O-GlcNAc Cycling Research Articles

O-GlcNAcylation of ribosome-associated proteins is concomitant with translational reprogramming during proteotoxic stress

Protein O-GlcNAc modification, similar to phosphorylation, supports cell survival by regulating key processes like transcription, cell division, trafficking, signaling, and stress tolerance. However, its role in protein homeostasis, particularly in protein synthesis, folding, and degradation remains poorly understood. Our previous research shows that O-GlcNAc cycling enzymes associate with the translation machinery during protein synthesis and modify ribosomal proteins. Protein translation is closely linked to 26S proteasome activity, which recycles amino acids and clears misfolded proteins during stress, preventing aggregation and cell death. In this study, we demonstrate that pharmacological perturbation of the proteasome—like that used in cancer treatment— leads to the increased abundance of OGT and OGA in a ribosome-rich fraction, concurrent with O-GlcNAc modification of core translational and ribosome-associated proteins. This interaction is synchronous with eIF2α-dependent translational reprogramming. We also found that protein ubiquitination depends partly on O-GlcNAc metabolism in MEFs, as OGT-depleted cells show decreased ubiquitination under stress. Using an O-GlcNAc-peptide enrichment strategy followed by LC-MS/MS, we identified 84 unique O-GlcNAc sites across 55 proteins, including ribosomal proteins, nucleolar factors, and the 70-kDa heat shock protein family. Hsp70 and OGT colocalize with the translational machinery in an RNA-independent manner, aiding in partial protein translation recovery during sustained stress. O-GlcNAc cycling on ribosome-associated proteins collaborates with Hsp70 to restore protein synthesis during proteotoxicity, suggesting a role in tumor resistance to proteasome inhibitors.

O-GlcNAc informatics: advances and trends.

As a post-translational modification, protein glycosylation is critical in health and disease. O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), as an intracellular monosaccharide modification on proteins, was discovered 40 years ago. Thanks to technological advances, the physiological and pathological significance of O-GlcNAcylation has been gradually revealed and widely appreciated, especially in recent years.O-GlcNAc informatics has been quickly evolving. Clearly, O-GlcNAc informatics tools have not only facilitatedO-GlcNAc functional studies, but also provided us a unique perspective on proteinO-GlcNAcylation. In this article, we review O-GlcNAc-focused software tools and servers that have been developed forO-GlcNAc research over the past four decades. Specifically, we will (1) survey bioinformatics tools that have facilitated O-GlcNAc proteomics data analysis, (2) introduce databases/servers forO-GlcNAc proteins/sites that have been experimentally identified by individual research labs, (3) describe software tools that have been developed to predictO-GlcNAc sites, and (4) introduce platforms cataloging proteins that interact with the O-GlcNAc cycling enzymes (i.e., O-GlcNAc transferaseand O-GlcNAcase). We hope these resources will provide useful information to both experienced researchers and new incomers to the O-GlcNAc field. We anticipate that this review provides a framework to stimulate the future development of more sophisticated informatic tools for O-GlcNAc research.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

Chronic rapid eye movement sleep deprivation aggravates the pathogenesis of Alzheimer’s disease by decreasing brain O-GlcNAc cycling in mice

This study investigated the role of O-GlcNAc cycling in Alzheimer’s disease-related changes in brain pathophysiology induced by chronic REM sleep deprivation (CSD) in mice. CSD increased amyloid beta (Aβ) and p-Tau accumulation and impaired learning and memory (L/M) function. CSD decreased dendritic length and spine density. CSD also increased the intensity of postsynaptic density protein-95 (PSD-95) staining. All of these Alzheimer’s disease (AD) pathogenic changes were effectively reversed through glucosamine (GlcN) treatment by enhancing O-GlcNAcylation. Interestingly, the lelvel of O-GlcNAcylated-Tau (O-Tau) exhibited an opposite trend compared to p-Tau, as it was elevated by CSD and suppressed by GlcN treatment. CSD increased neuroinflammation, as indicated by elevated levels of glial fibrillary acidic protein and IBA-1-positive glial cells in the brain, which were suppressed by GlcN treatment. CSD promoted the phosphorylation of GSK3β and led to an upregulation in the expression of endoplasmic reticulum (ER) stress regulatory proteins and genes. These alterations were effectively suppressed by GlcN treatment. Minocycline not only suppressed neuroinflammation induced by CSD, but it also rescued the decrease in O-GlcNAc levels caused by CSD. Minocycline also reduced AD neuropathy without affecting CSD-induced ER stress. Notably, overexpressing O-GlcNAc transferase in the dentate gyrus region of the mouse brain rescued CSD-induced cognitive dysfunction, neuropathy, neuroinflammation, and ER stress responses. Collectively, our findings reveal that dysregulation of O-GlcNAc cycling underlies CSD-induced AD pathology and demonstrate that restoration of OGlcNAcylation protects against CSD-induced neurodegeneration.

