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DeLamar Professor and Director
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Baltimore MD 21205
Roles of cytoplasmic and nuclear glycosylation in transcription, oncogene function, neurodegenerative disease, and in diabetes
In the early 1980’s, the Hart laboratory discovered a new type of protein modification (O-GlcNAc), present on proteins within the nucleus and cytoplasm of cells, in which a glucose-derived sugar (N-acetylglucosamine; simply glucose with a nitrogen and an acetyl group attached) is attached to serine or threonine side chains of proteins, exactly analogous to phosphorylation. After twenty-six years of research, it is now known that this modification (termed O-GlcNAc) is nearly as common as phosphorylation, often competes with it at the same or proximal sites on proteins, and serves to regulate cellular functions in response to nutrients and stress by cycling on and off sites on proteins exactly like phosphorylation. O-GlcNAc is required for life at the single cell level in mammals. Recent studies, have shown that O-GlcNAc plays an important role in diabetes and glucose toxicity, Alzhemier’s disease and in the functions of oncogenes and tumor suppressors important to cancer. Since the cycling of O-GlcNAc is similar to phosphate cycling, and since they have similar abundance and distribution in cells, and since they can be attached competitively to the same or proximal sites, it was postulated that O-GlcNAc and phosphate have a ‘yin-yang’ relationship in the regulation of cellular processes. Very recent studies have established that the crosstalk between GlcNAcylation and phosphorylation is extensive and results not only from competition at the same or proximal sites, but also by the cycling enzymes for each PTM regulating the other’s activities. Recent Reviews: Science 291, 2376-2378; Nature 446, 1017-1022; Ann. Rev. Biochem. 80:825-58; Nature Reviews Cancer 11, 678-684; Cell, 143, 672-676. Some On-Going Projects: Cancer & O-GlcNAcylation. Protein phosphorylation-mediated regulation of signaling, growth and transcription is not only key to normal cellular regulation, but also is commonly dysregulated in cancers. Many anti-cancer drugs are specifically directed at kinases. Protein GlcNAcylation (O-GlcNAc) is nearly as abundant as Ser(Thr) phosphorylation. O-GlcNAc cycles like phosphorylation, and is often competitive with it. O-GcNAc regulates signaling, transcription, and cytoskeletal functions in response to nutrients and stress. Recent glycoproteomic studies have revealed surprisingly extensive crosstalk or interplay between GlcNAcylation and phosphorylation. This crosstalk results not only from competition for occupancy at the same or proximal sites, but also by each modification regulating the activities of the other’s enzymes. For example, O-GlcNAc Transferase is regulated by phosphorylation and kinases are regulated by GlcNAcylation. The goal of our study is to elucidate the global crosstalk between GlcNAcylation and phosphorylation at the individual site level and to understand the extent and mechanisms as to how GlcNAcylation regulates kinases. Aim 1: Elucidate the Dynamic Crosstalk Between GlcNAcylation and Phosphorylation. Aim 2: Regulation of Kinases by GlcNAcylation. We will continue to identify GlcNAcylated kinases and study their regulation by O-GlcNAc, focusing initially on CAMKIV, ERK5, PKCa and Src. Aim 3: Study the Roles of O-GlcNAc in Cytokinesis. Prostate Cancer & O-GlcNAcylation. Using well-defined normal prostate (PrEC), prostate cancer non-aggressive (LNCaP), and prostate cancer aggressive (PC-3) cells, we are addressing four aims: Aim 1) Systematically compare site-specific O-GlcNAcylation/phosphorylation of nucleocytoplasmic proteins and the enzymes controlling O-GlcNAc cycling as a function of prostate cancer phenotype. Aim 2) Compare subcellular localiza¬tions, expression levels, & molecular associations of O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) in PrEC, LNCaP and PC3 cell lines. Aim 3) How does heat stress affect the O-GlcNAcylation of cellular proteins and the en¬zymes of O-GlcNAc cycling in these cells, PrEC, LNCaP and PC3? Aim 4) Evaluate how specific al¬terations in O-GlcNAcylation affect growth properties, aneuploidy and expression and activities of androgen and estrogen receptors in the prostate cancer cells and their responses to stress. Diabetes & O-GlcNAcylation. The modification of proteins by b-N-Acetylglucosamine (O-GlcNAc) has extensive crosstalk with Ser(Thr)-protein phosphorylation to regulate signaling and gene expression in response to nutrients/stress. The hexosamine biosynthetic pathway and its endpoint product, O-GlcNAc, plays important roles in glucose toxicity and insulin resistance. O-GlcNAcylation plays a direct role in insulin signaling and insulin resistance at many points in the insulin signaling pathway, particularly on insulin receptor substrate proteins (IRS). Another key sensor of the cell’s energy state is the 5’-AMP-dependent protein kinase, AMPK. AMPK isoforms are O-GlcNAcylated and many key AMPK substrates of are also regulated by O-GlcNAc. Hyperglycemia-induced and insulin stimulated O-GlcNAcylation of the transcription factor, Sp1 underlie deregulated transcription that contributes to glucose toxicity and metabolic disease. This project is elucidating molecular events leading to insulin resistance and glucose toxicity by focusing on the roles of O-GlcNAc on proteins regulating cellular metabolism in response to nutrients, including IRS-1, AMPK and Sp1. Specific Aim 1: a. Elucidate the Regulation of Insulin Receptor Substrate (IRS) by Crosstalk Between O-GlcNAcylation and Phosphorylation. b. Systematic Analysis of the Kinetics of Insulin Signaling by Concomitantly Quantifying Changes at the Individual Site Level on key proteins in the insulin signaling pathway in O-GlcNAcylation and Phosphorylation using Novel MS Methods. Specific Aim 2. Continue to Elucidate the Roles of the Crosstalk Between O-GlcNAc and the AMP-Dependent Protein Kinase (AMPK) in Cellular Regulation. a. Characterization of AMPK’s modification by O-GlcNAc, including site mapping. b. Analysis of site-specific and global regulation of AMPK’s activity, targeting and subcellular localization by O-GlcNAc. c. Analysis of AMPK’s regulation of O-GlcNAc transferase activity, targeting and subcellular localization. Specific Aim 3. Elucidate the Roles of GlcNAcylation of the General Transcription Factor Sp1 in its Activity, Promoter Specificity, Localization, Molecular Associations and Turnover. a. Map and quantify site occupancy of O-GlcNAc and phosphate on Sp1 in different cell types and as a function of diabetic state. b. How does nutrient-mediated O-GlcNAcylation of Sp1 affect its activity in living cells? c. Do different glycoforms of Sp1 have distinct promoter specificities? d. Do different glycoforms of Sp1 have different molecular associations, or subcellular localizations or degradation rates? The results of these studies are not only elucidate novel key mechanisms underlying deregulation in diabetes and the metabolic syndrome, but also will uncover novel avenues for therapeutics. O-GlcNAcylation and Cardiovascular Disease. Diabetes is a major risk factor for cardiovascular disease, culminating in myocardial infarction, and heart failure. Prolonged hyper-O-GlcNAcylation, due to nutrient excess and hyperglycemia, is a major molecular cause of glucose toxicity and insulin resistance. Increased O-GlcNAcylation directly contributes to diabetic cardiomyopathy and to dysfunctional mitochondria, perhaps contributing to excessive production of reactive oxygen species (ROS). Even though O-GlcNAcylation clearly plays an important role in diabetic cardiovascular disease, virtually nothing is known about O-GlcNAcylation in the cardiomyocyte. This project is elucidating the roles of O-GlcNAc in diabetic cardiomyopathy and will define the “O-GlcNAcome” of the cardiomyocyte at the site-specific level. Specific Aims: Aim 1: Quantify the Site-Specific Crosstalk Between O-GlcNAcylation and Phosphorylation in the cardiomyocyte proteome and in purified cardiomyocyte mitochondria from Normal and Diabetic Rats. Using chemico-enzymatic photocleavable tag enrichment combined with electron transfer dissociation (ETD) tandem mass spectrometry, we will quantify site occupancy for both O-GlcNAc and phosphate in cardiomyocyte contractile and mitochondrial proteins from normal and diabetic rats. Aim 2: Determine the Specific Roles of O-GlcNAcylation in normal cardiomyocyte mitochondria, and the sites of action and mechanisms of diabetes-induced dysfunction, leading to ROS production. We will specifically alter O-GlcNAcylation using methods developed during the past 20-years, and correlate alterations with specific mitochondrial function. Aim 3: Elucidate the properties and regulation of cardiomyocyte mitochondrial isoforms of O-GlcNAc Transferase and O-GlcNAcase. Virtually nothing is known about the mitochondrial isoforms of O-GlcNAc Transferase (OGT) or O-GlcNAcase (OGA). We will elucidate their localization, activities, molecular associations and kinetic activities in mitochondria from normal and diabetic rats. Aim 4: Evaluate the affects and roles of diabetes-induced mitochondrial dysfunction and increased O-GlcNAcylation of cardiomyocyte contractile machinery on cardiac physiology and function. We will systematically evaluate the importance of the crosstalk between O-GlcNAcylation and phosphorylation of cardiomyocyte contractile and mitochondrial proteins on the physiological functions of cardiomyocytes. These studies will open a new paradigm for understanding the regulation of cardiac functions and in diabetic cardiomyopathies. They will lead to totally unexplored avenues of possible therapeutic interventions. Mechanisms of Glucose Toxicity Diabetes is a leading cause of blindness, cardiovascular disease, end-stage renal disease and debilitating neuropathies. Hyperglycemia underlies each of these pleiotropic maladies. However, the biochemical and physiological mechanisms for glucose toxicity remain unclear. Several biochemical mechanisms of glucose toxicity have been proposed, including increased flux through the hexosamine and polyol pathways, increased non-enzymatic, chemical glycation, and activation of protein kinase C isoforms. It has been proposed that glucose-induced production of reactive oxygen species (ROS) by the mitochondria is a unifying process affecting these disparate mechanisms of glucose toxicity. Our working hypothesis is that hyperglycemia-induced protein O-GlcNAcylation is a major fundamental mechanism leading to ROS production and to other mechanisms of glucose toxicity important to the morbidity and mortality of diabetes. We are jointly investigating mechanisms of glucose toxicity in well-defined animal model systems and in patient samples by a multi-disciplinary team comprised of both basic scientists and clinical experts with highly complimentary expertise. Our team is working synergistically to systematically evaluate the biochemical and physiological roles of enzymatic protein O-GlcNAcylation in diabetic neuropathy, diabetic cardiomyopathy, diabetic retinopathy, in glucose-induced beta-cell destruction, and in mitochondrial ROS production.
