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
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This work was supported by the Intramural Research Program of the National Institutes of Health National Institute of Occupational Safety and Health.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.160465
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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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