IκB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-κB activity by glycine chloramine through methionine oxidation

摘要

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IκBα, the inhibitor of NF-κB (nuclear factor κB), and to prevent activation of the NF-κB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20–200 μM) and HOCl (50 μM) caused oxidation of IκBα methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IκBα. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IκBα degradation in cells treated with TNFα (tumour necrosis factor α) and inhibited nuclear translocation of NF-κB. IκBα modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IκBα persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-κB activation via reversible IκBα oxidation.