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O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.

We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.

Increased activity of the main carbohydrate pathways (glycolysis, pentose phosphate, and hexosamine biosynthetic pathways) is one of the hallmarks of metabolic diseases such as cancer. Sulfoquinovosyl diacylglycerol is a sulfoglycolipid found in the human diet that possesses anticancer activity that is absent when its carbohydrate moiety (glucose 6-sulfonate or sulfoquinovose) is removed. This work used bacterial systems to further understand the metabolism of this sugar through three main carbohydrate processing pathways and how this could influence its biological activity. Using 13C NMR spectroscopy and enzyme assays, we showed that glucose 6-sulfonate cannot enter the pentose phosphate pathway, hence decreasing pentose and nucleotide biosyntheses. In glycolysis, glucose 6-sulfonate only provides one pyruvate per monosaccharide molecule, decreasing the flux of this pathway by half when compared to glucose 6-phosphate. Glucose 6-sulfonate can enter the hexosamine biosynthetic pathway by producing glucosamine 6-sulfonate, which is a reported antibacterial agent that competitively inhibits hexosamine production. All these interactions with carbohydrate routes might help explain the observed anticancer activity that glucose 6-sulfonate has in vitro. This adds to our knowledge of how vegetables rich in glucose 6-sulfonate can also act as metabolic inhibitors of pathways that are increased in metabolic diseases.