O-linked β-N-acetylglucosamine Transferase Research Articles

Splicing Analysis Unveils Convergent Transcriptomic Pathways in U2AF1 S34 and Q157 Mutant Myeloid Neoplasia

Splicing factor mutations (SFMT) are frequent in MDS and their pathogenic consequences may include missplicing and changes in alternative splicing (AS). However, divergent sequence-sequence effects on misspliced genes were recorded in U2AF1MT at the two canonical hotspots (S34, Q157). Most of the current studies have been focused on the identification of distinctive features of splicing dysfunction between the two hotspots, unveiling various unique hotspot specific splicing events. Classical instances of missplicing events include those in ATG7,H2AFY and STRAP specific to U2AF1S34 MDS. However, whereas we and others have demonstrated certain unique clinical features of MDS patients carrying S34 vs Q1571, these two alterations produce a generally similar disease phenotype and likely rely on a common transformation mechanism. This conclusion suggests that missplicing events might converge in pathways relevant to MDS pathogenesis, which may be much more widespread than what is believed. While defining a topology of AS events (ASE), our analysis of the global landscape of RNA splicing in myeloid neoplasia (MN)2 also opened the possibility to search for subsets of patients sharing RNA splicing features and gene expression (GE). We now curated RNA-Seq data from diagnostic samples of 1258 MN patients by comparing RNA splicing profiles of clonal primary cells with S34 (n=19) and Q157 (n=26) (VAFs >10%, both) in search for overlapping ASE representing new cooperative mechanisms germane to leukemogenesis. ASE were filtered based on percent spliced in (PSI) (variation threshold > 0.01). Limma analysis for differential GE and PSI was applied. All data were analyzed on the genome assembly GRCh37/hg19. Patients without SFMT plus a cohort of 63 age-matched controls were used to detect exclusive ASE common in U2AF1 S34 and Q157. Pathway analysis showed an enrichment of chromatin remodeling, histone demethylases and signal transduction genes. A total of 17564 ASE were called with 30% (n=5106) of them found to be significant (qvalues ≤ .05 and inclusion level difference ≥ 5%). In-depth analysis of ASE showed that 70% of significant PSI changes resulted from exon skipping (ES) followed by intron retention (17%) and alternative 3′ and 5′ splice site events (14%). No significant mutually exclusive ASE was found. Considering that ES might result in truncated proteins and consequent lack of functional domains, we mapped ASE to protein domains. Top PSI changes which were common between S34 and Q157 were found in chromatin remodeling (EZH2,JMJD1C, ARID2/4B, CREBBP), O-linked β-N-acetylglucosamine transferase (OGT), a partner of TET2, GTPase activating (GNAS, ARHGAP9, HBS1L), RNA processing/ binding (RBM5, DDX21, DHX38,HNRNPM, PRPF3), unfolded protein response (XBP1), and ubiquitination (CUL3, NAE1, UBQLN1) genes. Lesions affecting chromosome 7 either structurally (cytogenetic alterations) or by mutations and CNV (EZH2 among others) are associated with adverse prognosis in MN. In total, 8% of the patients had deletion 7 and 4% carried EZH2MT. EZH2 mRNA expression levels were significantly lower in U2AF1MTvs controls (LFC=-0.88; P= 1e-11). Of note is that only 6 cases carried EZH2MT, of whom none harbored deletion 7. This suggests that EZH2 low expression levels might be due to other mechanisms. Looking at most relevant findings, we identified a significant and single ES (ex12) in EZH2 flanking the pre-SET domain (cysteine rich, CXC: aa503-605) crucial to maintain histone methyltransferase activity. Beside somatic mutations, EZH2 function can be altered by other mechanisms, e.g., cooperative mutations. The presence of an ES possibly suggests that an alternative mechanism of gene inactivation beside mutations in the CXC/SET domain and chromosome loss might play a role. Similarly, a CREBBP ES (ex11) was detected upstream the kinase inducible interacting (KIX) domain, a docking site for transcriptional activators (e.g., MLL, c-Myb sites). Finally, U2AF1MT have been also associated with deletion 20q or with a hyperactive long GNAS isoform. We found indeed an ES in GNAS and other GTPases, further emphasizing the broad dysfunctions in U2AF1 cooperative genes. In conclusion, our study describes convergent splicing features common to both U2AF1 S34 and Q157 to conceptualize the essential mechanisms contributing to clonal expansion due to lesions in U2AF1.

