O-GlcNAcase Research Articles

Silencing of O-GlcNAc transferase attenuated O-GlcNAcylation and metastatic potentials of melanoma cells through suppression of Akt-NFkB signaling pathway.

O-GlcNAcylation is an important biological process in regulating the function of many nucleocytoplasmic proteins in cells.Enhancement of O-GlcNAcylation was associated with cancer development and progression.Here, we demonstrated the involvement of O-GlcNAcylation in melanoma metastasis.Using the data from GEO database, we found that O-GlcNAcylation and its related enzymes, including glutamine fructose-6-phosphate amidotransferase (GFAT), O-GlcNAc transferase (OGT), and O-GlcNAcase (OGA); were elevated in metastatic melanoma compared with primary tumors and normal tissues.Functional analyses in melanoma cell lines--MNT-1, SK-MEL-28, and A-375 showed that suppression of O-GlcNAcylation by siRNA against OGT significantly reduces the migration and invasion abilities of the cells.Phosphorylation of Akt and NFkB was drastically suppressed after knockdown of OGT, suggesting the role of O-GlcNAcylation in regulating the Akt-NFkB signaling pathway.In addition, we found that the NFkB target genes, such as ZEB-2 and MCT-1, were significantly upregulated in metastatic tumors compared with primary tumors.MCT-1 expression in melanoma tissues was also correlated with O-GlcNAcylation level.Taken together, we have demonstrated in this study the possible role of O-GlcNAcylation in controlling melanoma metastasis via upregulating MCT-1 expression through activation of Akt-NFkB signaling pathway.

Brain Transcriptome Changes Associated With an Acute Increase of Protein O-GlcNAcylation and Implications for Neurodegenerative Disease.

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) has been considered as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer's disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the potential of using OGA inhibition to treat neurodegenerative diseases. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using the OGA inhibitor Thiamet G (TG), in normal mouse brains. We hypothesized that the transcriptome signature in response to a 3 h TG treatment (50 mg/kg) provides a comprehensive view of the effect of OGA inhibition. We then performed mRNA sequencing of the brain using NovaSeq PE 150 (n = 5 each group). We identified 1234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. AMPK subunit Prkab2 was one gene in the kinase activity pathway, and the increase after acute and chronic treatment was confirmed using qPCR. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the impact of OGA inhibition in the treatment of AD. In particular, OGA inhibitors appear to have downstream effects related to bioenergetics which may limit their therapeutic benefits.

Evaluation of Baseline Diffusion Tensor Imaging Biomarkers in phase 2 multi‐center PROSPECT‐ALZ study

AbstractBackgroundDiffusion Tensor Imaging (DTI) biomarkers are increasingly utilized in clinical trials of Alzheimer’s disease (AD). They have shown promise in detecting AD related changes in white‐matter (WM) integrity (Raghavan et al., 2020) and gray‐matter (GM) microstructure (Ofori et al., 2019). Moreover, DTI‐derived connectome has been shown to be related to tau progression (Vogel et al., 2020). Our study evaluated baseline GM microstructure and WM integrity measures in the PROSPECT‐ALZ trial (NCT05063539) of LY3372689, an O‐GlcNAcase (OGA) inhibitor.MethodsPROSPECT‐ALZ is a phase 2, randomized, double‐blind, multicenter, placebo‐controlled trial in participants with early symptomatic AD and presence of tau pathology measured using flortaucipir PET. Participants who received DTI scans (n = 223, age=73.40 (6.0), MMSE=25.6 (2.3), N female=124) were divided into groups based on visual assessment of flortaucipir images (Fleisher et al., 2021) (negative, n=56 vs positive, n = 144) and cognition (MMSE≤24, n = 81 vs MMSE>24 & MMSE≤29, n=133). Group differences in DTI scalars fractional‐anisotropy (FA), mean‐diffusivity (MD), and freewater‐fraction (FW) were evaluated in 16 GM regions and 9 WM tracts using ANCOVA (α = 0.05, LSmean±SE), adjusting for age and gender. FA and MD values were FW corrected (Conklin et al., CTAD 2023).ResultsVisually positive participants exhibited reduced FA in the hippocampus relative to negative participants (0.289±0.006 vs. 0.322±0.010). Positive participants exhibited increased MD in the hippocampus (1.224E‐03±2.328E‐05 vs. 1.055E‐03±3.692E‐05), isthmus cingulate (9.324E‐04±1.971E‐05 vs 8.440E‐04±3.126E‐05), medial temporal cortex (9.751E‐04±1.801E‐05 vs 8.772E‐04±2.855E‐05), and whole temporal cortex (1.050E‐03±1.707E‐05 vs 9.628E‐04±2.706E‐05). Finally, positive subjects exhibited increased FW in the hippocampus (0.554±0.006 vs 0.511±0.010), isthmus cingulate (0.457±0.006 vs 0.430±0.009), medial temporal cortex (0.475±0.005 vs 0.453±0.008), and whole temporal cortex (0.497±0.005 vs 0.472±0.008). No significant regional differences were found in WM. For MMSE, MD (1.243E‐03±3.141E‐05 vs 1.140E‐03±2.419E‐05) and FW (0.561±0.009 vs 0.529±0.007) in the hippocampus were significantly elevated in the more impaired group.ConclusionDTI was successfully implemented in phase 2 multi‐center PROSPECT‐ALZ study. Cross‐sectional analysis suggests that DTI measures can differentiate individuals based on global tau burden and cognition, presumably capturing differences in cortical microstructure. Future work will focus on multimodal evaluation of longitudinal changes in DTI, tau, and cognition.