Protective effect of increased O-GlcNAc cycling against 6-OHDA induced Parkinson’s disease pathology

This study aimed to elucidate the role of O-GlcNAc cycling in 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD)-like neurodegeneration and the underlying mechanisms. We observed dose-dependent downregulation of O-GlcNAcylation, accompanied by an increase in O-GlcNAcase following 6-OHDA treatment in both mouse brain and Neuro2a cells. Interestingly, elevating O-GlcNAcylation through glucosamine (GlcN) injection provided protection against PD pathogenesis induced by 6-OHDA. At the behavioral level, GlcN mitigated motor deficits induced by 6-OHDA, as determined using the pole, cylinder, and apomorphine rotation tests. Furthermore, GlcN attenuated 6-OHDA-induced neuroinflammation and mitochondrial dysfunction. Notably, augmented O-GlcNAcylation, achieved through O-GlcNAc transferase (OGT) overexpression in mouse brain, conferred protection against 6-OHDA-induced PD pathology, encompassing neuronal cell death, motor deficits, neuroinflammation, and mitochondrial dysfunction. These collective findings suggest that O-GlcNAcylation plays a crucial role in the normal functioning of dopamine neurons. Moreover, enhancing O-GlcNAcylation through genetic and pharmacological means could effectively ameliorate neurodegeneration and motor impairment in an animal model of PD. These results propose a potential strategy for safeguarding against the deterioration of dopamine neurons implicated in PD pathogenesis.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

Forskolin rescues hypoxia-induced cognitive dysfunction in zebrafish with potential involvement of O-GlcNAc cycling regulation

Repeated sublethal hypoxia exposure induces brain inflammation and affects the initiation and progression of cognitive dysfunction. Experiments from the current study showed that hypoxic exposure downregulates PKA/CREB signaling, which is restored by forskolin (FSK), an adenylate cyclase activator, in both Neuro2a (N2a) cells and zebrafish brain. FSK significantly protected N2a cells from hypoxia-induced cell death and neurite shrinkage. Intraperitoneal administration of FSK for 5 days on zebrafish additionally led to significant recovery from hypoxia-induced social interaction impairment and learning and memory (L/M) deficit. FSK suppressed hypoxia-induced neuroinflammation, as indicated by the observed decrease in NF-κB activation and GFAP expression. We further investigated the potential effect of FSK on O-GlcNAcylation changes induced by hypoxia. Intriguingly FSK induced marked upregulation of the protein level of O-GlcNAc transferase catalyzing addition of the GlcNAc group to target proteins, accompanied by elevated O-GlcNAcylation of nucleocytoplasmic proteins. The hypoxia-induced O-GlcNAcylation decrease in the brain of zebrafish was considerably restored following FSK treatment. Based on the collective results, we propose that FSK rescues hypoxia-induced cognitive dysfunction, potentially through regulation of HBP/O-GlcNAc cycling.

Repeated sleep deprivation decreases the flux into hexosamine biosynthetic pathway/O-GlcNAc cycling and aggravates Alzheimer’s disease neuropathology in adult zebrafish

