Nuclear Matrix Protein (NRP/B) Modulates the Nuclear Factor (Erythroid-derived 2)-related 2 (NRF2)-dependent Oxidative Stress Response*

Reactive molecules have diverse effects on cells and contribute to several pathological conditions. Cells have evolved complex protective systems to neutralize these molecules and restore redox homeostasis. Previously, we showed that association of nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2 (NRF2) with the nuclear matrix protein NRP/B was essential for the transcriptional activity of NRF2 target genes in tumor cells. The present study demonstrates the molecular mechanism by which NRP/B, via NRF2, modulates the transcriptional activity of antioxidant response element (ARE)-driven genes. NRP/B is localized in the nucleus of primary brain tissue and human neuroblastoma (SH-SY5Y) cells. Treatment with hydrogen peroxide (H2O2) enhances the nuclear colocalization of NRF2 and NRP/B and induces heme oxygenase 1 (HO1). Treatment of NRP/B or NRF2 knockdowns with H2O2 induced apoptosis. Co-expression of NRF2 with members of the Kelch protein family, NRP/B, MAYVEN, or MAYVEN-related protein 2 (MRP2), revealed that the NRF2-NRP/B complex is important for the transcriptional activity of ARE-driven genes HO1 and NAD(P)H:quinine oxidoreductase 1 (NQO1). NRP/B interaction with Nrf2 was mapped to NRF2 ECH homology 4 (Neh4)/Neh5 regions of NRF2. NRP/B mutations that resulted in low binding affinity to NRF2 were unable to activate NRF2-modulated transcriptional activity of the ARE-driven genes, HO1 and NQO1. Thus, the interaction of NRP/B with the Neh4/Neh5 domains of NRF2 is indispensable for activation of NRF2-mediated ARE-driven antioxidant and detoxifying genes that confer cellular defense against oxidative stress-induced damage.

small MAF proteins (21). The Kelch domain of KEAP1 interacts with Neh2 suppressing NRF2 activity. Neh4 and Neh5 interact with the CREB-binding protein (cAMP responsive elementbinding protein) to synergistically induce strong NRF2 transcriptional activation (22). Under conditions of oxidative stress, NRF2 is phosphorylated and released by KEAP1 into the nucleus where it associates with NRP/B and activates phase II detoxifying and antioxidant genes (17,20). The molecular mechanism by which NRF2 activates ARE-driven genes has remained elusive.
In the present study, we describe the mechanism of NRP/Bmodulated activation of NRF2 target genes. We also identify an interaction between the NRP/B BTB domain and the NRF2 Neh4/Neh5 region that may play a crucial role in modulating NRF2-dependent ARE-driven phase II detoxifying and antioxidant genes.
GST Pull-down Assay-HEK293T cells transfected with pCMV4-NRP/B or pMyc-NRP/B constructs were lysed in RIPA buffer and solubilized for 1 h at 4°C. Protein extracts were pre-cleaned by incubation with gluthatione-Sepharose beads for 30 min at 4°C. After centrifugation, the supernatants were incubated with GST or GST-NRF2 truncation proteins at room temperature for 3 min. After washing the precipitates were subjected to immunoblot analysis using anti-FLAG or anti-Myc antibody.
Immunoprecipitation-HEK293T cells were seeded in 6-well plates overnight and co-transfected with DNA constructs. 24 h after transfection, the cultures were washed twice with PBS and lysed with ice-cold lysis buffer (RIPA). Cell extracts were solubilized for 30 min at 4°C. After pre-clearing, the extracts were immunoprecipitated with anti-NRF2 antibody. Precipitates were blotted and probed with anti-Myc antibody.
