No abstract

Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.[Key Words: ARE; electrophile; Keap1; Nrf2; oxidative stress] Received August 14, 1998; revised version accepted November 10, 1998.Both electrophiles and reactive oxygen species (ROS) contribute to DNA damage and consequent development of malignancy, as well as to many other diseases (Miller 1970, Sims et al. 1974Ames 1983). To counteract these insults, terrestrial organisms have developed elaborate defense mechanisms (Bannai 1984;Prestera et al. 1993a;Primiano et al. 1997), which include the coordinated induction of a battery of specific genes that encode phase II detoxifying enzymes and oxidative stressinducible proteins (Buetler et al. 1995;Hayes and Pulford 1995). An illustration of how these defense mechanisms might work has been provided in model studies of carcinogenesis. Development of cancer in animals fed with strong chemical carcinogens is inhibited by the administration of low, but tolerable, concentrations of electrophilic agents (Wattenberg 1978). This protective phenomenon is referred to as the electrophile counterattack response (Prestera et al. 1993a), and is thought to be mediated by the induction of genes encoding phase II enzymes (Primiano et al. 1997) and antioxidative stress proteins. This coordinated response is principally regulated through cis elements, called antioxidant responsive elements (AREs; Rushmore et al. 1991) or electrophile responsive elements (EpREs;Friling et al. 1990), associated with these target genes.The ARE consensus sequence shows striking similarity to a binding motif referred to as the Maf recognition element (MARE), also known as the erythroid transcription factor NF-E2 binding sequence Motohashi et al. 1997). MAREs are specifically recognized by either homodimers of Maf family members or by heterodimeric proteins composed of CNC (Cap'n'Collar) and small Maf partners (Igarashi et al. 1994;Blank and Andrews ...

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.

Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione Stransferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient mice, we show here that Nrf2 also controls the expression of a group of electrophileand oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system x c Ϫ ) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells. The lack of induction of system x c Ϫ activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2 regulates a wideranging metabolic response to oxidative stress.

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central ␣-helix followed by a mini antiparallel ␤-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the ␤-propeller structure of Keap1. In contrast, the central ␣-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two ␤-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central ␣-helix in a correct position to benefit ubiquitin signaling.

Background : Transcription factor Nrf2 regulates the expression of a set of detoxifying and anti-oxidant enzyme genes. Several lines of evidence suggest that electrophiles and reactive oxygen species liberate Nrf2 from its cytoplasmic repressor Keap1 and provoke the accumulation of Nrf2 in the nucleus. To elucidate the molecular mechanisms as to how Nrf 2 is activated by inducers, we examined the cytoplasmicnuclear shuttling and turnover of Nrf2.

Background: Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes. The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors. To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2.

Background:  The transcription factor Nrf2 and its negative regulator Keap1 play important roles in transcriptional induction of a set of detoxifying and anti‐oxidant enzymes. To gain an insight into our present enigma as to how cells receive oxidative and electrophilic signals and transduce them to Nrf2, we have developed a zebrafish model system for molecular toxicological studies. Results: We systematically cloned zebrafish cytoprotective enzyme cDNAs and found their expression to be efficiently induced by electrophilic agents. We consequently identified the presence of Nrf2 and Keap1 in zebrafish. Both loss‐ and gain‐of‐function analyses demonstrated that Nrf2 is the primary regulator of a subset of cytoprotective enzyme genes, while Keap1 suppresses Nrf2 activity in zebrafish. An ETGE motif, critical for the Nrf2–Keap1 interaction, was identified in the Neh2 domain of Nrf2 by reverse two‐hybrid screening and found to be indispensable for the regulation of Nrf2 activity in zebrafish. Conclusion: Taken together, these results indicate that the Nrf2‐Keap1 system is highly conserved among vertebrates and that the interface between Nrf2 and Keap1 forms an important molecular basis of this regulatory system.