Johns Hopkins Biochemistry, Cellular and Molecular Biology Graduate Program

Research Topic: O-GlcNAc, A Novel Regulator of the Cellular Stress Response and Cell Surival.

O-GlcNAc

Hundreds, if not thousands, of key cellular proteins in the nucleus and cytoplasm of metazoans are modified by O-linked β-N-acetylglucosamine (O-GlcNAc) (Figure 1). Deletion of the UDP-GlcNAc: polypeptide O-β-N-acetyl-glucosaminyltransferase (OGT), the enzyme that adds O-GlcNAc, is lethal in single cells highlighting the importance of this simple post-translational modification. O-GlcNAc is thought to act as a modulator of protein function, in a manner analogous to protein phosphorylation; the addition of O-GlcNAc to the protein backbone is dynamic, responding to morphogens, the cell cycle, and changes in glucose metabolism. O-GlcNAc occurs at sites on the protein backbone that are similar to those modified by protein kinases; and is reciprocal with phosphorylation on some well studied proteins, including RNA Pol II, estrogen receptor-b, SV-40 large T-antigen, endothelial nitric oxide synthase, and the c-Myc proto-oncogene product. These data suggest that one mechanism by which O-GlcNAc modulates cellular function is by competing with phosphorylation (Figure 2). A clear role for O-GlcNAc in cellular regulation has not emerged, although modulation of O-GlcNAc levels are implicated in the etiology of Type II Diabetes, cancer, and neurodegenerative diseases.

In response to multiple forms of cellular stress, levels of the O-GlcNAc protein modification are elevated rapidly and dynamically on myriad nuclear and cytoplasmic proteins. Several studies demonstrate that elevation of O-GlcNAc prior to heat stress, oxidative stress, hypoxia, trauma hemorrhage, and ischemia reperfusion injury is protective, suggesting that increased O-GlcNAc in response to stress is a survival response of cells injury. Importantly, raising O-GlcNAc levels post-injury is protective in models of ischemia reperfusion injury, suggesting that modulation of O-GlcNAc may lead to the development of useful cardio-protective agents. In response to lethal doses of stress, global O-GlcNAc levels decline; and interestingly, in models of chronic stress such as diabetes O-GlcNAc sensitizes cells to cell death. These data highlight the importance of understanding the mechanisms by which O-GlcNAc regulates stress tolerance, and how these are mis-regulated in models of diabetes (Figure 3).

The exact molecular mechanism(s) by which O-GlcNAc regulates protein function leading to cellular protection have not been identified. However, O-GlcNAc has been shown to regulate the following pathways in a manner consistent with stress tolerance: 1) heat shock protein expression; 2) protein solubility; 3) cytosolic Ca2+ influx; 4) calpain activity; 5) p38 MAP kinase phosphorylation; 6) circulating IL-6 and TNF-a levels; and 7) maintenance of mitochondrial membrane potential, which is possibly dependent on VDAC. Thus, there is growing evidence to suggest that O-GlcNAc is a novel endogenously recruitable protective agent that modulates numerous proteins and cellular pathways to affect cellular survival.

Our long-range goal is to determine how stress-induced changes in the O-GlcNAc protein modification lead to increased cell/tissue survival in response to injury, in order to develop novel strategies for the treatment of numerous diseases, including ischemia reperfusion injury. Current research in the lab focus's on:

1) 1 Characterizing the molecular mechanisms by which O-GlcNAc regulates heat shock protein expression;

2) The development of novel cells lines for studying the O-GlcNAc modification;

3) Identifying proteins that are O-GlcNAc modified in response to different forms of cellular injury;

4) Understanding the signal transduction pathways that regulate O-GlcNAc modification in response to cellular injury;

5) Determining the molecular mechanisms by which stress-induced O-GlcNAcylation regulates the function of a protein (or group of proteins) leading to enhanced cellular survival.

Publications:

C. Gewinner, G. W. Hart, N.E. Zachara , R. N. Cole, C. Beisenherz-Huss, and B. Groner. (2004) The coactivator of transcription CREB binding protein interacts preferentially with the glycosylated form of Stat5. J. Biol. Chem., 279, 3563-3572.
PubMed Reference

N.E. Zachara , W. D. Cheung, and G. W. Hart (2004). O-GlcNAc, an essential post-translational modification of nucleocytoplasmic proteins. Current Organic Chemistry, 8, 369-383.
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N. O'Donnell, N.E. Zachara , G. W. Hart, and J. D. Marth. (2004) OGT-dependent X-chromosome-linked intracellular protein glycosylation is essential for mammalian viability and cellular metabolism. Mol. Cell. Biol., 24, 1680-1690.
PubMed Reference

N.E. Zachara , N. O'Donnell, J. J. Mercer, J. D. Marth, and G. W. Hart. (2004) Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J. Biol. Chem., 279, 30133-30142.
PubMed Reference

N.E. Zachara and G. W. Hart. (2004) O-GlcNAc a sensor of cellular state: The role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta., 1673, 13-28.
PubMed Reference

N.E. Zachara and G. W. Hart. (2004) O-GlcNAc, a nutritional effector molecule, modulates proteasome function. Trends in Cell Biology, 14, 218-221.
PubMed Reference

N.E. Zachara , W. D. Cheung, and G. W. Hart. (2004) Nucleocytoplasmic glycosylation, O-GlcNAc: identification and site mapping. In Signal Transduction Protocols, Methods in Molecular Biology, R. Dickson and M. Mendenhall (eds), Humana Press, Totowa, USA. Volume 284, pp.175-194.
PubMed Reference

N.E. Zachara and G. W. Hart. (2004) Protein glycosylation. In The Encyclopedia of Biochemistry, W. Lennarz and M. D. Lane (eds), Academic Press/Elsevier Science, New York, USA.Volume 3: 504-509.

C. Slawson, N.E. Zachara , K. Vosseller, W. Cheung, M. D. Lane, and G. W. Hart (2005) Perturbations in O-GlcNAc protein modification cause severe defects in mitotic progression and cytokinesis. J. Biol. Chem., 280, 32944-32956.
PubMed Reference

N.E. Zachara and G. W. Hart. (2006) Cell Signaling, the Essential Role of O-GlcNAc! Biochim. Biophys. Acta, 1761: 599-617.
PubMed Reference

N.E. Zachara . (2007) The sweet nature of cardioprotection. Amer J Phys (Heart and Circulatory Physiology; 293: H1324-H1326.
PubMed Reference

S. P. Jones, N.E. Zachara , Y. Teshima, G. W. Hart, and E. Marban. (2008) Endogenously-recruitable cardioprotection by N-acetylglucosamine linkage to cellular proteins. Circulation, 117(9):1172-82.
PubMed Reference

N.E. Zachara (2008). Detection and Analysis of Nuclearcytoplasmic Glycoproteins. In The Nucleus: Principles and Protocols, Meth Mol Biol. Hancock R (ed) Humana Press (Totowa, USA). Volume 464, pp. 227-254.
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