Identification of O-GlcNAc Modification Targets in Mouse Retinal Pericytes: Implication of p53 in Pathogenesis of Diabetic Retinopathy

Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.     

O-GlcNAc transferase is in a functional complex with protein phosphatase 1 catalytic subunits

A hallmark of signal transduction is the dynamic and inducible post-translational modification of proteins. In addition to the well characterized phosphorylation of proteins, other modifications have been shown to be regulatory, including O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modifies serine and threonine residues on a myriad of nuclear and cytosolic proteins, and for several proteins there appears to be a reciprocal relationship between phosphorylation and O-GlcNAc modification. Here we report further evidence of this yin-yang relationship by demonstrating that O-GlcNAc transferase, the enzyme that adds O-GlcNAc to proteins, exists in stable and active complexes with the serine/threonine phosphatases PP1beta and PP1gamma, enzymes that remove phosphate from proteins. The existence of this complex highlights the importance of understanding the dynamic relationship between O-GlcNAc and phosphate in modulating protein function in many cellular processes and disease states such as Alzheimer's disease and type II diabetes.     

N-乙酰氨基葡萄糖转移酶（OGT）研究进展

O-GlcNAc是一种广泛存在于蛋白质丝/苏氨酸残基上的动态、可逆的蛋白翻译后修饰,它广泛分布在细胞浆和细胞核中,参与调节多种细胞途径。研究表明蛋白的O-GlcNAc糖基化与神经退行性疾病、糖尿病和癌症等疾病相关。在体内,O-GlcNAc动态修饰由N-乙酰氨基葡萄糖转移酶（OGT）和N-乙酰氨基葡萄糖苷酶（OGA）协同完成。近年来,OGT逐渐成为糖生物学领域的研究热点,在其结构、作用机制及晶体学方面取得了快速发展。     

Hsp90 regulates O-linked β-N-acetylglucosamine transferase: a novel mechanism of modulation of protein O-linked β-N-acetylglucosamine modification in endothelial cells

O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is involved in many important cellular processes. Increased O-GlcNAc has been implicated in major diseases, such as diabetes and its complications and cardiovascular and neurodegenerative diseases. Recently, we reported that O-GlcNAc modification occurs in the proteasome and serves to inhibit proteasome function by blocking the ATPase activity in the 19S regulatory cap, explaining, at least in part, the adverse effects of O-GlcNAc modification and suggesting that downregulating O-GlcNAc might be important in the treatment of human diseases. In this study, we report on a novel mechanism to modulate cellular O-GlcNAc modification, namely through heat shock protein 90 (Hsp90) inhibition. We observed that O-linked β-N-acetylglucosamine transferase (OGT) interacts with the tetratricopeptide repeat binding site of Hsp90. Inhibition of Hsp90 by its specific inhibitors, radicicol or 17-N-allylamino-17-demethoxygeldanamycin, destabilized OGT in primary endothelial cell cultures and enhanced its degradation by the proteasome. Furthermore, Hsp90 inhibition downregulated O-GlcNAc protein modifications and attenuated the high glucose-induced increase in O-GlcNAc protein modification, including high glucose-induced increase in endothelial or type 3 isoform of nitric oxide synthase (eNOS) O-GlcNAcylation. These results suggest that Hsp90 is involved in the regulation of OGT and O-GlcNAc modification and that Hsp90 inhibitors might be used to modulate O-GlcNAc modification and reverse its adverse effects in human diseases.     

Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki rats

Many nuclear and cytoplasmic proteins are O-glycosylated on serine or threonine residues with the monosaccharide beta-N-acetylglucosamine, which is then termed O-linked N-acetylglucosamine (O-GlcNAc). It has been shown that abnormal O-GlcNAc modification (O-GlcNAcylation) of proteins is one of the causes of insulin resistance and diabetic complications. In this study, in order to examine the relationship between O-GlcNAcylation of proteins and glucose-stimulated insulin secretion in noninsulin-dependent type (type 2) diabetes, we investigated the level of O-GlcNAcylation of proteins, especially that of PDX-1, and the expression of O-GlcNAc transferase in Goto-Kakizaki (GK) rats, which are an animal model of type-2 diabetes. By immunoblot and immunohistochemical analyses, the expression of O-GlcNAc transferase protein and O-GlcNAc-modified proteins in whole pancreas and islets of Langerhans of 15-week-old diabetic GK rats and nondiabetic Wistar rats was examined. The expression of O-GlcNAc transferase at the protein level and O-GlcNAc transferase activity were increased significantly in the diabetic pancreas and islets. The diabetic pancreas and islets also showed an increase in total cellular O-GlcNAc-modified proteins. O-GlcNAcylation of PDX-1 was also increased. In the diabetic GK rats, significant increases in the immunoreactivities of both O-GlcNAc and O-GlcNAc transferase were observed. PUGNAc, an inhibitor of O-GlcNAcase, induced an elevation of O-GlcNAc level and a decrease of glucose-stimulated insulin secretion in isolated islets. These results indicate that elevation of the O-GlcNAcylation of proteins leads to deterioration of insulin secretion in the pancreas of diabetic GK rats, further providing evidence for the role of O-GlcNAc in the insulin secretion.     

O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress

Myriad nuclear and cytoplasmic proteins in metazoans are modified on Ser and Thr residues by the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc). The rapid and dynamic change in O-GlcNAc levels in response to extracellular stimuli, morphogens, the cell cycle and development suggests a key role for O-GlcNAc in signal transduction pathways. Modulation of O-GlcNAc levels has profound effects on the functioning of cells, in part mediated through a complex interplay between O-GlcNAc and O-phosphate. In many well-studied proteins, the O-GlcNAc modification and phosphorylation are reciprocal. That is, they occur on different subsets of the protein population, as the site of attachment occurs on the same or adjacent Ser/Thr residues. Recently, O-GlcNAc has been implicated in the etiology of type II diabetes, the regulation of stress response pathways, and in the regulation of the proteasome.     

Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics

The molecular complexity that defines different cell types and their biological responses occurs at the level of the cell's proteome. The recent increase in availability of genomic sequence information is a valuable tool for the field of proteomics. While most proteomic studies focus on differential expression levels, post-translational modifications such as phosphorylation, glycosylation, and acetylation, provide additional levels of functional complexity to the cell's proteome. The reversible post-translational modification O-linked beta-N-acetylglucosamine (O-GlcNAc) is found on serines and threonines of nuclear and cytoplasmic proteins. It appears to be as widespread as phosphorylation. While phosphorylation is recognized as a fundamental mechanism for controlling protein function, less is known about the specific roles of O-GlcNAc modification. However, evidence is building that O-GlcNAc may compete with phosphate at some sites of attachment. Aberrant O-GlcNAc modification has been linked to several disease states, including diabetes and Alzheimer's disease. Regulated enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal (O-GlcNAcase) of the modification have been cloned and OGT is required for life at the single cell level. Here we review the properties of O-GlcNAc that suggest it is a regulatory modification analogous to phosphorylation. We also discuss the use of comparative functional proteomics to elucidate functions for this ubiquitous intracellular carbohydrate modification.     

Intracellular glycosylation and development

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification of cytoplasmic and nuclear proteins. Although the function of this abundant modification is yet to be definitively elucidated, all O-GlcNAc proteins are phosphoproteins. Further, the serine and threonine residues substituted with O-GlcNAc are often sites of, or close to sites of, protein phosphorylation. This implies that there may be a dynamic interplay between these two post-translational modifications to regulate protein function. In this review, the functions of some of the proteins that are modified by O-GlcNAc will be considered in the context of the potential role of the O-GlcNAc modification. Furthermore, predictions will be made as to how cellular function and developmental regulation might be affected by changes in O-GlcNAc levels.     

