N-Tosyl-L-phenylalanine chloromethyl ketone inhibits NF-κB activation by blocking specific cysteine residues of IκB kinase β and p65/RelA

Abstract

N-Tosyl- L-phenylalanine chloromethyl ketone (TPCK), a serine/cysteine protease inhibitor, has been reported to inhibit expression of inflammatory mediators by blocking nuclear factor-κB (NF-κB) activation. We examined the effect of TPCK on the NF-κB activation pathway in HeLa cells by measuring the activity of IκB kinase (IKK) and p65/RelA-DNA binding. TPCK inhibited tumor necrosis factor-R-induced IKK activation and directly blocked IKK activity in vitro. TPCK-induced inhibition of NF-κB and IKK activation was abrogated by addition of the thiol-reducing agent dithiothreitol, suggesting that the effect of TPCK occurred through modification of a thiol group in IKK. Consistent with this, an IKKβ mutant in which Cys-179 was substituted with alanine was not more susceptible to TPCK. Our result also showed that TPCK inhibits the DNA binding of transiently expressed p65/RelA in HeLa cells. Inhibition of p65/RelA-DNA binding was recovered in the presence of dithiothreitol, and substitution of Cys-38 with Ser in p65/RelA rendered the protein resistant to inhibition by TPCK. Mass spectrometry analysis of IKKβ and p65/RelA isolated from cells treated with TPCK by UPLC-ESI-Q-TOF tandem MS revealed the labeling of Cys-179 of IKKβ and Cys-38 of p65/RelA with a tosylphenylalanylmethyl group. These results suggest that TPCK inhibits NF-κB activation by directly modifying thiol groups on two different targets: Cys-179 of IKKβ and Cys-38 of p65/RelA. ©2009 American Chemical Society.