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1.  O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Core Ribosomal Proteins 
Molecular Biology of the Cell  2010;21(12):1922-1936.
At least 20 core ribosome proteins are modified by O-GlcNAc. O-GlcNAcase is localized to the nucleolus and O-GlcNAc transferase is excluded from the nucleolus. Both enzymes associate with active polysomes. Overexpression of OGT disrupts ribosomal subunit homeostasis. Data suggest that O-GlcNAc regulates translation and ribosome biogenesis.
Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.
PMCID: PMC2883937  PMID: 20410138
2.  dbOGAP - An Integrated Bioinformatics Resource for Protein O-GlcNAcylation 
BMC Bioinformatics  2011;12:91.
Protein O-GlcNAcylation (or O-GlcNAc-ylation) is an O-linked glycosylation involving the transfer of β-N-acetylglucosamine to the hydroxyl group of serine or threonine residues of proteins. Growing evidences suggest that protein O-GlcNAcylation is common and is analogous to phosphorylation in modulating broad ranges of biological processes. However, compared to phosphorylation, the amount of protein O-GlcNAcylation data is relatively limited and its annotation in databases is scarce. Furthermore, a bioinformatics resource for O-GlcNAcylation is lacking, and an O-GlcNAcylation site prediction tool is much needed.
We developed a database of O-GlcNAcylated proteins and sites, dbOGAP, primarily based on literature published since O-GlcNAcylation was first described in 1984. The database currently contains ~800 proteins with experimental O-GlcNAcylation information, of which ~61% are of humans, and 172 proteins have a total of ~400 O-GlcNAcylation sites identified. The O-GlcNAcylated proteins are primarily nucleocytoplasmic, including membrane- and non-membrane bounded organelle-associated proteins. The known O-GlcNAcylated proteins exert a broad range of functions including transcriptional regulation, macromolecular complex assembly, intracellular transport, translation, and regulation of cell growth or death. The database also contains ~365 potential O-GlcNAcylated proteins inferred from known O-GlcNAcylated orthologs. Additional annotations, including other protein posttranslational modifications, biological pathways and disease information are integrated into the database. We developed an O-GlcNAcylation site prediction system, OGlcNAcScan, based on Support Vector Machine and trained using protein sequences with known O-GlcNAcylation sites from dbOGAP. The site prediction system achieved an area under ROC curve of 74.3% in five-fold cross-validation. The dbOGAP website was developed to allow for performing search and query on O-GlcNAcylated proteins and associated literature, as well as for browsing by gene names, organisms or pathways, and downloading of the database. Also available from the website, the OGlcNAcScan tool presents a list of predicted O-GlcNAcylation sites for given protein sequences.
dbOGAP is the first public bioinformatics resource to allow systematic access to the O-GlcNAcylated proteins, and related functional information and bibliography, as well as to an O-GlcNAcylation site prediction tool. The resource will facilitate research on O-GlcNAcylation and its proteomic identification.
PMCID: PMC3083348  PMID: 21466708
3.  Protein O-GlcNAcylation is a Novel Cytoprotective Signal in Cardiac Stem Cells 
Stem cells (Dayton, Ohio)  2012;31(4):765-775.
Clinical trials demonstrate the regenerative potential of cardiac stem cell (CSC) therapy in the post-infarcted heart. Despite these encouraging preliminary clinical findings, the basic biology of these cells remains largely unexplored. The principal requirement for cell transplantation is to effectively prime them for survival within the unfavorable environment of the infarcted myocardium. In the adult mammalian heart, the β-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a unique post-translational modification that confers cardioprotection from various otherwise lethal stressors. It is not known whether this signaling system exists in cardiac stem cells. In the present study, we demonstrate that protein O-GlcNAcylation is an inducible stress response in adult murine Sca-1+/lin− CSCs and exerts an essential pro-survival role. Post-hypoxic CSCs responded by time-dependently increasing protein O-GlcNAcylation upon reoxygenation. We utilized pharmacological interventions for loss- and gain-of-function, i.e., enzymatic inhibition of OGT (adds the O-GlcNAc modification to proteins) by TT04, or inhibition of OGA (removes O-GlcNAc) by thiamet-G (ThG). Reduction in the O-GlcNAc signal (via TT04, or OGT gene deletion using Cre-mediated recombination) significantly sensitized CSCs to post-hypoxic injury, whereas augmenting O-GlcNAc levels (via ThG) enhanced cell survival. Diminished O-GlcNAc levels render CSCs more susceptible to the onset of post-hypoxic apoptotic processes via elevated PARP cleavage due to enhanced caspase-3/7 activation, whereas promoting O-GlcNAcylation can serve as a pre-emptive anti-apoptotic signal regulating the survival of CSCs. Thus, we report the primary demonstration of protein O-GlcNAcylation as an important pro-survival signal in CSCs, which could enhance CSC survival prior to in vivo autologous transfer.
