期刊
BIOCHEMISTRY
卷 48, 期 30, 页码 7271-7278出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi900660f
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资金
- Korea Government (MEST) [R11-2002-098-07001-0, R15-2006-020]
- National Research Foundation of Korea [R11-2002-098-07001-0] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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-kappa B (NF-kappa B) activation. We examined the effect of TPCK on the NF-kappa B activation pathway in HeLa cells by measuring the activity of I kappa B kinase (IKK) and p65/Re1A-DNA binding. TPCK inhibited tumor necrosis factor-alpha-induced IKK activation and directly blocked IKK activity in vitro. TPCK-induced inhibition of NF-kappa B 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 IKK beta 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/Re1A in HeLa cells. Inhibition of p65/Re1A-DNA binding was recovered in the presence of dithiothreitol, and substitution of Cys-38 with Ser in p65/Re1A rendered the protein resistant to inhibition by TPCK. Mass spectrometry analysis of IKK beta and p65/Re1A isolated from cells treated with TPCK by UPLC-ESI-Q-TOF tandem MS revealed the labeling of Cys-179 of IKK beta and Cys-38 of p65/Re1A with a tosylphenylalanylmethyl group. These results suggest that TPCK inhibits NF-kappa B activation by directly modifying thiol groups on two different targets: Cys-179 of IKK beta and Cys-38 of p65/Re1A.
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