O-linked N-acetylglucosamine Modification Research Articles

Centrosomes: Til O-GlcNAc Do Us Apart.

The centrosome apparatus is vital for spindle assembly and chromosome segregation during mitotic divisions. Its replication, disjunction and separation have to be fine-tuned in space and time. A multitude of post-translational modifications (PTMs) have been implicated in centrosome modulation, including phosphorylation, ubiquitination and acetylation. Among them is the emerging O-linked N-acetylglucosamine (O-GlcNAc) modification. This quintessential PTM has a sole writer, O-GlcNAc transferase (OGT), and the only eraser, O-GlcNAcase (OGA). O-GlcNAc couples glucose metabolism with signal transduction and forms a yin-yang relationship with phosphorylation. Evidence from proteomic studies as well as single protein investigations has pinpointed a role of O-GlcNAc in centrosome number and separation, centriole number and distribution, as well as the cilia machinery emanating from the centrosomes. Herein we review our current understanding of the sweet modification embedded in centrosome dynamics and speculate that more molecular details will be unveiled in the future.

Role of O-linked N-acetylglucosamine in the homeostasis of metabolic organs, and its potential links with diabetes and its complications.

Recent studies using genetically manipulated mouse models have shown the pivotal role of O‐linked N‐acetylglucosamine modification (O‐GlcNAcylation) in the metabolism of multiple organs. The molecular mechanism involves the sensing of glucose flux by the hexosamine biosynthesis pathway, which leads to the adjustment of cellular metabolism to protect against changes in the environment of each organ through O‐GlcNAcylation. More recently, not only glucose, but also fluxes of amino acids and fatty acids have been reported to induce O‐GlcNAcylation, affecting multiple cellular processes. In this review, we discuss how O‐GlcNAcylation maintains homeostasis in organs that are affected by diabetes mellitus: skeletal muscle, adipose tissue, liver and pancreatic β‐cells. Furthermore, we discuss the importance of O‐GlcNAcylation in the pathogenesis of diabetic complications. By elucidating the molecular mechanisms whereby cellular homeostasis is maintained, despite changes in metabolic flux, these studies might provide new targets for the treatment and prevention of diabetes and its complications.

MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

1713-P: Adipocyte O-GlcNAc Transferase Regulate Adipogenesis through De Novo Lipogenesis in Mice

O-linked N-acetylglucosamine (O-GlcNAc) modification is one of the post-translational modifications which modulates protein fate and function, and it is mediated by O-GlcNAc transferase (OGT). The substrate of OGT, UDP-GlcNAc, is biosynthesized through the branch of glycolysis. Thus O-GlcNAcylation has been regarded as a nutrient sensor. Metabolic regulation in white adipose tissues (WAT) is dramatically changed in the process of obesity. We previously reported that the deletion of Ogt in adipose tissues (Ogt-FKO) decrease WAT mass without affecting glucose tolerance in mice under normal condition. However, the role of O-GlcNAcylation in WAT in the process of being obesity remains unknown. In this study, we investigated the role of O-GlcNAcylation in WAT under diet induced obesity. Despite no significant differences in food intake, body weight gain by high-fat diet was suppressed in Ogt-FKO mice along with the significant decrease of WAT mass. However, Ogt-FKO mice were severely impaired glucose tolerance and decreased insulin sensitivity in liver, which was confirmed by hyper-insulinemic euglycemic clamp. Histologically, we observed smaller adipocytes and fibrosis in WAT of Ogt-FKO mice. In addition, gene expression of F4/80, CD11c, MCP-1 and Col1a1 were significantly higher in Ogt-FKO mice, suggesting increased inflammation and fibrosis. Also, the expression of genes associated to fatty acid synthesis such as SREBP-1c, FAS, SCD1 and SCD2 were reduced in Ogt-FKO mice. Furthermore, Ogt-FKO mice showed lower serum concentration of adiponectin and leptin (46.43 ± 3.62 vs. 8.42 ± 1.53ng/ml) along with their gene expression, which is consistent with severe atrophy of WAT in Ogt-FKO mice. In conclusion, loss of O-GlcNAcylation in WAT exhibited obesity resistance and impaired glucose tolerance because of lipoatrophy under diet induced obesity, and the results suggest that O-GlcNAcylation in WAT plays important role for fatty acid synthesis and functional adipocyte formation. Disclosure K. Morino: None. A. Tsuji: None. N. Ohashi: None. S. Ida: None. R.J. Perry: Research Support; Self; AstraZeneca. S. Ugi: None. Y. Fujita: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. H. Maegawa: Speaker’s Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Sanofi K.K., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science (18K16226)

