Role Of O-GlcNAc Research Articles

Decreased O‐GlcNAcylation and mitochondrial dysfunction are involving in low glucose induced Alzheimer’s disease‐like phenotype in induced pluripotent stem cell‐derived neurons

AbstractBackgroundEvidence supporting that glucose metabolic abnormalities occur prior to Alzheimer’s disease (AD) symptoms. Reduced O‐GlcNAc (OGN) levels likely arise from impaired glucose metabolism or availability and correlates with AD pathogenesis. So far, the mechanism of sAD and the role of OGN in AD pathology remained largely unknown due to a lack of human sAD model.MethodWe generated human cortical neurons from human‐induced pluripotent stem cells and treated neurons with glucose reduction media. FluoroJADE staining and cell viability assays were used to study the effects of low glucose on the degenerative status and cell viability of neurons. Abnormal protein production, phosphorylation, and accumulation were detected by western blotting and immunofluorescence staining using antibody against p‐tau and beta‐amyloid. Neurite and synaptic structure were observed by immunofluorescence staining, while neuronal network activity was detected by multi‐electrode array electrophysiological analyses. Mitochondrial abnormalities, including increased oxidative stress, reduced membrane potential, and mitochondrial dysfunction, were evaluated using CM‐H2DCFDA, JC‐1 and Seahorse, respectively.ResultWe show that lowering glucose level to 2mM leads to dramatic increases of neuron degeneration on day 3 and 5 of treatment and decreased cell viability on day 7. Interestingly, long‐term low glucose treatment induces AD features in neurons, including abnormal hyperphosphorylated tau accumulation and increasing beta‐amyloid production. Besides, glucose deficiency also causes decreased neurite coverage, synapse density, and neuron network activity. Furthermore, we find that O‐GlcNAc levels are significantly reduced soon after low glucose treatment and remain low until the end of experiment. Raising O‐GlcNAc levels by thiamet‐G, inhibitor of O‐GlcNAcase, even in low glucose treated neurons, rescues low glucose‐induced AD phenotypes. Moreover, our data shows that O‐GlcNAc dysregulation causes mitochondrial abnormalities, which occur before any other degenerative phenotype appeared, and may be one of the underlying mechanisms of sAD onset and pathogenesis.ConclusionWe established a human neuron model in which the pathological features are reproduced by glucose deficiency. This platform can serve as a tool for better understanding molecular processes involved in neurodegeneration in sAD. Our results also suggest that dysregulated O‐GlcNAc levels and mitochondrial dysfunction by glucose deficiency are involved in the onset and progression of sAD.

O-Glcnacylation Modulates Erythropoiesis: O-Glcnac Transferase (OGT) Is an Essential Gene

Post-translational modifications (PTMs) can regulate protein activity; one such modification is O-GlcNAcylation. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. The O-GlcNAc PTM is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A balance in protein levels exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may be exploited therapeutically to alter gene expression. We previously demonstrated that O-GlcNAc plays a role in regulating human γ-globin gene transcription during development in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice and derivative immortalized bone marrow cells. OGT and OGA interact with Mi2β, a component of the GATA-1-FOG-Mi2b repressor complex that binds the -566 GATA site of the Aγ-globin promoter when γ-globin gene expression is silent. O-GlcNAcylation of Mi2βstimulates formation of this repressor complex, whereas removal of this PTM results in activation of Aγ-globin gene expression. Thus, O-GlcNAc cycling is a novel γ-globin regulatory mechanism, which might be modulated to increase fetal hemoglobin (HbF).Since O-GlcNAcylation involves input from multiple metabolic pathways, the modification acts as a general sensor of a cellular homeostasis. Thus, in response to environmental cues, the addition and removal of O-GlcNAc from proteins may be variably altered with potential effects on biochemical and transcriptional pathways essential for erythropoiesis. To better understand the mechanism as to how O-GlcNAcylation regulates β-like globin gene switching and whether it plays a role in erythropoiesis, we developed several new, innovative mouse models. These include erythroid-specific OGT conditional knockout mice, and transgenic mice with erythroid-specific enforced expression of human OGT or OGA to examine the effect of OGT loss-of-function or OGT/OGA gain-of-function on erythroid development in vivo . We utilized erythropoietin receptor promoter-Cre (ErGFPcre) mice or produced new tamoxifen-inducible μLCR-β-globin promoter-Cre (μLCR-β promoter-ERT2CreERT2) mice that were bred with floxed OGT mice to produce early erythroid progenitor-specific or tamoxifen-inducible erythroid-specific knockouts of OGT, respectively. In all instances, the final mice also contained the human β-YAC reporter transgene. Knockout of OGT using ErGFPcre results in fetal death during embryogenesis. Although conceptuses may be recovered up through embryonic day E12, they completely lack red blood cells and some organ anomalies are observed. An intermediate, anemic phenotype is observed when the knockout is incomplete. Adult floxed OGT, μLCR-β promoter-ERT2CreERT2 β-YAC mice treated with tamoxifen develop an anemia that is dose-time-dependent and ultimately correlates with the level of OGT still present. We present phenotypic and molecular data related to the hematopoietic system, including anemia, blood cell histology and morphology, standard blood indices and β-like globin gene expression during embryonic, fetal and adult stages of erythropoiesis for both the LOF and GOF mice.Understanding the molecular and cellular components of erythropoiesis is critical for treating anemia and other hematopoietic diseases which affect roughly 3 million Americans and 28% of the population worldwide. In a more general sense, our work may provide insight into the role of O-GlcNAc in regulating developmental and differentiation processes. DisclosuresNo relevant conflicts of interest to declare.

