Cysteine 38 in p65/NF-kappa B plays a crucial role in DNA binding inhibition by sesquiterpene lactones

Sesquiterpene lactones (SLs) have potent antiinflammatory properties. We ha ve shown previously that they exert this effect in part by inhibiting activ ation of the transcription factor NF-kappaB, a central regulator of the imm une response. We have proposed a molecular mechanism for this inhibition ba sed on computer molecular modeling data. In this model, SLs directly alkyla te the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Neverthele ss, an experimental evidence for the proposed mechanism was lacking. Moreov er, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit I kappaB-alp ha degradation. Here we report the construction of p65/NF-kappaB point muta nts that lack the cysteine residues alkylated by SLs in our model. In contr ast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,1120) - -> Ser mutants is no longer inhibited by SLs. In addition, we provide evide nce that parthenolide uses a similar mechanism to other SLs in inhibiting N F-kappaB. Contrary to previous reports, we show that parthenolide, like oth er SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Alth ough a slight inhibition of I kappaB degradation was detected for all SLs, the amount of remaining I kappaB was too low to explain the observed NF-kap paB inhibition.