PMCID: PMC3606688  PMID: 23335157
Adult stem cells; Cardiac; Cell biology; Hypoxia; Tissue-specific stem cells
4.  O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter? 
O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury.
PMCID: PMC3389386  PMID: 22757958
cardiovascular system; diabetes; inflammation; O-linked N-acetylglucosamine (O-GlcNAc); oxidative stress; phosphorylation; AGE, advanced glycation end product; BEMAD, β-elimination followed by Michael addition with dithiothreitol; DOCA, deoxycorticosterone acetate; eNOS, endothelial NO synthase; ET-1, endothelin-1; FoxO1, forkhead box O1; GFAT, glutamine:fructose-6-phosphate amidotransferase; GlcNAc, N-acetylglucosamine; GPX1, glutathione peroxidase 1; HBP, hexosamine biosynthesis pathway; I/R, ischaemia/reperfusion; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; mPTP, mitochondrial permeability transition pore; NF-κB, nuclear factor κB; OGA, β-N-acetylglucosaminidase; O-GlcNAc, O-linked GlcNAc; O-GlcNAc-P, phosphorylated O-GlcNAc; OGT, O-GlcNAc transferase; PP1, protein phosphatase 1; PTM, post-translational modification; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; RAGE, receptor for AGEs; ROS, reactive oxygen species; shRNA, short hairpin RNA; SOD, superoxide dismutase; TPR, tetratricopeptide repeat; VSMC, vascular smooth muscle cell
The Journal of biological chemistry  2006;282(6):3918-3928.
Transforming growth factor-βactivated kinase 1 (TAK1), a member of the MAPKKK family, was initially described to play an essential role in the TGF beta-signaling pathway, but recent evidence has emerged implicating TAK1 in the IL-1 and TNF pathways. Notably, two homologous proteins, TAB2 and TAB3, have been identified as adaptors linking TAK1 to the upstream adaptors TRAFs. However, it remains unclear whether the interaction between TAB2/TAB3 and TAK1 is necessary for its kinase activation and subsequent activation of the IKK and MAPK pathways. Here, we characterized the TAB2/TAB3-binding domain in TAK1 and further examined the requirement of this interaction for IL-1, TNF, and RANKL signaling. Through deletion mapping experiments, we demonstrated that the binding motif for TAB2/TAB2 is a non-contiguous region located within the last C-terminal 100 residues of TAK1. However, residues 479–553 of TAK1 appear to be necessary and sufficient for TAB2/TAB3 interaction. Conversely, residues 574–693 of TAB2 were shown to interact with TAK1. A green fluorescent protein (GFP) fusion protein containing the last 100 residues of TAK1 (TAK1-C100) abolished the interaction of endogenous TAB2/TAB3 with TAK1, the phosphorylation of TAK1 and prevented the activation of IKK and MAPK induced by IL-1, TNF, and RANKL. Furthermore, TAK1-C100 blocked RANKL-induced nuclear accumulation of NFATc1 and consequently osteoclast differentiation consistent with the ability of a catalytically inactive TAK1 to block RANKL-mediated signaling. Significantly, our study provides evidence that the TAB2/TAB3 interaction with TAK1 is crucial for the activation of signaling cascades mediated by IL-1, TNF, and RANKL.
