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

Alfonso J Garcı́A-Piñeres,Vı́Ctor Castro,Gerardo Mora,Thomas J Schmidt,Elisabeth Strunck,Heike L Pahl,Irmgard Merfort

doi:10.1074/jbc.m101985200

Abstract

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-kappaB, 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-kappaB, 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 IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> 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-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.

Highlights

Sesquiterpene lactones (SLs) have potent antiinflammatory properties
Cell extracts were prepared and assayed for NF-␬B DNA binding by Electrophoretic Mobility Shift Assays (EMSA)
The use of equal amounts of I␬B kinase complex (IKC) in each sample was confirmed by performing a Western blot against IKK-␤. These results show that the IKC is not inhibited by parthenolide at concentrations that completely inhibit NF-␬B DNA binding activity

Summary

Introduction

Sesquiterpene lactones (SLs) have potent antiinflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-␬B, a central regulator of the immune response. Transfected cells expressing either wild type (wt) or mutant proteins were incubated with increasing concentrations of different SLs, and their DNA binding was assessed by an EMSA. If parthenolide inhibits NF-␬B by preventing I␬B degradation, de novo NF-␬B DNA binding should be observed after treatment with DOC.

Results

Conclusion