This study investigated chronic and repeated sleep deprivation (RSD)-induced neuronal changes in hexosamine biosynthetic pathway/O-linked N-acetylglucosamine (HBP/O-GlcNAc) cycling of glucose metabolism and further explored the role of altered O-GlcNAc cycling in promoting neurodegeneration using an adult zebrafish model. RSD-triggered degenerative changes in the brain led to impairment of memory, neuroinflammation and amyloid beta (Aβ) accumulation. Metabolite profiling of RSD zebrafish brain revealed a significant decrease in glucose, indicating a potential association between RSD-induced neurodegeneration and dysregulated glucose metabolism. While RSD had no impact on overall O-GlcNAcylation levels in the hippocampus region, changes were observed in two O-GlcNAcylation-regulating enzymes, specifically, a decrease in O-GlcNAc transferase (OGT) and an increase in O-GlcNAcase (OGA). Glucosamine (GlcN) treatment induced an increase in O-GlcNAcylation and recovery of the OGT level that was decreased in the RSD group. In addition, GlcN reversed cognitive impairment by RSD. GlcN reduced neuroinflammation and attenuated Aβ accumulation induced by RSD. Repeated treatment of zebrafish with diazo-5-oxo-l-norleucine (DON), an inhibitor of HBP metabolism, resulted in cognitive dysfunction, neuroinflammation and Aβ accumulation, similar to the effects of RSD. The pathological changes induced by DON were restored to normal upon treatment with GlcN. Both the SD and DON-treated groups exhibited a common decrease in glutamate and γ-aminobutyric acid compared to the control group. Overexpression of OGT in zebrafish brain rescued RSD-induced neuronal dysfunction and neurodegeneration. RSD induced a decrease in O-GlcNAcylation of amyloid precursor protein and increase in β-secretase activity, which were reversed by GlcN treatment. Based on the collective findings, we propose that dysregulation of HBP and O-GlcNAc cycling in brain plays a crucial role in RSD-mediated progression of neurodegeneration and Alzheimer’s disease pathogenesis. Targeting of this pathway may, therefore, offer an effective regulatory approach for treatment of sleep-associated neurodegenerative disorders.

Cardioprotective O-GlcNAc signaling is elevated in murine female hearts via enhanced O-GlcNAc transferase activity

The post-translational modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc-cycling in response to I/R injury have not been comprehensively assessed. The cycling of O-GlcNAc is regulated by the collective efforts of two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and hydrolysis of O-GlcNAc respectively. It has previously been shown that baseline heart physiology and pathophysiology are impacted by sex. Here, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation both basally and in ischemic hearts. To address this question, we subjected male and female wild type murine hearts to ex vivo ischemia or ischemia-reperfusion injury. We assessed hearts for protein O-GlcNAcylation, abundance of OGT, OGA, and glutamine:fructose-6-phosphate aminotransferase (GFAT2), activity of OGT and OGA, and UDP-GlcNAc levels. Our data demonstrate elevated O-GlcNAcylation in female hearts both basally and during ischemia. We show OGT activity was enhanced in female hearts in all treatments, suggesting a mechanism for these observations. Furthermore, we found ischemia led to reduced O-GlcNAcylation and OGT specific activity. Our findings provide a foundation for understanding molecular mechanisms that regulate O-GlcNAcylation in the heart and highlight the importance of sex as a significant factor when assessing key regulatory events that control O-GlcNAc-cycling. These data suggest the intriguing possibility that elevated O-GlcNAcylation in females contributes to reduced ischemic susceptibility.

Hexosamine biosynthetic pathway and O-GlcNAc cycling of glucose metabolism in brain function and disease.

Impaired brain glucose metabolism is considered a hallmark of brain dysfunction and neurodegeneration. Disruption of the hexosamine biosynthetic pathway (HBP) and subsequent O-linked N-acetylglucosamine (O-GlcNAc) cycling has been identified as an emerging link between altered glucose metabolism and defects in the brain. Myriads of cytosolic and nuclear proteins in the nervous system are modified at serine or threonine residues with a single N-acetylglucosamine (O-GlcNAc) molecule by O-GlcNAc transferase (OGT), which can be removed by β-N-acetylglucosaminidase (O-GlcNAcase, OGA). Homeostatic regulation of O-GlcNAc cycling is important for the maintenance of normal brain activity. Although significant evidence linking dysregulated HBP metabolism and aberrant O-GlcNAc cycling to induction or progression of neuronal diseases has been obtained, the issue of whether altered O-GlcNAcylation is causal in brain pathogenesis remains uncertain. Elucidation of the specific functions and regulatory mechanisms of individual O-GlcNAcylated neuronal proteins in both normal and diseased states may facilitate the identification of novel therapeutic targets for various neuronal disorders. The information presented in this review highlights the importance of HBP/O-GlcNAcylation in the neuronal system and summarizes the roles and potential mechanisms of O-GlcNAcylated neuronal proteins in maintaining normal brain function and initiation and progression of neurological diseases.

Abstract P1166: O-GlcNAcylation Regulates Protein Synthesis In Cardiomyocytes Through Effects On The Translational Regulator Eif2α

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.

