O-GlcNAcase Inhibitor Research Articles

O-GlcNAcase inhibition by Thiamet G opposes acute cardiac decompensation in rats with chronic heart failure

Exacerbation of heart failure (HF), better known as acute decompensated HF, is characterized by dyspnea, edema and fatigue, which is associated with a high morbi-mortality. O-linked β-N-acetylglucosamine (O-GlcNAc) is a key regulator of the cellular stress response and increased O-GlcNAc levels exerts protective effects in models of acute vascular injury and ischemia-reperfusion injury. However, whether increasing O-GlcNAc exerts similar protective effects in acute decompensation of HF is unknown. We evaluated the cardiac effects of increasing O-GlcNAc levels by inhibiting O-GlcNAcase with Thiamet G (TG) in a rat model of acute decompensated HF. Three months after coronary artery ligation resulting in established HF, rats received 2.5 ml water (control) or 1.8 g/kg NaCl (dissolved in 2.5 ml water) provoking acute decompensation. Left ventricular (LV) fractional shortening and cardiac output (echography) as well as LV tissue perfusion (MRI) were assessed 1 and 14 days after NaCl-loading, and TG was administered 12 hours after NaCl (25 mg/kg IP). NaCl provoked acute decompensation in rats with HF. Indeed, 1 day after NaCl, the HF-related impairments of LV fractional shortening, cardiac output and myocardial tissue perfusion were aggravated. Fourteen days after NaCl, cardiac output partially recovered, but fractional shortening and myocardial perfusion remained impaired. As soon as 12 hours after administration, TG improved fractional shortening, cardiac output and tended to increase myocardial perfusion. Moreover, 14 days after TG, cardiac output and myocardial perfusion were still improved. These data show that in an experimental model salt-induced acute decompensation of HF, a curative treatment with TG improves both cardiac output as well as myocardial perfusion. The cellular mechanisms, i.e. post-translational protein O-GlcNAcylation, implicated in the effects of TG, are currently under investigation.

Disruption of O-GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization.

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O-GlcNAcylation-the addition of a single sugar residue (O-linked β-N-acetylglucosamine) on proteins-is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O-GlcNAcylation is mediated by O-GlcNAc transferase (OGT), which adds O-GlcNAc onto proteins, and O-GlcNAcase (OGA), which removes it. Here we investigated O-GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O-GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O-GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O-GlcNAc function, we disrupted O-GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet-G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O-GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O-GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.

Diabetic Hyperglycemia Regulates Potassium Channels and Arrhythmias in the Heart via Autonomous CaMKII Activation by O-Linked Glycosylation

Diabetic cardiomyopathy is associated with altered cardiac repolarization, K+ current density and arrhythmia risk. However, little is known about how acute hyperglycemia regulates cardiac K+ channels and arrhythmias. We measured action potentials (APs) and K+ currents in isolated mouse ventricular myocytes exposed to high glucose (30 mM) versus control (5.5 mM glucose+24.5 mM mannitol to match osmolarity). Acute hyperglycemia reduced transient outward K+ current (Ito) amplitude but enhanced Ito recovery from inactivation and increased inward rectifier K+ current (IK1). These changes were prevented by inhibitors of O-linked N-acetylglucosamine modification (O-GlcNAcylation), Osmi-I and DON. Conversely, O-GlcNAcase inhibitor thiamet-G produced the same K+ current effects as hyperglycemia. Thus O-GlcNAcylation is necessary and sufficient for these K+ channel effects. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by CaMKII inhibitor AIP, in CaMKIIδ-knockout myocytes and in mice in which a known O-GlcNAcylated Serine on CaMKII was mutated (S280A knock-in). Acute hyperglycemia slightly prolonged AP duration, increased delayed afterdepolarizations and inducibility of AP-duration alternans during tachypacing in WT only (not in CaMKIIδ-KO or S280A-KI). Thus, acute hyperglycemia-induced O-GlcNAcylation of CaMKII and/or K+ channels may contribute to arrhythmogenesis. In chronic diabetic models (streptozotocin & db/db) Ito and IK1 were both downregulated (as in heart failure and chronic CaMKII activation). AP duration was significantly prolonged in diabetes, and delayed afterdepolarizations and alternans were enhanced at normoglycemia and worsened by hyperglycemia. We conclude that K+ channels are differently modulated by acute versus chronic hyperglycemia and O-GlycNAcylation-dependent CaMKII activation. Acute hyperglycemia enhances K+ channel function via O-GlcNAcylation of CaMKII at Ser280, which may also promote arrhythmogenic SR Ca2+ leak. However, chronic hyperglycemia and CaMKII activation appear to downregulate K+ channel expression, which also promotes arrhythmia susceptibility.

