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

Abstract

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. 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-B, 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 IB-␣ degradation. Here we report the construction of p65/ NF-B 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 3 Ser and Cys 38,120 3 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-B. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-B most probably by alkylating p65 at Cys 38 . Although a slight inhibition of IB degradation was detected for all SLs, the amount of remaining IB was too low to explain the observed NF-B inhibition.The transcription factor NF-B promotes the expression of over 150 target genes in response to inflammation, viral and bacterial infections, and other stressful situations (1, 2). Both the nature of the NF-B inducers and the function of its target genes highlight its role, as a central mediator of the human immune response.NF-B is most frequently composed of a p50 and a p65 subunit retained in the cytoplasm in an inactive form by binding to IB, an inhibitory subunit. Inducers of NF-B, such as bacterial lipopolysaccharides (LPS) 1 or inflammatory cytokines, activate the IB kinase complex (IKC), which phosphorylates IB on serines 32 and 36. Phosphorylation causes IB ubiquitinylation and its subsequent degradation by the 26-S proteasome. Degradation of the inhibitor allows NF-B to translocate to the nucleus, where it stimulates transcription of its target genes.2 Genes that are regulated by NF-B include, for example, proinflammatory and inflammatory cytokines such as interleukin-1, -2, -4, and -6 or TNF-␣, as well as genes encoding immunoreceptors, cell adhesion molecules, acute phase proteins, and enzymes such as cyclooxygenase-II. Because of its central role in regulating inflammatory responses, a pharmacological inhibition of NF-B activation could be beneficial in the treatment of inflammation (4). Using helenalin as a model, we have shown that SLs inhibit neither IB degradation nor NF-B nuclear translocation. SLs interact directly with NF-B. DNA binding of NF-B is prevented by selectively alkylating cysteine sulfhydryl groups in its p65 subunit (5, 6). There are strong indications that this is a general mechanism for SLs, which possess ␣,␤-or ␣,␤,␥-unsaturated carbonyl structures such as ␣-methylene-␥-lactones or ␣,␤-unsubstituted...

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“…Because the effects of TQ could be reversed by reducing agent, it is possible that this agent modifies a cysteine residue in p65. These results are consistent with those previously reported from our laboratory and others with caffeic acid phenethyl ester (37), plumbagin (38), and sesquiterpene lactone parthenolide (40). A Cys 38 residue has been identified in p65 subunits of NF-nB that is crucial for DNA binding (40).…”

“…It has been reported that the cysteine residue located at position 38 in p65 is highly susceptible to various agents (38)(39)(40). Whether Cys 38 is a target for TQ was investigated.…”

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“…Some SLs can directly inhibit NF-kB DNA binding, primarily through interaction with Cys-38 in the DNA-binding loop of RelA (Garcı´a-Pin˜eres et al, 2001, 2004. Most SLs can also inhibit DNA binding by p50 and c-Rel through an analogous Cys residue in the DNA-binding loop, and mutations of this Cys residue to Ser generally make p50, RelA or c-Rel refractory to inhibition by such thiol-reactive compounds.…”