Neuronal O-GlcNAcylation Improves Cognitive Function in the Aged Mouse Brain.

SUMMARYMounting evidence in animal models indicates potential for rejuvenation of cellular and cognitive functions in the aging brain. However, the ability to utilize this potential is predicated on identifying molecular targets that reverse the effects of aging in vulnerable regions of the brain, such as the hippocampus. The dynamic post-translational modification O-linked N-Acetylglucosamine (O-GlcNAc) has emerged as an attractive target for regulating aging-specific synaptic alterations as well as neurodegeneration. While speculation exists about the role of O-GlcNAc in neurodegenerative conditions, such as Alzheimer’s disease, its role in physiological brain aging remains largely unexplored. Here, we report that countering age-related decreased O-GlcNAc transferase (OGT) expression and O-GlcNAcylation ameliorates cognitive impairments in aged mice. Mimicking an aged condition in young adults by abrogating OGT, using a temporally controlled neuron-specific conditional knockout mouse model, recapitulated cellular and cognitive features of brain aging. Conversely, overexpressing OGT in mature hippocampal neurons using a viral-mediated approach enhanced associative fear memory in young adult mice. Excitingly, in aged mice overexpressing neuronal OGT in the aged hippocampus rescued in part age-related impairments in spatial learning and memory as well as associative fear memory. Our data identify O-GlcNAcylaton as a key molecular mediator promoting cognitive rejuvenation.

O-GlcNAc: A Sweetheart of the Cell Cycle and DNA Damage Response.

The addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) to and from the Ser and Thr residues of proteins is an emerging post-translational modification. Unlike phosphorylation, which requires a legion of kinases and phosphatases, O-GlcNAc is catalyzed by the sole enzyme in mammals, O-GlcNAc transferase (OGT), and reversed by the sole enzyme, O-GlcNAcase (OGA). With the advent of new technologies, identification of O-GlcNAcylated proteins, followed by pinpointing the modified residues and understanding the underlying molecular function of the modification has become the very heart of the O-GlcNAc biology. O-GlcNAc plays a multifaceted role during the unperturbed cell cycle, including regulating DNA replication, mitosis, and cytokinesis. When the cell cycle is challenged by DNA damage stresses, O-GlcNAc also protects genome integrity via modifying an array of histones, kinases as well as scaffold proteins. Here we will focus on both cell cycle progression and the DNA damage response, summarize what we have learned about the role of O-GlcNAc in these processes and envision a sweeter research future.

O-Linked N-Acetylglucosamine Transiently Elevates in HeLa Cells during Mitosis.

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by O-GlcNAc. Although the role of O-GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of O-GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall O-GlcNAc levels in HeLa cells using double thymidine block. O-GlcNAc levels in G1, S, G2 and M phase were measured. We observed that O-GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G2/M fraction of the synchronized cells. Our data verify that O-GlcNAc is elevated during mitosis, but also emphasize that O-GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for O-GlcNAc studies.

O-GlcNAcylation regulates endoplasmic reticulum exit sites through Sec31A modification in conventional secretory pathway.