Structural studies of a surface-entropy reduction mutant of O-GlcNAcase.

The enzyme O-GlcNAcase catalyses the removal of the O-GlcNAc co/post-translational modification in multicellular eukaryotes. The enzyme has become of acute interest given the intimate role of O-GlcNAcylation in tau modification and stability; small-molecular inhibitors of human O-GlcNAcase are under clinical assessment for the treatment of tauopathies. Given the importance of structure-based and mechanism-based inhibitor design for O-GlcNAcase, it was sought to test whether different crystal forms of the human enzyme could be achieved by surface mutagenesis. Guided by surface-entropy reduction, a Glu602Ala/Glu605Ala variant [on the Gly11-Gln396/Lys535-Tyr715 construct; Roth et al. (2017), Nature Chem. Biol. 13, 610-612] was obtained which led to a new crystal form of the human enzyme. An increase in crystal contacts stabilized disordered regions of the protein, enabling 88% of the structure to be modelled; only 83% was possible for the wild-type construct. Although the binding of the C-terminus was consistent with the wild type, Lys713 in monomer A was bound in the -1 subsite of the symmetry-related monomer A and the active sites of the B monomers were vacant. The new crystal form presents an opportunity for enhanced soaking experiments that are essential to understanding the binding mechanism and substrate specificity of O-GlcNAcase.

Glucosamine improves survival in a mouse model of sepsis and attenuates sepsis-induced lung injury and inflammation

The aim of the current study was to investigate the effects of glucosamine (GlcN) on septic lethality and sepsis-induced inflammation using animal models of mice and zebrafish. GlcN pretreatment improved survival in the cecal ligation and puncture (CLP)-induced sepsis mouse model and attenuated lipopolysaccharide (LPS)-induced septic lung injury and systemic inflammation. GlcN suppressed LPS-induced M1-specific but not M2-specific gene expression. Furthermore, increased expressions of inflammatory genes in visceral tissue of LPS-injected zebrafish were suppressed by GlcN. GlcN suppressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and NF-κB in lung tissue. LPS triggered a reduction in O-GlcNAc levels in nucleocytoplasmic proteins of lung, liver, and spleen after 1 day, which returned to normal levels at day 3. GlcN inhibited LPS-induced O-GlcNAc down-regulation in mouse lung and visceral tissue of zebrafish. Furthermore, the O-GlcNAcase (OGA) level was increased by LPS, which were suppressed by GlcN in mouse and zebrafish. OGA inhibitors suppressed LPS-induced expression of inflammatory genes in RAW264.7 cells and the visceral tissue of zebrafish. Stable knockdown of Oga via short hairpin RNA led to increased inducible nitric oxide synthase (iNOS) expression in response to LPS with or without GlcN in RAW264.7 cells. Overall, our results demonstrate a protective effect of GlcN on sepsis potentially through modulation of O-GlcNAcylation of nucleocytoplasmic proteins.

O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis

The addition of a single β-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after β-estradiol (E2) addition. We observed that during G1E-ER4 differentiation, overall O-GlcNAc levels decrease, and physical interactions of GATA-1 with both OGT and OGA increase. RNA-Seq-based transcriptome analysis of G1E-ER4 cells differentiated in the presence of the OGA inhibitor Thiamet-G (TMG) revealed changes in expression of 433 GATA-1 target genes. ChIP results indicated that the TMG treatment decreases the occupancy of GATA-1, OGT, and OGA at the GATA-binding site of the lysosomal protein transmembrane 5 (Laptm5) gene promoter. TMG also reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. Sustained treatment of G1E-ER4 cells with TMG before differentiation reduced hemoglobin-positive cells and increased stem/progenitor cell surface markers. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for O-GlcNAcylation in regulating hematopoietic cell fate.

