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PMID: 14764894 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 101 ·No. 7 ·2004-02-17 ·Pages 2040-5

Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW, Talalay P

Abstract

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.

MeSH Terms
Adaptor Proteins, Signal Transducing Amino Acid Sequence Animals Antioxidants/metabolism Carrier Proteins/chemistry,genetics,metabolism Cysteine/genetics,metabolism Cytoskeletal Proteins DNA-Binding Proteins/chemistry,genetics,metabolism Disulfides/chemistry,metabolism Fibroblasts Gene Deletion Gene Silencing Kelch-Like ECH-Associated Protein 1 Mice Models, Biological Molecular Sequence Data NF-E2-Related Factor 2 Oxidation-Reduction Oxidative Stress Protein Binding Reducing Agents/metabolism Response Elements/genetics Trans-Activators/chemistry,genetics,metabolism Up-Regulation
Chemicals
Adaptor Proteins, Signal Transducing Antioxidants Carrier Proteins Cytoskeletal Proteins DNA-Binding Proteins Disulfides Keap1 protein, mouse Kelch-Like ECH-Associated Protein 1 NF-E2-Related Factor 2 Nfe2l2 protein, mouse Reducing Agents Trans-Activators Cysteine
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Wakabayashi Nobunao
The Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of Pharmacology and Molecular Sciences, School of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA.
Dinkova-Kostova Albena T
Holtzclaw W David
Kang Moon-Il
Kobayashi Akira
Yamamoto Masayuki
Kensler Thomas W
Talalay Paul
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0307301101
Published
2004-02-17
Epub
2004-00-05
Pages
2040-5
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC357048
Subset
IM
Grants
NCI NIH HHS · R01 CA094076 · United States
NCRR NIH HHS · 1S10-RR14702 · United States
NCI NIH HHS · CA 94076 · United States
External Links
PubMed source DOI: 10.1073/pnas.0307301101 PMC Full Text View journal details
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