Changes in O‐GlcNAcylation Alter Mitochondrial Function

Abstract

Mitochondrial dysfunction is a common characteristic of many diseases, such as Alzheimer's disease, diabetes, and cancer. Normal mitochondrial and cellular function is regulated by post-translational modifications (PTM) of proteins, which alter protein function to adapt to changes in the cellular environment. One PTM that plays a key role in protein function is the attachment of a single N-acetylglucosamine (O-GlcNAc) sugar to nuclear, mitochondrial, or cytoplasmic proteins at their serine or threonine residues. This process, known as O-GlcNAcylation, is catalyzed by only two enzymes: O-GlcNAc transferase (OGT), which attaches the sugar, and O-GlcNAcase (OGA), which removes the sugar. O-GlcNAcylation is sensitive to changes in various metabolic pathways, such as the metabolism of glucose, fatty acids, and amino acids. Thus, this modification is necessary for maintaining cellular homeostasis. Our previous data showed that changes in O-GlcNAcylation, such as loss of OGT or inhibition of OGA via thiamet-G (TMG) treatment in the liver, showed disruption of the electron transport chain (ETC); however, we still do not know which complexes are most affected by these changes. One potential explanation lies in the relationship between O-GlcNAcylation and acetylation, as acetylation is a highly abundant mitochondrial PTM responsible for the regulation of many components of the ETC. A variety of cell line knockouts and animal models were studied to more fully understand the role of O-GlcNAcylation in regulating mitochondrial function and acetylation. We generated a stable cell line in SH-SY5Y human neuroblastoma cells with shRNA mediated knockdown of OGT. These cells demonstrated an increase in acetylation in the mitochondria as a result of the loss of OGT. Further analysis also showed changes in the expressions of two proteins that are critical for mitochondrial acetylation: acetyl-coenzyme A synthetase 2-like (ACSS1) and NAD-dependent deacetylase sirtuin-3 (SIRT3). Taken together, these findings suggest that reduced O-GlcNAcylation alters the mitochondrial proteome including proteins that are regulated via acetylation. We also observed that oxidative phosphorylation rates were altered in the OGT knockdown cell lines, demonstrating alterations in mitochondrial function due to the reduction of OGT. To further elucidate the role of O-GlcNAcylation in the electron transport chain, ETC complex assays were run to assess changes in complex function due to different levels of O-GlcNAcylation. SH-SY5Y cells treated with and without TMG expressed different levels of complex activity, providing key insights about the importance of O-GlcNAcylation in the ETC. Based on these findings, more studies are needed to understand how O-GlcNAcylation and acetylation work in tandem to maintain electron transport chain function.