins encoded by mitochondrial DNA, such as MTCO1 (cytochrome oxidase 1), COX2 (cytochrome oxidase 2), and MT-ND4 (NADH:ubiquinone oxidoreductase core subunit 4), were also shown to be O-GlcNAcylated, confirming that O-GlcNAc cycling must occur within mitochondria [ 7 ].…”

O‐GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro‐survival mechanism in cancer cells

“…While the biological role of O-GlcNAcylation remains less understood compared to other cell signalling PTMs such as phosphorylation, thousands of O-GlcNAc substrates have been identified or predicted [6][7][8][9][10][11]. O-GlcNAcylation is a ubiquitous PTM implicated in various aspects of cellular functions, including gene transcription, cell signalling, and stress response [2,4,12].…”

“…For this ubiquitous PTM, an unbiased proteomics-level investigation would be ideal for uncovering novel functions of interest. Advances in mass spectrometry have indeed allowed the proteomic profiling of many O-GlcNAc substrates in a range of studies and models [6][7][8][9][10][11]. Bioscience and added to the culture media at the appropriate concentration, with BSA serving as the vehicle control.…”