Novel O-linked β-N-acetylglucosamine Transferase (OGT) Inhibitors from Tinospora Cordifolia: An In-Silico Approach

Diabetes mellitus (DM) characterized by excess blood sugar, is a multifactorial metabolic disease that has reached epidemic proportions worldwide. The International Diabetes Foundation (IDF) estimates that approximately 537 million adults will be living with DM in 2021. The total number of people living with DM is projected to rise to 783 million by 2045. According to the absolute or relative lack of insulin signaling, DM is classified into two major forms: Type 1 diabetes mellitus (T1DM) and Type 2 diabetes mellitus (T2DM). One of the proteins that play a major role in DM is O-linked β-N-acetylglucosamine Transferase (OGT) a glucose-dependent human enzyme that catalyzes the addition of UDP-GlcNAc on the serine and threonine residues of nuclear and cytoplasmic proteins. While this protein plays a vital role in cell cycle regulation and glucose metabolism, an aberration of it could be lethal, and up until now, there have been no reports of small molecule and potent plant-based inhibitors of OGT. In this study, we put molecular docking, ADME/Tox analysis, and MM/GBSA studies to use in identifying novel potent inhibitors of OGT from compounds of Tinospora cordifolia, and we compare our results with that of an established OGT inhibitor, OSMI-1. Based on docking scores and ligand-protein interactions, we predict four (4) top compounds; Apigenin, Bergenin, Diosmetin, and Syringin. In conclusion, the results from the ADME/Tox analysis have led to the prediction that a T. cordifolia compound (Bergenin) has better drug-like characteristics than the standard compound, OSMI-1.

The Beginner's Guide to O-GlcNAc: From Nutrient Sensitive Pathway Regulation to Its Impact on the Immune System.

The addition of N-acetyl glucosamine (GlcNAc) on the hydroxy group of serine/threonine residues is known as O-GlcNAcylation (OGN). The dynamic cycling of this monosaccharide on and off substrates occurs via O-linked β-N-acetylglucosamine transferase (OGT) and O-linked β-N-acetylglucosaminase (OGA) respectively. These enzymes are found ubiquitously in eukaryotes and genetic knock outs of the ogt gene has been found to be lethal in embryonic mice. The substrate scope of these enzymes is vast, over 15,000 proteins across 43 species have been identified with O-GlcNAc. OGN has been known to play a key role in several cellular processes such as: transcription, translation, cell signaling, nutrient sensing, immune cell development and various steps of the cell cycle. However, its dysregulation is present in various diseases: cancer, neurodegenerative diseases, diabetes. O-GlcNAc is heavily involved in cross talk with other post-translational modifications (PTM), such as phosphorylation, acetylation, and ubiquitination, by regulating each other’s cycling enzymes or directly competing addition on the same substrate. This crosstalk between PTMs can affect gene expression, protein localization, and protein stability; therefore, regulating a multitude of cell signaling pathways. In this review the roles of OGN will be discussed. The effect O-GlcNAc exerts over protein-protein interactions, the various forms of crosstalk with other PTMs, and its role as a nutrient sensor will be highlighted. A summary of how these O-GlcNAc driven processes effect the immune system will also be included.

Nanomolar inhibition of human OGA by 2-acetamido-2-deoxy-d-glucono-1,5-lactone semicarbazone derivatives.

