Cited by 1,947Genomics, phytochemicals, and oxidative stressRedox biology and oxidative stressGlutathione Transferases and Polymorphisms

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants

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Dinkova-Kostova, Albena T., Holtzclaw, W. David, Cole, Robert N., Itoh, Ken, Wakabayashi, Nobunao, Katoh, Yasutake, Yamamoto, Masayuki, Talalay, Paul·2002·Proceedings of the National Academy of Sciences

Abstract

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [ 3 H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C 257 , C 273 , C 288 , and C 297 ) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

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Crossref · 10.1073/pnas.172398899

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  • Cys273 and Cys288 are required for sensing oxidative stress under both basal and stress conditions, whereas Cys151 may be required only during oxidative stress conditions.

    “Cys273 and Cys288 are required for sensing oxidative stress under both basal and stress conditions, whereas Cys151 may be required only during oxidative stress conditions [53,54].”

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Authors
Dinkova-Kostova, A. T., Holtzclaw, W. D., Cole, R. N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M., & Talalay, P.
Affiliations
  • The Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of Pharmacology and Molecular Sciences, and Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and Center for TARA and Institute for Basic Medical Sciences, Tsukuba University, Tsukuba 305-8577, Japan
  • Johns Hopkins University (US)
Venue
Proceedings of the National Academy of Sciences
Published
2002-08-22
Volume
99
Issue
18
Pages
11908-11913
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
OpenAlex
W2097634036
Cited by
1,947
References
32
Citations by year
2026: 58 · 2025: 60 · 2024: 62 · 2023: 62 · 2022: 83 · 2021: 74 · 2020: 107 · 2019: 80 · 2018: 80 · 2017: 85 · 2016: 115 · 2015: 101 · 2014: 89 · 2013: 103 · 2012: 95
FWCI
21.56
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion)
Funders
  • National Institutes of Health (1s10rr014702-01)
  • National Cancer Institute (R01CA094076, CA 94076)
  • National Center for Research Resources (1S10-RR14702)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics, phytochemicals, and oxidative stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glutathione Transferases and Polymorphisms
Keywords
Keap1, Nrf2, cysteine thiol groups, antioxidant response element, protein disulfide bonds, sulforaphane
MeSH terms
Kelch-Like ECH-Associated Protein 1, Amino Acid Sequence, Animals, Carcinogens, Carrier Proteins, Cytoskeletal Proteins, DNA-Binding Proteins, Electrophoresis, Polyacrylamide Gel, Enzyme Induction, Kinetics, Molecular Sequence Data, Protein Binding, Sulfhydryl Compounds, Trans-Activators, Oxidants, Adaptor Proteins, Signal Transducing, NF-E2-Related Factor 2, Mice

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