The History Of The Antioxidant Response Elements Biology Essay

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), also known as NFE2L2, has emerged as a transcription factor that plays an important part in the maintenance of cellular homeostasis (Li et al. 2012). Nrf2 originates from studies of β-globin gene expression with the description of the locus control region as possessing important regulatory properties and is related to transcription factor activating protein 1 (AP-1) (Tuan et al., 1985). It belongs to the cnc ("cap ‘n’ collar") subfamily of the basic region leucine zipper transcription factors. (Zhang. D. 2006). The CNC-basic leucine zipper family (CNC-bZIP), a subfamily of bZIP proteins, play critical roles in the regulation of expression of genes involved in diverse biological activities, e.g. proliferation, apoptosis, differentiation, and stress responses (Mandy et al. 1999). Until now, six members in this family have been reported: NF-F2, Nrf1, Nrf2, Nrf3, Bach1, and Bach2, each of which have different biological roles (Zhang. D. 2006). The structure of the Nrf2 protein has been extensively examined. Nrf2 contains six NRF2-ECH homology domains designated as Neh1-Neh6 (Itoh et al. 1999). Neh1 is situated in the c- terminal half of the molecule and contains a CNC basic leucine zipper domains, which is substantial for DNA binding and dimerization with other b-Zip proteins e.g. small Maf proteins and the antioxidant response element (ARE) in the regulatory sites of target genes (Itoh et al. 1997). The Neh2 domain is located in the proximal N terminus region that serves as binding sites for the Nrf2 inhibitor protein Keap1 (Katoh et al. 2005). It contains seven lysine residues for ubiquitin conjugation which confers negative regulation of the Nrf2 activity through proteasome-mediated degradation of Nrf2 (Zhang et al., 2004). Neh2 domain has a serine residue, Ser40, which is critical for NRF2 release from Keap 1, however it is not required for NRF2 stabilization and accumulation in the nucleus (Bloom et al. 2002). It has been reported that electrophiles damage the interaction between Keap1 and the Neh2 domain either by stimulating phosphorylation of Nrf2 or by directly modifying cysteine residues in Keap1 (Huang et al. 2002). Moreover, researchers have found that Keap1 may stay bound to the Neh2 domain during oxidative stress. This make stabilize the Nrf2 leading to an increased transcriptional response (Zhang et al., 2004). Neh 3 domain found at the C-terminal end of the protein is necessary for transcriptional activity of Nrf2 by recruiting chromodomain helicase DNA-binding protein 6 (CHD6) (Nioi et al., 2005). Neh3 possess 40 amino acids and the deletion of the final 16 amino acids produces in a molecule that is transcriptionally silent but localizes normally to the nucleus and binds to DNA (Camellia A. 2008). Both Neh4 and Neh5 are considered as transactivation domains that rich in acidic residues and interact with CREB-binding protein (CBP) to organize the start of transcription (Katoh et al. 2001). Neh6 domain lies in the central part of Nrf2, which contributes to redox-independent negative control of Nrf2 (McMahon et al., 2004).

Kelch-like ECH-associating protein 1(Keap1)