Immunoblot Analysis-Cell cultures were lysed with cold lysis buffer (100 mM KCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl 2 , 1.2 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100, and 1 mM EGTA). The lysates were transferred to a fresh tube and solubilized for 1 h at 4°C and clarified by centrifugation at 12,000 ϫ g for 20 s at 4°C. Total cell extract protein concentration was determined using the Bradford assay. Equal amounts of proteins were electrophoresed on SDS-PAGE gels, blotted onto polyvinylidene difluoride membrane, and incubated with anti-NRP/B (VD2), anti-NRF2, anti-FLAG, anti-Myc, or anti-CSK antibodies. After washing, the blots were incubated with horseradish peroxidase-conjugated anti-IgG antibody.
Immunocytochemistry-Cell cultures were washed with PBS, fixed with 4% paraformaldehyde, and treated with 0.5% Triton X-100 in PBS for 30 min. After 3 washes with PBS, cells were treated with 10% goat serum in PBS for 2 h and incubated with anti-NRP/B (VD2) and/or anti-NRF2 antibodies for 1 h at room temperature. After washing with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated IgG and/or Texas Red-conjugated IgG antibodies for 1 h. The cells were then washed, mounted on slides, and imaged by confocal microscopy (Zeiss LSM 510 Meta).
RNA Isolation-Total RNA isolation was performed using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Briefly, cells were seeded on 10-cm dishes and treated accordingly. One ml of TRIzol reagent was added, followed by addition of 0.2 ml of chloroform after 15 min. Samples were vigorously inverted by hand for 15 s, incubated at room temperature for 3 min, and centrifuged at 12,000 ϫ g for 15 min at 4°C. 0.5 ml of isopropyl alcohol was added to the supernatant. After a 10-min incubation at room temperature, samples were centrifuged at 12,000 ϫ g for 10 min at 4°C. The pellets were washed with 75% ethanol, dissolved in RNase-free water, and incubated at 60°C for 10 min. Total RNA concentration was measured and the samples were stored at Ϫ80°C for use in Northern blotting and RT-PCR analysis.
RT-PCR Analysis-Transcript levels were semi-quantified by one-step RT-PCR according to the manufacturer's instructions (Clontech) using primers specific for HO1 and GAPDH ( Table  1). The temperature conditions were 50°C for 1 h, 94°C for 5 min, followed by 25 cycles of 94°C for 30 min, 68°C for 30 min, and 68°C for 60 min with an extension of 68°C for 2 min.
Northern Blot Analysis-20 g of total RNA was electrophoresed on a 1% agarose gel, and transferred onto Hybond-Nϩ (Amersham Biosciences) in 20ϫ SSC buffer overnight. Membranes were cross-linked and pre-hybridized in pre-hybridization solution (5ϫ SSPE, 2% SDS, and 5ϫ Denhardt's reagent containing 100 g/ml of denatured salmon testes DNA) at 65°C for 6 h. Purified PCR-generated NRP/B (BTB domain) serving as a probe, was labeled with ␣-32 P using the NEblot kit (catalog number N1500L, New England Biolabs) according to the manufacturer's instructions. The membranes were incubated with hybridization solution containing the NRP/B BTB domain-labeled probe at 65°C overnight, and then washed in washing solution (0.5ϫ SSPE, 1% SDS) for 20 min, and twice in washing solution (0.1ϫ SSPE, 1% SDS) for 20 min each. Membranes were exposed to x-ray film at Ϫ80°C.
Apoptotic DNA Ladder-DNA fragmentation in apoptotic cells was detected using the Apoptotic DNA Ladder Detection Kit (Chemicon International). The procedure was performed per the manufacturer's instructions. Briefly, cells were treated with an apoptosis inducing agent appropriate to the purposes of our experiment. Treated cells were washed with PBS, collected, and centrifuged. The pellets were resuspended in TE lysis buffer and treated with RNase A and proteinase K. DNA was precipitated with isopropyl alcohol and redissolved in DNA Suspension Buffer. DNA fragmentation was visualized on a 1% agarose gel stained with ethidium bromide.
