Search

The primary cilium plays critical roles in homeostasis and development of neurons. Recent studies demonstrate that cilia length is regulated by the metabolic state of cells, as dictated by processes such as glucose flux and O-GlcNAcylation (OGN). The study of cilia length regulation during neuron development, however, has been an area left largely unexplored. This project aims to elucidate the roles of O-GlcNAc in neuronal development through its regulation of the primary cilium. Here, we present findings suggesting that OGN levels negatively regulate cilia length on differentiated cortical neurons derived from human-induced pluripotent stem cells. In neurons, cilia length increased significantly during neurons maturation (after day 35), while OGN levels began to drop. Long-term perturbation of OGN via drugs, which inhibit or promote its cycling, during neuron development also have varying effects. Diminishing OGN levels increases cilia length until day 25, when neural stem cells expand and undergo early neurogenesis, before causing cell cycle exit defects and multinucleation. Elevating OGN levels induces greater primary cilia assembly but ultimately results in the development of premature neurons, which have higher insulin sensitivity. These results indicate that OGN levels and primary cilia length are jointly critical in proper neuron development and function. Understanding the interplays between these two nutrient sensors, O-GlcNAc and the primary cilium, during neuron development is important in paving connections between dysfunctional nutrient-sensing and early neurological disorders.

Background: Heart failure is a leading cause of death worldwide and is associated with the rising prevalence of obesity, hypertension, and diabetes. O -GlcNAcylation (the attachment of O -linked β-N-acetylglucosamine [ O -GlcNAc] moieties to cytoplasmic, nuclear, and mitochondrial proteins) is a posttranslational modification of intracellular proteins and serves as a metabolic rheostat for cellular stress. Total levels of O -GlcNAcylation are determined by nutrient and metabolic flux, in addition to the net activity of 2 enzymes: O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA). Failing myocardium is marked by increased O -GlcNAcylation, but whether excessive O -GlcNAcylation contributes to cardiomyopathy and heart failure is unknown. Methods: We developed 2 new transgenic mouse models with myocardial overexpression of OGT and OGA to control O -GlcNAcylation independent of pathologic stress. Results: We found that OGT transgenic hearts showed increased O -GlcNAcylation and developed severe dilated cardiomyopathy, ventricular arrhythmias, and premature death. In contrast, OGA transgenic hearts had lower O -GlcNAcylation but identical cardiac function to wild-type littermate controls. OGA transgenic hearts were resistant to pathologic stress induced by pressure overload with attenuated myocardial O -GlcNAcylation levels after stress and decreased pathologic hypertrophy compared with wild-type controls. Interbreeding OGT with OGA transgenic mice rescued cardiomyopathy and premature death, despite persistent elevation of myocardial OGT. Transcriptomic and functional studies revealed disrupted mitochondrial energetics with impairment of complex I activity in hearts from OGT transgenic mice. Complex I activity was rescued by OGA transgenic interbreeding, suggesting an important role for mitochondrial complex I in O -GlcNAc–mediated cardiac pathology. Conclusions: Our data provide evidence that excessive O -GlcNAcylation causes cardiomyopathy, at least in part, attributable to defective energetics. Enhanced OGA activity is well tolerated and attenuation of O -GlcNAcylation is beneficial against pressure overload–induced pathologic remodeling and heart failure. These findings suggest that attenuation of excessive O -GlcNAcylation may represent a novel therapeutic approach for cardiomyopathy.

Familial dysautonomia (FD), a rare neurodevelopmental and neurodegenerative disorder affects the sympathetic and sensory nervous system. Although almost all patients harbor a mutation in ELP1, it remains unresolved exactly how function of sympathetic neurons (symNs) is affected; knowledge critical for understanding debilitating disease hallmarks, including cardiovascular instability or dysautonomic crises, that result from dysregulated sympathetic activity. Here, we employ the human pluripotent stem cell (hPSC) system to understand symN disease mechanisms and test candidate drugs. FD symNs are intrinsically hyperactive in vitro, in cardiomyocyte co-cultures, and in animal models. We report reduced norepinephrine transporter expression, decreased intracellular norepinephrine (NE), decreased NE re-uptake, and excessive extracellular NE in FD symNs. SymN hyperactivity is not a direct ELP1 mutation result, but may connect to NET via RAB proteins. We found that candidate drugs lowered hyperactivity independent of ELP1 modulation. Our findings may have implications for other symN disorders and may allow future drug testing and discovery.