PERM1 regulates mitochondrial energetics through O-GlcNAcylation in the heart

PERM1 was initially identified as a new downstream target of PGC-1α and ERRs that regulates mitochondrial bioenergetics in skeletal muscle. Subsequently, we and other groups demonstrated that PERM1 is also a positive regulator of mitochondrial bioenergetics in the heart. However, the exact mechanisms of regulatory functions of PERM1 remain poorly understood. O-GlcNAcylation is a post-translational modification of proteins that are regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. O-GlcNAcylation is a powerful signaling mechanism mediating cellular responses to stressors and nutrient availability, which, among other targets, may influence cardiac metabolism. We hypothesized that PERM1 regulates mitochondrial energetics in cardiomyocytes through modulation of O-GlcNAcylation. We found that overexpression of PERM1 decreased the total levels of O-GlcNAcylated proteins, concomitant with decreased OGT and increased OGA expression levels. Luciferase gene reporter assay showed that PERM1 significantly decreases the promoter activity of Ogt without changing the promoter activity of Oga. The downregulation of OGT by PERM1 overexpression was mediated through its interaction with E2F1, a known transcription repressor of Ogt. A deliberate increase of O-GlcNAcylation through Oga silencing in cardiomyocytes decreased the basal and maximal mitochondrial respiration and ATP production rates, all of which were completely restored by PERM1 overexpression. Furthermore, excessive O-GlcNAcylation caused by the loss of PERM1 led to the increase of O-GlcNAcylated PGC-1α, a master regulator of mitochondrial bioenergetics, concurrent with the dissociation of PGC-1α from PPARα, a well-known transcription factor that regulates fatty acid β-oxidation. We conclude that PERM1 positively regulates mitochondrial energetics, in part, via suppressing O-GlcNAcylation in cardiac myocytes.

Rescuable sleep and synaptogenesis phenotypes in a Drosophila model of O-GlcNAc transferase intellectual disability.

O-GlcNAcylation is an essential intracellular protein modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Recently, missense mutations in OGT have been linked to intellectual disability, indicating that this modification is important for the development and functioning of the nervous system. However, the processes that are most sensitive to perturbations in O-GlcNAcylation remain to be identified. Here, we uncover quantifiable phenotypes in the fruit fly Drosophila melanogaster carrying a patient-derived OGT mutation in the catalytic domain. Hypo-O-GlcNAcylation leads to defects in synaptogenesis and reduced sleep stability. Both these phenotypes can be partially rescued by genetically or chemically targeting OGA, suggesting that a balance of OGT/OGA activity is required for normal neuronal development and function.

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

Opportunities for Therapeutic Modulation of O-GlcNAc.