PMCID: PMC3197015  PMID: 17158449
6.  O-GlcNAc Modification of NFκB p65 Inhibits TNF-α-Induced Inflammatory Mediator Expression in Rat Aortic Smooth Muscle Cells 
PLoS ONE  2011;6(8):e24021.
We have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling.
Methodology/Principal Findings
Quiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10−4 M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65.
There is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury.
PMCID: PMC3164132  PMID: 21904602
7.  Three Decades of Research on O-GlcNAcylation – A Major Nutrient Sensor That Regulates Signaling, Transcription and Cellular Metabolism 
Even though the dynamic modification of polypeptides by the monosaccharide, O-linked N-acetylglucosamine (O-GlcNAcylation) was discovered over 30 years ago, its physiological significance as a major nutrient sensor that regulates myriad cellular processes has only recently been more widely appreciated. O-GlcNAcylation, either on its own or by its interplay with other post-translational modifications, such as phosphorylation, ubiquitination, and others, modulates the activities of signaling proteins, regulates most components of the transcription machinery, affects cell cycle progression and regulates the targeting/turnover or functions of myriad other regulatory proteins, in response to nutrients. Acute increases in O-GlcNAcylation protect cells from stress-induced injury, while chronic deregulation of O-GlcNAc cycling contributes to the etiology of major human diseases of aging, such as diabetes, cancer, and neurodegeneration. Recent advances in tools to study O-GlcNAcylation at the individual site level and specific inhibitors of O-GlcNAc cycling have allowed more rapid progress toward elucidating the specific functions of O-GlcNAcylation in essential cellular processes.
PMCID: PMC4209869  PMID: 25386167
O-GlcNAcylation; O-GlcNAc transferase; O-GlcNAcase; signaling; transcription; diabetes; cancer; Alzheimer’s disease
8.  O-Linked β-N-Acetylglucosamine (O-GlcNAc): Extensive Crosstalk with Phosphorylation to Regulate Signaling and Transcription in Response to Nutrients and Stress 
Biochimica et biophysica acta  2009;1800(2):96.
Since its discovery in the early 1980s, O-linked-β-N-acetylglucosamine (O-GlcNAc), a single sugar modification on the hydroxyl group of serine or threonine residues, has changed our views of protein glycosylation. While other forms of protein glycosylation modify proteins on the cell surface or within luminal compartments of the secretory machinery, O-GlcNAc modifies myriad nucleocytoplasmic proteins. GlcNAcylated proteins are involved in transcription, ubiquitination, cell cycle, and stress responses. GlcNAcylation is similar to protein phosphorylation in terms of stoichiometry, localization and cycling. To date, only two enzymes are known to regulate GlcNAcylation in mammals: O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc, and β-N-acetylglucosaminidase (O-GlcNAcase), a neutral hexosaminidase responsible for O-GlcNAc removal. OGT and O-GlcNAcase are regulated by RNA splicing, by nutrients, and by post-translational modifications. Their specificities are controlled by many transiently associated targeting subunits. As methods for detecting O-GlcNAc have improved our understanding of O-GlcNAc's functions has grown rapidly.
Scope of Review
In this review, the functions of GlcNAcylation in regulating cellular processes, its extensive crosstalk with protein phosphorylation, and regulation of OGT and O-GlcNAcase will be explored.
Major Conclusions
GlcNAcylation rivals phosphorylation in terms of its abundance, protein distribution and its cycling on and off of proteins. GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling, transcription and the cytoskeleton in response to nutrients and stress.
General Significance
Abnormal crosstalk between GlcNAcylation and phosphorylation underlies dysregulation in diabetes, including glucose toxicity, and defective GlcNAcylation is involved in neurodegenerative disease and cancer and most recently in AIDS.
PMCID: PMC2815129  PMID: 19647786
O-GlcNAc; GlcNAcylation; Phosphorylation; OGT; OGA; Stress; glucosamine; Alzheimer's Disease; signaling; diabetes; O-GlcNAcase; O-GlcNAc transferase
9.  O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells 
PLoS ONE  2013;8(7):e69150.
