Cysteine 38 in p65/NF-κB Plays a Crucial Role in DNA Binding Inhibition by Sesquiterpene Lactones

• Published in: The Journal of biological chemistry 2001-10, Vol.276 (43), p.39713-39720
• Main Authors: Garcı́a-Piñeres, Alfonso J., Castro, Vı́ctor, Mora, Gerardo, Schmidt, Thomas J., Strunck, Elisabeth, Pahl, Heike L., Merfort, Irmgard
• Format: Article
• Language: English
• Subjects: anti-inflammatory agents; DNA-binding protein; I^KB^a protein; Sesquiterpene lactones
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M101985200

Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-κB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-κB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IκB-α degradation. Here we report the construction of p65/NF-κB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys38 → Ser and Cys38,120 → Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-κB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-κB most probably by alkylating p65 at Cys38. Although a slight inhibition of IκB degradation was detected for all SLs, the amount of remaining IκB was too low to explain the observed NF-κB inhibition.