O-Linked β-N-acetylglucosamine (O-GlcNAc) is an essential, dynamic monosaccharide post-translational modification (PTM) found on serine and threonine residues of thousands of nucleocytoplasmic proteins. The installation and removal of O-GlcNAc is controlled by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery four decades ago, O-GlcNAc has been found on diverse classes of proteins, playing important functional roles in many cellular processes. Dysregulation of O-GlcNAc homeostasis has been implicated in the pathogenesis of disease, including neurodegeneration, X-linked intellectual disability (XLID), cancer, diabetes, and immunological disorders. These foundational studies of O-GlcNAc in disease biology have motivated efforts to target O-GlcNAc therapeutically, with multiple clinical candidates under evaluation. In this review, we describe the characterization and biochemistry of OGT and OGA, cellular O-GlcNAc regulation, development of OGT and OGA inhibitors, O-GlcNAc in pathophysiology, clinical progress of O-GlcNAc modulators, and emerging opportunities for targeting O-GlcNAc. This comprehensive resource should motivate further study into O-GlcNAc function and inspire strategies for therapeutic modulation of O-GlcNAc.

Multi-omics after O-GlcNAc alteration identified cellular processes promoting aneuploidy after loss of O-GlcNAc transferase

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address the pleotropic nature of O-GlcNAc, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. These data show how a multi-Omics platform can disentangle the pleotropic nature of O-GlcNAc to discern how OGT fine-tunes multiple cellular pathways involved in aneuploidy.

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

O-GlcNAc participates in the meiosis of aging oocytes by mediating mitochondrial function.

O-GlcNAc plays an important role in many age-related diseases. This study shows that O-GlcNAc participates in oocyte aging and that reducing O-GlcNAc levels in aging oocytes improves oocyte quality. With an increase in the mean age at parturition worldwide, female reproductive aging has become a key health problem. Advanced maternal age is reflected by decreased oocyte quality; however, the molecular mechanisms of oocyte aging are uncharacterized. O-linked N-acetylglucosamine (O-GlcNAc), a dynamic posttranslational modification, plays a critical role in the development of many age-related diseases; yet, it remains unclear whether and how O-GlcNAc participates in oocyte aging. Here, we found that global O-GlcNAc was elevated in normal biological aging mice oocytes (9 months), which were characterized by meiotic maturation failure and impaired mitochondrial function. Specifically, O-GlcNAc targeted the mitochondrial fission protein dynamic-related protein 1 to mediate mitochondrial distribution in the process of aging. Using the O-GlcNAcase (OGA) pharmacological inhibitor Thiamet-G and Oga knockdown (Oga-KD) to mimic the age-related high O-GlcNAc in young oocytes from 6-8 week-old mice mimicked the phenotype of oocyte aging. Moreover, reducing O-GlcNAc levels in aging oocytes restored spindle organization to improve oocyte quality. Our results demonstrate that O-GlcNAc is a key regulator of meiotic maturation that participates in the progression of oocyte aging.

Discovery and clinical translation of ceperognastat, an O‐GlcNAcase (OGA) inhibitor, for the treatment of Alzheimer's disease