Enhancement of O-linked N-acetylglucosamine modification promotes metastasis in patients with colorectal cancer and concurrent type 2 diabetes mellitus.

Reversible post-translational modification of serine and threonine residues by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation has been indicated to regulate the activities of a number of different proteins. Augmented O-GlcNAcylation contributes to the etiologies of type 2 diabetes mellitus (T2DM) and cancer. Moreover, diabetic conditions increase the risk of colorectal cancer. However, the effect of O-GlcNAcylation in patients with colorectal cancer and concurrent T2DM has not been elucidated. The current study evaluated the level of O-GlcNAcylation in patients with colorectal cancer with or without T2DM. Notably, O-GlcNAcylation levels were significantly higher in tissues from patients with T2DM compared with those in patients without T2DM, and higher in cancer tissues compared with corresponding adjacent tissues. O-GlcNAcylation and cancer stage were more strongly correlated in cancer tissues from patients with T2DM compared with those from patients without T2DM. Additionally, distant metastasis was significantly correlated with O-GlcNAcylation in cancer tissues from patients with T2DM. β-catenin levels in colorectal cancer tissues were the highest in patients with advanced-stage cancer and concurrent T2DM. In SW480 human colon cancer cells, thiamet G (TMG) treatment and OGA silencing, which increased O-GlcNAcylation, significantly increased β-catenin and SNAIL in high-glucose, but not during normal-glucose conditions. These data suggest that O-GlcNAcylation is closely associated with distant metastasis, most likely through upregulation of the β-catenin/SNAIL signaling pathway in colorectal cancer patients with T2DM.

O-GlcNAcylation of myosin phosphatase targeting subunit 1 (MYPT1) dictates timely disjunction of centrosomes

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)-protein phosphatase 1cβ (PP1cβ) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc-profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions.

Chemoproteomic Profiling of O-GlcNAcylation in Caenorhabditis elegans.

Genetic studies have revealed essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in Caenorhabditis elegans. However, large-scale identification of O-GlcNAcylated proteins and mapping the modification sites in C. elegans remain relatively unexplored. By using a chemoproteomic strategy, we herein report the identification of 108 high-confidence O-GlcNAcylated proteins and 64 modification sites in C. elegans. Furthermore, quantitative proteomics upon altering O-GlcNAcylation show that the abundance of a large number of proteins are affected by O-GlcNAc. These proteins are involved in regulating reproduction and lifespan, which may correlate with the previously observed phenotypes in genetic studies. The data set in this study reveals the O-GlcNAc modification landscape in C. elegans and provides a valuable resource for dissecting the biological function of O-GlcNAcylation.

Diabetic Hyperglycemia Regulates Potassium Channels and Arrhythmias in the Heart via Autonomous CaMKII Activation by O-Linked Glycosylation