O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer's disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Increasing the stoichiometry of O-GlcNAc on proteins before analysis Basic Protocol 2: Detection of proteins modified by O-GlcNAc using antibodies Basic Protocol 3: Detection of proteins modified by O-GlcNAc using the lectin sWGA Support Protocol 1: Control for O-linked glycosylation Basic Protocol 4: Detection and enrichment of proteins using WGA-agarose Support Protocol 2: Digestion of proteins with hexosaminidase Alternate Protocol: Detection of proteins modified by O-GlcNAc using galactosyltransferase Support Protocol 3: Autogalactosylation of galactosyltransferase Support Protocol 4: Assay of galactosyltransferase activity Basic Protocol 5: Characterization of labeled glycans by β-elimination and chromatography Basic Protocol 6: Detection of O-GlcNAc in 96-well plates Basic Protocol 7: Assay for OGT activity Support Protocol 5: Desalting of O-GlcNAc transferase Basic Protocol 8: Assay for O-GlcNAcase activity.

Heart failure is a leading cause of death worldwide and is associated with the rising prevalence of obesity, hypertension, and diabetes.We developed 2 new transgenic mouse models with myocardial overexpression of OGT and OGA to controlWe found that OGT transgenic hearts showed increasedOur data provide evidence that excessive

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

See Hart and Huang (doi:10.1093/brain/awaa398) for a scientific commentary on this article. Lee et al. show that O-GlcNAcylation, an evolutionarily conserved post-translational modification, is critical for the physiological functioning and survival of dopaminergic neurons. Upregulating O-GlcNAcylation mitigates neurodegeneration, synaptic impairments and motor deficits in a mouse model of Parkinson’s disease., The dopamine system in the midbrain is essential for volitional movement, action selection, and reward-related learning. Despite its versatile roles, it contains only a small set of neurons in the brainstem. These dopamine neurons are especially susceptible to Parkinson’s disease and prematurely degenerate in the course of disease progression, while the discovery of new therapeutic interventions has been disappointingly unsuccessful. Here, we show that O-GlcNAcylation, an essential post-translational modification in various types of cells, is critical for the physiological function and survival of dopamine neurons. Bidirectional modulation of O-GlcNAcylation importantly regulates dopamine neurons at the molecular, synaptic, cellular, and behavioural levels. Remarkably, genetic and pharmacological upregulation of O-GlcNAcylation mitigates neurodegeneration, synaptic impairments, and motor deficits in an animal model of Parkinson’s disease. These findings provide insights into the functional importance of O-GlcNAcylation in the dopamine system, which may be utilized to protect dopamine neurons against Parkinson’s disease pathology.

In the early 1980s, while using purified glycosyltransferases to probe glycan structures on surfaces of living cells in the murine immune system, we discovered a novel form of serine/threonine protein glycosylation (O-linked β-GlcNAc; O-GlcNAc) that occurs on thousands of proteins within the nucleus, cytoplasm, and mitochondria. Prior to this discovery, it was dogma that protein glycosylation was restricted to the luminal compartments of the secretory pathway and on extracellular domains of membrane and secretory proteins. Work in the last 3 decades from several laboratories has shown that O-GlcNAc cycling serves as a nutrient sensor to regulate signaling, transcription, mitochondrial activity, and cytoskeletal functions. O-GlcNAc also has extensive cross-talk with phosphorylation, not only at the same or proximal sites on polypeptides, but also by regulating each other's enzymes that catalyze cycling of the modifications. O-GlcNAc is generally not elongated or modified. It cycles on and off polypeptides in a time scale similar to phosphorylation, and both the enzyme that adds O-GlcNAc, the O-GlcNAc transferase (OGT), and the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), are highly conserved from C. elegans to humans. Both O-GlcNAc cycling enzymes are essential in mammals and plants. Due to O-GlcNAc's fundamental roles as a nutrient and stress sensor, it plays an important role in the etiologies of chronic diseases of aging, including diabetes, cancer, and neurodegenerative disease. This review will present an overview of our current understanding of O-GlcNAc's regulation, functions, and roles in chronic diseases of aging.