AbstractINTRODUCTIONThe aggregation and spread of hyperphosphorylated, pathological tau in the human brain is hypothesized to play a key role in Alzheimer's disease (AD) as well as other neurogenerative tauopathies. O‐GlcNAcylation, an important post‐translational modification of tau and many other proteins, is significantly decreased in brain tissue of AD patients relative to healthy controls. Increased tau O‐GlcNAcylation has been shown to reduce tau pathology in mouse in vivo tauopathy models. O‐GlcNAcase (OGA) catalyzes the removal of O‐GlcNAc from tau thereby driving interest in OGA inhibition as a potential therapeutic approach to reduce tau pathology and slow the progression of AD.METHODSA multidisciplinary approach was used to identify ceperognastat (LY3372689) as a potent OGA inhibitor, including an extensive discovery effort with synthetic chemistry, structure‐based drug design, and in vivo OGA enzyme occupancy studies. Preclinical studies assessed the target engagement, inhibition of OGA enzyme activity, OGA enzyme occupancy, and changes in tau O‐GlcNAc. Four clinical Phase 1 studies of ceperognastat in healthy participants were performed to assess clinical safety and tolerability, pharmacokinetics (PK), and enzyme occupancy.RESULTSCeperognastat is a potent, central nervous system (CNS)‐penetrant, low‐dose inhibitor of OGA, which can achieve > 95% OGA enzyme occupancy in animal and human brain. Overall, ceperognastat had an acceptable safety profile in Phase 1 clinical studies with no serious adverse events reported following single and multiple dosing. The PK, enzyme occupancy, and safety profile supported Phase 2 development of ceperognastat.DISCUSSIONCeperognastat is an orally available, highly potent, CNS‐penetrant OGA inhibitor that achieved high (> 80%) OGA enzyme occupancy and increased brain O‐GlcNAc‐tau preclinically. Ceperognastat demonstrated > 95% OGA enzyme occupancy in Phase 1 trials. These occupancy data informed the dose selection for the Phase 2 clinical program.Highlights Ceperognastat is a highly potent, CNS‐penetrant OGA inhibitor. Ceperognastat is both orally available and CNS‐penetrant even when given at low doses. Ceperognastat can achieve > 95% OGA enzyme occupancy in the animal and human brain. Ceperognastat had an acceptable safety profile in Phase 1 clinical studies.

Acute increase of protein O-GlcNAcylation in mice leads to transcriptome changes in the brain opposite to what is observed in Alzheimer's Disease.

ABSTRACTEnhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) is explored as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer’s disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the mechanistic path of using OGA inhibition to treat AD. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using OGA inhibitor Thiamet G (TG), on normal mouse brains. We hypothesized that the transcritome signature in respones to TG treatment provides a comprehensive view of the effect of OGA inhibition. We sacrificed the mice and dissected their brains after 3 hours of saline or 50 mg/kg TG treatment, and then performed mRNA sequencing using NovaSeq PE 150 (n=5 each group). We identified 1,234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the potential benefits of OGA inhibition in the treatment of AD. In particular, although OGA inhibitors are promising to treat AD, their downstream chronic effects related to bioenergetics may be a limiting factor.Abstract Figure

SMAD4 promotes EMT in COPD airway remodeling induced by cigarette smoke through interaction with O-GlcNAc transferase

Cigarette smoke (CS) is a prevalent chemical indoor air contaminant known to be the primary cause of EMT during airway remodeling in COPD. While some evidence indicates the involvement of SMAD4 in EMT across certain diseases, its specific role in CS-induced EMT in airway remodeling associated with COPD is not established. In our research, we observed a substantial upregulation in SMAD4 expression, O-GlcNAcylation and EMT in patients with COPD, as well as in vitro and in vivo COPD models induced by CS, than those of the controls. Downregulation of SMAD4 resulted in a reduction in CS-induced EMT in vitro and in vivo. As a post-translational modification of proteins, O-GlcNAcylation is dynamically controlled by the duo of enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA). We further discovered the enhancement of O-GlcNAcylation levels induced by CS was due to an elevated OGT expression, as the expression of OGA remained unchanged. Using an OGT inhibitor (OSMI-1) counteracted the effects of SMAD4 on EMT. Whereas, overexpressing OGT increased SMAD4 expression and promoted EMT. OGT-mediated SMAD4 O-GlcNAcylation shielded SMAD4 from proteasomal degradation by reducing its ubiquitination, thereby aiding in SMAD4 stabilization in response to EMT induced by CS. Overall, this research uncovers a fresh pathway for CS-induced EMT in the airway remodeling of COPD and offers valuable insights.

Abstract Tu072: O-GlcNAcylation underlies the activation of sodium-glucose cotransporter 1 in diabetic hearts

