Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis.

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

Transcriptional regulation of O-GlcNAc homeostasis is disrupted in pancreatic cancer.

Many intracellular proteins are reversibly modified by O-linked GlcNAc (O-GlcNAc), a post-translational modification that dynamically regulates fundamental cellular processes in response to diverse environmental cues. Accumulating evidence indicates that both excess and deficiency of protein O-GlcNAcylation can have deleterious effects on the cell, suggesting that maintenance of O-GlcNAc homeostasis is essential for proper cellular function. However, the mechanisms through which O-GlcNAc homeostasis is maintained in the physiologic state and altered in the disease state have not yet been investigated. Here, we demonstrate the existence of a homeostatic mechanism involving mutual regulation of the O-GlcNAc-cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) at the transcriptional level. Specifically, we found that OGA promotes Ogt transcription through cooperation with the histone acetyltransferase p300 and transcription factor CCAAT/enhancer-binding protein ß (C/EBPß). To examine the role of mutual regulation of OGT and OGA in the disease state, we analyzed gene expression data from human cancer data sets, which revealed that OGT and OGA expression levels are highly correlated in numerous human cancers, particularly in pancreatic adenocarcinoma. Using a KrasG12D -driven primary mouse pancreatic ductal adenocarcinoma (PDAC) cell line, we found that inhibition of extracellular signal-regulated kinase (ERK) signaling decreases OGA glycosidase activity and reduces OGT mRNA and protein levels, suggesting that ERK signaling may alter O-GlcNAc homeostasis in PDAC by modulating OGA-mediated Ogt transcription. Our study elucidates a transcriptional mechanism that regulates cellular O-GlcNAc homeostasis, which may lay a foundation for exploring O-GlcNAc signaling as a therapeutic target for human disease.

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis.

AIMS/HYPOTHESIS: O-GlcNAcylation is implicated in modulating mitochondrial function, which is closely involved in regulating muscle metabolism. The presence of O-GlcNAcase (OGA), the enzyme involved in the removal of O-GlcNAc, in mitochondria was recently confirmed in rats. In the present study, we investigated the regulation of myogenesis and muscle insulin sensitivity to OGA in mice, with a focus on mitochondria. METHODS: C57BL/6J mice fed a high-fat diet for 4 months were used to observe mitochondrial density, activity and O-GlcNAcylation in muscle. Small interfering RNA and overexpression vectors were used to modulate protein content in vitro. RESULTS: High-fat feeding decreased the OGA level and largely increased mitochondrial O-GlcNAcylation in mouse skeletal muscle that was accompanied by decreased levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), decreased mitochondrial density and disrupted mitochondrial complex activities. Knockdown of OGA in C2C12 myoblasts promoted PGC-1α degradation, resulting in the suppression of mitochondrial biogenesis and myogenesis, whereas neither knockdown of O-GlcNAc transferase nor overexpression of OGA had significant effects on myogenesis. Mitochondrial dysfunction as evidenced by decreased ATP content and increased reactive oxygen species production, and increased lipid and protein oxidation was observed in both myoblasts and myotubes after OGA knockdown. Meanwhile, elevated O-GlcNAcylation through either OGA knockdown or treatment with the OGA inhibitor PUGNAc and the O-GlcNAc transferase substrate D-GlcNAc suppressed myotube insulin signalling transduction and glucose uptake. OGA overexpression had no significant effect on insulin sensitivity but sufficiently improved the insulin resistance induced by D-GlcNAc treatment. CONCLUSIONS/INTERPRETATION: These data suggest that OGA can modulate mitochondrial density via PGC-1α and mitochondrial function via protein O-GlcNAcylation. In this manner, OGA appears to play a key role in myogenesis and the development of muscle insulin resistance.

A mutant O-GlcNAcase as a probe to reveal global dynamics of protein O-GlcNAcylation during Drosophila embryonic development.

O-GlcNAcylation is a reversible type of serine/threonine glycosylation on nucleocytoplasmic proteins in metazoa. Various genetic approaches in several animal models have revealed that O-GlcNAcylation is essential for embryogenesis. However, the dynamic changes in global O-GlcNAcylation and the underlying mechanistic biology linking them to embryonic development is not understood. One of the limiting factors towards characterizing changes in O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. In the present study, harnessing the unusual properties of an O-GlcNAcase (OGA) mutant that binds O-GlcNAc (O-N-acetylglucosamine) sites with nanomolar affinity, we uncover changes in protein O-GlcNAcylation as a function of Drosophila development.

The non-catalytic domains of O-GlcNAc cycling enzymes present new opportunities for function-specific control.

O-GlcNAcylation is an essential protein glycosylation governed by two O-GlcNAc cycling enzymes: O-GlcNAc transferase (OGT) installs a single sugar moiety N-acetylglucosamine (GlcNAc) on protein serine and threonine residues, and O-GlcNAcase (OGA) removes them. Aberrant O-GlcNAcylation has been implicated in various diseases. However, the large repertoire of more than 1000 O-GlcNAcylated proteins and the elusive mechanisms of OGT/OGA in substrate recognition present significant challenges in targeting the dysregulated O-GlcNAcylation for therapeutic development. Recently, emerging evidence suggested that the non-catalytic domains play critical roles in regulating the functional specificity of OGT/OGA via modulating their protein interactions and substrate recognition. Here, we discuss recent studies on the structures, mechanisms, and related tools of the OGT/OGA non-catalytic domains, highlighting new opportunities for function-specific control.

The Emerging Roles of Protein Interactions with O-GlcNAc Cycling Enzymes in Cancer.

