Spatial Redox Regulation of a Critical Cysteine Residue of NF-κB in Vivo

We demonstrate that the redox state of NF-␬B is spatially regulated by its subcellular localization

Takeyuki Nishi; Noriaki Shimizu; Masaki Hiramoto; Iwao Sato; Yuki Yamaguchi; Makoto Hasegawa; Shin Aizawa; Hirotoshi Tanaka; Kohsuke Kataoka; Hajime Watanabe; Hiroshi Handa

2002

Scholarcy highlights

  • The redox states of cysteine residues, which can change reversibly within cells, often greatly influence the various properties of proteins, such as protein stability, chaperone activity, enzymatic activity, and protein structure
  • B and C, the fluorescence intensity of Trx increased with time, depending on the concentration of TrxR. When both Cys-32 and Cys-35 of Trx were mutated to serine residues, fluorescence intensity did not increase to a detectable level over time. These results strongly suggest that the F5M labeling reaction is specific and permits the quantitative measurement of the protein redox states
  • E3330 had little effect on the redox states of p65. These results indicate that redox factor-1 is involved in p50 reduction in the nucleus but not in the reduction of cytoplasmic p50/p65 or nuclear p65
  • Ref-1 Activates NF-␬B through Reduction of p50 —We showed here that Ref-1 reduced Cys-62 of p50, which led to activation of NF-␬B DNA binding in vitro and in vivo
  • We showed that Trx and TrxR had no effect on the redox status of p50
  • Quantitative Western blotting suggests that Ref-1 exists at 0.5ϳ1 ϫ 106 molecules per cell or 100- to 200-fold molar excess to p50, whereas Trx exists at 0.1ϳ0.2 ϫ 106 molecules per cell or 20- to 40-fold molar excess in the nucleus of phorbol 12-myristate 13-acetate-stimulated
  • I␬B degradation reconfigures the DNA recognition loop such that Cys-62 is highly sensitive to oxidation, thereby requiring redox factor-1 to keep this cysteine reduced in the nucleus

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