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

Title
N-Tosyl-L-phenylalanine chloromethyl ketone inhibits NF-κB activation by blocking specific cysteine residues of IκB kinase β and p65/RelA
Authors
Ha K.-H.Byun M.-S.Choi J.Jeong J.Lee K.-J.Jue D.-M.
Ewha Authors
이공주
SCOPUS Author ID
이공주scopus
Issue Date
2009
Journal Title
Biochemistry
ISSN
0006-2960JCR Link
Citation
vol. 48, no. 30, pp. 7271 - 7278
Indexed
SCI; SCIE; SCOPUS WOS scopus
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.
DOI
10.1021/bi900660f
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약학대학 > 약학과 > Journal papers
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