O-GlcNAc Dynamics: The Sweet Side of Protein Trafficking Regulation in Mammalian Cells.

The transport of proteins between the different cellular compartments and the cell surface is governed by the secretory pathway. Alternatively, unconventional secretion pathways have been described in mammalian cells, especially through multivesicular bodies and exosomes. These highly sophisticated biological processes rely on a wide variety of signaling and regulatory proteins that act sequentially and in a well-orchestrated manner to ensure the proper delivery of cargoes to their final destination. By modifying numerous proteins involved in the regulation of vesicular trafficking, post-translational modifications (PTMs) participate in the tight regulation of cargo transport in response to extracellular stimuli such as nutrient availability and stress. Among the PTMs, O-GlcNAcylation is the reversible addition of a single N-acetylglucosamine monosaccharide (GlcNAc) on serine or threonine residues of cytosolic, nuclear, and mitochondrial proteins. O-GlcNAc cycling is mediated by a single couple of enzymes: the O-GlcNAc transferase (OGT) which catalyzes the addition of O-GlcNAc onto proteins, and the O-GlcNAcase (OGA) which hydrolyses it. Here, we review the current knowledge on the emerging role of O-GlcNAc modification in the regulation of protein trafficking in mammalian cells, in classical and unconventional secretory pathways.

Inhibiting O-GlcNAcylation impacts p38 and Erk1/2 signaling and perturbs cardiomyocyte hypertrophy

The dynamic cycling of O-linked GlcNAc (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase. O-GlcNAc cycling is important in homeostatic and stress responses, and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases, while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide "click" chemistry, we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis and also the noncanonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, decreased expression of cTnT (key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes.

Chemical Biology Approaches to Understanding Neuronal O-GlcNAcylation.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification in mammals, decorating thousands of intracellular proteins. O-GlcNAc cycling is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases. Notably, O-GlcNAcylation is abundant in the brain and numerous studies have linked aberrant O-GlcNAc signaling to various neurological conditions. However, the complexity of the nervous system and the dynamic nature of protein O-GlcNAcylation have presented challenges for studying of neuronal O-GlcNAcylation. In this context, chemical approaches have been a particularly valuable complement to conventional cellular, biochemical, and genetic methods to understand O-GlcNAc signaling and to develop future therapeutics. Here we review selected recent examples of how chemical tools have empowered efforts to understand and rationally manipulate O-GlcNAcylation in mammalian neurobiology.

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

Simple SummaryDynamic O-GlcNAc modification regulates the functions of proteins in a broad range of cellular processes. 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. Here, we highlight recent studies on the structural features of OGT and OGA, as well as the emerging roles and molecular mechanisms of their aberrant protein–protein interactions (PPIs) in rewiring cancer networks, to help identify key protein contacts and functional modules that drive malignancies and to promote cancer therapeutic innovations.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.

A nexus of lipid and O-Glcnac metabolism in physiology and disease.

Although traditionally considered a glucose metabolism-associated modification, the O-linked β-N-Acetylglucosamine (O-GlcNAc) regulatory system interacts extensively with lipids and is required to maintain lipid homeostasis. The enzymes of O-GlcNAc cycling have molecular properties consistent with those expected of broad-spectrum environmental sensors. By direct protein-protein interactions and catalytic modification, O-GlcNAc cycling enzymes may provide both acute and long-term adaptation to stress and other environmental stimuli such as nutrient availability. Depending on the cell type, hyperlipidemia potentiates or depresses O-GlcNAc levels, sometimes biphasically, through a diversity of unique mechanisms that target UDP-GlcNAc synthesis and the availability, activity and substrate selectivity of the glycosylation enzymes, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA). At the same time, OGT activity in multiple tissues has been implicated in the homeostatic regulation of systemic lipid uptake, storage and release. Hyperlipidemic patterns of O-GlcNAcylation in these cells are consistent with both transient physiological adaptation and feedback uninhibited obesogenic and metabolic dysregulation. In this review, we summarize the numerous interconnections between lipid and O-GlcNAc metabolism. These links provide insights into how the O-GlcNAc regulatory system may contribute to lipid-associated diseases including obesity and metabolic syndrome.