O-GlcNAcylation is a dynamic post-translational modification mediated by O-linked β-N-acetylglucosamine transferase (OGT) and O-GlcNAc hydrolase (OGA), that adds or removes a single β-N-acetylglucosamine (GlcNAc) moiety to or from serine/threonine residues of nucleocytosolic and mitochondrial proteins, respectively. The perturbed homeostasis of O-GlcNAc cycling results in several pathological conditions. Human OGA is a promising therapeutic target in diseases where aberrantly low levels of O-GlcNAc are experienced, such as tauopathy in Alzheimer's disease. A new class of potent OGA inhibitors, 2-acetamido-2-deoxy-d-glucono-1,5-lactone (thio)semicarbazones, have been identified. Eight inhibitors were designed and synthesized in five steps starting from d-glucosamine and with 15-55% overall yields. A heterologous OGA expression protocol with strain selection and isolation has been optimized that resulted in stable, active and full length human OGA (hOGA) isomorph. Thermal denaturation kinetics of hOGA revealed environmental factors affecting hOGA stability. From kinetics experiments, the synthesized compounds proved to be efficient competitive inhibitors of hOGA with Ki-s in the range of ∼30-250nM and moderate selectivity with respect to lysosomal β-hexosaminidases. In silico studies consisting of Prime protein-ligand refinements, QM/MM optimizations and QM/MM-PBSA binding free energy calculations revealed the factors governing the observed potencies, and led to design of the most potent analogue 2-acetamido-2-deoxy-d-glucono-1,5-lactone 4-(2-naphthyl)-semicarbazone 6g (Ki=36nM). The protocol employed has applications in future structure based inhibitor design targeting OGA.

Investigating the Impact of OGT Inhibition on Doxorubicin- and Docetaxel-Induced Cytotoxicity in PC-3 and WPMY-1 Cells.

Reduction in sensitivity in terms of cytotoxicity is responsible for therapy failure in patients undergoing chemotherapy with first-line anticancer drug molecules. A plethora of literature evidence points out that increased O-linked β-N-acetylglucosamine transferase (OGT) enzyme level/hyper-O-GlcNAcylation has direct implications in development of cancer and interferes with clinical outcomes of chemotherapy via interaction with oncogenic factors. The aim of this research was to evaluate the combination approach of anticancer drugs with an OGT inhibitor (OSMI-1) as an alternative way to resolve issues in the treatment of prostate cancer and assess the benefits offered by this approach. Effect of combination of doxorubicin and docetaxel with OSMI-1 on drug-induced cell death and synergism/antagonism was investigated using resazurin assay. Reduction in OGT enzyme level was evaluated using ELISA kit. Caspase-3/7 fluorescence assay was performed to detect apoptosis induction in PC-3 cells after treatment with the combinations of doxorubicin and OGT inhibitor to further understand the mechanism of cell death by concomitant treatment. Studies reveal that combination approach is indeed effective in terms of reducing the half-maximum growth inhibition value of doxorubicin when concomitantly treated with OSMI-1 and has synergistic effect in prostate cancer cells. PC-3 cells exhibited elevated levels of OGT enzyme in comparison to WPMY-1, and OSMI-1 has potential to inhibit OGT enzyme significantly. Data show that OSMI-1 alone and in combination with doxorubicin reduces OGT enzyme level significantly accompanied by increased apoptosis in prostate cancer cells. Combination of doxorubicin with OSMI-1 reduced the elevated OGT level which led to a drastic increase in sensitivity of PC-3 cells toward doxorubicin in comparison to doxorubicin alone. This finding provides important insight regarding alternative treatment strategies for effective management of cancer.

O-GlcNAcylation Is Essential for Autophagy in Cardiomyocytes.

Since both O-GlcNAcylation and autophagy sense intracellular nutrient level, the alteration of those two pathways plays substantial roles in the progression of heart failure. Hence, determining the relationship between O-GlcNAcylation and autophagy is imperative to understand, prevent, and treat heart failure. However, the mechanism on how O-GlcNAcylation regulates autophagy in the heart is poorly investigated. In this study, we demonstrated that O-GlcNAcylation is required for autophagy in cardiomyocytes by utilizing an O-linked β-N-acetylglucosamine transferase (OGT) cardiomyocyte-specific knockout mouse model for the first time. We also identified that OGT might regulate the initiation of autophagy in cardiomyocytes through promoting the activity of ULK1 by O-GlcNAcylation. In conclusion, our findings provide new insights into the molecular mechanisms underlying heart dysfunction and benefit the development of treatments for heart failure.