Hardivillй A, Hart GW, (2014) Nutrient Regulation of Transcription, Signaling, and Cell Physiology by O-GlcNAcylation. Cell Metabolism 20, 208–213
Bullen J, Balsbaugh J, Neumann D, Shabanowitz J, Hunt DF, Hart GW, (2014) Dynamic Crosstalk between two essential nutrient-sensitive enzymes: O-GlcNAc Transferase (OGT) and AMP-activated protein kinase (AMPK)” J Biol Chem. (2014) 289:10592-606
Hart GW, (2013) Nutrient Regulation of Immunity: O-GlcNAcylation Regulates Stimulus-Specific NF-?B–Dependent Transcription. Science Signaling 6 (290), pe26. [DOI: 10.1126/scisignal.2004596]
Erickson JR, Pereira L, Wang L, Han G, Ferguson A, Dao K,, Copeland RJ,, ;Despa F, Hart GW, Ripplinger CM, (2013) Diabetic Hyperglycemia activates CaMKII and Arrhythmias by O linked Glycosylation. Nature 502(7471):372-6.
Sakabe K and Hart GW,(2011) O-GlcNAc is Part of the Histone Code Proc. Natl. Acad. Sci. (USA) 107, 19915-19920
Tarrant MK, Rho H-S, Xie Z, Jiang YL, Gross C, Qian J, Ichikawa Y,, Matsuoka T, Zachara N, Etzkorn F,Hart GW, Jeong J-S, Blackshaw S, Zhu H, Cole PA, (2011) Multi-faceted Regulation of Protein Kinase CK2 by Phosphorylation and O-GlcNAcylation Revealed through Semisynthesis. Nature Chemical Biology 8(3):262-9
Hart GW, Slawson C, Ramirez-Correa G, and Lagerlof O, (2011) Crosstalk Between O-GlcNAcylation and Phosphorylation: Roles in Signaling, Transcription and Chronic Disease. Annual Review of Biochemistry 80:825-58
Zeidan Q, Wang Z, De Maio A and Hart GW, (2010) O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Many Core Ribosomal Proteins. Molecular Biology of the Cell 21, 1922-1936
Wang Z; Udeshi N; O’Malley M; Shabanowitz J; Hunt DF; and Hart GW (2010) Highly Specific Enrichment and Site-Mapping of O-GlcNAcylation Using Chemoenzymatic Tagging, Solid-Phase Photocleavage, and Electron Transfer Dissociation Mass Spectrometry. Molecular and Cellular Proteomics 9: 153-160.
Wang Z; Udeshi N; Slawson C; Compton P; Shabanowitz J; Hunt DF; and Hart GW, (2010) Extensive Crosstalk Between GlcNAcylation and Phosphorylation Regulates Cytokinesis Science Signaling 3, (104) ra2
Park Y, Saudek CD, and Hart GW, (2010) Increased Expression of ?-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals. Diabetes. 59(7):1845-50
Sakabe K and Hart GW, (2010) O-Glcnac TRANSFERASE REGULATES MITOTIC CHROMATIN DYNAMICS J. Biol. Chem. Nov 5; 285(45):34460-8 and Copeland RJ, (2010) Glycomics hits the big time. Cell 143, 672-676
Whelan SA, Dias W, Lakshmanan T, Lane MD, Hart GW, (2009) Regulation of Insulin Receptor 1 (IRS-1)/AKT Kinase Mediated Insulin Signaling by O-linked ?-N-acetylglucosamine (O-GlcNAc) in 3T3-L1 Adipocytes.J. Biol. Chem. 285, 5204-5211
Dias WB, Cheung WD, Wang Z, and Hart GW, (2009) Regulation of Calcium/Calmodulin-dependent Kinase IV by O-GlcNAc Modification. J. Biol. Chem. 284, 21327–21337