Disruption of O-GlcNAcylation Homeostasis Induced Ovarian Granulosa Cell Injury in Bovine

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous, reversible, and highly dynamic post-translational modification, which takes charge of almost all biological processes examined. However, little information is available regarding the molecular regulation of O-GlcNAcylation in granulosa cell function and glucose metabolism. This study focused on the impact of disrupted O-GlcNAc cycling on the proliferation and apoptosis of bovine granulosa cells, and further aimed to determine how this influenced glucose metabolism. Pharmacological inhibition of OGT with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) led to decreased cellular O-GlcNAc levels, as well as OGT and OGA protein expressions, whereas increasing O-GlcNAc levels with the OGA inhibitor, O-(2-acetamido-2-deoxy--gluco-pyranosylidene) (PUGNAc), resulted in elevated OGA protein expression and decreased OGT protein expression in granulosa cells. Dysregulated O-GlcNAc cycling reduced cell viability, downregulated the proliferation-related genes of CDC42 and PCNA transcripts, upregulated the pro-apoptotic genes of BAX and CASPASE-3 mRNA and the ratio of BAX/BCL-2, and increased the apoptotic rate. Glycolytic enzyme activities of hexokinase and pyruvate kinase, metabolite contents of pyruvate and lactate, mitochondrial membrane potential, ATP levels, and intermediate metabolic enzyme activities of succinate dehydrogenase and malate dehydrogenase involved in the tricarboxylic acid cycle, were significantly impaired in response to altered O-GlcNAc levels. Moreover, inhibition of OGT significantly increased the expression level of thioredoxin-interacting protein (TXNIP), but repression of OGA had no effect. Collectively, our results suggest that perturbation of O-GlcNAc cycling has a profound effect on granulosa cell function and glucose metabolism.

O-GlcNAcylation modulates liquid-liquid phase separation of SynGAP/PSD-95.

Liquid-liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O-GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex. We identified multiple O-GlcNAc modification sites for the endogenous SynGAP isolated from rat brain and the recombinantly expressed protein. Protein semisynthesis was used to generate site-specifically O-GlcNAcylated forms of SynGAP, and in vitro and cell-based LLPS assays demonstrated that T1306 O-GlcNAc of SynGAP blocks the interaction with PSD-95, thus inhibiting LLPS. Furthermore, O-GlcNAcylation suppresses SynGAP/PSD-95 LLPS in a dominant-negative manner, enabling sub-stoichiometric O-GlcNAcylation to exert effective regulation. We also showed that O-GlcNAc-dependent LLPS is reversibly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). These findings demonstrate that OGT- and OGA-catalysed O-GlcNAc cycling may serve as an LLPS-regulating post-translational modification.

Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila.

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.

Chronically Elevated O-GlcNAcylation Limits Nitric Oxide Production and Deregulates Specific Pro-Inflammatory Cytokines

Inflammation is the immune response to harmful stimuli, including pathogens, damaged cells and toxic compounds. However, uncontrolled inflammation can be detrimental and contribute to numerous chronic inflammatory diseases, such as insulin resistance. At the forefront of this response are macrophages, which sense the local microenvironment to respond with a pro-inflammatory, M1-polarized phenotype, or anti-inflammatory, M2-polarized phenotype. M1 macrophages upregulate factors like pro-inflammatory cytokines, to promote inflammatory signaling, and inducible Nitric Oxide Synthase (iNOS), to produce nitric oxide (NO). The generated NO can kill microorganisms to protect the body, but also signal back to the macrophage to limit pro-inflammatory cytokine production to maintain macrophage homeostasis. Thus, the tight regulation of iNOS in macrophages is critical for the immune system. Here, we investigated how elevation of the nutrient-sensitive posttranslational modification, O-GlcNAc, impacts M1 polarized macrophages. We identified increased gene expression of specific pro-inflammatory cytokines (Il-6, Il-1β, Il-12) when O-GlcNAc cycling was blocked. We further uncovered an interaction between O-GlcNAc and iNOS, with iNOS being an OGT target in vitro. Analysis of M1 polarized bone marrow derived macrophages deficient in the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), revealed decreased iNOS activity as measured by a reduction in NO release. Further, elevated O-GlcNAc acted on Il-6 expression through the iNOS pathway, as iNOS inhibitior L-NIL raised wildtype Il-6 expression similar to OGA deficient cells but had no further effect on the hyper-O-GlcNAcylated cells. Thus O-GlcNAc contributes to macrophage homeostasis through modulation of iNOS activity.