O-linked N-acetylglucosamine (O-GlcNAcylation) is an important post-translational modification on serine or threonine of proteins, mainly observed in nucleus or cytoplasm. O-GlcNAcylation regulates many cell processes, including transcription, cell cycle, neural development and nascent polypeptide chains stabilization. However, the facile identification of O-GlcNAc is a major bottleneck in O-GlcNAcylation research. Herein, we report that a lectin, Agrocybe aegerita GlcNAc-specific lectin (AANL), also reported as AAL2, can be used as a powerful probe for O-GlcNAc identification. Glycan array analyses and surface plasmon resonance (SPR) assays show that AANL binds to GlcNAc with a dissociation constant (KD) of 94.6 μM, which is consistent with the result tested through isothiocyanate (ITC) assay reported before (Jiang S, Chen Y, Wang M, Yin Y, Pan Y, Gu B, Yu G, Li Y, Wong BH, Liang Y, et al. 2012. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem J. 443:369–378.). Confocal imaging shows that AANL co-localizes extensively with NUP62, a heavily O-GlcNAcylated and abundant nuclear pore glycoprotein. Furthermore, O-GlcNAc-modified peptides could be effectively enriched in the late flow-through peak from simple samples by using affinity columns Sepharose 4B-(AANL) or POROS-(AANL). Therefore, using AANL affinity column, we identified 28 high-confidence O-linked HexNAc-modified peptides mapped on 17 proteins involving diverse cellular progresses, including transcription, hydrolysis progress, urea cycle, alcohol metabolism and cell cycle. And most importantly, major proteins and sites were not annotated in the dbOGAP database. These results suggest that the AANL lectin is a new useful tool for enrichment and identification of O-GlcNAcylated proteins and peptides.

Diabetes mellitus and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF in patients. However, the mechanism(s) underlying this clinical association is unknown. Elevated protein O-GlcNAcylation (OGN) and reactive oxygen species (ROS) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by OGN (OGN-CaMKII) and ROS (ox-CaMKII). We induced type 1 (T1D) and type 2 diabetes (T2D) in a portfolio of genetic mouse models capable of dissecting the role of OGN and ROS at CaMKII and the type 2 ryanodine receptor (RyR2), an intracellular Ca2+ channel implicated as an important downstream mechanism of CaMKII- mediated arrhythmias. Here we show that T1D and T2D significantly increased AF, similar to observations in patients, and this increase required CaMKII. While T1D and T2D both require ox-CaMKII to increase AF, they respond differently to loss of OGN-CaMKII or OGN inhibition. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF, and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by diverse mechanisms and targets, including CaMKII and RyR2. The proarrhythmic consequences of OGN- and ox-CaMKII differ between T1D and T2D. These results provide new and unanticipated insights into the mechanisms for increased AF in diabetes mellitus, and suggest successful future therapies will need to be different for AF in T1D and T2D.

In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Herein, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBP(T114A) CRISPR/Cas9 edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of β-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199of β-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199(gS199)-actin and in the phosphorylation of Ser199(pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

Increased protein O -GlcNAcylation (OGN) is a common feature of failing heart muscle. However, it is unknown if excessive OGN contributes to cardiomyopathy and heart failure. OGN levels are determined by the net activity of two enzymes: OGT ( O -GlcNAc transferase, adds OGN) and OGA ( O -GlcNAcase, removes OGN). We hypothesized that excessive myocardial OGN is a cause of cardiomyopathy. To test for a role of OGN in cardiomyopathy we developed new transgenic (TG) mouse models with myocardial overexpression of OGT or OGA. The OGT-TG hearts showed progressive decline in left ventricular (LV) systolic function, dilation, increased mass (Figure A, B) (Statistical Analysis ANOVA - *** = p

