Screening-based discovery of drug-like O-GlcNAcase inhibitor scaffolds

O-GlcNAcylation is an essential posttranslational modification in metazoa. Modulation of O-GlcNAc levels with small molecule inhibitors of O-GlcNAc hydrolase (OGA) is a useful strategy to probe the role of this modification in a range of cellular processes. Here we report the discovery of novel, low molecular weight and drug-like O-GlcNAcase inhibitor scaffolds by high-throughput screening. Kinetic and X-ray crystallographic analyses of the binding modes with human/bacterial O-GlcNAcases identify some of these as competitive inhibitors. Comparative kinetic experiments with the mechanistically related human lysosomal hexosaminidases reveal that three of the inhibitor scaffolds show selectivity towards human OGA. These scaffolds provide attractive starting points for the development of non-carbohydrate, drug-like OGA inhibitors.     

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Background

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.

Methods and results

Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.

Conclusions

The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.

General significance

Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.     

O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase

Increasing cellular O-GlcNAc levels through pharmacological inhibition of O-GlcNAcase, the enzyme responsible for removal of the O-GlcNAc post-translational modification, is being increasingly used to aid in discerning the roles played by this form of intracellular glycosylation. Interestingly, two forms of O-GlcNAcase have been studied; a full-length isoform that is better characterized, and a shorter nuclear-localized variant, arising from failure to splice out one intron, which has not been as well characterized. Given the increasing use of O-GlcNAcase inhibitors as research tools, we felt that a clear understanding of how these inhibitors affect both isoforms of O-GlcNAcase is important for proper interpretation of studies making use of these inhibitors in cell culture and in vivo. Here we describe an enzymatic characterization of the nuclear variant of human O-GlcNAcase. We find that this short nuclear variant of O-GlcNAcase, which has the identical catalytic domain as the full-length enzyme, has similar trends in a pH-rate profile and Taft linear free energy analysis as the full-length enzyme. These findings strongly suggest that both enzymes use broadly similar transition states. Consistent with this interpretation, the short isoform is potently inhibited by several previously described inhibitors of full-length O-GlcNAcase including PUGNAc, NAG-thiazoline, and the selective O-GlcNAcase inhibitor NButGT. These findings contrast with earlier studies and suggest that studies using O-GlcNAcase inhibitors in cultured cells or in vivo can be interpreted with the knowledge that both these forms of O-GlcNAcase are inhibited when present.     

Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells

Increased modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) has been implicated in the development of diabetic cardiomyopathy. We used the well-characterized ES cells (Nkx2.5GFP knock-in ES cells), to investigate the role of O-GlcNAcylation in cardiomyocyte development. O-GlcNAcylation decreased in differentiating ES cells, as did the expression of O-GlcNAc transferase. Increasing O-GlcNAcylation with glucosamine or by inhibiting N-acetylglucosaminidase (streptozotocin or PUGNAc) decreased the number of cardiomyocyte precursors and cardiac-specific gene expression. On the other hand, decreasing O-GlcNAcylation with an inhibitor of glutamine fructose-6-phosphate amidotransferase (6-diazo-5-oxo-norleucine) increased cardiomyocyte precursors. These results suggest that excessive O-GlcNAcylation impairs cardiac cell differentiation in ES cells.     

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (O-GlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced O-GlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer’s and Parkinson’s diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.     

Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain

The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent ‘pseudo’-AT.     

Murine platelets are not regulated by O-linked beta-N-acetylglucosamine

It is generally appreciated that platelets derived from diabetic patients display increased responsiveness to low levels of agonists. O-GlcNAcylation has been linked to hyperglycemia-related effects in other tissues; therefore we examined this modification in platelets to determine if O-GlcNAcylation affects platelet function. This post-translational modification consists of an N-acetylglucosamine attached to serine and/or threonine residues. We examined O-GlcNAc levels in platelets from a hyperglycemic murine model of Type I diabetes with known hypersensitivity to agonists and a Type II diabetes model (ob/ob) lacking detectable alterations in the aggregation profile. Neither model showed marked increases in protein O-GlcNAcylation. Treatment of platelets with multiple O-GlcNAcase inhibitors led to O-GlcNAc accumulation on multiple platelet proteins. However, the inhibitor-induced accumulation of this modification does not correlate with any gross alterations in platelet aggregation. These data suggest that while the modification occurs in platelets, their activity is not globally sensitive to O-GlcNAc levels.     

Monitoring protein O-linked β-N-acetylglucosamine status via metabolic labeling and copper-free click chemistry

O-Linked β-N-acetylglucosamine (O-GlcNAc) modification found on the serine and threonine residues of intracellular proteins is an inducible post-translational modification that regulates numerous biological processes. In combination with other cell biological and biochemical approaches, a robust and streamlined strategy for detecting the number and stoichiometry of O-GlcNAc modification can provide valuable insights for decoding the functions of O-GlcNAc at the molecular level. Here, we report an optimized workflow for evaluating the O-GlcNAc status of proteins using a combination of metabolic labeling and click chemistry-based mass tagging. This method is strategically complementary to the chemoenzymatic-based mass-tagging method.     

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca²⁺ with EGTA or blocking store operated Ca²⁺ entry with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.     

MicroRNA-539 Is Up-regulated in Failing Heart,and Suppresses O-GlcNAcase Expression

Derangements in metabolism and related signaling pathways characterize the failing heart. One such signal, O-linked β-N-acetylglucosamine (O-GlcNAc), is an essential post-translational modification regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase (OGA), which modulate the function of many nuclear and cytoplasmic proteins. We recently reported reduced OGA expression in the failing heart, which is consistent with the pro-adaptive role of increased O-GlcNAcylation during heart failure; however, molecular mechanisms regulating these enzymes during heart failure remain unknown. Using miRNA microarray analysis, we observed acute and chronic changes in expression of several miRNAs. Here, we focused on miR-539 because it was predicted to target OGA mRNA. Indeed, co-transfection of the OGA-3′UTR containing reporter plasmid and miR-539 overexpression plasmid significantly reduced reporter activity. Overexpression of miR-539 in neonatal rat cardiomyocytes significantly suppressed OGA expression and consequently increased O-GlcNAcylation; conversely, the miR-539 inhibitor rescued OGA protein expression and restored O-GlcNAcylation. In conclusion, this work identifies the first target of miR-539 in the heart and the first miRNA that regulates OGA. Manipulation of miR-539 may represent a novel therapeutic target in the treatment of heart failure and other metabolic diseases.     

Inhibition of mTOR affects protein stability of OGT

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.