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Research ArticleTOXICOLOGY

Critical Cysteine Residues of Kelch-Like ECH-Associated Protein 1 in Arsenic Sensing and Suppression of Nuclear Factor Erythroid 2-Related Factor 2

Xiaoqing He and Qiang Ma
Journal of Pharmacology and Experimental Therapeutics January 2010, 332 (1) 66-75; DOI: https://doi.org/10.1124/jpet.109.160465
Xiaoqing He
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Qiang Ma
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Abstract

Arsenic activates nuclear factor erythroid 2-related factor 2 (Nrf2) to induce phase II and antioxidative genes. Here we analyzed arsenic–Kelch-like ECH-associated protein 1 (Keap1) cysteine thiol interaction in Nrf2 activation. Arsenic-based Nrf2 activators, fluorescent biarsenical labeling reagent (FlAsH) and phenylarsine oxide (PAO), were used to probe binding of arsenic to Keap1. Strong fluorescence was observed on binding of FlAsH to purified Keap1. Pretreatment with arsenic, tert-butylhydroquinone (tBHQ), or 2,3-dimercaptopropanol significantly reduced the fluorescent signal. PAO affinity beads effectively pulled down Keap1 in vitro and from hepa1c1c7 cells. Arsenic, tBHQ, free PAO, or cadmium blocked Keap1 pulldown. Furthermore, arsenic and free PAO significantly reduced the free thiol contents of purified or endogenous Keap1. Thus, arsenic, FlAsH, and PAO, as well as tBHQ and cadmium, bind to Keap1 cysteine thiols in a similar fashion. All the domains of Keap1 bound PAO, and the linker region exhibited the highest binding activity. The function of arsenic-Keap1 interaction was evaluated in a reconstituted system that mimics endogenous Nrf2 regulation. Mutation of Cys273 or Cys288 in the linker region resulted in high level basal expression of Nrf2 protein. Mutation of Cys151 abolished Nrf2 activation by arsenic. Overexpression of C273A, C288A, or C151A altered the basal and arsenic-induced expression of Nrf2 target genes. The study shows an important role of Cys273 and Cys288 in the suppression of Nrf2 by Keap1 and a critical function of Cys151 in arsenic responsiveness. Our findings support a model in which arsenic binds to different sets of Keap1 cysteine residues to regulate divergent functions in Nrf2 signal transduction.

Footnotes

  • This work was supported by the Intramural Research Program of the National Institutes of Health National Institute of Occupational Safety and Health.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.109.160465

  • ABBREVIATIONS:

    Nrf2
    nuclear factor erythroid 2-related factor 2
    Keap1
    Kelch-like ECH-associated protein 1
    ARE
    antioxidant response element
    FlAsH
    fluorescent biarsenical labeling reagent
    PAO
    phenylarsine oxide
    LR
    linker region
    tBHQ
    tert-butylhydroquinone
    BAL
    2,3-dimercaptopropanol
    MG132
    N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
    Nqo1
    NAD(P)H:quinone oxidoreductase 1
    PBS
    phosphate-buffered saline
    NTR
    N-terminal region
    BTB
    broad complex/tramtrack/bric a brac
    CTR
    C-terminal region
    KelchC
    Kelch region plus C terminus of Keap1
    GFP
    green fluorescence protein
    DLG
    Asp-Leu-Gly, human Nrf2 peptide 23LWRODIDLG31
    ETGE
    Gly-Thr-Gly-Glu, human Nrf2 peptide 79ETGE82.

    • Received August 14, 2009.
    • Accepted October 6, 2009.
  • © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 332 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 332, Issue 1
1 Jan 2010
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Research ArticleTOXICOLOGY

Critical Cysteine Residues of Kelch-Like ECH-Associated Protein 1 in Arsenic Sensing and Suppression of Nuclear Factor Erythroid 2-Related Factor 2

Xiaoqing He and Qiang Ma
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 66-75; DOI: https://doi.org/10.1124/jpet.109.160465

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Research ArticleTOXICOLOGY

Critical Cysteine Residues of Kelch-Like ECH-Associated Protein 1 in Arsenic Sensing and Suppression of Nuclear Factor Erythroid 2-Related Factor 2

Xiaoqing He and Qiang Ma
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 66-75; DOI: https://doi.org/10.1124/jpet.109.160465
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