Diabetic cardiomyopathy is associated with altered cardiac repolarization, K+ current density and arrhythmia risk. However, little is known about how acute hyperglycemia regulates cardiac K+ channels and arrhythmias. We measured action potentials (APs) and K+ currents in isolated mouse ventricular myocytes exposed to high glucose (30 mM) versus control (5.5 mM glucose+24.5 mM mannitol to match osmolarity). Acute hyperglycemia reduced transient outward K+ current (Ito) amplitude but enhanced Ito recovery from inactivation and increased inward rectifier K+ current (IK1). These changes were prevented by inhibitors of O-linked N-acetylglucosamine modification (O-GlcNAcylation), Osmi-I and DON. Conversely, O-GlcNAcase inhibitor thiamet-G produced the same K+ current effects as hyperglycemia. Thus O-GlcNAcylation is necessary and sufficient for these K+ channel effects. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by CaMKII inhibitor AIP, in CaMKIIδ-knockout myocytes and in mice in which a known O-GlcNAcylated Serine on CaMKII was mutated (S280A knock-in). Acute hyperglycemia slightly prolonged AP duration, increased delayed afterdepolarizations and inducibility of AP-duration alternans during tachypacing in WT only (not in CaMKIIδ-KO or S280A-KI). Thus, acute hyperglycemia-induced O-GlcNAcylation of CaMKII and/or K+ channels may contribute to arrhythmogenesis. In chronic diabetic models (streptozotocin & db/db) Ito and IK1 were both downregulated (as in heart failure and chronic CaMKII activation). AP duration was significantly prolonged in diabetes, and delayed afterdepolarizations and alternans were enhanced at normoglycemia and worsened by hyperglycemia. We conclude that K+ channels are differently modulated by acute versus chronic hyperglycemia and O-GlycNAcylation-dependent CaMKII activation. Acute hyperglycemia enhances K+ channel function via O-GlcNAcylation of CaMKII at Ser280, which may also promote arrhythmogenic SR Ca2+ leak. However, chronic hyperglycemia and CaMKII activation appear to downregulate K+ channel expression, which also promotes arrhythmia susceptibility.

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O-GlcNAc-Cycling Enzymes.

The O-linked N-acetylglucosamine (O-GlcNAc) modification is an essential component in cell regulation. A single pair of human enzymes conducts this modification dynamically on a broad variety of proteins: O-GlcNAc transferase (OGT) adds the GlcNAc residue and O-GlcNAcase (OGA) hydrolyzes it. This modification is dysregulated in many diseases, but its exact effect on particular substrates remains unclear. In addition, no apparent sequence motif has been found in the modified proteins, and the factors controlling the substrate specificity of OGT and OGA are largely unknown. In this minireview, we will discuss recent developments in chemical and biochemical methods toward addressing the challenge of OGT and OGA substrate recognition. We hope that the new concepts and knowledge from these studies will promote research in this area to advance understanding of O-GlcNAc regulation in health and disease.

Histone O-GlcNAcylation and Potential Biological Functions

Histone modifications play an important role in the control of all DNA-based processes by altering the structure and function of chromatin. An O-linked N-acetylglucosamine (O-GlcNAc) modification is a type of post-translational modification of proteins, and gets attached to serine (Ser)/threonine (Thr) residues under the control of a sole pair of enzymes, i.e. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Recent evidence indicates that histone modifications include the O-GlcNAc modification. To date, 16 histone O-GlcNAc sites have been reported. Since O-GlcNAc modification is known as a nutrient-sensitive modification, it likely serves as a molecular mechanism linking nutrient and epigenetic status. Recently, with the acquisition of specific antibodies against O-GlcNAcylation of histone residues, functional analyses have been advanced. Here, we discuss the current knowledge of histone O-GlcNAc modification, with a view to elucidating its comprehensive biological role.

MicroRNA-200a/200b Modulate High Glucose-Induced Endothelial Inflammation by Targeting O-linked N-Acetylglucosamine Transferase Expression.

Background and Aims: Increased O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins by O-GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation.Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining.Results: HG upregulated OGT mRNA and protein expression and protein O-GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3′-untranslated region (3′-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O-GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3′-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O-GlcNAcylation levels; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Additionally, siRNA-mediated OGT depletion reduced HG-induced protein O-GlcNAcylation; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion, confirming that HG-induced endothelial inflammation is partially mediated via OGT-induced protein O-GlcNAcylation. These results were validated in vivo: tail-vein injection of miR-200a/200b mimics downregulated endothelial OGT and ICAM-1 expression in db/db mice.Conclusion: miR-200a/200b are involved in modulating HG-induced endothelial inflammation by regulating OGT-mediated protein O-GlcNAcylation, suggesting the therapeutic role of miR-200a/200b on vascular complications in diabetes.