The dynamic O-GlcNAc modification of intracellular proteins is an important nutrient sensor for integrating metabolic signals into vast networks of highly coordinated cellular activities. Dysregulation of the sole enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), and the associated cellular O-GlcNAc profile is a common feature across nearly every cancer type. Many studies have investigated the effects of aberrant OGT/OGA expression on global O-GlcNAcylation activity in cancer cells. However, recent studies have begun to elucidate the roles of protein-protein interactions (PPIs), potentially through regions outside of the immediate catalytic site of OGT/OGA, that regulate greater protein networks to facilitate substrate-specific modification, protein translocalization, and the assembly of larger biomolecular complexes. Perturbation of OGT/OGA PPI networks makes profound changes in the cell and may directly contribute to cancer malignancies. Herein, we highlight recent studies on the structural features of OGT and OGA, as well as the emerging roles and molecular mechanisms of their aberrant PPIs in rewiring cancer networks. By integrating complementary approaches, the research in this area will aid in the identification of key protein contacts and functional modules derived from OGT/OGA that drive oncogenesis and will illuminate new directions for anti-cancer drug development.

Pancreatic ß-cell hyper-O-GlcNAcylation leads to impaired glucose homeostasis in vivo.

Protein O-GlcNAcylation is a nutrient and stress-sensitive protein post-translational modification (PTM). The addition of an O-GlcNAc molecule to proteins is catalyzed by O-GlcNAc transferase (OGT), whereas O-GlcNAcase (OGA) enzyme is responsible for removal of this PTM. Previous work showed that OGT is highly expressed in the pancreas, and we demonstrated that hypo-O-GlcNAcylation in ß-cells cause severe diabetes in mice. These studies show a direct link between nutrient-sensitive OGT and ß-cell health and function. In the current study, we hypothesized that hyper-O-GlcNAcylation may confer protection from ß-cell failure in high-fat diet (HFD)-induced obesity. To test this hypothesis, we generated a mouse model with constitutive ß-cell OGA ablation (ßOGAKO) to specifically increase O-GlcNAcylation in ß-cells. Under normal chow diet, young male and female ßOGAKO mice exhibited normal glucose tolerance but developed glucose intolerance with aging, relative to littermate controls. No alteration in ß-cell mass was observed between ßOGAKO and littermate controls. Total insulin content was reduced despite an increase in pro-insulin to insulin ratio in ßOGAKO islets. ßOGAKO mice showed deficit in insulin secretion in vivo and in vitro. When young animals were subjected to HFD, both male and female ßOGAKO mice displayed normal body weight gain and insulin tolerance but developed glucose intolerance that worsened with longer exposure to HFD. Comparable ß-cell mass was found between ßOGAKO and littermate controls. Taken together, these data demonstrate that the loss of OGA in ß-cells reduces ß-cell function, thereby perturbing glucose homeostasis. The findings reinforce the rheostat model of intracellular O-GlcNAcylation where too much (OGA loss) or too little (OGT loss) O-GlcNAcylation are both detrimental to the ß-cell.

Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe.

The essential human O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.

OGA Inhibition Alters Energetics and Nutrient Sensing in Alzheimer's Disease Cytoplasmic Hybrids.

BACKGROUND: Alzheimer's disease (AD) features reductions in key bioenergetic fluxes and perturbed mitochondrial function. Cytoplasmic hybrids (cybrids) generated through the transfer of AD subject mitochondria to mtDNA-depleted SH-SY5Y neuroblastoma cells recapitulate some of these features in an in vitro setting. OBJECTIVE: For this study, we used the AD cybrid model to assess the impact of a nutrient-excess like-state via increasing O-GlcNAcylation on whole cell and mitochondrial homeostasis. METHODS: We induced increased O-GlcNAc by treating AD and control cybrid cell lines with Thiamet G (TMG), an inhibitor of the O-GlcNAcase enzyme that mediates removal of the nutrient-dependent O-GlcNAc modification. RESULTS: Relative to control cybrid cell lines, AD cybrid lines showed a blunted response to TMG-induced O-GlcNAcylation. At baseline, AD cybrid cell line mitochondria showed partial activation of several proteins that help maintain bioenergetic homeostasis such as AMP-Regulated Kinase suggesting that AD mitochondria initiate a state of nutrient stress promoting energetic compensation; however, this compensation reduces the capacity of cells to respond to additional nutrient-related stresses such as TMG treatment. Also, TMG caused disruptions in acetylation and Sirtuin 3 expression, while lowing total energetic output of the cell. CONCLUSION: Together, these findings suggest that modulation of O-GlcNAc is essential for proper energetic function of the mitochondria, and AD mitochondrial capacity to handle nutrient-excess is limited.

Regulation of cardiac O-GlcNAcylation: More than just nutrient availability.

The post-translational modification of serine and threonine residues of nuclear, cytosolic, and mitochondrial proteins by O-linked ß-N-acetyl glucosamine (O-GlcNAc) has long been seen as an important regulatory mechanism in the cardiovascular system. O-GlcNAcylation of cardiac proteins has been shown to contribute to the regulation of transcription, metabolism, mitochondrial function, protein quality control and turnover, autophagy, and calcium handling. In the heart, acute increases in O-GlcNAc have been associated with cardioprotection, such as those observed during ischemia/reperfusion. Conversely, chronic increases in O-GlcNAc, often associated with diabetes and nutrient excess, have been shown to contribute to cardiac dysfunction. Traditionally, many studies have linked changes in O-GlcNAc with nutrient availability and as such O-GlcNAcylation is often seen as a nutrient driven process. However, emerging evidence suggests that O-GlcNAcylation may also be regulated by non-nutrient dependent mechanisms, such as transcriptional and post-translational regulation. Therefore, the goals of this review are to provide an overview of the impact of O-GlcNAcylation in the cardiovascular system, how this is regulated and to discuss the emergence of regulatory mechanisms other than nutrient availability.