Rationale: Type-2 diabetes (T2D) greatly increases the risk for developing heart failure. Recent evidence indicates that the cardiac expression and function of the sodium-glucose cotransporter 1 (SGLT1) is elevated in disease states, including T2D, leading to structural and functional remodeling of the heart. The mechanisms underlying SGLT1 activation in the diabetic heart are currently unknown. Objective: To investigate if SGLT1 undergoes the O-linked attachment of β- N -acetylglucosamine (O-GlcNAcylation), a post-translational modification that is exacerbated in T2D, and its effect on SGLT1 activity in diabetic hearts. Methods/Results: SGLT1 co-immunoprecipitates with O-GlcNAc in heart homogenates and HL-1 cardiomyocyte lysates. Enhancing global protein O-GlcNAcylation in HL-1 cells via si-RNA-mediated silencing of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, resulted in increased amounts of O-GlcNAc that precipitate with SGLT1. SGLT1 function, measured as the SGLT1-mediated Na + influx, was significantly increased in HL-1 cells with silenced OGA. These data suggest that SGLT1 undergoes O-GlcNAcylation and this modification results in SGLT1 activation. Using rats that express human amylin in the pancreatic β-cells (HIP rats) as a model of late-onset T2D and their wild-type littermates as controls, we found that a larger fraction of SGLT1 co-immunoprecipitates with O-GlcNAc in hearts from T2D rats compared to controls. The SGLT1-mediated Na + influx was larger in myocytes from T2D vs . control rats. Increasing total O-GlcNAcylation with Thiamet-G in control myocytes resulted in larger Na + influx. In reverse experiments, inhibition of O-GlcNAcylation with 6-diazo-5-oxo-L-norleucine crystalline reduced SGLT1-mediated Na + uptake in myocytes from T2D rats. Inhibition of O-GlcNAcylation in T2D myocytes reduced Ca 2+ spark frequency and this effect was significantly less pronounced with SGLT1 blocked. These results suggest that O-GlcNAcylation affects myocyte Na + regulation by activating SGLT1, which contributes to myocyte Na + overload and its downstream effects on sarcoplasmic reticulum Ca 2+ leak in T2D. Conclusion: Exacerbated O-GlcNAcylation underlies SGLT1 activation in diabetic hearts. Preventing O-GlcNAcylation may limit myocyte Na + overload and the frequency of pro-arrhythmogenic Ca 2+ sparks in myocytes from T2D rats.

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.

Decoding the Role of O-GlcNAcylation in Hepatocellular Carcinoma.

Post-translational modifications (PTMs) influence protein functionality by modulating protein stability, localization, and interactions with other molecules, thereby controlling various cellular processes. Common PTMs include phosphorylation, acetylation, ubiquitination, glycosylation, SUMOylation, methylation, sulfation, and nitrosylation. Among these modifications, O-GlcNAcylation has been shown to play a critical role in cancer development and progression, especially in hepatocellular carcinoma (HCC). This review outlines the role of O-GlcNAcylation in the development and progression of HCC. Moreover, we delve into the underlying mechanisms of O-GlcNAcylation in HCC and highlight compounds that target O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) to improve treatment outcomes. Understanding the role of O-GlcNAcylation in HCC will offer insights into potential therapeutic strategies targeting OGT and OGA, which could improve treatment for patients with HCC.

O-GlcNAc Modification Is a Promising Therapeutic Target for Diabetic Retinopathy.

Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.

OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

Biological Functions and Potential Therapeutic Significance of O-GlcNAcylation in Hepatic Cellular Stress and Liver Diseases.

O-linked-β-D-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation), which is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a post-translational modification involved in multiple cellular processes. O-GlcNAcylation of proteins can regulate their biological functions via crosstalk with other post-translational modifications, such as phosphorylation, ubiquitination, acetylation, and methylation. Liver diseases are a major cause of death worldwide; yet, key pathological features of the disease, such as inflammation, fibrosis, steatosis, and tumorigenesis, are not fully understood. The dysregulation of O-GlcNAcylation has been shown to be involved in some severe hepatic cellular stress, viral hepatitis, liver fibrosis, nonalcoholic fatty acid liver disease (NAFLD), malignant progression, and drug resistance of hepatocellular carcinoma (HCC) through multiple molecular signaling pathways. Here, we summarize the emerging link between O-GlcNAcylation and hepatic pathological processes and provide information about the development of therapeutic strategies for liver diseases.

OGT and OGA gene-edited human induced pluripotent stem cells for dissecting the functional roles of O-GlcNAcylation in hematopoiesis.

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor O-GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of OGA-depleted (OGAi) and OGT-depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34+ hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O-GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O-GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O-GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of O-GlcNAcylation.