O-GlcNAcylation In Cells Research Articles

Global profiling of O-GlcNAcylated c-Rel-dependent gene expression

Abstract NF-κB c-Rel is a critical regulator of transcription in T cells. We previously showed that c-Rel is O-GlcNAcylated at a single residue, serine 350, and this posttranslational modification has a dual role in positive and negative regulation of c-Rel-dependent transcription. Here we performed a targeted RNA expression study to examine how c-Rel O-GlcNAcylation influences the transcriptional landscape of known NF-κB-dependent genes in T cells. We found groups of genes that were upregulated, downregulated or unregulated upon increased O-GlcNAcylation, and several of these genes were dependent on c-Rel serine 350 as analyzed by mutational studies. We also developed a novel site-specific antibody targeting serine 350 O-GlcNAcylated c-Rel. Our functional studies show that this novel antibody is capable of detecting hyperglycemia-induced c-Rel O-GlcNAcylation in cells isolated from diabetes patients and identify O-GlcNAcylation-dependent DNA binding of c-Rel by chromatin immunoprecipitation. Knowledge gained from the gene expression study and the use of S350 site-specific anti-O-GlcNAcylated c-Rel antibody have the potential to reveal the role of c-Rel O-GlcNAcylation in regulating T cell function in autoimmune diabetes.

Islet O-GlcNAcylation Is Required for Lipid Potentiation of Insulin Secretion through SERCA2

During early obesity, pancreatic β cells compensate for increased metabolic demand through a transient phase of insulin hypersecretion that stabilizes blood glucose and forestalls diabetic progression. We find evidence that β cell O-GlcNAcylation, a nutrient-responsive post-translational protein modification regulated by O-GlcNAc transferase (OGT), is critical for coupling hyperlipidemia to β cell functional adaptation during this compensatory prediabetic phase. In mice, islet O-GlcNAcylation rises and falls in tandem with the timeline of secretory potentiation during high-fat feeding while genetic models of β-cell-specific OGT loss abolish hyperinsulinemic responses to lipids, invivo and invitro. We identify the endoplasmic reticulum (ER) Ca2+ ATPase SERCA2 as a β cell O-GlcNAcylated protein in mice and humans that is able to rescue palmitate-stimulated insulin secretion through pharmacological activation. This study reveals an important physiological role for β cell O-GlcNAcylation in sensing and responding to obesity, with therapeutic implications for managing the relationship between type 2 diabetes and its most common risk factor.

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

An Isotope-Coded Photocleavable Probe for Quantitative Profiling of Protein O-GlcNAcylation.

O-linked N-acetylglucosamine ( O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.

Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates.

The modification of proteins with O-linked N-acetylglucosamine ( O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.

Calpain I Activation Causes GLUT3 Proteolysis and Downregulation of O-GlcNAcylation in Alzheimer's Disease Brain.

Impairment of cerebral glucose uptake/metabolism in individuals with Alzheimer's disease (AD) is believed to lead to downregulation of protein O-GlcNAcylation, which contributes to tau pathogenesis through tau hyperphosphorylation. Level of glucose transporter 3 (GLUT3), a neuronal specific glucose transporter, is decreased in AD brain, which may contribute to impaired brain glucose uptake/metabolism. However, what causes the reduction of GLUT3 in AD brain is not fully understood. Here, we report 1) that decrease of GLUT3 is associated with the reduction of protein O-GlcNAcylation in AD brain, 2) that GLUT3 level is negatively correlated with calpain I activation in human brain, 3) that calpain I proteolyzes GLUT3 at the N-terminus in vitro, and 4) that activation of calpain I is negatively correlated with protein O-GlcNAcylation in AD brain. Furthermore, we found that overexpression of GLUT3 enhances protein O-GlcNAcylation in N2a cells. Overexpression of calpain I suppresses protein O-GlcNAcylation in these cells. These findings suggest a novel mechanism by which calpain I overactivation leads to GLUT3 degradation and the consequent down-regulation of protein O-GlcNAcylation in AD brain.

Discovery of Cell-Permeable O-GlcNAc Transferase Inhibitors via Tethering in Situ Click Chemistry.

O-GlcNAc transferase (OGT) is a key enzyme involved in dynamic O-GlcNAcylation of nuclear and cytoplasmic proteins similar to phosphorylation. Discovery of cell-permeable OGT inhibitors is significant to clarify the function and regulatory mechanism of O-GlcNAcylation. This will establish the foundation for the development of therapeutic drugs for relevant diseases. Here, we report two cell-permeable OGT inhibitors (APNT and APBT), developed from low-activity precursors (IC50 > 1 mM) via "tethering in situ click chemistry (TISCC)". Both of them were able to inhibit O-GlcNAcylation in cells without significant effects on cell viability. Unusual noncompetitive inhibition of OGT was helpful to discover novel inhibitors and explore the regulatory mechanism of OGT. The development of these molecules validates that TISCC can be utilized to discover novel lead compounds from components that exhibited very weak binding to the target.

Hsp90 Inhibition Leads to the Degradation of O‐Linked N‐Acetylglucosamine Transferase and to the Reduction of O‐GlcNAcylation

O‐linked N‐acetylglucosamine (O‐GlcNAc), which is increased in conditions such as hyperglycemia, has been implicated in many major human diseases such as diabetes and cardiovascular diseases. O‐GlcNAc transferase (OGT) catalyzes the addition of O‐GlcNAc to the serine/threonine residues of the protein substrates. Recently, we found that O‐GlcNAcylation inhibits proteasome function by the modification of Rpt2. In this study, we found a novel mechanism to modulate O‐GlcNAcylation in the cell. Exposure of bovine aortic endothelial (BAE) cells and human lung microvascular endothelial (HLMVE) cells to Hsp90 inhibitors, radicicol or 17‐AAG, resulted in significant reduction of OGT expression, and consequently, the reduction of O‐GlcNAcylation, suggesting that OGT might be a client protein of Hsp90. Hsp90 inhibition attenuated the increased O‐GlcNAcylation in cells exposed to high glucose, glucosamine or O‐GlcNAcase inhibitor, PUGNAc. The half‐life of OGT was reduced dramatically by Hsp90 inhibition. The degradation of OGT was proteasome‐dependent. Inhibition of proteasome by MG132, epoxomycin or Bortezomib, in conjunction with Hsp90 inhibition, led to the accumulation of OGT in the detergent‐insoluble fraction of the cell lysates, but not in the supernatant. These results strongly suggest that Hsp90 inhibitors might be administered to reverse the adverse effects of O‐GlcNAcylation in human diseases.