As discussed, Nrf2 has six homologous regions named Neh1 to Neh6. The Neh2 domain located in the N-terminus of Nrf2 is known to have a negative regulatory role for the transactivating activity of Nrf2. Neh2 interacts with cytosolic protein, Kelch like ECH-associated protein 1 (Keap1) and negatively controls Nrf2 function (Itoh et al., 1999). Keap1 which was cloned by Yamamoto and coworkers using the Neh2 domain of Nrf2 as bait in a yeast two-hybrid screening system (Itoh et al., 1999), is an actin-binding protein and has been thought to supress the activity of Nrf2 by simply sequestering the protein in the cytoplasm (yeong et al. 2010). Studies revealed that Knockout of Keap1 will result in activation of Nrf2 signalling (Okawa et al. 2006). Itoh and his collagenous have identified that the structure of murine Keap1 to be composed of 624 amino acids and there is around 95% homology between human and mouse (Itoh et al., 1999). Molecular analysis revealed that human Keap1 contains five major domains: an N-terminal region (NTR), broad complex, tramtrack, and bric-a-brac domain (BTB), an cysteine-rich intervening region (IVR), the double glycine repeat region (DGR) or Kelch domain, and a C terminal Kelch region (CTR)(Itoh et al., 2004). The N-terminal and BTB regions were implicated in homodimerization and heterodimerization of the Keap1 protein (Zipper et al. 2002). The intervening region which is rich in cysteine residues was demonstrated to be essential for the activity of Keap1 (Zhang et al. 2003). Both of BTB and IVR domains thought to be involved in proteosome-dependent Nrf2 degradation (Kobayashi et al. 2004). The DGR or Kelch domain binds to the Neh2 domain of Nrf2, establishing Nrf2 onto actin cytoskeleton (Kang et al. 2004). The C-terminal Kelch region of Keap1 was proved to bind to the Neh2 domain of Nrf2 (Tong et al., 2006). Since Keap1 was initially described as a cytoplasmic factor that sequester Nrf2 in the cytoplasm under the unstimulated condition (Zipper et al. 2002), it was believed that upon exposure of cells to oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus where it forms a heterodimer bound with its obligatory partner Maf protein, and eventually activates antioxidant response elements (ARE)-dependent gene expression (Zhang D. 2006). Based on recent laboratory findings, Keap1 was demonstrated not just passively sequester Nrf2 in the cytoplasm, Keap1 was also proved to plays an active role in targeting Nrf2 for ubiquitination and proteasomal degradation ((Zhang D. 2006). In addition, Keap1 was identified to be associated with Cullin-3, a scaffold protein responsible for formation of an ubiquitin ligase E3 complex (Kobayashi et al. 2004). Thus, Keap1 may not only affect Nrf2 localization, but also actively targets Nrf2 to degradation (Rong et al. 2010).

2.2.1.3. Antioxidant response elements (ARE)

The Antioxidant response elements or ARE is enhancer sequence or regulatory element that mediates transcriptional activation of genes encoding phase II detoxification enzymes and antioxidant proteins (Jong et al. 2004). Proteins that are members of the battery of ARE genes involve those associated with glutathione biosynthesis, redox proteins with active sulfydryl moieties and drug metabolizing enzymes (Rushmore et al.1991). ARE was first identified in the 5′-flanking region of a 41bp DNA sequence of the rat Gsta2 subunit gene (TAATGGTGACAAAGCA) (Rushmore et al., 1990), containing 41-bp DNA and this gene enhancer is substantial for the inducible expression of Gsta2 due to its responsiveness to not only phenolic antioxidants and planar aromatic compounds, but also hydrogen peroxide and reactive oxygen species (ROS) (Rushmore et al., 1991). Furthermore, mutational analysis of the rat GST A1 promoter sequence assays delineated that the core ARE sequence was defined as 5‟-TGACnnnGC-3‟ or 3’-YCACTGnnnCG-5’ (Rushmore et al., 1991). In addition to being antioxidant-inducible, studies have demonstrated that the ARE sequence is found in numerous genes, and contribute to the basal and inducible expression of downstream genes (Jong et al. 2004). These genes includes, rat GSTA2, mouse GST A1(Rushmore et al. 1991), rat GST P1 (Okuda et al. 1989), rat NQO1, human NQO1(Favreau et al. 1991), human glutamate cysteine ligase catalytic subunit (GCLC) and modulatory (GCLM) subunits (Moinova et al 1998), mouse ferritin-L, mouse metallothionein-1, and mouse UDP glucuronyl transferase (UGT) (Jong et al. 2004). Wasserman and co-workers was found that the core sequence of ARE was suggested to be 5′-A/GTGAC/GNNNGCa/c-3′ (Wasserman et al. 1997). However, as more AREs are demonstrated in a variety of phase 2 genes, great variability in the core sequence of ARE was found, suggesting that consensus ARE sequences may be dependent on the specific investigated gene (Kyeong et al. 2010).

