Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells

Abstract

Edited by Gerald W. HartO-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.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism

“…However, genetic ablation of OGT dramatically lowers the basal level of O-GlcNAc producing severe metabolic phenotypes (28 -30), and OGT or OGA overexpression impairs respiration (9). Recently, RNAi-mediated knockout of the mitochondrial form of OGT (mOGT), which is only expressed in a few mammals, including primates (31), in HeLa cells caused mitochondrial fragmentation and reduced membrane potential, whereas knock-out of both mOGT and the nuclear and cytoplasmic form of OGT led to compensatory increase in mitochondrial respiration partially due to the decrease in mitochondrial content (32). Even though TMG treatment lowered mitochondrial respiration in this study, ROS was not increased suggesting an intervention that raises both O-GlcNAc and OGA expression promotes a healthier metabolic phenotype than genetic manipulation of OGT or OGA alone.…”

T cell development and the physiological role of O‐GlcNAc

“…Though less energy efficient than oxidative phosphorylation, only producing two ATP versus 36 ATP, glycolysis generates metabolic intermediates that provide building blocks for synthesis of amino acids, fatty acids and nucleotides, all necessary for growth [22]. Studies that have either overexpressed OGT and OGA, or knocked down endogenous OGT have all highlighted the role of O-GlcNAc cycling in mitochondrial morphology and cellular respiration [23] which can contribute to determining cell state. Due to their unique metabolic profiles, altered mitochondrial dynamics and structure are hallmarks of stem cells.…”

“…As specification proceeds, remodeling mitochondrial activity helps signal differentiation. Studies that have either overexpressed OGT and OGA, or knocked down endogenous OGT have all highlighted the role of O-GlcNAc cycling in mitochondrial morphology and cellular respiration [23] which can contribute to determining cell state.…”

Assembly and Characterization of Biocompatible Coenzyme Q10‐Enriched Lipid Nanoparticles

“…On the day of the assay, the cells were washed three times with XF Media (Seahorse Bioscience), glucose (4.5 g/L), 1 mM sodium pyruvate, and 1 mM glutamine. OCRs were analyzed at baseline for at least four cycles, after which 1 μm oligomycin, 1 μm FCCP, and 1 μm rotenone/antimycin A was sequentially injected into each well to assess ATP‐linked OCR, maximal OCR, and mitochondrially‐derived OCR, respectively (Lujan et al, 2016; Sacoman et al, 2017; Zhang et al, 2019). In the present study, at the end of each assay, cells in the XF24 cell culture microplate were fixed in 4% paraformaldehyde for 30 min and washed sequentially with PBS and 1.25 μg/mL 4′ 6‐diamidino‐2‐phenylindole (DAPI) to stain nuclei.…”