Luciferase Assay-SH-SY5Y or HEK293T cells were co-transfected with a combination of plasmids as indicated in figure legends using Lipofectamine 2000 or Lipofectatime. In addition, pCMV ␤-galactosidase was co-transfected for the purpose of controlling the transcriptional activity of the promoter(s). Cell lysates were prepared and luciferase and ␤-galactosidase activities were quantified using a Luciferase assay kit (Promega, Madison, WI) according to the manufacturer's instructions. The effects of the proteins on the promoter(s) transcriptional activity were normalized to the ratio of the luciferase versus ␤-galactosidase activity.
Statistical Analysis-Data are presented as the mean Ϯ S.D. The Student's t test and one-way analysis of variance were used to assess the significance of independent experiments. p Ͻ 0.05 represents the statistical significance.

Expression of NRP/B in Neuronal Cells-NRP/B has been
shown to be expressed in various tissues and cells, particularly neuronal cells (17,19,20). We determined the molecular mechanism by which NRP/B mediates cellular protection against agents of oxidative stress in the brain. We examined expression of NRP/B in SH-SY5Y and PC12 cells by Northern and Western blot analyses. HCT-116 cells reported to express NRP/B (28) served as a positive control. Using a human NRP/B probe, Northern blotting showed that NRP/B mRNA was detected in HCT-116 and SH-SY5Y cells (Fig. 1A). Protein extracts from HCT-116, SH-SY5Y, and PC12 cells were subjected to Western blot analysis using a monoclonal antibody against NRP/B (DV2) (17). This analysis showed that NRP/B was expressed in all cell lines tested (Fig. 1B). NRP/B expression was detected in PC12 by Western blot, but not by Northern blot (Fig. 1, A and B) due to the high specificity of the human NRP/B probe and the high contingency of the Northern blot condition. The human NRP/B probe used did not hybridize rat NRP/B mRNA in PC12 cells (Fig. 1A), indicating that the Northern blot condition was highly specific.
Immunohistochemical staining with anti-NRP/B (VD2) antibody was carried out to detect expression and localization of NRP/B in human brain specimens. NRP/B was expressed in the nucleus of the brain specimen (Fig. 1C). In addition to our previous report (17), expression of NRP/B mRNA and protein was detected in human SH-SY5Y. Thus, we selected SH-SY5Y cells for further analysis of NRP/B function and mechanism in the oxidative stress response. (20). We examined the effect of H 2 O 2 on NRF2 and NRP/B localization in SH-SY5Y cells. SH-SY5Y cells were treated with 5 M H 2 O 2 for 12 h, fixed, and immunostained with anti-NRF2 and anti-NRP/B (VD2) antibodies ( Fig. 2A). Fixed cells stained with either mouse or rabbit IgG antibody served as control (supplemental Fig. S1). Colocalization of NRF2 and NRP/B intensified in the nucleus following H 2 O 2 treatment ( Fig. 2A).

H 2 O 2 Increased the Colocalization of NRF2 and NRP/B in SH-SY5Y Cells-H 2 O 2 treatment has been shown to increase the co-localization of NRP/B and Nrf2 in breast cancer cells
NRF2 and NRP/B Conferred Cellular Protection against H 2 O 2 via HO1 Activation-To define the mechanism by which the association of NRF2 and NRP/B induces HO1 activation, we examined the effect of H 2 O 2 treatment on HO1 promoter activity by luciferase assay. SH-SY5Y cells were transfected with the HO1 promoter and treated with dimethyl sulfoxide or 5 M H 2 O 2 in a time-dependent manner as indicated (Fig. 2B). Cell lysates were prepared and luciferase activity was measured. Six hours after treatment, relative luciferase activity was up to 7-fold higher in cells treated with H 2 O 2 compared with those treated with dimethyl sulfoxide (Fig. 2B). Next, we measured the transcription levels of HO1 mRNA induced by H 2 O 2 treatment. SH-SY5Y cells were harvested 0, 6, and 12 h after treatment with H 2 O 2 . Total RNA was purified and semi-quantitative RT-PCR analysis showed that HO1 mRNA was up-regulated over the course of H 2 O 2 treatment (Fig. 2C). We then examined whether NRF2 and NRP/B expression protected cells from apoptosis induced by H 2 O 2 treatment. SH-SY5Y cells were treated with siNRP/B, siNRF2, or siCon (control). After 24 h, cells were treated with 10 M H 2 O 2 for 12 h. DNA fragmentation resulting from H 2 O 2 -induced apoptosis was detected using the DNA ladder kit. Intense DNA fragmentation was observed in siNRP/B-and siNRF2-treated cells when com-pared with siCon-treated controls in response to H 2 O 2 treatment (Fig. 2D). These data provide strong evidence that expression of both NRP/B and NRF2 is crucial for cellular protection against H 2 O 2 -induced apoptosis.