Experience-driven synaptic plasticity is believed to underlie adaptive behavior by rearranging the way neuronal circuits process information. We have previously discovered that O-GlcNAc transferase (OGT), an enzyme that modifies protein function by attaching β-N-acetylglucosamine (GlcNAc) to serine and threonine residues of intracellular proteins (O-GlcNAc), regulates food intake by modulating excitatory synaptic function in neurons in the hypothalamus. However, how OGT regulates excitatory synapse function is largely unknown. Here we demonstrate that OGT is enriched in the postsynaptic density of excitatory synapses. In the postsynaptic density, O-GlcNAcylation on multiple proteins increased upon neuronal stimulation. Knockout of the OGT gene decreased the synaptic expression of the AMPA receptor GluA2 and GluA3 subunits, but not the GluA1 subunit. The number of opposed excitatory presynaptic terminals was sharply reduced upon postsynaptic knockout of OGT. There were also fewer and less mature dendritic spines on OGT knockout neurons. These data identify OGT as a molecular mechanism that regulates synapse maturity.

O-GlcNAcylation is a dynamic post-translational modification that is responsive to nutrient availably via the hexosamine biosynthetic pathway and its endproduct UDP-GlcNAc. O-GlcNAcylation serves as a nutrient sensor to regulate the activities of many proteins involved in nearly all biological processes. Within the last decade, OGT, OGA and O-GlcNAcylation have been shown to be at the nexus of epigenetic marks controlling gene expression during embryonic development, cell differentiation, in the maintenance of epigenetic states and in the etiology of epigenetic related diseases. OGT O-GlcNAcylates histones and epigenetic writers/erasers, and regulates gene activation, as well as gene repression. Here, we highlight recent work documenting the important roles O-GlcNAcylation and its cycling enzymes play in the nutrient regulation of epigenetic partners controlling gene expression.

As a dynamic post-translational modification, O-linked β- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

Glycosylation plays important roles in many cellular processes, such as signal transduction, cell cycle progression and transcriptional regulation. However, the identification and analysis of glycosylation are severely hampered by the low specificity or avidity of antiglycan antibodies and lectins. We have reported that a lectin AANL, which has high specificity for terminal GlcNAc glycans and contains six carbohydrate binding sites (CBSs), was used to enrich O-GlcNAcylated peptides. To further improve AANL binding specificity, we designed a CBS-homogenization strategy and restructured six mutant lectins, known as AANL1-AANL6. Affinity chromatography with GlcNAc and isothermal titration calorimetry analysis indicated that the two mutants (AANL3 and AANL6) all maintained GlcNAc binding activity. AANL6 and AANL3 showed higher specificity for terminal GlcNAc glycans than AANL, as shown by the hemagglutination assay, cell binding assays and glycan microarray analysis, and AANL6 exhibited the highest specificity. The binding activity of AANL6 for O-GlcNAcylated peptides was shown by surface plasmon resonance assays. By AANL6 affinity chromatography enrichment and mass spectrometry analysis, 79 high-confidence and 21 putative O-GlcNAcylated sites were identified on 85 peptides mapped onto 54 proteins. Most of these sites were new sites compared with reported data. These results indicate that the enrichment capacity of AANL6 is higher than that of wild-type AANL. In conclusion, the CBS-homogenization mutation strategy was successful, and AANL6 was identified as a powerful tool for O-GlcNAcylation enrichment. Our research suggests that the CBS-homogenization strategy is valuable for improving the specificity of lectins with multiple CBSs.

O-GlcNAc, first characterized more than 30 years ago, is an O-linked β-N-acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residues. O-GlcNAc has been found on over 4,000 cytoplasmic and nuclear proteins. The addition of O-GlcNAc to proteins is catalyzed by O-GlcNAc transferase (OGT), while its removal is catalyzed by O-linked N-acetyl-β-D-glucosaminidase (OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, such as signal transduction, transcription, cell cycle progression and protein-protein interactions. O-GlcNAc plays important roles in human diseases, such as cancer, diabetes and neurological disorders. Protein phosphorylation is a post-translational modification that serves as a rapid and reversible means to modulate protein activity and transduce signals. The regulation of phosphorylation is a central mechanism in cell health and disease. O-GlcNAcylation has extensive interplay with phosphorylation at the si...