Suppressed OGT expression inhibits cell proliferation while inducing cell apoptosis in bladder cancer

BackgroundThis study aimed to explore hyper-O-linked N-acetylglucosaminylation (O-GlcNAcylation) with an elevation of the expression of O-linked-β-N-acetylglucosamine transferase (OGT) in human bladder cancer.MethodsImmunohistochemical staining for OGT and O-GlcNAcylation was performed in 20 paired human bladder cancer and adjacent normal tissues, as well as in human bladder cancer tissue microarrays (N = 169). The expression level of OGT and O-GlcNAcylation in cell lines were detected using the Western blot analysis. The effects of O-GlcNAcylation on the cell proliferation of bladder cancer were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clone formation assays. Cell apoptosis and cell cycle analysis were detected using flow cytometry. The autophagy of bladder cancer cells was investigated using the Western blot analysis, and GFP–LC3 plasmid was used to detect the autophagic flux. MTT assay was performed to detect the sensitivity of bladder cancer cells to cisplatin after OGT knockdown.ResultsThe expression of OGT and the O-GlcNAcylation were upregulated in bladder cancer tissues and cell lines. O-GlcNAcylation and OGT were observed in nucleus and cytoplasm and found to be higher in muscle-invasive bladder cancer (MIBC) than in non-muscle-invasive bladder cancer (NMIBC). Reducing hyper-O-GlcNAcylation by OGT knockdown inhibited the proliferation of bladder cancer cells in vitro and xenograft tumor growth in vivo, triggered apoptosis, as well as led to cell cycle arrest. It also increased autophagy in bladder cancer cells. This study demonstrated increased autophagy pro-survival, but not pro-death. Reducing hyper-O-GlcNAcylation by OGT knockdown facilitated the chemosensitivity of bladder cancer cells to cis-platinum.ConclusionsThe data indicated that hyper-O-GlcNAcylation enhanced oncogenic phenotypes and was involved in DNA damage response in bladder cancer.

Alpha-lipoic acid reduces retinal cell death in diabetic mice

Oxidative stress plays an important role in the development of diabetic retinopathy. Here, we examined whether α-lipoic acid (α-LA), a natural antioxidant, attenuated retinal injury in diabetic mice. The α-LA was orally administered to control mice or mice with streptozotocin-induced diabetes. We found that α-LA reduced oxidative stress, decreased and increased retinal 4-hydroxy-2-nonenal and glutathione peroxidase, respectively, and inhibited retinal cell death. Concomitantly, α-LA reversed the decreased activation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase, and increased the levels of peroxisome proliferator-activated receptor delta and sirtuin3 in diabetic mouse retinas, similar to results shown after metformin treatment of retinal pigment epithelial cells (RPE) exposed to high glucose. Moreover, α-LA lowered the levels of O-linked β-N-acetylglucosamine transferase (OGT) and thioredoxin-interacting protein (TXNIP) in diabetic retinas that were more pronounced after metformin treatment of RPE cells. Importantly, α-LA lowered interactions between AMPK and OGT as shown by co-immunoprecipitation analyses, and this was accompanied by less cell death as measured by double immunofluorescence staining by terminal deoxynucleotide transferase-mediated dUTP nick-end labelling and OGT or TXNIP in retinal ganglion cells. Consistently, α-LA lowered the levels of cleaved poly(ADP-ribose) polymerase and pro-apoptotic marker cleaved caspase-3 in diabetic retinas. Our results indicated that α-LA reduced retinal cell death partly through AMPK activation or OGT inhibition in diabetic mice.

Correction: A study of the structural properties of sites modified by the O-linked 6-N-acetylglucosamine transferase.

[This corrects the article DOI: 10.1371/journal.pone.0184405.].

A study of the structural properties of sites modified by the O-linked 6-N-acetylglucosamine transferase.