Specific Activation of NRF2 Target Genes by NRP/B-We previously cloned and characterized several Kelch-related family members including NRP/B (19), MRP2 (24), and MAYVEN (29). These proteins have similar domain organization (Fig. 3A) consisting of a N-terminal BTB domain and a C-terminal Kelch repeat domain separated by an IVS. BTB domain mediates homo-and heterodimerization (12) whereas, the C-terminal Kelch repeat region functions to bind actin tails, and is involved in protein-protein interactions (30). KEAP1, another Kelch family member, functions as transcriptional repressor for NRF2 (8), whereas NRP/B is an enhancer for NRF2 activity (17,20). MRP2 is involved in glycogen synthase kinase 3␤-mediated neuronal differentiation (24) and process elongation in oligodendrocytes (25,31). MAYVEN is also essential for FYN-modulated process elongation in oligodendrocytes (25). Whereas KEAP1, MRP2, and MAYVEN are endogenously localized in the cytoplasm (8,24,25), NRP/B is endogenously localized in the nucleus (19,32). We examined the differential effects of these proteins on NRF2mediated HO1 transcriptional activity using a luciferase reporter assay. The HO1 promoter plasmid was co-transfected with NRP/B, MRP2, KEAP1, MAYVEN, or mock (control). Cell lysates were prepared and luciferase activity was measured after 24 h. Relative luciferase activity was up to 2-fold higher in cells transfected with NRP/B compared with cells transfected with mock or MRP2, and up to 4-fold higher when compared with cells transfected with KEAP1 or MAYVEN (Fig. 3B).
A similar set of experiments performed using cells cotransfected with the HO1 promoter and NRF2 in conjunction with NRP/B, MAYVEN, MRP2, KEAP1, or mock (control) in a dose-dependent manner (Fig. 3C) showed a more than 10-fold increase in HO1 transcriptional activity in cells transfected with NRP/B as compared with those transfected with MAYVEN, MRP2, KEAP1, or mock (Fig. 3C). NRP/B was important in up-regulating HO1 transcriptional activity, whereas KEAP1 showed an inhibitory effect on the activity of HO1. MAYVEN and MRP2 did not significantly effect the transcriptional activity as compared with control counterparts. These data together with previous reports (17,20) indicate that NRP/B specifically activates NRF2 target genes.
Mapping Interacting Domains of NRF2 and NRP/B-We previously showed that the NRF2-NRP/B complex was crucial for induction of the NRF2 target gene, NQO1 (17,20). In this study, FIGURE 1. Expression and localization of NRP/B in neuronal origin-derived cells and human brain specimen. A, for Northern blot analysis, total RNA was purified from HCT-116, SH-SY5Y, and PC12 cells using TRIzol reagent. Ten g of RNA was blotted on a Hybond-N ϩ membrane and hybridized with an ␣-32 P-labeled human NRP/B probe. B, total cell lysates were prepared from several cell lines, and 100-g samples were blotted on a polyvinylidene difluoride membrane. The blot was incubated with anti-NRP/B (VD2) or anti-c-src tyrosine kinase (CSK) antibodies. CSK was used to confirm equal protein loading. C, localization of NRP/B in sample specimens of normal brains. Normal brain specimens were obtained from the Cooperative Human Tissue Network. The paraffin-embedded samples were immunostained with anti-NRP/B (VD2) antibody. WB, Western blot.