Effects of Global O-GlcNAcylation on Galectin Gene-expression Profiles in Human Cancer Cell Lines.

The effects of O-linked β-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA) inhibitors on galectin gene expression profiles were examined in MCF7, HT-29, and HL-60 cancer cell lines. Cell cultures were treated for 24 h with OGA inhibitor thiamet G or OGT inhibitor 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-glucopyranose, and global O-GlcNAc levels and expression of galectin genes were determined using an immunodot blot assay and real-time quantitative polymerase chain reaction. Two galectin genes, LGALS3 in MCF7 cells and LGALS12 in HL-60 cells, were up-regulated by O-GlcNAc, whereas other cell-specific galectins were unresponsive to changes in O-GlcNAc level. Of interest, basal levels of O-GlcNAc in resting HL-60 and HT-29 cells were significantly higher than those in cells differentiated into neutrophilic or enterocytic lineages, respectively. O-GlcNAc-mediated signaling pathways may be involved in regulating the expression of only a limited number of galectin genes. Additional O-GlcNAc-dependent mechanisms may work at the protein level (galectin secretion and intracellular localization) and warrant further investigation.

Modification of the Thioglycosyl-Naphthalimides as Potent and Selective Human O-GlcNAcase Inhibitors.

β-N-Acetylhexosaminidases are widely distributed exoglycosidases and have attracted significant attention due to their important roles in the field of pesticide and drug discovery. Remarkably, human O-GlcNAcase (hOGA) and human β-N-acetylhexosaminidase (HsHex) possess the same catalytic mechanism but play different physiological actions in vivo. In this Letter, we aim to improve the inhibitory potency and selectivity of previously reported thioglycosyl-naphthalimides against hOGA. The rational compound design led to the synthesis of 13r bearing a 4-piperidylnaphthalimide moiety as a highly potent hOGA inhibitor (K i = 0.6 μM against hOGA) with good selectivity (K i > 100 μM against HsHexB). Furthermore, to investigate the basis for the potency and selectivity of 13r against hOGA, the possible inhibitory mechanisms of selected inhibitors (15b, 13b, and 13r) against hOGA and HsHexB were studied using molecular docking and MD simulations. These 4-substituted naphthalimide thioglycosides may potentially serve as useful tools for the further study of the function of hOGA.

Novel glucopyranoside C2-derived 1,2,3-triazoles displaying selective inhibition of O-GlcNAcase (OGA)

O-GlcNAcylation or O-GlcNAc modification is a post-translational modification of several proteins responsible for fundamental cellular processes. Dysregulation of the O-GlcNAc pathway has been linked to the etiology of several diseases such as neurodegenerative and cardiovascular diseases, type 2 diabetes and cancer. O-GlcNAcase (OGA) catalyzes the removal of O-GlcNAc from the modified proteins and several carbohydrate-based OGA inhibitors have been synthesized to understand the role of O-GlcNAc-modified proteins in physiological and pathological conditions. However, many of the inhibitors lack selectivity for OGA over lysosomal hexosaminidases A and B. Aiming the selectively inhibition of OGA, we propose herein the synthesis of twelve novel glucopyranoside derivatives exploring the bioisosteric replacement of the GlcNAc 2-acetamide group by 1,4-disubstituted 1,2,3-triazole ring, bearing a variety of central chains with different shapes. Compounds were readily prepared through "Copper(I) Catalyzed Azide/Alkyne Cycloaddition" (CuAAC) reaction between a sugar azide and different terminal alkynes. Initial Western Blot analyses and further inhibitory assays proved that compounds 6a (IC50 = 0.50 ± 0.02 μM, OGA), 6k (IC50 = 0.52 ± 0.01 μM, OGA) and 6l (IC50 = 0.72 ± 0.02 μM, OGA) were the most potent and selective compounds of the series. Structure-activity relationship analyses and molecular docking simulations demonstrated that the bridge of two-carbon atoms between the C-4 position of the triazole and the phenyl ring (6a), which may be replaced by heteroatoms such as N (6k) or O (6l), is fundamental for accommodation and inhibition within OGA catalytic pocket.