Protein O-GlcNAcylation (O-GlcNAc) is an essential post-translational modification (PTM) in higher eukaryotes. The O-linked β-N-acetylglucosamine transferase (OGT), targets specific Serines and Threonines (S/T) in intracellular proteins. However, unlike phosphorylation, fewer than 25% of known O-GlcNAc sites match a clear sequence pattern. Accordingly, the three-dimensional structures of O-GlcNAc sites were characterised to investigate the role of structure in molecular recognition. From 1,584 O-GlcNAc sites in 620 proteins, 143 were mapped to protein structures determined by X-ray crystallography. The modified S/T were 1.7 times more likely to be annotated in the REM465 field which defines missing residues in a protein structure, while 7 O-GlcNAc sites were solvent inaccessible and unlikely to be targeted by OGT. 132 sites with complete backbone atoms clustered into 10 groups, but these were indistinguishable from clusters from unmodified S/T. This suggests there is no prevalent three-dimensional motif for OGT recognition. Predicted features from the 620 proteins were compared to unmodified S/T in O-GlcNAcylated proteins and globular proteins. The Jpred4 predicted secondary structure shows that modified S/T were more likely to be coils. 5/6 methods to predict intrinsic disorder indicated O-GlcNAcylated S/T to be significantly more disordered than unmodified S/T. Although the analysis did not find a pattern in the site three-dimensional structure, it revealed the residues around the modification site are likely to be disordered and suggests a potential role of secondary structure elements in OGT site recognition.

A Putative O-Linked β- N -Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme

Most species of filamentous cyanobacteria are capable of gliding motility, likely via a conserved type IV pilus-like system that may also secrete a motility-associated polysaccharide. In a subset of these organisms, motility is achieved only after the transient differentiation of hormogonia, which are specialized filaments that enter a nongrowth state dedicated to motility. Despite the fundamental importance of hormogonia to the life cycles of many filamentous cyanobacteria, the molecular regulation of hormogonium development is largely undefined. To systematically identify genes essential for hormogonium development and motility in the model heterocyst-forming filamentous cyanobacterium Nostoc punctiforme, a forward genetic screen was employed. The first gene identified using this screen, designated ogtA, encodes a putative O-linked β-N-acetylglucosamine transferase (OGT). The deletion of ogtA abolished motility, while ectopic expression of ogtA induced hormogonium development even under hormogonium-repressing conditions. Transcription of ogtA is rapidly upregulated (1 h) following hormogonium induction, and an OgtA-GFPuv fusion protein localized to the cytoplasm. In developing hormogonia, accumulation of PilA but not HmpD is dependent on ogtA Reverse transcription-quantitative PCR (RT-qPCR) analysis indicated equivalent levels of pilA transcript in the wild-type and ΔogtA mutant strains, while a reporter construct consisting of the intergenic region in the 5' direction of pilA fused to gfp produced lower levels of fluorescence in the ΔogtA mutant strain than in the wild type. The production of hormogonium polysaccharide in the ΔogtA mutant strain is reduced compared to that in the wild type but comparable to that in a pilA deletion strain. Collectively, these results imply that O-GlcNAc protein modification regulates the accumulation of PilA via a posttranscriptional mechanism in developing hormogonia.IMPORTANCE Filamentous cyanobacteria are among the most developmentally complex prokaryotes. Species such as Nostoc punctiforme develop an array of cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogonia, that function in dispersal as well as the establishment of nitrogen-fixing symbioses with plants and fungi. These symbioses are major contributors to global nitrogen fixation. Despite the fundamental importance of hormogonia to the life cycle of filamentous cyanobacteria and the establishment of symbioses, the molecular regulation of hormogonium development is largely undefined. We employed a genetic screen to identify genes essential for hormogonium development and motility in Nostoc punctiforme The first gene identified using this screen encodes a eukaryotic-like O-linked β-N-acetylglucosamine transferase that is required for accumulation of PilA in hormogonia.

Aspartate Glycosylation Triggers Isomerization to Isoaspartate.

O-Linked β-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor 1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. We report here that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells but was previously thought to be exclusively non-enzymatic. Our findings suggest it may also be enzyme-catalyzed. In addition to OGT, enzymes that may catalyze aspartate to isoaspartate isomerization include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.

