15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-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-kappaB. Here we explore the possibility that the NF-kappaB subunit p50 may be a target for the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(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(2) was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ(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(2)-treated p50, respectively. The interaction between p50 and 15d-PGJ(2) was relevant in intact cells. 15d-PGJ(2) effectively inhibited cytokine-elicited NF-kappaB activity in HeLa without reducing IkappaBalpha degradation or nuclear translocation of NF-kappaB subunits. 15d-PGJ(2) reduced NF-kappaB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-kappaB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ(2) resulted in the formation of a 15d-PGJ(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(2) that results in inhibition of DNA binding.
Cernuda-Morollón
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E
Departamento de Estructura y Función de Proteinas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas (C.S.I.C.), Madrid, Spain.
Pineda-Molina
E
E
Cañada
F J
FJ
Pérez-Sala
D
D
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000494:pharmacology*; D011485:Protein Binding
C097240:15-deoxy-delta(12,14)-prostaglandin J2; D016328:NF-kappa B; D004247:DNA; D005978:Glutathione; D015230:Prostaglandin D2