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15-Deoxy- Delta super(12,14)-prostaglandin J sub(2) Inhibition of NF- Kappa B-DNA Binding through Covalent Modification of the p50 Subunit
Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF- Kappa B. Here we explore the possibility that the NF- Kappa B subunit p50 may be a target for the cyclopentenone 15-deoxy- Delta super(12,1...
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Published in: | The Journal of biological chemistry 2001-09, Vol.276 (38), p.35530-35536 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Online Access: | Get full text |
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Summary: | Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF- Kappa B. Here we explore the possibility that the NF- Kappa B subunit p50 may be a target for the cyclopentenone 15-deoxy- Delta super(12,14)-prostaglandin J sub(2) (15d-PGJ sub(2)). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ sub(2) was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ sub(2) was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ sub(2)-treated p50, respectively. The interaction between p50 and 15d-PGJ sub(2) was relevant in intact cells. 15d-PGJ sub(2) effectively inhibited cytokine-elicited NF- Kappa B activity in HeLa without reducing I Kappa B alpha degradation or nuclear translocation of NF- Kappa B subunits. 15d-PGJ sub(2) reduced NF- Kappa B DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF- Kappa B proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ sub(2) resulted in the formation of a 15d-PGJ sub(2)-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ sub(2) that results in inhibition of DNA binding. |
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ISSN: | 0021-9258 |