How the glycosyltransferase OGT catalyzes amide bond cleavage.

The essential human enzyme O-GlcNAc transferase (OGT), known for modulating the functions of nuclear and cytoplasmic proteins through Ser/Thr glycosylation, was unexpectedly implicated in the proteolytic maturation of the cell cycle regulator host cell factor-1 (HCF-1). Here we show that HCF-1 cleavage occurs via glycosylation of a glutamate side chain followed by on-enzyme formation of an internal pyroglutamate, which undergoes spontaneous backbone hydrolysis.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

O‐GlcNAc Regulates p53 in Ovarian Cancer

O‐GlcNAcylation is a dynamic post‐translational modification (PTM) that consists of an addition of a single N‐acetylglucosamine sugar to serine and threonine residues in nuclear, cytoplasmic and mitochondrial proteins. The enzyme O‐linked β‐N‐acetylglucosamine transferase (OGT) adds the modification, while enzyme O‐GlcNAcase (OGA) removes the modification. O‐GlcNAc regulates enzymatic activity, localization, stability and protein‐protein interactions on a wide range of proteins such as transcription factors, structural proteins and kinases. In cancer, when comparing O‐GlcNAc levels in normal and tumor cells, tumor samples present higher O‐GlcNAcylation in vitro and in vivo. This aberrant O‐GlcNAc pattern contributes to changes in important signaling pathways and could contribute to the progression of the disease. Interestingly, the tumor suppressor p53 is modified by O‐GlcNAc. The transcription factor p53 regulates diverse cellular processes like cell survival and the cell cycle. Most solid tumors contain mutations in p53 leading to increased degradation or loss of p53 function, which promotes malignant tumor growth. Ovarian cancer has one of the highest frequencies of p53 mutation rates. The goal of this work is to understand the relationship between O‐GlcNAcylation and p53 function in ovarian cancer. First, O‐GlcNAcylation, OGT and OGA protein and mRNA levels were measured in a series of human ovarian cancer cell lines and no correlation was found between p53 genetic status and O‐GlcNAcylation. Overexpression or silencing of p53 in a p53 wild‐type cell line (A2780) had little effect on O‐GlcNAcylation. On the other hand, pharmacological inhibition of OGA with the inhibitor Thiamet‐G (TMG) leads to an increase in nuclear localization of p53 and increased mRNA levels of p53‐target genes (p21 and Bax). Overexpression of OGA or OGT leads to increased p53 protein but not mRNA levels and a concurrent increase in p53‐targets (Mdm‐2, p21 and Bax) expression. Also, p53 acetylation on lysine 382, a transcriptional activating modification, is higher in OGA/OGT overexpressing cells. In conclusion, O‐GlcNAcylation regulates p53 function through stabilization and activation.Support or Funding InformationSupport and funding: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Science Without Borders Program, NIH NIDDK R01 DK 100595.

Pharmacological Inhibition of O-GlcNAcase Does Not Increase Sensitivity of Glucocorticoid Receptor-Mediated Transrepression.

Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked β-N-acetylglucosamine transferase (OGT), which adds an O-linked β-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid–induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.

Gene and protein expression of O-GlcNAc-cycling enzymes in human laryngeal cancer.

Aberrant protein O-GlcNAcylation may contribute to the development and malignant behavior of many cancers. This modification is controlled by O-linked β-N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA). The aim of this study was to determine the expression of O-GlcNAc cycling enzymes mRNA/protein and to investigate their relationship with clinicopathological parameters in laryngeal cancer. The mRNA levels of OGT and MGEA5 genes were determined in 106 squamous cell laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent laryngeal mucosa (NCLM) controls using quantitative real-time PCR. The level of OGT and OGA proteins was analyzed by Western blot. A positive expression of OGT and MGEA5 transcripts and OGT and OGA proteins was confirmed in 75.5 and 68.9 % and in 43.7 and 59.4 % samples of SCLC, respectively. Higher levels of mRNA/protein for both OGT and OGA as well as significant increases of 60 % in total protein O-GlcNAcylation levels were noted in SCLC compared with NCLM (p < 0.05). As a result, an increased level of OGT and MGEA5 mRNA was related to larger tumor size, nodal metastases, higher grade and tumor behavior according to TFG scale, as well as incidence of disease recurrence (p < 0.05). An inverse association between OGT and MGEA5 transcripts was determined with regard to prognosis (p < 0.05). In addition, the highest OGT and OGA protein levels were observed in poorly differentiated tumors (p < 0.05). No correlations with other parameters were noted, but the results showed a trend of more advanced tumors to be more frequently OGT and OGA positive. The results suggest that increased O-GlcNAcylation may have an effect on tumor aggressiveness and prognosis in laryngeal cancer.