Interplay Between Phosphorylation and O-GlcNAcylation of Sarcomeric Proteins in Ischemic Heart Failure.

Post-translational modifications (PTMs) of sarcomeric proteins could participate to left ventricular (LV) remodeling and contractile dysfunction leading in advanced heart failure (HF) with altered ejection fraction. Using an experimental rat model of HF (ligation of left coronary artery) and phosphoproteomic analysis, we identified an increase of desmin phosphorylation and a decrease of desmin O-N-acetylglucosaminylation (O-GlcNAcylation). We aim to characterize interplay between phosphorylation and O-GlcNAcylation for desmin in primary cultures of cardiomyocyte by specific O-GlcNAcase (OGA) inhibition with thiamet G and silencing O-GlcNAc transferase (OGT) and, in perfused heart perfused with thiamet G in sham- and HF-rats. In each model, we found an efficiency of O-GlcNAcylation modulation characterized by the levels of O-GlcNAcylated proteins and OGT expression (for silencing experiments in cells). In perfused heart, we found an improvement of cardiac function under OGA inhibition. But none of the treatments either in in vitro or ex vivo cardiac models, induced a modulation of desmin, phosphorylated and O-GlcNAcylated desmin expression, despite the presence of O-GlcNAc moities in cardiac desmin. Our data suggests no interplay between phosphorylation and O-GlcNAcylation of desmin in HF post-myocardial infarction. The future requires finding the targets in heart involved in cardiac improvement under thiamet G treatment.

Evaluation of a PET Radioligand to Image O-GlcNAcase in Brain and Periphery of Rhesus Monkey and Knock-Out Mouse.

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 μSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.

The effects of O-GlcNAc alteration on Alzheimer-like neurodegeneration in SK-N-SH cells.

O-GlcNAcylation is a highly dynamic post-translational modification that plays a key role in regulating phosphorylation of protein and cell survival. The proteins O-GlcNAcylation level is regulated dynamically by O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA). Although previous studies have suggested the role of O-GlcNAcylation in neurodegenerative diseases, the mechanism of O-GlcNAcylation in Alzheimer's disease (AD) remains unclear. The decrease of O-GlcNAcylation by alloxan, an OGT inhibitor, and increase by NAG-thiazolines (NAG-Ae), an O-GlcNAcase inhibitor were tested to investigate the effects of O-GlcNAc alteration on AD like neurodegeneration in SK-N-SH cells. The level of O-GlcNAcylation was decreased in alloxan treated cells and increased in NAG-Ae treated cells. Meanwhile, it was observed that both abnormal phosphorylation of NFs in cell bodies and apoptosis induced by alloxan treatment can be resisted by pretreatment or simultaneous treatment with appropriate NAG-Ae. These results demonstrated that increasing O-GlcNAc with NAG-Ae protected AD like neurodegeneration from NFs hyperphosphorylation and the cell loss, suggesting the role of O-GlcNAc in the pathogenesis and therapy of AD.

