Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects

Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G₁/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.     

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings express-ing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca^2 ]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H202. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca^2 ]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.     

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.     

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.     

E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.     

Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation

Ser(Thr)-O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous modification of nucleocytoplasmic proteins. Extensive crosstalk exists between O-GlcNAcylation and phosphorylation, which regulates signaling in response to nutrients/stress. The development of novel O-GlcNAc detection and enrichment methods has improved our understanding of O-GlcNAc functions. Mass spectrometry has revealed O-GlcNAc’s many interactions with phosphorylation-mediated signaling. However, mechanisms regulating O-GlcNAcylation and phosphorylation are quite different. Phosphorylation is catalyzed by hundreds of distinct kinases. In contrast, in mammals, uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyl transferase (OGT) and β-D-N-acetylglucosaminidase (OGA) are encoded by single highly conserved genes. Both OGT’s and OGA’s specificities are determined by their transient associations with many other proteins to create a multitude of specific holoenzymes. The extensive crosstalk between O-GlcNAcylation and phosphorylation represents a new paradigm for cellular signaling.     

Multiple Tissue-specific Roles for the O-GlcNAc Post-translational Modification in the Induction of and Complications Arising from Type II Diabetes

In this minireview, we will highlight work in the last 30 years that has clearly demonstrated that the O-GlcNAc modification is nutrient-responsive and plays multiple roles in metabolic regulation of signaling and gene expression. Further, we will examine recent studies that have investigated the impact of O-GlcNAc in a variety of glucose- and insulin-responsive tissues and the roles attributed to O-GlcNAc in the induction of insulin resistance and glucose toxicity, the hallmarks of type II diabetes mellitus. We will also summarize potential causal roles for the O-GlcNAc modification in complications associated with diabetes.