1.2.1 Nrf2

The discovery of Nrf2 originates from studies of β-globin gene expression with the characterization of the locus control region as having critical regulatory properties (Tuan et al., 1985; Forrester et al., 1986). This regulatory region contains a tandem AP1-NFE2 (activating protein 1 and nuclear factor erythroid 2) motif, originally termed as the DNase hypersensitive site 2, which has a strong enhancer activity (Moi & Kan, 1990; Ikuta & Kan, 1991). Subsequent work to clone the transcription factors that bind to this AP1-NFE2 site have identified several members belonging to the cap ‘n’ collar (CNC) subfamily of the basic leucine zipper (bZIP) transcription factors; among the first identified were p45- NFE2 ( Chan et al., 1993), Nrf1 (Chan et al., 1993) and Nrf2 (Moi et al., 1994). Nrf3 (Kobayashi et al., 1999) and two distantly related proteins, i.e., Bach1 (Oyake et al., 1996) and Bach2 (Muto et al., 1998), are other CNC family proteins discovered and characterized later. These proteins function as heterodimeric transcription factors by dimerizing with other bZIP proteins such as small Mafs (sMafs)(Igarashi et al., 1994). Targeted gene disruption of Nrf1 in mice resulted in embryonic lethality with severe anemia and liver abnormality being suggested to be the primary culprits (Chan et al., 1998). Nevertheless, Nrf2 disrupted mice were normal, fertile, and did not show a phenotype of developmental deficits (Chan et al., 1996; Itoh et al., 1997), suggesting that Nrf2 is not essential for murine development and survival.

1.2.2 Keap1

Detailed analysis of Nrf2 activity and structure across various species has identified six evolutionarily conserved domains, named Neh (Nrf2-ECH<chicken Nrf2>homologous domain)(Itoh et al., 1997). A summary of roles and functions that has been described for these conserved domains is depicted in Figure 1. The domain in the N terminus, Neh2, was discovered as having a negative regulatory role for the transactivating activity of Nrf2 (Itoh et al., 1999). Deletion of Neh2 was found to remarkably increase Nrf2’s transactivation activity, which hinted that the Neh2 may contain a critical interaction site to which the negative regulator of Nrf2 binds. Using a yeast two hybridization system and Neh2 as a bait, Keap1 (Kelch-like ECH-associating protein 1), a zinc metalloprotein, was identified to be the major protein, represented 80% of the independent clones isolated, interacting with the Neh2 domain. Based on Keap1 cDNA sequences, the primary structure of murine Keap1 was predicted to be composed of 624 amino acids and there is ~95% homology between human and mouse (Itoh et al., 1999).

Molecular dissecting analysis suggests that Keap1 consists of five domains: the Nterminal region (NTR), the BTB/POZ (Bric-a-brac, tramtrack, broad-complex/poxvirus zinc finger), the intervening region (IVR), the double glycine repeat (DGR) or Kelch domain, and the C-terminal region (CTR)(Itoh et al., 2004A). The BTB/POZ domain is involved in protein homodimerization and heterodimerization, making homomeric and heteromeric multimers of Keap1 (Yoshida et al., 1999). In addition, both BTB and IVR domains are involved in proteosome-dependent Nrf2 degradation (Kobayashi, et al., 2004). The DGR or Kelch domain binds to the Neh2 domain of Nrf2, anchoring Nrf2 onto actin cytoskeleton (Itoh et al., 1999; Kang et al., 2004). From crystal structure analysis of the Kelch domain of human Keap1, it was revealed to have six structurally similar β- propeller blades (Li et al., 2004B). These inter- and intra-blades, which are tied firmly by hydrogen bonds, are believed to construct the complex structure of Keap1-Nrf2 and its anchorage to actin. The C-terminal region of Keap1 was also shown to bind to the Neh2 domain of Nrf2 (Tong et al., 2006A). A "two-site molecular recognition model" has thus been proposed for Keap1-Nrf2 complex whereby the two motifs, namely DLG and ETGE, in the Neh2 of Nrf2 independently associate with the Keap1-DC (DGR and CTR)(Tong et al., 2006B). This double tethering of Nrf2 with Keap1 is thought to contribute to the overall stability of Keap1-Nrf2 complex.