Gideon J. Davies

Angewandte Chemie International EditionVolume 57, Issue 39 p. 12620-12620 Author ProfileFree Access Gideon J. Davies First published: 13 August 2018 https://doi.org/10.1002/anie.201803117AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract “I would have liked to have discovered gene editing. My favorite author (fiction) is Jane Austen …” Find out more about Gideon J. Davies in his Author Profile. Gideon J. Davies The author presented on this page has published more than 10 articles since 2002 in Angewandte Chemie, most recently: “A Convenient Approach to Stereoisomeric Iminocyclitols: Generation of Potent Brain-Permeable OGA Inhibitors”: M. Bergeron-Brlek et al., Angew. Chem. Int. Ed. 2015, 54, 15429; Angew. Chem. 2015, 127, 15649. The work of G. J. Davies has been featured on the cover of Angewandte Chemie: “A Glycosynthase Catalyst for the Synthesis of Flavonoid Glycosides”: M. Yang, G. J. Davies, B. G. Davis, Angew. Chem. Int. Ed. 2007, 46, 3885; Angew. Chem. 2007, 119, 3959. Date of birth: July 6, 1964 Position: Professor of Chemistry, University of York E-mail: gideon.davies@york.ac.uk Homepage: http://www.york.ac.uk/chemistry/staff/academic/d-g/gdavies/ Education: 1995 BSc (Hons) Biochemistry, University of Bristol 1990 PhD supervised by Herman Watson and Len Hall, University of Bristol 1990–1991 Postdoctoral work with Keith Wilson, EMBL Hamburg 1991–1996 Postdoctoral work in York, with periods in Grenoble and Uppsala Awards: 2006 Whistler Prize, International Carbohydrate Organization; 2010 GlaxoSmithKline Award, Biochemical Society; 2010 Gabor Medal, The Royal Society; 2010 Fellow of the Royal Society; 2014 Khorana Prize, Royal Society of Chemistry; 2015 Davy Medal, The Royal Society; 2017 Ken Murray Research Professorship, The Royal Society Current research interests: Mammalian glycobiology; O-GlcNAc and neurodegeneration; conformational analysis of enzyme reaction coordinates; enzymes for biofuels and polysaccharide degradation. Hobbies: Walking, sailing, hockey, cricket, chemistry of Vitis vinifera I would have liked to have discovered gene editing. My favorite author (fiction) is Jane Austen. The most amusing chemistry adventure in my career was exploding logwood chips over my parents' bathroom walls; the concept of pH-sensitive wallpaper never took off, sadly. My favorite food is the culinary alchemy of poulet aux vin jaune et aux morilles. If I won the lottery, I would buy a yacht and sail the world (or maybe just its warmer parts), in between tending my vines. When I'm frustrated, I walk to quiet outdoor places and take in nature. The most important thing I learned from my parents is a passion for learning. If I could have dinner with three famous scientists from history, they would be Hypatia, Richard Feynman, Joseph Banks. My favorite place on earth is the west coast of Scotland—after a storm when the sky is blue and the salmon are leaping up rain-fed waterfalls. The biggest problem that scientists face is a lack of quality time. My 5 top papers: 1“Substrate Distortion by a ß-Mannanase: Snapshots of the Michaelis and Covalent-Intermediate Complexes Suggest a B2,5 Conformation for the Transition State”: V. M.-A. Ducros, D. L. Zechel, G. N. Murshudov, H. J. Gilbert, L. Szabó, D. Stoll, S. G. Withers, G. J. Davies, Angew. Chem. Int Ed 2002, 41, 2824; Angew. Chem. 2002, 114, 2948. (The unusual conformational itinerary, through a boat conformation, of enzymes acting on the sugar mannose.) 2“Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate”: D. J. Vocadlo, G. J. Davies, R. Laine, S. G. Withers, Nature 2001, 412, 835. (Only by applying analysis to lysozyme did people take notice of the evidence for covalent intermediates on retaining glycosidases.) 3“A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo”: S. A. Yuzwa et al., Nat. Chem. Biol. 2008, 4, 483. (Toxic tau species can be reduced using a mechanism and structure-based inhibitor of the O-GlcNAc hydrolase.) 4“The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases”: K. E. H. Frandsen et al., Nat. Chem. Biol. 2016, 12, 298. (Deep chemical insight, from a European consortium, into the action of recently discovered copper-containing lytic polysaccharide monooxygenases.) 5“Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes”: J. Jiang et al., ACS Central Science 2016, 2, 351. (European collaboration that produced mechanism-based probes that function as clinical diagnostics for Pompe disease.) Volume57, Issue39September 24, 2018Pages 12620-12620 ReferencesRelatedInformation