Dynamic cycling of O-linked β-N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins serves as a nutrient sensor to regulate numerous biological processes. However, mitochondrial protein O-GlcNAcylation and its effects on function are largely unexplored. In this study, we performed a comparative analysis of the proteome and O-GlcNAcome of cardiac mitochondria from rats acutely (12 h) treated without or with thiamet-G (TMG), a potent and specific inhibitor of O-GlcNAcase. We then determined the functional consequences in mitochondria isolated from the two groups. O-GlcNAcomic profiling finds that over 88 mitochondrial proteins are O-GlcNAcylated, with the oxidative phosphorylation system as a major target. Moreover, in comparison with controls, cardiac mitochondria from TMG-treated rats did not exhibit altered protein abundance but showed overall elevated O-GlcNAcylation of many proteins. However, O-GlcNAc was unexpectedly down-regulated at certain sites of specific proteins. Concomitantly, TMG treatment resulted in significantly increased mitochondrial oxygen consumption rates, ATP production rates, and enhanced threshold for permeability transition pore opening by Ca(2+). Our data reveal widespread and dynamic mitochondrial protein O-GlcNAcylation, serving as a regulator to their function.

Summary The cycling (addition and removal) of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine residues of nuclear and cytoplasmic proteins serves as a nutrient sensor via the hexosamine biosynthetic pathway's production of UDP-GlcNAc, the donor for the O-GlcNAc transferase (OGT). OGT is exquisitely sensitive both in terms of its catalytic activity and by its specificity to the levels of this nucleotide sugar. UDP-GlcNAc is a major node of metabolism whose levels are coupled to flux through the major metabolic pathways of the cell. O-GlcNAcylation has extensive crosstalk with protein phosphorylation to regulate signalling pathways in response to flux through glucose, amino acid, fatty acid, energy and nucleotide metabolism. Not only does O-GlcNAcylation compete for phosphorylation sites on proteins, but also over one-half of all kinases appear to be O-GlcNAcylated, and many are regulated by O-GlcNAcylation. O-GlcNAcylation is also fundamentally important to nutrient regulation of gene expression. OGT is a polycomb gene. Nearly all RNA polymerase II transcription factors are O-GlcNAcylated, and the sugar regulates their activities in many different ways, depending upon the transcription factor and even upon the specific O-GlcNAc site on the protein. O-GlcNAc is part of the histone code, and the sugar affects the modification of histones by other epigenetic marks. O-GlcNAcylation regulates DNA methylation by the TET family of proteins. O-GlcNAc modification of the basal transcription machinery is required for assembly of the pre-initiation complex in the transcription cycle. Dysregulated O-GlcNAcylation is directly involved in the aetiology of the major chronic diseases associated with ageing.

SUMMARY To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology., Graphical Abstract, In Brief Comprehensive proteogenomic characterization in 103 treatment-naive clear cell renal cell carcinoma patient samples highlights tumor-specific alterations at the proteomic level that are unrevealed by transcriptomic profiling and proposes a revised subtyping scheme based on integrated omics analysis.

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.

Diabetic retinopathy is a leading cause of blindness worldwide. Despite laser and surgical treatments, anti-angiogenic and other therapies, and strict metabolic control, many patients progress to visual impairment and blindness. New insights are needed into the pathophysiology of diabetic retinopathy in order to develop new methods to improve the detection and treatment of disease and the prevention of blindness. Hyperglycemia and diabetes result in increased flux through the hexosamine biosynthetic pathway, which, in turn, results in increased post-translational modification of Ser/Thr residues of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is involved in regulation of many nuclear and cytoplasmic proteins in a manner similar to protein phosphorylation. Altered O-GlcNAc signaling has been implicated in the pathogenesis of diabetes and may play an important role in the pathogenesis of diabetic retinopathy. The goal of this review is to summarize the biology of the hexosamine biosynthesis pathway and O-GlcNAc signaling, to present the current evidence for the role of OGlcNAc signaling in diabetes and diabetic retinopathy, and to discuss future directions for research on O-GlcNAc in the pathogenesis of diabetic retinopathy.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

