Jin Won Cho

Modification of p53 with O -linked N -acetylglucosamine regulates p53 activity and stability

Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.

NFκB activation is associated with its O-GlcNAcylation state under hyperglycemic conditions

The transcription factor NFκB is activated by phosphorylation and acetylation and plays important roles in inflammatory and immune responses in the cell. Additionally, posttranslational modification of the NFκB p65 subunit by O-linked N-acetylglucosamine (O-GlcNAc) has been reported, but the modification site of O-GlcNAc on NFκB p65 and its exact function have not been elucidated. In this work, we show that O-GlcNAcylation of NFκB p65 decreases binding to IκBα and increases transcriptional activity under hyperglycemic conditions. Also, we demonstrate that both Thr-322 and Thr-352 of NFκB p65 can be modified with O-GlcNAc, but modification on Thr-352, not Thr-322, is important for transcriptional activation. Our findings suggest that site-specific O-GlcNAcylation may be a reason why NFκB activity increases continuously under hyperglycemic conditions.

Protein N-glycosylation, protein folding, and protein quality control

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.

Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition.

Protein O‐phosphorylation often occurs reciprocally with O‐GlcNAc modification and represents a regulatory principle for proteins. O‐phosphorylation of serine by glycogen synthase kinase‐3β on Snail1, a transcriptional repressor of E‐cadherin and a key regulator of the epithelial–mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O‐phosphorylation‐mediated degradation, O‐GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E‐cadherin mRNA expression. Hyperglycaemic condition enhances O‐GlcNAc modification and initiates EMT by transcriptional suppression of E‐cadherin through Snail1. Thus, dynamic reciprocal O‐phosphorylation and O‐GlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.

O-GlcNAc protein modification in cancer cells increases in response to glucose deprivation through glycogen degradation

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

Down-regulation of Sp1 Activity through Modulation of O-Glycosylation by Treatment with a Low Glucose Mimetic, 2-Deoxyglucose

2-Deoxyglucose (2-DG), a nonmetabolizable glucose analogue, blocks glycolysis at the phosphohexose isomerase step and has been frequently used as a glucose starvation mimetic in studies of a wide variety of physiological dysfuctions. However, the effect of 2-DG on protein glycosylation and related signal pathways has not been investigated in depth. In HeLa, an HPV18-positive cervical carcinoma line, 2-DG treatment down-regulates human papillomavirus early gene transcription. This down-regulation was also achieved by low glucose supply or hypoxia, suggesting that this is a response commonly modulated by cellular glucose or energy level. We investigated how 2-DG and low glucose affect transcriptional activity. Human papillomavirus gene transcription was only marginally affected by the inhibition of ATP synthesis or the supplementation of pyruvate to 2-DG-treated cells, suggesting that poor ATP generation is involved only to a limited extent. 2-DG treatment also inhibited activation of p21 WAF1 promoter, which is controlled by p53 and/or Sp1. In a reporter assay using p21 WAF1 promoter constructs, 2-DG exerted a strong inhibitory effect on Sp1 activity. DNA binding activity of Sp1 in 2-DG-treated HeLa cells was intact, whereas it was severely impaired in cells incubated in a low glucose medium or in hypoxic condition. Unexpectedly, Sp1 was heavily modified with GlcNAc in 2-DG-treated cells, which is at least partially attributed to the inhibitory effect of 2-DG on N-acetyl-β-d-glucosaminidase activity. Our results suggest that 2-DG, like low glucose or hypoxic condition, down-regulates Sp1 activity, but through hyper-GlcNAcylation instead of hypo-GlcNAcylation.