Parallel study on protein O-GlcNAcylation in prostate cancer cell with a sensitive microarray biochip

Although a variety of approaches have been developed to analyze protein O-GlcNAcylation, efficient investigations on O-GlcNAcylation of proteins of interest in high-throughput manner are still in high demand to further explore its functionality. In this work, we first develop a powerful microarray platform for a sensitive, specific and high-throughput analysis of protein O-GlcNAcylation. The developed array biochip is then utilized to parallelly analyze the O-GlcNAcylation of three oncogenic transcription factors C-Myc, NF-κB and p53 in normal prostate epithelial cell (RWPE-1) and prostate cancer cell line (PC-3). The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are also monitored by the microarray platform. The experimental results show that the overall O-GlcNAcylation and OGT expression level are obviously elevated in PC-3 as compared to RWPE-1. The protein expression-normalized O-GlcNAcylation of C-Myc and NF-κB in PC-3 is significantly higher than that in RWPE-1, while opposite result is observed from p53. In addition, the biological behaviors including proliferation and migration of PC-3 cells are also studied when OGA inhibitor Thiamet G is applied to elevate the total O-GlcNAcylation level.

O-GlcNAc transferase associates with the MCM2\u20137 complex and its silencing destabilizes MCM\u2013MCM interactions

O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O-GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.

O-deGlcNAcylation is required for Entamoeba histolytica-induced HepG2 cell death

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. E. histolytica can induce host-cell apoptosis by initiating various intracellular signaling mechanisms closely associated with tissue pathogenesis and parasitic immune evasion. O-GlcNAcylation, similar to phosphorylation, is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, whether O-GlcNAc alterations in host cells affect E. histolytica-induced cell death and which signal molecules participate in E. histolytica-induced deglycosylation remain unknown. In this study, co-incubation of HepG2 cells with E. histolytica increased DNA fragmentation and LDH release as compared with control cells. Additionally, Gal-lectin-mediated amoebic adherence of live trophozoites to HepG2 cells decreased O-GlcNAcylated protein levels within 5 min. We also observed a rapid decrease in cellular OGT protein level, but not OGA, in HepG2 cells in a contact-dependent manner. Furthermore, HepG2 pretreatment with OGA inhibitors or OGA siRNA prevented E. histolytica-induced O-deGlcNAcylation, DNA fragmentation, and LDH release. Our results suggested that E. histolytica-induced O-deGlcNAcylation in HepG2 cells was an important process required for hepatocyte cell death induced by E. histolytica adherence.

Angiotensin-(1-7) Attenuates Protein O-GlcNAcylation in the Retina of Mice Fed a High-Fat Diet

Increased retinal protein O-GlcNAcylation occurs in response to hyperglycemia and contributes to diabetic retinopathy (DR). Alternatively, systemic pharmacological blockade of the Renin-Angiotensin System (RAS) lowers the incidence of DR. The classic RAS that systemically regulates blood pressure is defined by the ACE/AngII/AT1R axis; this is counter-regulated by actions of the ACE2/Angiotensin-(1-7) (Ang1-7)/Mas axis of the RAS. Increased endogenous production of Ang1-7 is responsible for many of the beneficial effects of cardioprotective RAS blockers such as ACE inhibitors; however the mechanism whereby DR outcomes are improved remains unresolved. The hypothesis here is that Ang1-7 acts to reduce retinal O-GlcNAcylation by activating Protein Kinase A (PKA) and thereby inhibiting the rate-limiting enzyme of the hexosamine biosynthetic pathway, glutamine-fructose-6-phosphate amidotransferase (GFAT). Mice fed a 60% high fat diet (HFD) for 11 weeks and treated with the ACE inhibitor captopril (50 mg/L) for 3 weeks exhibited a Mas receptor-dependent attenuation of retinal protein O-GlcNAcylation as compared to vehicle treated controls. To determine the mechanism by which Ang1-7 regulates O-GlcNAcylation, R28 retinal neurons were treated with Ang1-7 and the adenylate cyclase activator forskolin. Both Ang1-7 and forskolin increased cAMP concentrations and attenuated levels of protein O-GlcNAcylation induced by treatment with the O-GlcNAcase inhibitor thiamet G. Classically, cAMP activates PKA, which phosphorylates and inhibits GFAT. Overall, these studies establish the first link between dysregulation of the well-studied physiological RAS and diabetes-induced retinal tissue damage as a consequence of excessive protein O-GlcNAcylation. Disclosure S. Dierschke: None. A.C. Arnold: None. A.J. Barber: None. M.D. Dennis: None.