The post-translational modification of serine and threonine residues of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is highly ubiquitous, dynamic and inducible. Protein O-GlcNAcylation serves as a key regulator of critical biological processes including transcription, translation, proteasomal degradation, signal transduction and apoptosis. Increased O-GlcNAcylation is directly linked to insulin resistance and to hyperglycemia-induced glucose toxicity, two hallmarks of diabetes and diabetic complications. In this review, we briefly summarize what is known about protein O-GlcNAcylation and nutrient metabolism, as well as discuss the commonly used tools to probe changes of O-GlcNAcylation in cultured cells and in animal models. We then focus on some key proteins modified by O-GlcNAc, which play crucial roles in the etiology and progression of diabetes and diabetic complications. Proteomic approaches are also highlighted to provide a system view of protein O-GlcNAcylation. Finally, we discuss how aberrant O-GlcNAcylation on certain proteins may be exploited to develop methods for the early diagnosis of pre-diabetes and/or diabetes.

O-linked β-d-N-acetyl glucosamine (O-GlcNAc) addition (O-GlcNAcylation), a post-translational modification of serine/threonine residues of proteins, is involved in diverse cellular metabolic and signaling pathways. Aberrant O-GlcNAcylation underlies the initiation and progression of multiple chronic diseases including diabetes, cancer, and neurodegenerative diseases. Numerous methods have been developed for the analysis of protein O-GlcNAcylation, but instead of discussing the classical biochemical techniques, this unit covers O-GlcNAc characterization by combining several enrichment methods and mass spectrometry detection techniques [including collision-induced dissociation (CID), higher energy collision dissociation (HCD), and electron transfer dissociation (ETD) mass spectrometry]. © 2017 by John Wiley & Sons, Inc. Keywords: BEMAD; CID; enrichment; ETD; HCD; mass spectrometry; O-GlcNAc; O-GlcNAcome; O-GlcNAcomics; site mapping

The enzymatic addition of a single β-D-N-acetylglucosamine sugar molecule on serine and/or threonine residues of protein chains is referred to as O-GlcNAcylation. This novel form of post-translational modification, first reported in 1984, is extremely abundant on nuclear and cytoplasmic proteins and has site specific cycling dynamics comparable to that of protein-phosphorylation. A nutrient and stress sensor, O-GlcNAc abnormalities underlie insulin resistance and glucose toxicity in diabetes, neurodegenerative disorders and dysregulation of tumor suppressors and oncogenic proteins in cancer. Recent advances have helped understand the biochemical mechanisms of GlcNAc addition and removal and have opened the door to developing key inhibitors towards this type of protein modification. Advanced methods in detecting and measuring O-GlcNAcylation have assisted in delineating its biological roles in a variety of cellular processes and diseased states. Availability of facile glycomic techniques are allowing for the exponential growth in the study of protein O-GlcNAacylation and are helping to elucidate key biological roles of this novel PTM.

O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications including cardiomyopathy. However, mitochondrial O-GlcNAcylation, especially in response to chronic hyperglycemia in diabetes, has been poorly explored. We performed a comparative O-GlcNAc profiling of mitochondria from control and streptozotocin (STZ)-induced diabetic rat hearts by using an improved β-elimination/Michael addition with isotopic DTT reagents (BEMAD) followed by tandem mass spectrometric analysis. In total, 86 mitochondrial proteins, involved in diverse pathways, were O-GlcNAcylated. Among them, many proteins have site-specific alterations in O-GlcNAcylation in response to diabetes, which suggests that protein O-GlcNAcylation is a novel layer of regulation mediating adaptive changes in mitochondrial metabolism during the progression of diabetic cardiomyopathy.