O-GlcNAc modification of PPARγ reduces its transcriptional activity

The peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is a key regulator of adipogenesis and is important for the homeostasis of the adipose tissue. The β-O-linked N-acetylglucosamine (O-GlcNAc) modification, a posttranslational modification on various nuclear and cytoplasmic proteins, is involved in the regulation of protein function. Here, we report that PPARγ is modified by O-GlcNAc in 3T3-L1 adipocytes. Mass spectrometric analysis and mutant studies revealed that the threonine 54 of the N-terminal AF-1 domain of PPARγ is the major O-GlcNAc site. Transcriptional activity of wild type PPARγ was decreased 30% by treatment with the specific O-GlcNAcase (OGA) inhibitor, but the T54A mutant of PPARγ did not respond to inhibitor treatment. In 3T3-L1 cells, an increase in O-GlcNAc modification by OGA inhibitor reduced PPARγ transcriptional activity and terminal adipocyte differentiation. Our results suggest that the O-GlcNAc state of PPARγ influences its transcriptional activity and is involved in adipocyte differentiation.

Heat shock protein 60 modified with O-linked N-acetylglucosamine is involved in pancreatic β-cell death under hyperglycemic conditions

The objective of this study was to identify proteins modified with O‐linked N‐acetylglucosamine (O‐GlcNAc) in pancreatic β‐cells and to understand their roles in cell death under hyperglycemic conditions. Here we report that heat shock protein 60 (HSP60) is modified with O‐GlcNAc. Levels of O‐GlcNAcylated HSP60 increased twofold in response to hyperglycemic conditions. HSP60 is a chaperonin known to bind to Bax in the cytoplasm under normoglycemic conditions. Under hyperglycemic conditions, Bax detached from O‐GlcNAcylated HSP60 and translocated to mitochondria. Hyperglycemic conditions were also associated with cytochrome c release, caspase‐3 activation, and cell death, suggesting that elevated O‐GlcNAcylation of HSP60 interferes with HSP60–Bax interactions, leading to pancreatic β‐cell death.

Carbohydrate Arrays for Functional Studies of Carbohydrates

Carbohydrates, as components of glycoproteins, glycolipids and proteoglycans, play an important biological role as recognition markers through carbohydrate-protein interactions. For the most part, biophysical and biochemical methods have been used to analyze these biomolecular interactions. In contrast, less attention has been given to the development of high-throughput procedures to elucidate carbohydrate-protein recognition events. Recently, carbohydrate arrays were developed and employed as a novel high-throughput analytic tool for monitoring carbohydrate-protein interactions. This technique has been used to profile protein binding and enzymatic activity. The results have shown that carbohydrate binding to the corresponding lectins is highly selective and that the relative binding affinities are well correlated with those obtained from solution-based assays. In addition, this effort demonstrated that carbohydrate arrays could be also utilized to identify and characterize novel carbohydrate-binding proteins or carbohydrate-processing enzymes. Finally, the results of this investigation showed that lectin-carbohydrate binding affinities could be quantitatively assessed by determining IC50 values for soluble carbohydrates with the carbohydrate arrays. The results of these studies suggest that carbohydrate arrays have the potential of playing an important role in basic researches, the diagnoses of diseases and drug discovery.

Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells.

Increased modification of proteins with O‐linked N‐acetylglucosamine (O‐GlcNAc) has been implicated in the development of diabetic cardiomyopathy. We used the well‐characterized ES cells (Nkx2.5GFP knock‐in ES cells), to investigate the role of O‐GlcNAcylation in cardiomyocyte development. O‐GlcNAcylation decreased in differentiating ES cells, as did the expression of O‐GlcNAc transferase. Increasing O‐GlcNAcylation with glucosamine or by inhibiting N‐acetylglucosaminidase (streptozotocin or PUGNAc) decreased the number of cardiomyocyte precursors and cardiac‐specific gene expression. On the other hand, decreasing O‐GlcNAcylation with an inhibitor of glutamine fructose‐6‐phosphate amidotransferase (6‐diazo‐5‐oxo‐norleucine) increased cardiomyocyte precursors. These results suggest that excessive O‐GlcNAcylation impairs cardiac cell differentiation in ES cells.