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.     

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.     

Increasing O-GlcNAc levels: An overview of small-molecule inhibitors of O-GlcNAcase

The O-GlcNAc modification is found on many nucleocytoplasmic proteins. The dynamic nature of O-GlcNAc, which in some ways is reminiscent of phosphorylation, has enabled investigators to modulate the stoichiometry of O-GlcNAc on proteins in order to study its function. Although several genetic and pharmacological methods for manipulating O-GlcNAc levels have been described, one of the most direct approaches of increasing global O-GlcNAc levels is by using small-molecule inhibitors of O-GlcNAcase (OGA). As the interest in increasing O-GlcNAc levels has grown, so too has the number of OGA inhibitors. This review provides an overview of the available methods of increasing O-GlcNAc levels, with a special emphasis on inhibition of OGA by small molecules. Known inhibitors of OGA are discussed with particular attention on those most suitable for cell-based biological studies. Several examples in which OGA inhibitors have been used to study the functional role of the O-GlcNAc modification in biological systems are discussed, highlighting the pros and cons of different inhibitors.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

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.     

Tracking of fast moving neuronal vesicles with ageladine A

O-Linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins is a ubiquitous post-translational modification in multicellular organisms studied so far. Since aberrant O-GlcNAcylation has a link with insulin resistance, it is important to establish the status of O-GlcNAcylation in differentiation of mesenchymal cells such as preadipocytes. In this study, we found a differentiation-dependent drastic increase in the level of O-GlcNAcylation in mouse 3T3-L1 preadipocytes. The occurrence of the increase in O-GlcNAcylation, which correlated with the expression of C/EBPα, was in part due to increased expression of O-GlcNAc transferase. In addition to the well-known O-GlcNAcylated proteins such as nucleoporins and vimentin, pyruvate carboxylase, long chain fatty acid-CoA ligase 1, and Ewing sarcoma protein were identified as the proteins which are heavily O-GlcNAcylated with the adipocyte differentiation. Both adipocyte differentiation and the differentiation-dependent increase in O-GlcNAcylation were blocked by 6-diazo-5-oxo-norleucine. These results suggest that O-GlcNAcylation particilates, at least in part, in adipogenesis.     

In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of β-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutininprecipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the Ser⁴¹ residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s). [BMB Reports 2014; 47(10): 593-598]     

HLA class II allele frequencies in the Lebanese population

Aims

Being one of the most polymorphic genetic systems , the Human Leukocyte Antigen system is divided into class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DP, -DQ and -DR). This study is the first and largest of its kind to describe the distribution of HLA-DQB1 and HLA-DRB1 alleles in Lebanon and the region.

Methods

Respectively, 560 and 563 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-DQB1 and HLA-DRB1 alleles using the polymerase chain reaction/sequence specific priming (PCR-SSP) method.

Results

Our data were compared to that of several populations with interesting common findings between the Lebanese, Jordanian, Bahraini, Saudi, Kuwaiti, Tunisian, Korean, Japanese, Thai, Irish, Bulgarian and Polish populations.

Conclusion

These data about the Lebanese population are going to aid future researchers to study the relation of HLA-DQB1 and HLA-DRB1 alleles with major and common diseases in the Lebanese population and will add to the available international literature associated with these loci. In addition it will serve as a reference for the future national bone marrow registry program in our country. We also reviewed the literature for the described association between HLA-DRB1 and -DQB1 loci and different disease entities.     

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.     

Cross-talk between Two Essential Nutrient-sensitive Enzymes: O-GlcNAc TRANSFERASE (OGT) AND AMP-ACTIVATED PROTEIN KINASE (AMPK)*

Nutrient-sensitive pathways regulate both O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK), cooperatively connecting metabolic homeostasis to regulation of numerous intracellular processes essential for life. Similar to phosphorylation, catalyzed by kinases such as AMPK, O-GlcNAcylation is a highly dynamic Ser/Thr-specific post-translational modification of nuclear, cytoplasmic, and mitochondrial proteins catalyzed exclusively by OGT. OGT and AMPK target a multitude of intracellular proteins, with the net effect to protect cells from the damaging effects of metabolic stress. Despite hundreds of studies demonstrating significant overlap in upstream and downstream signaling processes, no study has investigated if OGT and AMPK can directly regulate each other. We show acute activation of AMPK alters the substrate selectivity of OGT in several cell lines and nuclear localization of OGT in C2C12 skeletal muscle myotubes. Nuclear localization of OGT affects O-GlcNAcylation of numerous nuclear proteins and acetylation of Lys-9 on histone 3 in myotubes. AMPK phosphorylates Thr-444 on OGT in vitro; phosphorylation of Thr-444 is tightly associated with AMPK activity and nuclear localization of OGT in myotubes, and phospho-mimetic T444E-OGT exhibits altered substrate selectivity. Conversely, the α- and γ-subunits of AMPK are O-GlcNAcylated, O-GlcNAcylation of the γ1-subunit increases with AMPK activity, and acute inhibition of O-GlcNAc cycling disrupts activation of AMPK. We have demonstrated significant cross-talk between the O-GlcNAc and AMPK systems, suggesting OGT and AMPK may cooperatively regulate nutrient-sensitive intracellular processes that mediate cellular metabolism, growth, proliferation, and/or tissue function.     

Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 °C.     

Extracellular O-linked β-N-acetylglucosamine: Its biology and relationship to human disease

The O-linked β-N-acetylglucosamine(O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, O-GlcNAc transferase(OGT). Recently, an atypical O-GlcNAc transferase, extracellular O-linked β-N-acetylglucosamine(EOGT), which is responsible for the modification of extracellular O-GlcNAc, was identified. Although both OGT and EOGT are regulated through the common hexosamine biosynthesis pathway, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. In Drosophila, loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy, a membrane-anchored apical extracellular matrix protein, was identified as a major O-GlcNAcylated protein, and EOGT mediates Dumpy-dependent cell adhesion. In mammals, extracellular O-GlcNAc was detected on extracellular proteins including heparan sulfate proteoglycan 2, Nell1, laminin subunit alpha-5, Pamr1, and transmembrane proteins, including Notch receptors. Although the physiological function of O-GlcNAc in mammals has not yet been elucidated, exome sequencing identified homozygous EOGT mutations in patients with Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects. This review summarizes the current knowledge of extracellular O-GlcNAc and its implications in the pathological processes in Adams-Oliver syndrome.