1.2.3 ARE

The antioxidant responsive element or ARE was first identified to be a DNA consensus motif (gene enhancer) on the 5’-flanking region of the rat glutathione stransferase Ya (GST Ya) whose function was executed upon exposure to electrophilic and planar aromatic compounds and phenolic antioxidants (Friling et al., 1990; Rushmore et al., 1990). Further sequence analysis by means of gene reporter assays delineated that the core ARE is essentially represented by 5’-RGTGACnnnGC-3’ or 3’-YCACTGnnnCG-5’ (Rushmore et al., 1991). In addition to being antioxidant-inducible, this ARE could as well contribute to the basal (constitutive) expression of this rat GST isoform. At that time, the transcription factor(s) responsible to interact with this consensus motif remained unknown. Nevertheless, the high similarity between this ARE sequence and the NFE2- AP1 motif, which was found to bind Nrf2 and other AP-1 family of transcription factors, as well as the TRE [phorbol-12-O-tetradecanoate-13-acetate (TPA)-responsive element] type-Maf recognition element (T-MARE) has been noted (Kataoka et al., 1994; Xie et al. 1995; Prestera et al., 1993).

The first demonstration that ARE as the cognate enhancer of Nrf2 came from the work of Venugopal and Jaiswal (1996). They identified a cis-element resembling the documented ARE sequence in 5’-flanking regulatory region of the human NQO1 [NAD(P)H 0xidoreductase 1] can physically bind Nrf1 and Nrf2 which corresponded with an increased transactivation activity and NQO1 gene induction. Further evidence was subsequently provided by Itoh and coworkers (1997) who observed an impaired constitutive and butylated hydroxyanisole (BHA)-induced expression of the phase II enzymes GSTs Ya and Yb in Nrf2-disrupted mice. Along with this, binding of Nrf2-MafK heterodimer to ARE of the GST Ya gene that was identified earlier (Rushmore et al., 1990) has also been convincingly shown. Questions remain as to whether the ARE sequences between different species are similar and whether there exists an indispensable nucleotide sequence in the core ARE of all ARE-regulated genes. It was found that an active ARE located in the promoter of human glutamate cysteine ligase modulatory subunit (GCLM) gene has a variant ARE sequence (Erickson et al., 2002). These findings called upon a revision of the core ARE to 5’-RTKAYnnnGCR-3’.

1.2.4 Nrf2 transcription complex

Upon activation, cytosolic Nrf2 liberates itself from physical entrapment and/or negative regulation of Keap1 and accumulates in the cell nucleus to bind to its cognate enhancer ARE, thereby inducing a battery of target cytoprotective genes. The small Maf (sMaf) proteins consisting of MafK and MafG are the most common heterodimer partner recruited by Nrf2 to the transcription initiation complex (Itoh et al., 1997). The exact role of sMaf in Nrf2 transactivation activity on ARE remains controversial, with studies showing their participation as positive (Itoh et al., 1997) as well as negative regulators (Venugopal & Jaiswal, 1996). Other bZIP proteins, such as AP-1 (JunD, c-Jun/c-Fos, FRA-1), ATF4 and PMF-1, were suggested mainly by the gene-promoter analysis to also interact/partner with Nrf2, affecting ARE-regulated genes (Jaiswal, 2004). Similar to sMaf proteins, whether these stress-responsive proteins participate in Nrf2 transactivation as co-activators or co-repressors have been controversial with mixed reports. Other than this, the cyclic AMP responsive element binding protein (CBP)/p300 is a histone acetyltransferase-derived co-activator protein identified to be part of the transcription complex of Nrf2 (Katoh et al., 2001). It is noteworthy that the AP-1 proteins, sMafs, and other bZip proteins can also bind to ARE independent of Nrf2 (Venugopal & Jaiswal, 1996; Tsuji, 2005; Yang et al., 2006), although the nucleotide sequence of cisacting element may preferentially select certain sets/combinations of these proteins (Yamamoto et al., 2006). The versatility of ARE to interact with multiple transcription factors enables upregulation of ARE-mediated genes in response to various signals and inducers of cellular stress. Whether there exist hierarchical, temporal and spatial, and cell- and/or species-specific regulation of the ARE-mediated genes by various transcription factors, further investigation is warranted.