O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.
PMCID: PMC3730543  PMID: 23935944
10.  O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver 
Diabetes  2011;60(5):1399-1413.
Carbohydrate-responsive element–binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction.
O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBPOG in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice.
Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBPOG in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice.
Taken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions.
PMCID: PMC3292313  PMID: 21471514
11.  Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers 
PLoS ONE  2012;7(10):e48218.
O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle.
PMCID: PMC3480486  PMID: 23110217
12.  Human OGA binds substrates in a conserved peptide recognition groove 
Biochemical Journal  2010;432(Pt 1):1-7.
Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.
PMCID: PMC2973230  PMID: 20863279
β-N-acetylglucosaminidase; O-linked N-acetylglucosamine (O-GlcNAc); peptide recognition groove; protein glycosylation; CpNagJ, Clostridium perfringens NagJ; CREB, cAMP-response-element-binding protein; Fmoc, fluoren-9-ylmethoxycarbonyl; FoxO1, forkhead box O1; GST, glutathione transferase; HAT, histone acetyltransferase; HEK, human embryonic kidney; LC, liquid chromatography; 4MU-GlcNAc, 4-methylumbelliferyl-β-N-acetylglucosamine; OGA, O-GlcNAcase or β-N-acetylglucosaminidase; hOGA, human OGA; OgOGA, Oceanicola granulosus OGA; OGT, O-GlcNAc transferase; O-GlcNAc, O-linked N-acetylglucosamine; pNP-GlcNAc, p-nitrophenyl-β-N-acetylglucosamine; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; RMSD, root mean square deviation; TAB1, transforming growth factor-β-activated protein kinase 1-binding protein 1
13.  Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease 
Brain  2009;132(7):1820-1832.
It has been established for a long time that brain glucose metabolism is impaired in Alzheimer's disease. Recent studies have demonstrated that impaired brain glucose metabolism precedes the appearance of clinical symptoms, implying its active role in the development of Alzheimer's disease. However, the molecular mechanism by which this impairment contributes to the disease is not known. In this study, we demonstrated that protein O-GlcNAcylation, a common post-translational modification of nucleocytoplasmic proteins with β-N-acetyl-glucosamine and a process regulated by glucose metabolism, was markedly decreased in Alzheimer's disease cerebrum. More importantly, the decrease in O-GlcNAc correlated negatively with phosphorylation at most phosphorylation sites of tau protein, which is known to play a crucial role in the neurofibrillary degeneration of Alzheimer's disease. We also found that hyperphosphorylated tau contained 4-fold less O-GlcNAc than non-hyperphosphorylated tau, demonstrating for the first time an inverse relationship between O-GlcNAcylation and phosphorylation of tau in the human brain. Downregulation of O-GlcNAcylation by knockdown of O-GlcNAc transferase with small hairpin RNA led to increased phosphorylation of tau in HEK-293 cells. Inhibition of the hexosamine biosynthesis pathway in rat brain resulted in decreased O-GlcNAcylation and increased phosphorylation of tau, which resembled changes of O-GlcNAcylation and phosphorylation of tau in rodent brains with decreased glucose metabolism induced by fasting, but not those in rat brains when protein phosphatase 2A was inhibited. Comparison of tau phosphorylation patterns under various conditions suggests that abnormal tau hyperphosphorylation in Alzheimer's disease brain may result from downregulation of both O-GlcNAcylation and protein phosphatase 2A. These findings suggest that impaired brain glucose metabolism leads to abnormal hyperphosphorylation of tau and neurofibrillary degeneration via downregulation of tau O-GlcNAcylation in Alzheimer's disease. Thus, restoration of brain tau O-GlcNAcylation and protein phosphatase 2A activity may offer promising therapeutic targets for treating Alzheimer's disease.
PMCID: PMC2702834  PMID: 19451179
tau phosphorylation; O-GlcNAcylation; glucose metabolism; protein phosphatase 2A; neurofibrillary degeneration
14.  O-GlcNAcylation: A New Cancer Hallmark? 
O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression.