The molecular mechanisms linking glucose metabolism with active transcription remain undercharacterized in mammalian cells. Using nuclear factor-κB (NF-κB) as a glucose-responsive transcription factor, we show that cells use the hexosamine biosynthesis pathway and O-linked β- N -acetylglucosamine ( O -GlcNAc) transferase (OGT) to potentiate gene expression in response to tumor necrosis factor (TNF) or etoposide. Chromatin immunoprecipitation assays demonstrate that, upon induction, OGT localizes to NF-κB–regulated promoters to enhance RelA acetylation. Knockdown of OGT abolishes p300-mediated acetylation of RelA on K310, a posttranslational mark required for full NF-κB transcription. Mapping studies reveal T305 as an important residue required for attachment of the O -GlcNAc moiety on RelA. Furthermore, p300 fails to acetylate a full-length RelA(T305A) mutant, linking O- GlcNAc and acetylation events on NF-κB. Reconstitution of RelA null cells with the RelA(T305A) mutant illustrates the importance of this residue for NF-κB–dependent gene expression and cell survival. Our work provides evidence for a unique regulation where attachment of the O- GlcNAc moiety to RelA potentiates p300 acetylation and NF-κB transcription.

The RNA polymerase II C-terminal domain (CTD), which serves as a scaffold to recruit machinery involved in transcription, is modified post-translationally. Although the O-GlcNAc modification of RNA polymerase II CTD was documented in 1993, its functional significance remained obscure. We show that O-GlcNAc transferase (OGT) modified CTD serine residues 5 and 7. Drug inhibition of OGT and OGA (N-acetylglucosaminidase) blocked transcription during preinitiation complex assembly. Polymerase II and OGT co-immunoprecipitated, and OGT is a component of the preinitiation complex. OGT shRNA experiments showed that reduction of OGT causes a reduction in transcription and RNA polymerase II occupancy at several B-cell promoters. These data suggest that the cycling of O-GlcNAc on and off of polymerase II occurs during assembly of the preinitiation complex. Our results define unexpected roles for both the CTD and O-GlcNAc in the regulation of transcription initiation in higher eukaryotes.

O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

Dynamic posttranslational modification of serine and threonine residues of nucleocytoplasmic proteins by β- N -acetylglucosamine (O-GlcNAc) is a regulator of cellular processes such as transcription, signaling, and protein–protein interactions. Like phosphorylation, O-GlcNAc cycles in response to a wide variety of stimuli. Although cycling of O-GlcNAc is catalyzed by only two highly conserved enzymes, O-GlcNAc transferase (OGT), which adds the sugar, and β- N -acetylglucosaminidase (O-GlcNAcase), which hydrolyzes it, the targeting of these enzymes is highly specific and is controlled by myriad interacting subunits. Here, we demonstrate by multiple specific immunological and enzymatic approaches that histones, the proteins that package DNA within the nucleus, are O-GlcNAcylated in vivo. Histones also are substrates for OGT in vitro. We identify O-GlcNAc sites on histones H2A, H2B, and H4 using mass spectrometry. Finally, we show that histone O-GlcNAcylation changes during mitosis and with heat shock. Taken together, these data show that O-GlcNAc cycles dynamically on histones and can be considered part of the histone code.

Mitosis must faithfully divide the genome such that each progeny inherits the same genetic material. DNA condensation is crucial in ensuring that chromosomes are correctly attached to the mitotic spindle for segregation, preventing DNA breaks or constrictions from the contractile ring. Histones form an octameric complex of basic proteins important in regulating DNA organization and accessibility. Histone post-translational modifications are altered during mitosis, although the roles of these post-translational modifications remain poorly characterized. Here, we report that N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme catalyzing the addition of O-GlcNAc moieties to nuclear and cytoplasmic proteins at serine and threonine residues, regulates some aspects of mitotic chromatin dynamics. OGT protein amounts decrease during M phase. Modest overexpression of OGT alters mitotic histone post-translational modifications at Lys-9, Ser-10, Arg-17, and Lys-27 of histone H3. Overexpression of OGT also prevents mitotic phosphorylation of coactivator-associated arginine methyltransferase 1 (CARM1) and prevents its correct cellular localization during mitosis. Moreover, OGT overexpression results in an increase in abnormal chromosomal bridge formation. Together, these results show that regulating the amount of OGT during mitosis is important in ensuring correct chromosomal segregation during mitosis.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a sugar attachment to serine or threonine hydroxyl moieties on nuclear and cytoplasmic proteins. In many ways, O-GlcNAcylation is similar to phosphorylation because both post-translational modifications cycle rapidly in response to internal or environmental cues. O-GlcNAcylated proteins are involved in transcription, translation, cytoskeletal assembly, signal transduction, and many other cellular functions. O-GlcNAc signaling is intertwined with cellular metabolism; indeed, the donor sugar for O-GlcNAcylation (UDP-GlcNAc) is synthesized from glucose, glutamine, and UTP via the hexosamine biosynthetic pathway. Emerging research indicates that O-GlcNAc signaling and its crosstalk with phosphorylation are altered in metabolic diseases, such as diabetes and cancer.