Next we examined the interaction of the NRP/B BTB domain with NRF2. For this purpose, HEK293T cells were transfected with NRP/B constructs containing the BTB domain (NRP/B BTB), the BTB domain and central linker region (NRP/B BTB-IVS), and the central linker region alone (NRP/B IVS). Cell extracts were prepared, immunoprecipitated with anti-NRF2 antibody, and subjected to Western blot analysis using anti-FLAG antibody. Immunoprecipitation assays showed that the NRP/B BTB and NRP/B BTB-IVS regions strongly interacted with NRF2, whereas an NRP/B IVS was less obvious (Fig. 4D). Thus, NRF2 may interact with the BTB domain of NRP/B. Taken together our data indicate that interaction of NRP/B with the region encompassing the Neh4 domain of NRF2 may interact to induce NRF2-mediated HO1 activity. Homology Modeling of the NRP/B BTB Domain-A homology model of the NRP/B BTB domain containing residues 1 to 147 was generated using the program MODELLER (33). The crystal structure of BCL6 BTB domain (Protein Data Bank code 1R29), which shares 33% sequence identity with the NRP/B BTB domain, was used as a template structure (Fig. 5A). The homology model was selected from three computed models based upon the lowest values of the MODELLER objective function. The secondary structure of the model contains the BTB core elements of three ␤-strands (␤2-␤4) forming a mixed ␤-sheet, and five ␣-helices (␣2-␣6) (Fig. 5A). A short ␣3 10 helix interrupts the ␣3-␤4 loop. The region from Asp-30 to Arg-43 of NRP/B forms an N-terminal ␣-helical extension of the core BTB domain. The ␤1 strand observed in the crystal structure of the BCL6 BTB domain is not present in the NRP/B BTB homology model.
As shown in Fig. 5B, Asp-47 is a conserved amino acid and sits in the loop region connecting the ␣1 and ␤2 secondary structure elements. Its side chain makes a hydrogen bond to the backbone of Arg-61. Amino acid position 61 is invariably an Kelch-related protein family consists of two main domains, BTB (responsible for homo-or hetero-dimerization) and Kelch repeats (responsible for actin binding). B, differential effect of Kelch-related proteins on HO1 promoter activity. NRP/B, MRP2, KEAP1, MAYVEN, or mock were co-transfected with HO1 promoter in SH-SY5Y cells. Following 24 h, cell extracts were prepared. HO1 promoter (luciferase) activity was measured, and then the relative luciferase activity versus ␤-galactosidase activity (derived from three individual experiments) was expressed in arbitrary units. The bars in the graphs represent the mean Ϯ S.D. *, p Ͻ 0.05. C, differential effect of Kelch-related proteins on NRF2-mediated HO1 promoter activity, SH-SY5Y cells. NRP/B, MRP2, KEAP1, MAYVEN, or mock were co-transfected with HO1 promoter and NRF2 in SH-SY5Y cells. Following 24 h, cell extracts were prepared. HO1 promoter activity was measured, and then the relative luciferase activity versus ␤-galactosidase activity (derived from three individual experiments) was expressed in arbitrary units. The bars in the graphs represent the mean Ϯ S.D. **, p Ͻ 0.01. arginine or a lysine. Arg-61 in the NRP/B BTB domain is part of the ␣2 helix and makes up to three hydrogen bonds, one to the carbonyl oxygen of Asp-47 in the ␣1-␤2 loop, one to Ser-82 and one to Asp-84 in the ␣3-␤4 loop. The side chain of His-60 at the N-terminal end of ␣2 hydrogen bonds to the backbone oxygen of Leu-44 in the ␣1-␤2 loop. These interactions serve to link some of the core secondary structure elements and stabilize the overall BTB domain fold. The mutations D47A, H60A, and R61D would break this hydrogen bonding network, disturb the fold, and disrupt the BTB domains ability to associate with interacting proteins such as NRF2.