PMCID: PMC3740238  PMID: 23964270
O-glycosylation; O-GlcNAc; post-translational modification; cancer; metastasis; cell cycle; epigenetics; transcription factors
15.  TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells 
Molecular cancer research : MCR  2011;9(8):1042-1053.
Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, but the underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase 1 (TAK1) signaling in tumor cells promotes bone destruction by metastatic breast carcinoma cells, controlling expression of pro-metastatic factors, including MMP-9 and COX2. Suppression of TAK1 signaling by dominant-negative (dn) TAK1 in breast carcinoma MDA-MB-231 cells impairs bone colonization by carcinoma cells and bone osteolysis in the intra-cardiac injection model. Mechanistic studies showed that inhibition of TAK1 by dn-TAK1 or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9, COX2/PTGS2, PTHrP, and IL8, but did not affect activation of p38MAPK by TGF-β. TAK1 signaling is mediated by TAK1-binding partners TAB1, TAB2 and TAB3. Carcinoma cells express elevated mRNA levels of TAB2 and TAB3, whereas the TAB1 expression is noticeably low. Accordingly, depletion of TAB2 by siRNA reduced expression of MMP-9 and COX2. Together, these studies demonstrate that the TAK1-TAB2/TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of pro-invasive and osteolytic factors. These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis.
PMCID: PMC3157546  PMID: 21700681
Invasion; metastasis; bone metastasis; TAK1; TGF-beta
16.  Activation of the Transcriptional Function of the NF-κB Protein c-Rel by O-GlcNAc Glycosylation 
Science signaling  2013;6(290):ra75.
The transcription factor nuclear factor κB (NF-κB) rapidly reprograms gene expression in response to various stimuli, and its activity is regulated by several posttranslational modifications, including phosphorylation, methylation, and acetylation. The addition of O-linked β-N-acetylglucosamine (a process known as O-GlcNAcylation) is an abundant posttranslational modification that is enhanced in conditions such as hyperglycemia and cellular stress. We report that the NF-κB subunit c-Rel is modified and activated by O-GlcNAcylation. We identified serine 350 as the site of O-GlcNAcylation, which was required for the DNA binding and transactivation functions of c-Rel. Blocking the O-GlcNAcylation of this residue abrogated c-Rel–mediated expression of the cytokine-encoding genes IL2, IFNG, and CSF2 in response to T cell receptor (TCR) activation, whereas increasing the extent of O-GlcNAcylation of cellular proteins enhanced the expression of these genes. TCR- or tumor necrosis factor (TNF)–induced expression of other NF-κB target genes, such as NFKBIA (which encodes IκBα) and TNFAIP3 (which encodes A20), occurred independently of the O-GlcNAcylation of c-Rel. Our findings suggest a stimulus-specific role for hyperglycemia-induced O-GlcNAcylation of c-Rel in promoting T cell–mediated autoimmunity in conditions such as type 1 diabetes by enhancing the production of T helper cell cytokines.
PMCID: PMC4066889  PMID: 23982206
17.  Receptor Activator of NF-κB Ligand (RANKL) Activates TAK1 Mitogen-Activated Protein Kinase Kinase Kinase through a Signaling Complex Containing RANK, TAB2, and TRAF6 
Molecular and Cellular Biology  2002;22(4):992-1000.
The receptor activator of NF-κB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-κB through IκB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts.
PMCID: PMC134634  PMID: 11809792
18.  O-Linked β-N-Acetylglucosaminylation in Mouse Embryonic Neural Precursor Cells 
Journal of neuroscience research  2009;87(16):3535-3545.