Thesis NRF2

1.2.4 Antioxidant responsive element

The ARE is a cis-acting enhancer sequence that mediates transcriptional activation of genes in cells exposed to oxidative stress. Proteins that are members of the ARE-gene battery include those associated with glutathione biosynthesis, redox proteins with active sulfydryl moieties and drug-metabolizing enzymes. The cis-acting element was first identified within the 5‟ flanking region of a 41bp DNA sequence in the rat GSTA2 gene containing 41-bp DNA and was later designated as the ARE due to its responsiveness to phenolic antioxidants (Rushmore et al., 1991). The core DNA sequence essential for the response to these chemicals was determined through deletion and mutational analysis and was firstly defined as 5‟-TGACnnnGC-3‟ (Favreau & Pickett, 1991). However, the same study also showed that nucleotides situated immediately 5‟ to the „core‟ ARE were also required for basal and inducible expression of the gene. Consistent with this finding, research from another group proposed the extended ARE core sequence as 5‟-TMAnnRTGAYnnnnGCRwwww-3‟ (M=A/C; R-A/G; Y=C/T; and W=A/T), demonstrating the importance of flanking sequence for the context-specific regulation of gene transcription (Wasserman & Fahl, 1997). In 2003, through point mutations across the whole ARE in the mouse Nqo1 promoter, Nioi et al. found that the 3‟tetra-nucleotide „wwww‟ is required for neither basal nor inducible gene expression. The same study also revealed that nucleotides previously suggested to be redundant (which are shown as „n‟ in the sequence mentioned above) are required for gene induction, and on the other hand, the core sequence that had been reported previously to be essential before was found to be dispensable in the case of mouse Nqo1 (Nioi et al., 2003). Take together, these studies indicated that the sequence of ARE in the promoter of different genes may be distinct. In addition to the genes that encode the rat GSTA2 and mouse Gsta1 proteins, genes encoding the rat and human NQO1 proteins (Favreau & Pickett, 1991; Jaiswal, 1991), glutamate cysteine ligase catalytic subunit (GCLC) and modulatory (GCLM) subunits (Moinova & Mulcahy, 1998; Mulcahy et al., 1997; Wild et al., 1998), and HO-1 (Inamdar et al., 1996) were also found to be transcriptionally regulated via the ARE. Table 1.3 show the ARE sequence present in the promoter region of different ARE-driven genes.

The CNC-bZIP Nrf2 transcription factor

1.3.1 CNC-bZIP family of transcription factors

The CNC-basic leucine zipper (CNC-bZIP) family, a subfamily of bZIP proteins, play important roles in mammalian development and the regulation of expression of genes involved in various biological processes, including proliferation, apoptosis, differentiation, and stress responses. The first isolated CNC-bZIP protein was the nuclear factor-erythroid 2 p45-subunit (NF-E2 p45) (Chang et al., 1993). Subsequently, another three closely related transcription factors Nrf1 (Chan et al., 1993), Nrf2 (Moi et al., 1994) and Nrf3 (Derjuga et al., 2004) were cloned. In addition, two distantly related proteins were also isolated and named Bach1 and Bach2 (Oyake et al., 1996). The CNC-bZIP transcription factors are composed of two conserved structural domains, named the „CNC‟ domain and bZIP domain, with the CNC domain situated just N-terminal to the bZIP domain (Chan et al., 1993; Chan et al., 1998; Moi et al., 1994). Members of the CNC-bZIP family form heterodimers with the bZIP small Maf proteins, composed of MafK, MafF and MafG, to bind to DNA sequences with different specificity (Motohashi et al., 1997). Both the CNC and the bZIP domains are responsible for the DNA binding property and binding specificity of the transcription factors.

1.3.2 Stucture of Nrf2

The CNC bZIP transcription factor Nrf2 contains six domains, namely Neh1-Neh6 (Figure 1.8), which are conserved amongst species (Itoh et al., 1995; Itoh et al., 1999). The Neh1 domain comprises a bZIP region fused to a CNC region and is responsible for its ability to dimerize with small Maf proteins and its ability to bind DNA as an obligate heterodimer. The N-terminal Neh2 domain is required for redox-sensitive negative control of the CNC-bZIP factor (Itoh et al., 1999). The C-terminal Neh3 domain interacts with chromodomain helicase DNA-binding protein 6 (CHD6) and therefore might associate with the transcriptional apparatus (Nioi et al., 2005). Both Neh4 and Neh5 are transactivation domains that interact with CREB-binding protein (CBP) (Katoh et al., 2001). The central Neh6 domain contributes to redox-independent negative control of Nrf2 (McMahon et al., 2004).

Thesis: books mechanism

Antioxidant Responsive Element

The antioxidant responsive element (ARE) is a cis-acting regulatory element or enhancer sequence, which is found in promoter regions of genes encoding phase II detoxification enzymes and antioxidant proteins. Okuda and coworkers (1989) first described an enhancer element, which is similar to TPA-responsive element (TRE) or AP-1 site in the rat glutathione S-transferase (GST)-P gene. This was followed by the characterization of a similar cis-acting regulatory element or enhancer sequence in rat GST Ya (Rushmore et al., 1990), mouse GST Ya (Friling et al., 1990), and human NAD(P)H:quinone oxidoreductase-1 (NQO1) (Li and Jaiswal, 1992). Collectively, these studies provided a common mechanism for phase II detoxification gene induction. The core ARE sequence was defined as 5’-TGACnnnGCA- 3’ based on mutational analysis of the rat GST A1 promoter sequence (Rushmore et al., 1991). In addition, many studies have demonstrated that the ARE sequence is found in numerous genes, and plays an important role in down stream gene expression. The list of ARE-driven genes includes rat GST A1, mouse GST A1, rat GST P1, rat NQO1, human NQO1, human glutamate-cysteine ligase (GCL), mouse ferritin-L, mouse metallothionein-1, and mouse UDP glucuronyl transferase (UGT). It has been well described that the Ah receptor mediates the gene expression of phase I drug metabolizing enzymes through XRE. However, little was known about the transacting factor or binding protein for the

ARE sequence until Nrf2 was identified.

An Important Role of Nrf2-ARE

THE NRF2 TRANSCRIPTION FACTOR

Nrf2 was cloned by Kan and coworkers in 1996 as a factor that binds to the NF-E2 repeat of the -globin gene promoter (Moi et al., 1994). It belongs to the cnc ("cap ‘n’ collar") subfamily of the basic region leucine zipper transcription factors. So far, six members in this family have been identified: NF-F2, Nrf1, Nrf2, Nrf3, Bach1, and Bach2. In spite of the high homology in their DNA binding and leucine zipper domains, they have distinct biological roles. NF-E2 expression is erythroid-specific, and the NF-E2 knockout mouse suffers from mild anemia or excessive bleeding (Shivdasani et al., 1995). Nrf1 is expressed in virtually all tissues, and the absence of Nrf1 is lethal to embryonic development (Chan et al., 1998). Nrf2 is also ubiquitously expressed, but it is dispensable for normal development (Chan et al., 1996). However, the Nrf2 knockout mouse has decreased expression of both constitutive and inducible levels of phase II enzymes and endogenous antioxidants, as mentioned. Nrf3 is preferentially expressed in placenta, and the Nrf3 knockout mouse has no obvious phenotype (Derjuga et al., 2004). Several homologue domains were identified when different species of the Nrf2 genes, such as human, mouse, and chicken, were aligned (Fig. 1). They are designated as Neh 1–6. The N-terminal Neh2 domain contains seven lysine residues for ubiquitin conjugation, so it confers negative regulation of the Nrf2 activity through proteasome-mediated degradation of Nrf2 (Zhang et al., 2004). It also binds to the Kelch domain of Keap1 (Itoh et al., 1999). The Neh4 and Neh5 are two independent transactivation domains that are rich in acidic residues and interact with CREB-binding protein (CBP) (Katoh et al., 2001). The function of Neh6 remains largely unknown, although it is known to have a high content of serine residues. The Neh1 domain contains a CNC-type basic leucine zipper, which is necessary for DNA binding and dimerization with other transcription factors (Itoh et al., 1999; Nioi et al., 2005). In addition, there is a functional nuclear localization signal (NLS) within this domain (Jain et al., 2005). The C-terminal Neh3 is indispensable for transcriptional activity of Nrf2 by recruiting CHD6, a coactivator with both helicase domain and chromodomain (Nioi et al., 2005). However, the precise function of CHD6 is unclear.

KEAP1, A NEGATIVE REGULATOR OF NRF2

As discussed, Nrf2 is a critical factor regulating the cellular defense response when cells are under oxidative stress or are stimulated with chemopreventive compounds. The activity of Nrf2 is tightly regulated by a negative regulator named Keap1, which was cloned using the Neh2 domain of Nrf2 as bait in a yeast two-hybrid system by Yamamoto and coworkers (Itoh et al., 1999). Keap1 contains three major domains: an N-terminal BTB (broad complex, tramtrack, and bric-a-brac) domain, a linker region, and a C terminal Kelch domain (Fig. 1). The N-terminal BTB domain was implicated in homodimerization of the Keap1 protein (Zipper and Mulcahy, 2002). The linker region is a cysteine-rich domain that was proved to be indispensable for the activity of Keap1 (Zhang and Hannink, 2003). The C-terminal Kelch domain contains six conserved Kelch repeat sequences and binds to the Neh2 domain of Nrf2. Recently, the crystal structure of the Kelch domain has been solved and revealed a six -propeller structure (Li et al., 2004; Padmanabhan et al., 2006).

Keap1 was initially described as a cytoplasmic factor that binds to actin cytoskeleton and Nrf2 to retain Nrf2 in the cytoplasm. Upon exposure of cells to oxidative stress or chemopreventive compounds, Nrf2 dissociates from Keap1, translocates to the nucleus, forms a heterodimer with its obligatory partner Maf, and ultimately activates ARE-dependent gene expression. Recently, findings from our laboratory and others indicate that Keap1 does not just passively sequester Nrf2 in the cytoplasm but plays an active role in targeting Nrf2 for ubiquitination and proteasomal degradation (Cullinan et al., 2004; Kobayashi et al., 2004; Zhang et al., 2004; Furukawa and Xiong, 2005). In addition, both our in vivo data and the in vitro data from another group have challenged the Nrf2-Keap1 dissociation model (Zhang et al., 2004; Eggler et al., 2005). Both ubiquitin-mediated degradation of Nrf2 and failure of dissociation of the Nrf2-Keap1 complex in response to Nrf2-inducers will be discussed in the following sections.

MECHANISTIC STUDIES

Antioxidant Response Element (ARE)

It has long been known that many phase 2 genes, including GSTs and NQO, are regulated through a cis-acting element, the antioxidant response element (ARE), located in their promoters. ARE was first identified in the 5′-flanking region of the rat Gsta2 subunit gene (TAATGGTGACAAAGCA) [39] and this enhancer is essential for the inducible expression of Gsta2 in response not only to phenolic antioxidants and metabolizable planar aromatic compounds, but also hydrogen peroxide and ROS [40]. Similarly, ARE (TCACAGTGACTTGGCA) of the rat nqo1 gene is necessary for the inducible expression of this gene [41,42]; subsequently it was found that the rat nqo1 ARE was highly conserved in the human nqo1 gene (TCACAGTGACTCAGCA) [43]. In addition, GCLC and GCLM, which are genes encoding GSH biosynthetic enzyme subunits, contain multiple functional AREs; the human GCLM promoter retains tandem AREs, which are in opposite orientations [44,45]. In the promoter of the human GCLC gene, a reverse ARE (TCCCCGTGACTCAGCG) located at -3118 bp was identified as a functional ARE, responsible for induction in response to β-naphthoflavone and putative chemopreventive agents [46]. In an early study by Wasserman and Fahl et al. [47], the core sequence of ARE was proposed to be 5′-A/GTGAC/GNNNGCa/c-3′. However, as more AREs are identified in a wide array of phase 2 genes, great variability in the core sequence of ARE was found, indicating that consensus ARE sequences may be dependent on the specific interrogated gene.

Keap1 as a Protein Inhibitor of Nrf2

Nrf2 has six highly conserved homologous regions named Neh1 to Neh6 [80]. The Neh1 domain has a bZIP region interacting with partner proteins for heterodimerization [81]. The Neh3 domain interacts with chromodomain helicase DNA-binding protein 6 (CHD6) [82], and two acidic transactivation domains, Neh4 and Neh5, cooperatively bind with CBP (cAMP response elementbinding protein-binding protein) [81]. The Neh2 domain located in the N-terminus of Nrf2 is known to be a regulatory domain responding to oxidative stress: Neh2 interacts with cytosolic protein, Kelchlike ECH-associated protein 1 (Keap1) and negatively controls Nrf2 function [83]. Keap1, which was originally isolated as an Nrf2-binding protein, is an actin-binding protein and has been thought to inhibit the function of Nrf2 by simply sequestering the protein in the cytoplasm [83,84]. However, as described in Figure 1, recent advances in Nrf2-Keap biology revealed that Keap1 functions as an adaptor protein between Nrf2 and Cul3, a component of the E3 ligase complex, and this binding promotes the continuous degradation of Nrf2 by the proteasome under normal conditions [85-87]. Since the dissociation of Nrf2 from Keap1 is the primary mechanism for Nrf2 activation, it will be an intriguing problem to determine how these two molecules interact with each other in different cellular environments. Recently, Yamamoto and colleagues have proposed the "hinge and latch" model to explain the response of Nrf2-Keap1 complex to stimuli [88,89]. Nrf2 contains 2 Keap1 binding sites within the Neh2 domain: DLG motif and ETGE motif, which enable the formation of a complex of one molecule of Nrf2 and two molecules of Keap1 [90,91]. Of note, these two sites have different binding affinities: the affinity of DLG motif to Keap1 is much weaker than that of ETGE. This led to the hypothetical "latch" binding of DLG-Keap1, which can be easily disturbed by Keap1 conformational changes of [88-91]. Since DLG binding involves the subsequent Cul3-proteasomal degradation of Nrf2, alterations in the DLG-Keap1 binding can result in the rescue of Nrf2 from degradation and accumulation of this protein within the nucleus. In recognition that Keap1 is a cysteine-rich protein (human and murine Keap1 contain 27 and 25 cysteines, respectively), modifications in sulfhydryl residues of Keap1 protein were initially speculated to result in protein conformational changes [83]. In fact, oxidative stress conditions and many exogenous chemicals alter the reducing status of cysteine residues of Keap1 and lead to Nrf2 translocation. As reactive cysteine residues mediating Nrf2 activation, an initial study by Dinkova-Kostova et al [92], has identified Cys257, Cys273, Cys288, and Cys297 to be dexamethasone-modified cysteine residues using mass spectrometry analysis. Subsequent independent studies confirmed that Cys273 and Cys288 are essential for Nrf2 activation in response to phase 2 enzyme inducers such as dithiolethiones and sulforaphane [91]. In addition to these, Cys151 was demonstrated to be required for the effect of sulforaphane and tBHQ [93-95]. Taken together, these results indicate that Keap1 is a sensor protein responding to oxidative and environmental stresses through dynamic changes in cysteine reducing status (Figure 1).

The Nrf2 System as a Potential Target for the Development of

Keap1

Keap1 plays a central role in the regulation of Nrf2 activity. Keap1 was isolated as a Nrf2-associating protein by using a yeast two-hybrid screening system (35). Knockout of Keap1 resulted in constitutive activation Nrf2 signaling (121, 156, 157).

Keap1 is a 69-kDa cytosolic protein with high homology to Drosophila actin-binding protein Kelch (61). Primary sequence and alignment analysis reveals that human Keap1 consists of five domains: (i) the N-terminal region (a.a. 1–66), (ii) broad complex, tramtrack, and bric a brac domain (BTB, a.a. 67–178), (iii) an cysteine-rich intervening region (IVR, a.a. 179–321), (iv) double glycine=Kelch repeat region (DGR a.a. 322–608), and (v) C-terminal region (CTR, a.a. 609–625) (29) (Fig. 3B).

Keap1 was observed to locate mainly in the cytoplasm, presumably by association with F-actin or myosin VIIa through the Kelch domain (61, 69, 70, 153).

Accordingly, Keap1 is hypothesized to be a cytosolic anchor protein that sequesters Nrf2 in cytoplasm under the unstimulated condition. Site-directed mutagenesis of a conserved serine (S104A) within the Keap1 BTB domain revealed that Nrf2 is indeed sequestered in cytoplasm by Keap1 (182).

A recent study using transgenic animaloriginated cells demonstrated that direct interaction between the Neh2 domain of Nrf2 and Kelch repeat domain of Keap1 is essential for retaining of Nrf2 in the cytoplasm by Keap1 (70). Keap1 also contains a strong nuclear exporting signal (NES) sequence in the IVR domain, which may play a major role in determining the subcellular distribution of Keap1 (72, 142, 154).

Keap1 is identified to be associatedwith Cullin-3, a scaffold protein responsible for formation of an ubiquitin ligase E3 complex (25, 36, 87, 177). It is now clear that Keap1 may not only dictate Nrf2 localization, but also actively targets Nrf2 to degradation.

Regulation of NF-E2-Related Factor 2