The roles of O-linked β-N-acetylglucosamine in cardiovascular physiology and disease

More than 1,000 proteins of the nucleus, cytoplasm, and mitochondria are dynamically modified by O-linked β-N-acetylglucosamine (O-GlcNAc), an essential post-translational modification of metazoans. O-GlcNAc, which modifies Ser/Thr residues, is thought to regulate protein function in a manner analogous to protein phosphorylation and, on a subset of proteins, appears to have a reciprocal relationship with phosphorylation. Like phosphorylation, O-GlcNAc levels change dynamically in response to numerous signals including hyperglycemia and cellular injury. Recent data suggests that O-GlcNAc appears to be a key regulator of the cellular stress response, the augmentation of which is protective in models of acute vascular injury, trauma hemorrhage, and ischemia-reperfusion injury. In contrast to these studies, O-GlcNAc has also been implicated in the development of hypertension and type II diabetes, leading to vascular and cardiac dysfunction. Here we summarize the current understanding of the roles of O-GlcNAc in the heart and vasculature.     

Cytosolic O-glycosylation is abundant in nerve terminals

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post-translational modification: beta-N-acetylglucosamine linked to hydroxyls of serines or threonines (O-GlcNAc(1)). O-GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O-GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O-GlcNAc transferase (OGT) and N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the two enzymes regulating O-GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O-GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron-specific O-GlcNAc modified proteins: collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1) and beta-synuclein. CRMP-2, which is involved in growth cone collapse, is a major O-GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O-GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O-GlcNAc may play a role in neurodegenerative disease.     

O-GlcNAc修饰与肿瘤发生及转移

O-连接的β-N-乙酰葡糖胺（O-GlcNAc）修饰是一种广泛存在于细胞浆和细胞核蛋白质丝/苏氨酸上的动态、可逆的翻译后修饰. 这种修饰与经典的糖基化不同而类似于磷酸化修饰,它在生命过程中发挥重要的调节作用. O-GlcNAc修饰作为潜在的营养感受器,可以调节转录、代谢等众多细胞进程,并与癌症等人类重大疾病密切相关. 本文主要综述了O-GlcNAc修饰与肿瘤形成和转移的关系,并对O-GlcNAc促进肿瘤形成与转移的潜在分子机制进行了探讨.     

Defining the regulated secreted proteome of rodent adipocytes upon the induction of insulin resistance

Insulin resistance defines the metabolic syndrome and precedes, as well is the hallmark of, type II diabetes. Adipocytes, besides being a major site for energy storage, are endocrine in nature and secrete a variety of proteins, adipocytokines (adipokines), that can modulate insulin sensitivity, inflammation, obesity, hypertension, food intake (anorexigenic and orexigenic), and general energy homeostasis. Recent data demonstrates that increased intracellular glycosylation of proteins via O-GlcNAc can induce insulin resistance and that a rodent model with genetically elevated O-GlcNAc levels in muscle and fat displays hyperleptinemia. The link between O-GlcNAc levels, insulin resistance, and adipocytokine secretion is further explored here. First, with the use of immortalized and primary rodent adipocytes, the secreted proteome of differentiated adipocytes is more fully elucidated by the identification of 97 and 203 secreted proteins, respectively. Mapping of more than 80 N-linked glycosylation sites on adipocytokines from the cell lines further defines this proteome. Importantly, adipocytokines that are modulated when cells are shifted from insulin responsive to insulin resistant conditions are determined. By the use of two protocols for inducing insulin resistance, classical hyperglycemia with chronic insulin exposure and pharmacological elevation of O-GlcNAc levels, several proteins are identified that are regulated in a similar fashion under both conditions including HCNP, Quiescin Q6, Angiotensin, lipoprotein lipase, matrix metalloproteinase 2, and slit homologue 3. Detection of these potential prognostic/diagnostic biomarkers for metabolic syndrome, type II diabetes, and the resulting complications of both diseases further establishes the central role of the O-GlcNAc modification of intracellular proteins in the pathophysiology of these conditions.