Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity

Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185, USA.
AJP Endocrinology and Metabolism (Impact Factor: 4.09). 08/2008; 295(1):E17-28. DOI: 10.1152/ajpendo.90281.2008
Source: PubMed

ABSTRACT O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification that, analogous to phosphorylation, cycles on and off serine and/or threonine hydroxyl groups. Cycling of O-GlcNAc is regulated by the concerted actions of O-GlcNAc transferase and O-GlcNAcase. GlcNAcylation is a nutrient/stress-sensitive modification that regulates proteins involved in a wide array of biological processes, including transcription, signaling, and metabolism. GlcNAcylation is involved in the etiology of glucose toxicity and chronic hyperglycemia-induced insulin resistance, a major hallmark of type 2 diabetes. Several reports demonstrate a strong positive correlation between GlcNAcylation and the development of insulin resistance. However, recent studies suggest that inhibiting GlcNAcylation does not prevent hyperglycemia-induced insulin resistance, suggesting that other mechanisms must also be involved. To date, proteomic analyses have identified more than 600 GlcNAcylated proteins in diverse functional classes. However, O-GlcNAc sites have been mapped on only a small percentage (<15%) of these proteins, most of which were isolated from brain or spinal cord tissue and not from other metabolically relevant tissues. Mapping the sites of GlcNAcylation is not only necessary to elucidate the complex cross-talk between GlcNAcylation and phosphorylation but is also key to the design of site-specific mutational studies and necessary for the generation of site-specific antibodies, both of which will help further decipher O-GlcNAc's functional roles. Recent technical advances in O-GlcNAc site-mapping methods should now finally allow for a much-needed increase in site-specific analyses to address the functional significance of O-GlcNAc in insulin resistance and glucose toxicity as well as other major biological processes.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. Both in vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study, we show that a therapeutically relevant concentration of Cr(3+) (100nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr(3+) is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase (GFAT), a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr(3+) attenuates reactive oxygen species (ROS) formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an anti-proliferative effect of Cr(3+) that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr(3+) to modulate aberrant VSMC function associated with hyperglycemia-induced vascular complications.
    AJP Cell Physiology 10/2014; 308(2):ajpcell.00256.2014. DOI:10.1152/ajpcell.00256.2014 · 3.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein O-GlcNAcylation, involving the attachment of single N-acetylglucosamine (GlcNAc) to the hydroxyl group of serine or threonine residues. Elucidation of O-GlcNAcylation sites on proteins is required in order to decipher its crucial roles in regulating cellular processes and aid in drug design. With an increasing number of O-GlcNAcylation sites identified by mass spectrometry (MS)-based proteomics, several methods have been proposed for the computational identification of O-GlcNAcylation sites. However, no development that focuses on the investigation of O-GlcNAcylated substrate motifs has existed. Thus, we were motivated to design a new method for the identification of protein O-GlcNAcylation sites with the consideration of substrate site specificity. In this study, 375 experimentally verified O-GlcNAcylation sites were collected from dbOGAP, which is an integrated resource for protein O-GlcNAcylation. Due to the difficulty in characterizing the substrate motifs by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. To construct the predictive models learned from the identified substrate motifs, we adopted Support Vector Machines (SVMs). A five-fold cross validation was used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 0.76, 0.80, and 0.78, respectively. Additionally, an independent testing set, which was really blind to the training data of predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (0.94) and outperform three other O-GlcNAcylation site prediction tools. This work proposed a computational method to identify informative substrate motifs for O-GlcNAcylation sites. The evaluation of cross validation and independent testing indicated that the identified motifs were effective in the identification of O-GlcNAcylation sites. A case study demonstrated that the proposed method could be a feasible means of conducting preliminary analyses of protein O-GlcNAcylation. We also anticipated that the revealed substrate motif may facilitate the study of extensive crosstalk between O-GlcNAcylation and phosphorylation. This method may help unravel their mechanisms and roles in signaling, transcription, chronic disease, and cancer.
    BMC Bioinformatics 12/2014; 15(Suppl 16):S1. DOI:10.1186/1471-2105-15-S16-S1 · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: O-GlcNAc signalling is regulated by an opposing pair of enzymes: O-GlcNAc transferase (OGT) instals, and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics and the regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches. This review charts the discovery and the functional and structural analysis of OGA, and summarises the insights gained from recent studies using OGA inhibition, gene knock-out and overexpression. We identify several areas of 'known unknowns' that would benefit from future research, such as the enigmatic C-terminal domain of OGA.
    Journal of Biological Chemistry 10/2014; DOI:10.1074/jbc.R114.609198 · 4.60 Impact Factor