In neural stem cells (NSCs), glycoconjugates and carbohydrate antigens are known not only to serve as excellent cell surface biomarkers for cellular differentiation and development but also to play important functional roles in determining cell fate. O-linked β-N-acetylglucosamine (O-GlcNAc), which modifies nuclear and cytoplasmic proteins on the serine and threonine residues, is also expected to play an important regulatory role. It is not known, however, whether O-GlcNAc is expressed in NSCs or what the function of this expression is. In this study, we evaluated the patterns and possible functions O-GlcNAcylation in mouse embryonic neuroepithelial cells (NECs), which are known to be rich in NSCs. We confirmed the expression of O-GlcNAc transferase, O-GlcNAcase, and several O-GlcNAcylated proteins in NECs. Treatment of NECs with O-GlcNAcase inhibitors, PUGNAc and streptozotocin, induced robust accumulation of O-GlcNAc in NECs and reduction of number of NECs. In O-GlcNAcase inhibitor-treated NECs, the Rasmitogen-activated protein kinase pathway and the phosphoinositide 3-kinase-Akt pathway, important for proliferation and survival, respectively, were intact, but caspase-3, an executioner for cell death, was activated. These results suggest the possibility that O-GlcNAc is involved in cell death signaling in NECs. Furthermore, for NECs, we identified an O-GlcNAc-modified protein, Sp1 transcription factor. Our study is the first to evaluate expression and functions of O-GlcNAc in NECs.
PMCID: PMC3956749  PMID: 19598243
development; carbohydrate; signal transduction
19.  O-GlcNAcylation and Inflammation: A Vast Territory to Explore 
O-GlcNAcylation is a reversible post-translational modification that regulates the activities of cytosolic and nuclear proteins according to glucose availability. This modification appears to participate in several hyperglycemia-associated complications. An important feature of metabolic diseases such as diabetes and obesity is the presence of a low-grade chronic inflammation that causes numerous complications. Hyperglycemia associated with the metabolic syndrome is known to promote inflammatory processes through different mechanisms including oxidative stress and abnormally elevated protein O-GlcNAcylation. However, the role of O-GlcNAcylation on inflammation remains contradictory. O-GlcNAcylation associated with hyperglycemia has been shown to increase nuclear factor κB (NFκB) transcriptional activity through different mechanisms. This could contribute in inflammation-associated diabetic complications. However, in other conditions such as acute vascular injury, O-linked N-acetyl glucosamine (O-GlcNAc) also exerts anti-inflammatory effects via inhibition of the NFκB pathway, suggesting a complex regulation of inflammation by O-GlcNAc. Moreover, whereas macrophages and monocytes exposed to high glucose for a long-term period developed a pro-inflammatory phenotype, the impact of O-GlcNAcylation in these cells remains unclear. A future challenge will be to clearly establish the role of O-GlcNAcylation in pro- and anti-inflammatory functions in macrophages.
PMCID: PMC4288382  PMID: 25620956
O-GlcNAc glycosylation; diabetes; metabolic syndrome; inflammation; cytokines; macrophages; nitric oxide; NFκB
20.  O-Linked N-Acetylglucosaminylation of Sp1 Inhibits the Human Immunodeficiency Virus Type 1 Promoter▿  
Journal of Virology  2009;83(8):3704-3718.
Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5′ long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5′ LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-d-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4+ T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.
PMCID: PMC2663287  PMID: 19193796
21.  Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis 
Science signaling  2010;3(104):ra2.
Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
PMCID: PMC2866299  PMID: 20068230
22.  O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway 
Cardiovascular Research  2010;89(3):614-622.
Glycosylation with β-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway.
Methods and results
Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 μM) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ETA antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan.
We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.
PMCID: PMC3028974  PMID: 20978008
β-N-acetylglucosamine; Endothelin-1; Vascular reactivity; RhoA/Rho-kinase pathway
23.  Novel interaction between the TAK1-TAB1-TAB2 and the RCAN1-calcineurin regulatory pathways defines a signaling nodal control point 
Nature cell biology  2009;11(2):154-161.
