Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-κB. Here we explore the possibility that the NF-κB subunit p50 may be a target for the cyclopentenone 15-deoxy-Δ^12,14-prostaglandin J₂(15d-PGJ₂). 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-PGJ₂ was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ₂ 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-PGJ₂-treated p50, respectively. The interaction between p50 and 15d-PGJ₂ was relevant in intact cells. 15d-PGJ₂ effectively inhibited cytokine-elicited NF-κB activity in HeLa without reducing IκBα degradation or nuclear translocation of NF-κB subunits. 15d-PGJ₂ reduced NF-κB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-κB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ₂ resulted in the formation of a 15d-PGJ₂-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ₂ that results in inhibition of DNA binding.

The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial–mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transport-deficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc.