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

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.

γ-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells

“…An alternative hypothesis of Nrf2 liberation, modification of Keap1, proposes that some reactive -SH groups on Keap1 act as oxidative stress sensors and that modification of them by ROS or electrophiles disrupts the Nrf2-Keap1 complex leading to Nrf2 dissociation. This hypothesis is supported by evidence of continuous Nrf2 activation by mutation of the active cysteine residues of Keap1 [67,70,71] and by the finding of conjugate formation between the electrophiles and the thiol group in Keap1 [72]. It seems that in the case of Nrf2 activation by HNE, Keap1 modification may be involved.…”

“…It is then translocated to the nucleus to form heterodimers with other leucine zipper proteins such as c-Jun and small Mafs. These complexes transactivate gene expression by binding to EpRE [65][66][67]. Two mechanisms have been proposed for Nrf2-Keap1 dissociation, i.e., Nrf2 phosphorylation and Keap1 modification.…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Nrf2 activation is prevented by binding to KEAP-1. Oxidative insults target KEAP-1 by targeting cysteines 273 and 288, which cause dissociation of KEAP-1 from Nrf2, allowing accumulation of Nrf2 in the nucleus and activation of target genes [174,175]. Investigations into the mechanisms whereby KEAP-1 promotes degradation of Nrf2 have unraveled that KEAP-1 acts as a redox sensitive adaptor for a Cul3-based E3 ligase [176].…”

Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells

“…Different mechanisms of Nrf2 activation have been proposed and shown to vary with different activators. According to one pathway, Keap1 appears to interact with Nrf2 activators such as phenolic antioxidants through its thio-groups, leading to the stabilization of the Nrf2 protein [17]. Whereas the phenolic antioxidant tert-butylhydroquinone (tBHQ) may stabilize Nrf2 by inhibiting its degradation via the Keap1-dependent E3 without disrupting the Nrf2/Keap1/Cul3 complex, toxic metals such as arsenic (As) and chromium (Cr(VI)) increase the half-life of the Nrf2 protein as well as dissociate Nrf2 from Keap1 and Cul3 in the nucleus [10,13].…”