The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in yeast up through mammals. The physiologic function of RCAN proteins remains an area of ongoing investigation because both positive and negative calcineurin regulatory effects have been reported. Here, we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 directly interacted with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signaling complex. Overexpression of TAK1 and TAB1, or active TAK1-ΔN, promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at two novel sites, serine 94 and 136, switching RCAN1 from an inhibitor to a facilitator of calcineurin-NFAT signaling, enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. Remarkably, Rcan1/2 or Tab2 deficient mouse embryonic fibroblast (MEF) cultures each failed to show an interaction between the TAK1-TAB1-TAB2 and the calcineurin-NFAT signaling modules. We also observed a reciprocal negative feedback mechanism whereby sustained calcineurin activation inhibited TAK1 signaling through dephosphorylation of TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by prohypertrophic stimuli. TAK1-dependent growth was also blocked by inhibition of calcineurin activity with Cain. Finally, a dominant interfering fragment of RCAN1 that disrupts the TAK1-TAB1-TAB2, calcineurin-NFAT complex also blocked cardiomyocyte hypertrophy to several stimuli. These results describe a novel signaling relationship between two central regulatory pathways whereby TAK1-TAB1-TAB2 selectively induces calcineurin-NFAT signaling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signaling by dephosphorylation of TAK1 and TAB1.
PMCID: PMC2656285  PMID: 19136967
24.  O-GlcNAcylation of AMPA Receptor GluA2 Is Associated with a Novel Form of Long-Term Depression at Hippocampal Synapses 
The Journal of Neuroscience  2014;34(1):10-21.
Serine phosphorylation of AMPA receptor (AMPAR) subunits GluA1 and GluA2 modulates AMPAR trafficking during long-term changes in strength of hippocampal excitatory transmission required for normal learning and memory. The post-translational addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) also occurs on serine residues. This, together with the high expression of the enzymes O-GlcNAc transferase (OGT) and β-N-acetylglucosamindase (O-GlcNAcase), suggests a potential role for O-GlcNAcylation in modifying synaptic efficacy and cognition. Furthermore, because key synaptic proteins are O-GlcNAcylated, this modification may be as important to brain function as phosphorylation, yet its physiological significance remains unknown. We report that acutely increasing O-GlcNAcylation in Sprague Dawley rat hippocampal slices induces an NMDA receptor and protein kinase C-independent long-term depression (LTD) at hippocampal CA3–CA1 synapses (O-GcNAc LTD). This LTD requires AMPAR GluA2 subunits, which we demonstrate are O-GlcNAcylated. Increasing O-GlcNAcylation interferes with long-term potentiation, and in hippocampal behavioral assays, it prevents novel object recognition and placement without affecting contextual fear conditioning. Our findings provide evidence that O-GlcNAcylation dynamically modulates hippocampal synaptic function and learning and memory, and suggest that altered O-GlcNAc levels could underlie cognitive dysfunction in neurological diseases.
PMCID: PMC3866478  PMID: 24381264
depression; hippocampus; LTD; LTP; O-GlcNAc; synaptic transmission
25.  TAK1 Expression in the Cochlea: A Specific Marker for Adult Supporting Cells 
Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β and BMP signaling, TAK1 mediates the activation of the c-Jun N-terminal kinase signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt/Fz signaling in mesenchymal stem cells. Expression of TAK1 in the cochlea has not been defined. Data mining of previously published murine cochlear gene expression databases indicated that TAK1, along with TAK1 interacting proteins 1 (TAB1), and 2 (TAB2), is expressed in the developing and adult cochlea. The expression of TAK1 in the developing cochlea was confirmed using RT-PCR and immunohistochemistry. Immunolabeling of TAK1 in embryonic, neonatal, and mature cochleas via DAB chromogenic and fluorescent immunohistochemistry indicated that TAK1 is broadly expressed in both the developing otocyst and periotic mesenchyme at E12.5 but becomes more restricted to specific types of supporting cells as the organ of Corti matures. By P1, TAK1 immunolabeling is found in cells of the stria vascularis, hair cells, supporting cells, and Kölliker’s organ. By P16, TAK1 labeling is limited to cochlear supporting cells. In the adult cochlea, TAK1 immunostaining is only present in the cytoplasm of Deiters’ cells, pillar cells, inner phalangeal cells, and inner border cells, with no expression in any other cochlear cell types. While the role of TAK1 in the inner ear is unclear, TAK1 expression may be used as a novel marker for specific sub-populations of supporting cells.
PMCID: PMC3123448  PMID: 21472480
cochlea; Deiters’ cell; phalangeal cell; pillar cell; development; gene expression

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