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

• DOI: 10.1073/pnas.0307301101
• Publication Date: 2004-02-17T20:32:02Z
• Authors: Nobunao Wakabayashi; Albena T. Dinkova...; W. David Holtzclaw; Moon-Il Kang; Akira Kobayashi; Masayuki Yamamoto; Thomas W. Kensler; Paul Talalay
• Keywords: Inducer; KEAP1

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