Multi-Omics after O-GlcNAc Alteration Identifies Cellular Processes Working Synergistically to Promote Aneuploidy.

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can synergistically fine-tune multiple cellular pathways.

A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

O-GlcNAc cycling mediates energy balance by regulating caloric memory.

Caloric need has long been thought a major driver of appetite. However, it is unclear whether caloric need regulates appetite in environments offered by many societies today where there is no shortage of food. Here we observed that wildtype mice with free access to food did not match calorie intake to calorie expenditure. While the size of a meal affected subsequent intake, there was no compensation for earlier under- or over-consumption. To test how spontaneous eating is subject to caloric control, we manipulated O-linked ß-N-acetylglucosamine (O-GlcNAc), an energy signal inside cells dependent on nutrient access and metabolic hormones. Genetic and pharmacological manipulation in mice increasing or decreasing O-GlcNAcylation regulated daily intake by controlling meal size. Meal size was affected at least in part due to faster eating speed. Without affecting meal frequency, O-GlcNAc disrupted the effect of caloric consumption on future intake. Across days, energy balance was improved upon increased O-GlcNAc levels and impaired upon removal of O-GlcNAcylation. Rather than affecting a perceived need for calories, O-GlcNAc regulates how a meal affects future intake, suggesting that O-GlcNAc mediates a caloric memory and subsequently energy balance.

The autophagy receptor p62/SQST-1 promotes proteostasis and longevity in C. elegans by inducing autophagy.

Autophagy can degrade cargos with the help of selective autophagy receptors such as p62/SQSTM1, which facilitates the degradation of ubiquitinated cargo. While the process of autophagy has been linked to aging, the impact of selective autophagy in lifespan regulation remains unclear. We have recently shown in Caenorhabditis elegans that transcript levels of sqst-1/p62 increase upon a hormetic heat shock, suggesting a role of SQST-1/p62 in stress response and aging. Here, we find that sqst-1/p62 is required for hormetic benefits of heat shock, including longevity, improved neuronal proteostasis, and autophagy induction. Furthermore, overexpression of SQST-1/p62 is sufficient to induce autophagy in distinct tissues, extend lifespan, and improve the fitness of mutants with defects in proteostasis in an autophagy-dependent manner. Collectively, these findings illustrate that increased expression of a selective autophagy receptor is sufficient to induce autophagy, enhance proteostasis and extend longevity, and demonstrate an important role for sqst-1/p62 in proteotoxic stress responses.

Elevated O-GlcNAcylation enhances pro-inflammatory Th17 function by altering the intracellular lipid microenvironment.

Chronic, low-grade inflammation increases the risk for atherosclerosis, cancer, and autoimmunity in diseases such as obesity and diabetes. Levels of CD4+ T helper 17 (Th17) cells, which secrete interleukin 17A (IL-17A), are increased in obesity and contribute to the inflammatory milieu; however, the relationship between signaling events triggered by excess nutrient levels and IL-17A-mediated inflammation is unclear. Here, using cytokine, quantitative real-time PCR, immunoprecipitation, and ChIP assays, along with lipidomics and MS-based approaches, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc, are present in naive CD4+ T cells from a diet-induced obesity murine model and that elevated O-GlcNAc levels increase IL-17A production. We also found that increased binding of the Th17 master transcription factor RAR-related orphan receptor γ t variant (RORγt) at the IL-17 gene promoter and enhancer, as well as significant alterations in the intracellular lipid microenvironment, elevates the production of ligands capable of increasing RORγt transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase 1 (ACC1), is O-GlcNAcylated and necessary for production of these RORγt-activating ligands. Our results suggest that increased O-GlcNAcylation of cellular proteins may be a potential link between excess nutrient levels and pathological inflammation.

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single ß-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with ß-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

The sweet side of the cell cycle.

Cell division (mitosis) and gamete production (meiosis) are fundamental requirements for normal organismal development. The mammalian cell cycle is tightly regulated by different checkpoints ensuring complete and precise chromosomal segregation and duplication. In recent years, researchers have become increasingly interested in understanding how O-GlcNAc regulates the cell cycle. The O-GlcNAc post-translation modification is an O-glycosidic bond of a single ß-N-acetylglucosamine sugar to serine/threonine residues of intracellular proteins. This modification is sensitive toward changes in nutrient levels in the cellular environment making O-GlcNAc a nutrient sensor capable of influencing cell growth and proliferation. Numerous studies have established that O-GlcNAcylation is essential in regulating mitosis and meiosis, while loss of O-GlcNAcylation is lethal in growing cells. Moreover, aberrant O-GlcNAcylation is linked with cancer and chromosomal segregation errors. In this review, we will discuss how O-GlcNAc controls different aspects of the cell cycle with a particular emphasis on mitosis and meiosis.

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2ß Protein at the Aγ-Globin Promoter.

One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2ß repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ß-globin locus yeast artificial chromosome (ß-YAC) bone marrow cells. When WT ß-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2ß at the (A)γ-globin promoter is increased. In addition, OGT and Mi2ß recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human ß-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2ß is modified with O-GlcNAc, and both OGT and OGA interact with Mi2ß, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2ß repressor complex at the -566 GATA motif within the promoter.

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.

O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis.

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single ß-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer's disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc.

Altering O-linked ß-N-acetylglucosamine cycling disrupts mitochondrial function.

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.

O-linked N-acetylglucosamine cycling regulates mitotic spindle organization.

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events.