The retinoblastoma-susceptibility gene product (pRB) is a classical tumor suppressor. pRB regulates a number of cellular processes including proliferation, differentiation, and apoptosis. One of the essential mechanisms by which pRB, and the related p107 and p130 family members, act is through its interactions with the E2F class of transcription factors. E2F-1 transcription is necessary for entry into S-phase during the cell-cycle. pRB binds E2F-1 and represses transcription via recruitment of a histone deacetylase complex and by preventing co-activator complexes from binding E2F-1. Current dogma suggests that phosphorylation of pRB during mid- to late-G1 leads to release of E2F-1 and E2F-1 dependent transcriptional activation of essential S-phase genes. Here we show that pRB, and the related p107 protein, are modified by O-linked β-N-acetylglucosamine (O-GlcNAc) in an in vitro transcription/translation system. Furthermore, we show in vivo that pRB is more heavily glycosylated in G1 of the cell-cycle when pRB is known to be in an active, hypophosphorylated state. Finally, we demonstrate that E2F-1 associated pRB is modified by O-GlcNAc. These studies suggest that regulation of pRB function(s) may be controlled by dynamic O-GlcNAc modification, as well as phosphorylation.

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.

OBJECTIVE O-linked β-N-acetylglucosamine (O-GlcNAc) plays an important role in the development of insulin resistance and glucose toxicity. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT), which attaches O-GlcNAc to serine and/or threonine residues of proteins and by O-GlcNAcase, which removes O-GlcNAc. We investigated the expression of these two enzymes in erythrocytes of human subjects with diabetes or pre-diabetes. RESEARCH DESIGN AND METHODS Volunteers with normal condition, pre-diabetes, and diabetes were recruited through a National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases) study and at the Johns Hopkins Comprehensive Diabetes Center. Erythrocyte proteins were extracted and hemoglobins were depleted. Global O-GlcNAcylation of erythrocyte proteins was confirmed by Western blotting using an O-GlcNAc–specific antibody. Relative OGT and O-GlcNAcase protein amounts were determined by Western blot analysis. Relative expression of O-GlcNAcase was compared with the level of A1C. RESULTS Erythrocyte proteins are highly O-GlcNAcylated. O-GlcNAcase expression is significantly increased in erythrocytes from both individuals with pre-diabetes and diabetes compared with normal control subjects. Unlike O-GlcNAcase, protein levels of OGT did not show significant changes. CONCLUSIONS O-GlcNAcase expression is increased in erythrocytes from both individuals with pre-diabetes and individuals with less well-controlled diabetes. These findings, together with the previous study that demonstrated the increased site-specific O-GlcNAcylation of certain erythrocyte proteins, suggest that the upregulation of O-GlcNAcase might be an adaptive response to hyperglycemia-induced increases in O-GlcNAcylation, which are likely deleterious to erythrocyte functions. In any case, the early and substantial upregulation of O-GlcNAcase in individuals with pre-diabetes may eventually have diagnostic utility.

Increased O-linked beta-N-acetylglucosamine (O-GlcNAc) is associated with insulin resistance in muscle and adipocytes. Upon insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313-5318), we focused on the pathway upstream of AKT. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y(608)XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to insulin resistance is by inhibiting phosphorylation at the Y(608)XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well.

At least 20 core ribosome proteins are modified by O-GlcNAc. O-GlcNAcase is localized to the nucleolus and O-GlcNAc transferase is excluded from the nucleolus. Both enzymes associate with active polysomes. Overexpression of OGT disrupts ribosomal subunit homeostasis. Data suggest that O-GlcNAc regulates translation and ribosome biogenesis., Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.