Role of cysteine residues of p65/NF-κB on the inhibition by the sesquiterpene lactone parthenolide and N-ethyl maleimide, and on its transactivating potential

Abstract

Sesquiterpene lactones (SLs) are potent anti-inflammatory substances. It was previously shown that the anti-inflammatory effect could be partly explained by the inhibition of the transcription factor NF-κB. Whether they inhibit the DNA binding of NF-κB, the activation of the IκB-kinase, or both is still a matter of debate. The data supporting these hypotheses were obtained using different cell systems. In this contribution we analyzed the mechanism of the sesquiterpene lactone-mediated inhibition using different cell systems, and showed that in all the cell lines analyzed, SLs inhibited both NF-κB binding and the IκB-kinase, but that the former played a more preponderant role in the inhibition. These results again confirm the importance of cysteine 38 in the inhibition and regulation of NF-κB's function. Moreover, we compared the selectivity of the SL parthenolide with that of N-ethyl maleimide (NEM). We showed that NEM directly alkylated p65 as well as p50 of NF-κB, whereas SLs possess a selectivity towards p65. Finally, we studied the transactivating properties of various p65 mutants, to analyze the effect of exchanged cysteine residues in the DNA binding domain of NF-κB/p65 on its function and demonstrated that the transactivating potential of the mutants did not correlate with their DNA binding strenght.