The conventional secretory pathway is indispensable for eukaryotic cells. Newly synthesized membrane and secretory proteins are released from the endoplasmic reticulum (ER) through ER-derived vesicles to their appropriate destination. Vesicle formation is important for steady protein trafficking. O-GlcNAcylation ( O-GlcNAc) is a unique protein glycosylation signature, whose dynamic regulation by O-GlcNAc transferase and O-GlcNAcase occurs exclusively for nuclear and cytoplasmic proteins. Because of this locally limited property, the role of O-GlcNAc in the conventional protein secretory pathway is unknown. We report that Sec31A on COPII vesicles, a specific coat-protein complex for anterograde trafficking in the ER-Golgi network, is O-GlcNAcylated on S964, which accelerates COPII vesicle formation through control of its binding affinity to apoptosis-linked gene 2, a calcium-binding protein. Together, O-GlcNAc on Sec31A regulates conventional secretory vesicle trafficking in the ER-Golgi network. These modifications accelerate COPII vesicle formation and accelerated anterograde transport of vesicles within the ER-Golgi networks.-Cho, H. J., Mook-Jung, I. O-GlcNAcylation regulates endoplasmic reticulum exit sites through Sec31A modification in conventional secretory pathway.

Sustained O‐GlcNAcylation Amplifies Erk Signaling

O‐linked N‐Acetylglucosamine, more commonly referred to as O‐GlcNAc, is a single glycosidic post‐translational modification. OGT (O‐GlcNAc Transferase) and OGA (O‐GlcNAcase) are the sole enzymes responsible for the addition (O‐GlcNAcylation) and removal of O‐GlcNAc, respectively. O‐GlcNAc, OGA, and OGT are present in all multicellular organisms and organ tissues. Importantly, OGT knockouts are embryonically lethal, demonstrating the requirement for O‐GlcNAcylation in early organismal development. Like most intracellular post‐translational modifications, O‐GlcNAc regulates a number of cellular pathways including mitochondrial respiration, cell cycle progression, and transcription. Recently, we demonstrated that prolonged treatment of SY5Y neuroblastoma cells with Thiamet‐G (TMG), an OGA inhibitor, caused a dramatic reprogramming of the transcriptome. Therefore, we performed next generation RNA sequencing to investigate the transcriptome changes from isolated liver tissues from mice treated with TMG for two weeks. We also performed the same analysis for liver tissue containing an OGT knockout (OGT‐KO) to compare the effects of sustained O‐GlcNAcylation to the loss of O‐GlcNAcylation. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differences in expression for gene sets between the two groups. Further analysis of the data demonstrated an inverse correlation of transcriptional changes for several gene families between TMG‐treated and OGT‐KO. MAPK/ERK signaling genes were among these families, which we found to be of interest. ERK (extracellular regulated protein kinase) phosphorylates a wide variety of protein substrates, regulating processes such as apoptosis, transcription, and cell cycle progression. OGT‐KO was predicted to down‐regulate this pathway while our previous data predicted TMG to up‐regulate the pathway. To test this observation, we grew HeLa cells in TMG for three weeks and serum starved them overnight. The following day, we returned the serum to the media and harvested the lysate at 0, 5,10, 20, 60, and 120 minute time points. When we performed a western blot analysis of the results, the TMG‐treated group demonstrated dramatic amplification of pERK. This observation demonstrates a novel role for O‐GlcNAc as a regulator of the ERK signaling pathway.Support or Funding InformationNIH NIDDK R01 DK100595This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag)

Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

Characterization of tools to detect and enrich human and mouse O-GlcNAcase.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential regulatory post-translational modification of thousands of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc is dynamically added and removed from proteins by the O-GlcNAc transferase and the O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAc-cycling is implicated in the etiology of numerous diseases including tumorigenesis, metabolic dysfunction, and neurodegeneration. To facilitate studies focused on the role of O-GlcNAc and OGA in disease, we sought to identify commercially available antibodies that enable the enrichment of full-length OGA from lysates of mouse and human origin. Here, we report that antibodies from Abcam and Bethyl Laboratories can be used to immunoprecipitate OGA to near-saturation from human and mouse cell lysates. However, Western blotting analysis indicates that both antibodies, as well as three non-commercially available antibodies (345, 346, 352), detect full-length OGA and numerous cross-reacting proteins. These non-specific signals migrate similarly to full-length OGA and are detected robustly, suggesting that the use of appropriate controls is essential to avoid the misidentification of OGA.

O‐GlcNAcylation modifies the metastatic properties of prostate cancer cells (789.5)

Prostate cancer is one of the most common forms of cancer in men and the second leading cause of cancer deaths in men in the United States. O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a ubiquitous and dynamic post‐translational modification that exists on serine and threonine residues of nuclear and cytoplasmic proteins. This modification is regulated by O‐GlcNAc transferase (OGT), which attaches O‐GlcNAc to proteins, and O‐GlcNAcase (OGA), which removes O‐GlcNAc. O‐GlcNAc serves as a nutrient sensor to regulate virtually all cellular processes as well as playing roles in the various diseases, including Alzheimer's disease, diabetes, and cancer. The roles of O‐GlcNAc in the regulation of prostate cancer remains unclear. Herein, we investigate how the regulation of O‐GlcNAc may affect prostate cancer, progression using normal prostate epithelial cell (PrEc) and prostate cancer cell lines (LNCaP and PC‐3) as models. Using western blotting techniques, we show that O‐GlcNAc, OGT, and OGA are highly elevated in LNCaP and PC‐3 when compared to PrEc. LNCaP exhibited the highest levels of O‐GlcNAc, OGT, and OGA. OGT and OGA are differentially localized in lines with different metastatic properties. Additionally, 2D‐gel electrophoretic analyses show significant differences in O‐GlcNAcylated protein profiles among the three cell lines. In order to answer how O‐GlcNAc affects the biological properties of these cancer cells, including shapes, proliferation, invasion and metastasis, several assays were used. The result of soft agar assays indicate that alterations in O‐GlcNAc influence the metastasis ability of prostate cancer.

Overexpression of O-GlcNAc by prostate cancer cells is significantly associated with poor prognosis of patients

O-linked β-N-acetylglucosamine (O-GlcNAc) is a glycan essential for fundamental cellular processes such as transcription/translation, nuclear transport, protein stability and protein-protein interactions. However, the role of O-GlcNAc in prostate cancer progression of patients remains poorly unknown. Here we investigated the clinicopathological significance of O-GlcNAc expression level in prostate cancer. O-GlcNAc expression level in prostate cancer cells was determined by immunohistochemistry of prostate biopsy specimens obtained from 56 patients later treated with hormone deprivation therapy comparing with adjacent normal prostate glands in the same sections. Overall survival was determined by the Kaplan-Meier and Cox proportional hazards methods with univariate and multivariate models. The effects of reduced O-GlcNAc expression level on proliferation and invasion of prostate cancer LNCaP cells were examined using small interfering RNA (siRNA) targeting O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAc biosynthesis. Defining cancer cells showing stronger cytoplasmic staining than normal prostate glands as overexpression of O-GlcNAc, 39% of prostate cancer patients were categorized as overexpression. The Kaplan-Meier and Cox proportional hazards methods with univariate model analysis revealed that O-GlcNAc overexpression was associated with overall survival (P=0.0012 for the Kaplan-Meier and P=0.0021 for Cox univariate hazard model analysis). Furthermore, O-GlcNAc was the only item in which a significant difference was observed at overall survival by multivariate analysis (P=0.0475). Finally, siRNA-mediated OGT knockdown in LNCaP cells resulted in decreased expression of O-GlcNAc and promoted decreased proliferation and tumor cell invasion compared with control siRNA-transfected LNCaP cells. These results indicate that O-GlcNAc expression level in prostate cancer cells is associated with poor prognosis of prostate cancer patients and likely enhances tumor cell proliferation and invasion.

Chronic ingestion of a Western diet increases O-linked-β-N-acetylglucosamine (O-GlcNAc) protein modification in the rat heart

AimsProtein O-GlcNAcylation is both a nutrient sensing and cellular stress response that mediates signal transduction in the heart. Chronically elevated O-GlcNAc has been associated with the development of cardiac dysfunction at both the cellular and organ levels in obesity, insulin resistance and diabetes. Development of these pathologies is often attributed to diets high in saturated fat and sugar (a “Western” diet; WES) but a role for O-GlcNAc in diet-induced cardiac dysfunction has not been established. The aims of this study were to examine the effect of chronic consumption of WES on cardiac O-GlcNAcylation and investigate associations of O-GlcNAc with cardiac function and markers of cellular stress. Main methodsYoung male rats received either a control diet (CON; n=9) or WES (n=8) diet for 52weeks. Key findingsThere was no evidence of cardiac dysfunction, advanced glycation endproduct (AGE) accumulation, pathological cardiac hypertrophy, calcium handling impairment, fibrosis or endoplasmic reticulum stress in WES hearts. However, cardiac O-GlcNAc protein, particularly in the higher molecular weight range, was significantly higher in WES hearts compared to CON (P<0.05). Protein levels of the enzymes that regulate O-GlcNAc attachment were not different between groups; thus, the increased O-GlcNAcylation in WES hearts appears to be due to increased nutrient availability rather than enzymatic regulation of cellular stress. SignificanceThese data suggest that diets high in saturated fat and sugar may contribute to the adverse effects of metabolic syndrome and diabetes by an O-GlcNAc-mediated process and that this may occur in the absence of overt cellular stress.