FOXP3 O‐GlcNAcylation Controls Regulatory T Cell Homeostasis and Suppressive Function

Regulatory T (Treg) cells are a distinct lineage of T lymphocytes that control immunological self‐tolerance and homeostasis. The use of Treg cells for immunotherapy has been challenging, partially because of the functional heterogeneity and instability of Treg cells. The transcription factor FOXP3 is crucial for Treg lineage specification during development and is continuously required for the suppressive function of mature Treg cells. FOXP3 can be regulated by various post‐translational modifications, which enable transient regulation of FOXP3 functionality in response to environmental cues. Here, we investigate the novel O‐linked N‐Acetylglucosamine (O‐GlcNAc) modification on FOXP3 in Treg cell function and stability.Protein O‐GlcNAcylation is enriched in induced Treg cells, compared to naïve T cells. Chemical augmentation of O‐GlcNAcylation promotes the induction of Treg cell signature genes. O‐GlcNAcylation, controlled by O‐GlcNAc cycling enzymes O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), modifies and stabilizes FOXP3 by counteracting with ubiquitination. Treg cell‐specific OGT deletion in mice does not affect Treg cell lineage specification, but results in fewer suppressive effector Treg cells and the development of an aggressive autoimmune syndrome. O‐GlcNAc‐deficient Treg cells show reduced FOXP3 protein expression and increased effector T‐cell signatures. In summary, we demonstrate that protein O‐GlcNAcylation, by modulating FOXP3, is indispensable for Treg cell suppressive function and lineage stability. Manipulating O‐GlcNAc levels may represent a novel approach to translate Treg cell immunotherapy into clinical practice.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MICRORNA-200A/200B MODULATE HIGH GLUCOSE-INDUCED ENDOTHELIAL INFLAMMATION VIA TARGETING O-LINKED N-ACETYLGLUCOSAMINE TRANSFERASE EXPRESSION

Enhanced O-linked N-acetylglucosamine (O-GlcNAc) modification by O-GlcNAc transferase (OGT) is associated with diabetic complications. Increase oxidative stress causes endothelial inflammation in diabetes. Members of microRNA-200 (miR-200) family are sensitive to oxidative stress. Here, we examined

Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using 2′,5′-ADP-Sepharose pull-down assay

Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2′,5′-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2′,5′-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions.

The sweet tooth of the circadian clock.

The endogenous circadian clock is a key regulator of daily metabolic processes. On the other hand, circadian clocks in a broad range of tissues can be tuned by extrinsic and intrinsic metabolic cues. The bidirectional interaction between circadian clocks and metabolism involves both transcriptional and post-translational mechanisms. Nuclear receptors exemplify the transcriptional programs that couple molecular clocks to metabolism. The post-translational modifications of the core clock machinery are known to play a key role in metabolic entrainment of circadian clocks. O-linked N-acetylglucosamine modification (O-GlcNAcylation) of intracellular proteins is a key mediator of metabolic response to nutrient availability. This review highlights our current understanding of the role of protein O-GlcNAcylation in mediating metabolic input and output of the circadian clock.

Pivotal Role of O-GlcNAc Modification in Cold-Induced Thermogenesis by Brown Adipose Tissue Through Mitochondrial Biogenesis.

Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism. O-linked N-acetylglucosamine (O-GlcNAc) modification is characterized by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (Ogt), subsequently modulating various cellular processes. However, little is known about the role of O-GlcNAc modification in adipose tissues. Here, we report the critical role of O-GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and BAT using adiponectin promoter-driven Cre recombinase resulted in severe cold intolerance with decreased uncoupling protein 1 (Ucp1) expression. Furthermore, Ogt deletion led to decreased mitochondrial protein expression in conjunction with decreased peroxisome proliferator-activated receptor γ coactivator 1-α protein expression. This phenotype was further confirmed by deletion of Ogt in BAT using Ucp1 promoter-driven Cre recombinase, suggesting that O-GlcNAc modification in BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under fasting conditions. This effect was mitigated after normal diet consumption but not after consumption of a fatty acid-rich ketogenic diet lacking carbohydrates, suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, O-GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT physiological responses.

