Modulation of transcription factor NF-kappa B binding activity by oxidation-reduction in vitro.

TLDR

NF‑κB is a key transcription factor that remains inactive in the cytosol bound to IκB and becomes DNA‑binding upon stimulus‑induced dissociation and nuclear translocation. The study shows that in vitro NF‑κB binding can be inhibited by agents that modify free sulfhydryl groups. Treatment with N‑ethylmaleimide or diamide abolishes binding, but the effect of diamide is reversed by 2‑mercaptoethanol, which also synergizes with deoxycholate and NP‑40 to activate cytosolic NF‑κB, suggesting redox state modulation as a post‑translational control mechanism.

Abstract

NF-kappa B is a widely used regulator of inducible and tissue-specific gene control. In the cytosol, when complexed to an inhibitory molecule, I kappa B, NF-kappa B is in an inactive form and cannot bind DNA. Activation of cells with appropriate stimuli results in the dissociation of NF-kappa B from I kappa B and its translocation to the nucleus as an active binding protein. We now demonstrate that NF-kappa B binding in vitro can be inhibited by agents that modify free sulfhydryls. Binding is eliminated after treatment with N-ethylmaleimide, an alkylating agent, and diamide, an oxidizing agent. The diamide effect can be reversed by 2-mercaptoethanol. Further, 2-mercaptoethanol acts synergistically with deoxycholate plus Nonidet P-40 in converting inactive cytosolic NF-kappa B to an active DNA-binding form. It is therefore possible that modulation of the redox state of NF-kappa B could represent a post-translational control mechanism for this factor.