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

Early intervention of tau pathology prevents behavioral changes in the rTg4510 mouse model of tauopathy.

Although tau pathology, behavioral deficits, and neuronal loss are observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Here we found that rTg4510 mice, which overexpress human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity (LMA), which correlates with neurofibrillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To further clarify the relationship between these endpoints, we treated the rTg4510 mice with either doxycycline to reduce mutant tau expression or an O-GlcNAcase inhibitor Thiamet G, which has been shown to ameliorate tau pathology in animal models. We found that both doxycycline and Thiamet G treatments starting at 2 months of age prevented the progression of hyperactivity, slowed brain atrophy, and reduced brain hyperphosphorylated tau. In contrast, initiating doxycycline treatment at 4 months reduced neither brain hyperphosphorylated tau nor hyperactivity, further confirming the relationship between these measures. Collectively, our results demonstrate a unique behavioral phenotype in the rTg4510 mouse model of tauopathy that strongly correlates with disease progression, and that early interventions which reduce tau pathology ameliorate the progression of the locomotor dysfunction. These findings suggest that better understanding the relationship between locomotor deficits and tau pathology in the rTg4510 model may improve our understanding of the mechanisms underlying behavioral disturbances in patients with tauopathies.

Sustained O‐GlcNAcylation Amplifies Erk Signaling

O‐linked N‐Acetylglucosamine, more commonly referred to as O‐GlcNAc, is a single glycosidic post‐translational modification. OGT (O‐GlcNAc Transferase) and OGA (O‐GlcNAcase) are the sole enzymes responsible for the addition (O‐GlcNAcylation) and removal of O‐GlcNAc, respectively. O‐GlcNAc, OGA, and OGT are present in all multicellular organisms and organ tissues. Importantly, OGT knockouts are embryonically lethal, demonstrating the requirement for O‐GlcNAcylation in early organismal development. Like most intracellular post‐translational modifications, O‐GlcNAc regulates a number of cellular pathways including mitochondrial respiration, cell cycle progression, and transcription. Recently, we demonstrated that prolonged treatment of SY5Y neuroblastoma cells with Thiamet‐G (TMG), an OGA inhibitor, caused a dramatic reprogramming of the transcriptome. Therefore, we performed next generation RNA sequencing to investigate the transcriptome changes from isolated liver tissues from mice treated with TMG for two weeks. We also performed the same analysis for liver tissue containing an OGT knockout (OGT‐KO) to compare the effects of sustained O‐GlcNAcylation to the loss of O‐GlcNAcylation. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differences in expression for gene sets between the two groups. Further analysis of the data demonstrated an inverse correlation of transcriptional changes for several gene families between TMG‐treated and OGT‐KO. MAPK/ERK signaling genes were among these families, which we found to be of interest. ERK (extracellular regulated protein kinase) phosphorylates a wide variety of protein substrates, regulating processes such as apoptosis, transcription, and cell cycle progression. OGT‐KO was predicted to down‐regulate this pathway while our previous data predicted TMG to up‐regulate the pathway. To test this observation, we grew HeLa cells in TMG for three weeks and serum starved them overnight. The following day, we returned the serum to the media and harvested the lysate at 0, 5,10, 20, 60, and 120 minute time points. When we performed a western blot analysis of the results, the TMG‐treated group demonstrated dramatic amplification of pERK. This observation demonstrates a novel role for O‐GlcNAc as a regulator of the ERK signaling pathway.Support or Funding InformationNIH NIDDK R01 DK100595This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.