Alterations in O‐linked‐β‐N‐acetylglucosamine levels during acute exercise

Acute bouts of exercise reduce damage to cardiomyocytes during ischemic events, including a reduction in infarct size and improvement in heart function. The mechanism underlying exercise-induced cardioprotection is still unknown. Previous reports have demonstrated increases in O-linked-β-N-aceytlglucosamine (O-GlcNAc) provide protection against ischemia-reperfusion injury, yet the role of O-GlcNAc in exercise has not been investigated. Our lab proposes O-GlcNAc may be a major factor in exercise-induced cardioprotection. We predicted an increase in O-GlcNAc levels in both cellular and nuclear compartments of male CD-1 mouse hearts following acute exercise (EX) compared to a sedentary control (SED). O-GlcNAc levels were not altered in either cytosolic or nuclear fractions of the 30 min EX group and SED group. O-GlcNAc transferase (OGT), the enzyme that attaches O-GlcNAc to proteins, was not different between 30 min groups. The 15 min groups did not demonstrate a difference in cytosolic levels of OGT, however, there was a significant decrease in OGT levels in the nuclear compartment of the EX group. Overall, the data suggest that cardiac O- GlcNAc and OGT levels either do not change or decrease during acute exercise.

Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc

Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.

O-GlcNAc modification of proteins affects volume regulation in Jurkat cells

An increasing amount of recent research has demonstrated that the hexosamine biosynthesis pathway (HBP) plays a significant role in the modulation of intracellular signaling transduction pathways, and affects cellular processes via modification of protein by O-linked beta-N-acetylglucosamine (O-GlcNAc). Besides the many known and postulated effects of protein O-GlcNAc modifications, there is little available data on the role of O-GlcNAc in cellular volume regulation. Our objective was to test the effect of increased O-GlcNAc levels on hypotonia-induced volume changes in Jurkat cells. We pretreated Jurkat cells for 1 h with glucosamine (GlcN), PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate) an inhibitor of O-GlcNAcase, or a high level of glucose to induce elevated levels of O-GlcNAc. We found that the response of Jurkat cells to hypotonic stress was significantly altered. The hypotonia induced cell-swelling was augmented in both GlcN and PUGNAc-treated cells and, to a lesser extent, in high glucose concentration-treated cells. Evaluated by NMR measurements, GlcN and PUGNAc treatment also significantly reduced intracellular water diffusion. Taken together, increased cell swelling and reduced water diffusion caused by elevated O-GlcNAc show notable analogy to the regulatory volume changes seen by magnetic resonance methods in nervous and other tissues in different pathological states. In conclusion, we demonstrate for the first time that protein O-GlcNAc could modulate cell volume regulation.

The sweet nature of cardioprotection

glucose, glutamine, and glucosamine are metabolites that can alter cardioprotection ([1][1], [3][2], [8][3], [9][4], [13][5]–[15][6]). Numerous studies suggest that one molecular mechanism by which these metabolites protect cells is though their conversion to uridine diphosphate- N -

Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain.

The covalent modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degradation, and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput analysis of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymatic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixtures and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins associated with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymatic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiological significance of O-GlcNAc across the proteome.

An N-terminal Region of Sp1 Targets Its Proteasome-dependent Degradation in Vitro

The transcription factor Sp1 is important for the expression of many cellular genes. Previously, it was shown that reduced O-glycosylation of Sp1 is associated with increased proteasome susceptibility. Sp1 undergoes proteasome-dependent degradation in cells stressed with glucose deprivation and adenylate cyclase activation, and this process is blocked in cells treated with glucosamine. In this study, using a reconstituted in vitro system, we identified the principal structural determinant in Sp1 that targets Sp1 for proteasome-dependent degradation. We found by using deletion analysis that the N-terminal 54 amino acids of Sp1 is required for Sp1 degradation. This element can act as an independent processing signal by directing degradation of an unrelated protein. Recognition of this Sp1 element by the proteasome-dependent system is saturable, and ubiquitination of this element is not required for recognition. Time course experiments revealed that Sp1 degradation is a two-step process. First, a discrete endoproteolytic cleavage occurs downstream of the target region immediately C-terminal to Leu56. The Sp1 sequence C-terminal to the cleavage site is subsequently degraded, whereas the N-terminal peptide remains intact. The identification of this Sp1 degradation-targeting signal will facilitate the identification of the critical proteins involved in the control of Sp1 proteasome-dependent degradation and the role of OGlcNAc in this process.