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

Defective Branched-Chain Amino Acid Catabolism Disrupts Glucose Metabolism and Sensitizes the Heart to Ischemia-Reperfusion Injury

Elevated levels of branched-chain amino acids (BCAAs) have recently been implicated in the development of cardiovascular and metabolic diseases, but the molecular mechanisms are unknown. In amouse model of impaired BCAA catabolism (knockout [KO]), we found that chronic accumulation of BCAAs suppressed glucose metabolism and sensitized the heart to ischemic injury. High levels of BCAAs selectively disrupted mitochondrial pyruvate utilization through inhibition of pyruvate dehydrogenase complex (PDH) activity. Furthermore, downregulation of the hexosamine biosynthetic pathway in KO hearts decreased protein O-linked N-acetylglucosamine (O-GlcNAc) modification and inactivated PDH, resulting in significant decreases in glucose oxidation. Although the metabolic remodeling in KO did not affect baseline cardiac energetics or function, it rendered the heart vulnerable to ischemia-reperfusion injury. Promoting BCAA catabolism or normalizing glucose utilization by overexpressing GLUT1 in the KO heart rescued the metabolic and functional outcome. These observations revealed a novel role of BCAA catabolism in regulating cardiac metabolism and stress response.

Schwann Cell O-GlcNAc Glycosylation Is Required for Myelin Maintenance and Axon Integrity.

Schwann cells (SCs), ensheathing glia of the peripheral nervous system, support axonal survival and function. Abnormalities in SC metabolism affect their ability to provide this support and maintain axon integrity. To further interrogate this metabolic influence on axon-glial interactions, we generated OGT-SCKO mice with SC-specific deletion of the metabolic/nutrient sensing protein O-GlcNAc transferase that mediates the O-linked addition of N-acetylglucosamine (GlcNAc) moieties to Ser and Thr residues. The OGT-SCKO mice develop tomaculous demyelinating neuropathy characterized by focal thickenings of the myelin sheath (tomacula), progressive demyelination, axonal loss, and motor and sensory nerve dysfunction. Proteomic analysis identified more than 100 O-GlcNAcylated proteins in rat sciatic nerve, including Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans. PRX lacking O-GlcNAcylation is mislocalized within the myelin sheath of these mutant animals. Furthermore, phenotypes of OGT-SCKO and Prx-deficient mice are very similar, suggesting that metabolic control of PRX O-GlcNAcylation is crucial for myelin maintenance and axonal integrity. The nutrient sensing protein O-GlcNAc transferase (OGT) mediates post-translational O-linked N-acetylglucosamine (GlcNAc) modification. Here we find that OGT functions in Schwann cells (SCs) to maintain normal myelin and prevent axonal loss. SC-specific deletion of OGT (OGT-SCKO mice) causes a tomaculous demyelinating neuropathy accompanied with progressive axon degeneration and motor and sensory nerve dysfunction. We also found Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans, is O-GlcNAcylated. Importantly, phenotypes of OGT-SCKO and Prx mutant mice are very similar, implying that compromised PRX function contributes to the neuropathy of OGT-SCKO mice. This study will be useful in understanding how SC metabolism contributes to PNS function and in developing new strategies for treating peripheral neuropathy by targeting SC function.

Human RNA Polymerase II Promoter Recruitment in Vitro Is Regulated by O-Linked N-Acetylglucosaminyltransferase (OGT)

Although the O-linked N-acetylglucosamine (O-GlcNAc) modification of the RNA polymerase II C-terminal domain was described 20 years ago, the function of this RNA polymerase II (pol II) species is not known. We show here that an O-GlcNAcylated pol II species (pol IIγ) exists on promoters in vitro Inhibition of O-GlcNAc-transferase activity and O-GlcNAcylation prevents pol II entry into the promoter, and O-GlcNAc removal from pol II is an ATP-dependent step during initiation. These data indicate that O-GlcNAc-transferase activity is essential for RNA pol II promoter recruitment and that pol II goes through a cycling of O-GlcNAcylation at the promoter. Mass spectrometry shows that serine residues 2 and 5 of the pol II C-terminal domain are O-GlcNAcylated, suggesting an overlap with the transcription factor IIH (TFIIH)-dependent serine 5 phosphorylation events during initiation and P-TEFb (positive transcriptional elongation factor b) events during elongation. These data provide unexpected and important insights into the role of a previously ill-defined species of RNA polymerase II in regulating transcription.