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.
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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-β- 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
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Acknowledgments
The authors acknowledge the generous gift of antibodies by Dr. Peter N. Kao (Anti-NF-90 and NF-45), Dr. Jean-Pierre Paccaud (Anti-Sec24), Dr. Katsuko Tani (Anti-p125i), and Dr. Taka-aki Tamura (anti-Tip49α, antiTip49β). The author’s work is supported by NIH grants HD R37-13563, CA R01-42486, DK-R01-61671, DK-R21/33-71280 (GWH), the National Heart, Lung, and Blood Institute, National Institutes of Health, contract No. N01-HV-28180 (GWH and AP), an NIH roadmap grant for Technology Centers of Networks and Pathways (U54RR020839), and an A*Star Research Grant to Johns Hopkins Singapore (NEZ). Under a licensing agreement between Covance Research Products and The Johns Hopkins University, Dr. Hart receives a share of royalties received by the university on sales of the CTD 110.6 antibody. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.
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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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Zachara, N.E., Molina, H., Wong, K.Y. et al. The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways. Amino Acids 40, 793–808 (2011). https://doi.org/10.1007/s00726-010-0695-z
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DOI: https://doi.org/10.1007/s00726-010-0695-z