Amino Acids

, Volume 40, Issue 3, pp 793–808

The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways

  • Natasha E. Zachara
  • Henrik Molina
  • Ker Yi Wong
  • Akhilesh Pandey
  • Gerald W. Hart
Original Article

Abstract

The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.

Keywords

O-GlcNAc Cellular stress Glycosylation Signal transduction Chaperone Cell stress 

Abbreviations

Carm1 CK II

Casein kinase II

DNA-PK

DNA-dependent protein kinase

DMSO

Dimethylsulfoxide

DON

6-diazo-5-oxonorleucine

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

FBS

Fetal bovine serum

HSP

Heat shock protein

MEFs

Mouse embryonic fibroblasts

O-GlcNAc

Monosaccharides of O-linked β-N-acetylglucosamine

O-GlcNAcase

O-GlcNAc hexosaminidase (EC 3.2.1.52)

OGT: UDP-GlcNAc

Polypeptide O-N-acetylglucosaminyltransferase (EC 2.4.1.94)

PBS

Phosphate-buffered saline

PUGNAc

O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate

TBS

Tris-buffered saline

Supplementary material

726_2010_695_MOESM1_ESM.pdf (45 kb)
(PDF 44 kb)
726_2010_695_MOESM2_ESM.xls (179 kb)
Supplementary Table 2 Raw Mass Spectrometry Data (XLS 179 kb)
726_2010_695_MOESM3_ESM.eps (4.4 mb)
Supplementary Fig. S1 NF45 does not appear to be O-GlcNAc modified. NF-90 was immunoprecipitated from Cos-7 cells and separated by SDS–PAGE (16.5cm, 7.5% gel). While NF-90 appeared in the complex, we could not detect it by blotting with CTD110.6 suggesting that it’s occurance in the CTD110.6 IP is due to the other O-GlcNAcylated proteins in this complex (EPS 4467 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Natasha E. Zachara
    • 1
    • 2
  • Henrik Molina
    • 1
    • 3
  • Ker Yi Wong
    • 2
  • Akhilesh Pandey
    • 1
    • 3
  • Gerald W. Hart
    • 1
  1. 1.The Department of Biological ChemistryThe Johns Hopkins University School of MedicineBaltimoreUSA
  2. 2.The Division of Biomedical SciencesJohns Hopkins SingaporeSingaporeSingapore
  3. 3.McKusick-Nathans Institute for Genetic Medicine, Department of Biological Chemistry and OncologyJohns Hopkins UniversityBaltimoreUSA

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