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.

O-GlcNAc Transferase Integrates Metabolic Pathways to Regulate the Stability of c-MYC in Human Prostate Cancer Cells

Metabolic disruptions that occur widely in cancers offer an attractive focus for generalized treatment strategies. The hexosamine biosynthetic pathway (HBP) senses metabolic status and produces an essential substrate for O-linked β-N-acetylglucosamine transferase (OGT), which glycosylates and thereby modulates the function of its target proteins. Here, we report that the HBP is activated in prostate cancer cells and that OGT is a central regulator of c-Myc stability in this setting. HBP genes were overexpressed in human prostate cancers and androgen regulated in cultured human cancer cell lines. Immunohistochemical analysis of human specimens (n = 1987) established that OGT is upregulated at the protein level and that its expression correlates with high Gleason score, pT and pN stages, and biochemical recurrence. RNA interference-mediated siliencing or pharmacologic inhibition of OGT was sufficient to decrease prostate cancer cell growth. Microarray profiling showed that the principal effects of OGT inhibition in prostate cancer cells were related to cell-cycle progression and DNA replication. In particular, c-MYC was identified as a candidate upstream regulator of OGT target genes and OGT inhibition elicited a dose-dependent decrease in the levels of c-MYC protein but not c-MYC mRNA in cell lines. Supporting this relationship, expression of c-MYC and OGT was tightly correlated in human prostate cancer samples (n = 1306). Our findings identify HBP as a modulator of prostate cancer growth and c-MYC as a key target of OGT function in prostate cancer cells.

Exercise and diabetes have opposite effects on the assembly and O-GlcNAc modification of the mSin3A/HDAC1/2 complex in the heart

BackgroundExercise causes physiological cardiac hypertrophy and benefits the diabetic heart. Mammalian switch-independent 3A (mSin3A) and histone deacetylases (HDACs) 1 and 2 regulate hypertrophic genes through associations with the DNA binding proteins repressor element-1 silencing transcription factor (REST) and O-linked β-N-acetylglucosamine transferase (OGT). O-linked β-N-acetylglucosamine (O-GlcNAc) is a glucose derivative that is chronically elevated in diabetic hearts, and a previous study showed that exercise reduces cardiac O-GlcNAc. We hypothesized that O-GlcNAc and OGT would physically associate with mSin3A/HDAC1/2 in the heart, and that this interaction would be altered by diabetes and exercise.Methods8-week-old type 2 diabetic db/db (db) and non-diabetic C57 mice were randomized to treadmill exercise or sedentary groups for 1 or 4 weeks.ResultsO-GlcNAc was significantly higher in db hearts and increased with exercise. Db hearts showed lower levels of mSin3A, HDAC1, and HDAC2 protein, but higher levels of HDAC2 mRNA and HDAC1/2 deacetylase activity. Elevated HDAC activity was associated with significantly blunted expression of α-actin and brain natriuretic peptide in db hearts. In sedentary db hearts, co-immunoprecipitation assays showed that mSin3A and OGT were less associated with HDAC1 and HDAC2, respectively, compared to sedentary C57 controls; however, exercise removed these differences.ConclusionsThese data indicate that diabetes and exercise oppositely affect interactions between pro-hypertrophic transcription factors, and suggest that an increase in total cardiac O-GlcNAc is a mechanism by which exercise benefits type 2 diabetic hearts.