The modification of proteins by ?-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 will elucidate 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 will not only elucidate novel key mechanisms underlying deregulation in diabetes and the metabolic syndrome, but also will uncover novel avenues for therapeutics.

Public Health Relevance

The modification of cellular proteins by a sugar is involved in cellular dysfunctions caused by diabetes. This study is revealing molecular mechanisms by which this sugar modification contributes to defective insulin signaling and to the toxic effects of high blood glucose. Data from these experiments will reveal totally new avenues for developing treatments for diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK061671-11
Application #
8462963
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Sechi, Salvatore
Project Start
2002-05-01
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
11
Fiscal Year
2013
Total Cost
$325,105
Indirect Cost
$126,870
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Bullen, John W; Balsbaugh, Jeremy L; Chanda, Dipanjan et al. (2014) Cross-talk between two essential nutrient-sensitive enzymes: O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK). J Biol Chem 289:10592-606
Hardivillé, Stéphan; Hart, Gerald W (2014) Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation. Cell Metab 20:208-13
Ma, Junfeng; Hart, Gerald W (2013) Protein O-GlcNAcylation in diabetes and diabetic complications. Expert Rev Proteomics 10:365-80
Copeland, Ronald J; Han, Guanghui; Hart, Gerald W (2013) O-GlcNAcomics-Revealing roles of O-GlcNAcylation in disease mechanisms and development of potential diagnostics. Proteomics Clin Appl :
Hart, Gerald W (2013) Nutrient regulation of immunity: O-GlcNAcylation regulates stimulus-specific NF-?B-dependent transcription. Sci Signal 6:pe26
Erickson, Jeffrey R; Pereira, Laetitia; Wang, Lianguo et al. (2013) Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation. Nature 502:372-6
Hart, Gerald W (2013) How sugar tunes your clock. Cell Metab 17:155-6
Banerjee, Partha S; Hart, Gerald W; Cho, Jin Won (2013) Chemical approaches to study O-GlcNAcylation. Chem Soc Rev 42:4345-57
Slawson, Chad; Hart, Gerald W (2011) O-GlcNAc signalling: implications for cancer cell biology. Nat Rev Cancer 11:678-84
Wells, Lance; Slawson, Chad; Hart, Gerald W (2011) The E2F-1 associated retinoblastoma-susceptibility gene product is modified by O-GlcNAc. Amino Acids 40:877-83

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