Involvement of NRP/B BTB Domain in NRF2-mediated ARE-driven Transcriptional Activity-Homology modeling showed that Asp-47, His-60, and Arg-61 of the NRP/B BTB domain have important roles in maintaining the overall fold (Fig.  5B). Here, we examined whether mutations D47A, H69A, and R61D altered ARE-driven transcriptional activity. The triple mutant D47A, H69A,R61D, designated NRP/B Mut1, was generated, and its expression was verified by Western blotting using anti-Myc antibody. The HO1 promoter in conjunction with NRF2 was co-transfected with wild-type NRP/B (NRP/B WT), NRP/B Mut1, KEAP1, or Mock (control) in HEK293T cells. Cell lysates were prepared and luciferase activity was measured 24 h after transfection. Relative luciferase activity was ϳ5-fold higher in cells transfected with NRP/B (wild-type) (17) as compared with cells receiving NRP/B Mut1 or mock (Fig. 5C). KEAP1 inhibited HO1 promoter activity as expected (Fig. 5C).
We also examined the effect of these mutations on transcriptional activity of the NQO1 promoter and ARE-driven luciferase activity. NQO1 promoter (34) or AREdriven luciferase (27) in conjunction with NRF2 was co-transfected with NRP/B WT, NRP/B Mut1, KEAP1, or mock (control) in HEK293T cells. NRP/B Mut1 was unable to induce NRF2-mediated transcriptional activity of NQO1 (Fig. 5D) and ARE-driven gene expression (Fig. 5E).
To further examine the effect of the mutations in the BTB domain of NRP/B on its association with NRF2, NRF2 was cotransfected with either NRP/B WT or NRP/B Mut1. Protein extracts were prepared, immunoprecipitated with anti-NRF2 antibody, and subjected to Western blot analysis using anti-Myc antibody. For expression verification, cell extracts were blotted and probed with anti-NRF2 or anti-Myc antibody. As shown in Fig. 5F, NRP/B Mut1 had lower binding affinity with NRF2 as compared with the NRP/B WT counterpart (Fig. 5F). Taken together, these results indicate that the NRP/B BTB domain played an important role in modulating ARE-driven antioxidant gene expression in an NRF2-dependent fashion.

DISCUSSION
In the present study we elucidated the molecular mechanism by which NRP/B activates transcriptional activity of AREdriven gene(s) through the NRF2 pathway. We observed that the NRP/B BTB domain interacts with the activation domains, Neh4/Neh5, of NRF2. Mutations that are predicted to disturb the secondary structure of the NRP/B BTB domain fail to induce NRF2 target genes (NQO1 and HO1) and ARE-driven transcriptional activity. This study illustrates that the association of the NRP/B with the Nhe4/Neh5 domains of NRF2 is indispensable for activation of NRF2-mediated ARE-driven phase II enzymes that confer cellular protection against oxidative stress-induced damage.
More than 60 Kelch-related proteins have been identified in organisms from viruses to mammals. Forming multiprotein complexes through contact sites within their BTB and Kelch domains, this family of proteins regulates cell morphology and organization and gene expression (14,35). Previously, we cloned and characterized several members of the Kelch-related protein family, namely, NRP/B, MRP2, and MAYVEN. These proteins and another Kelch-related protein, KEAP1 have similar domain organization (Fig. 3A). We investigated the functions of these proteins in the context of oxidative stress responses. Despite similar domain organization their cellular localizations were found to be different. While MAYVEN, MRP2, and KEAP1 are localized in the cytoplasm, NRP/B is confined to the nucleus (17,20,24). In the presence of NRF2, NRP/B actively induced the transcriptional activity of HO1, whereas KEAP1 inhibited this activity (Fig. 3, B and C). Our previous report showed that GFP-NRF2 shuttled from the nucleus to the cytoplasm when it was cotransfected with KEAP1 and inhibited transcriptional activity of the NRF2 target gene, NQO1 (17). However, the phenomenon did not occur when NRF2 was contransfected with MAYVEN or MRP2 (data not shown). These findings illustrated a role for NRP/B and KEAP1 in modulating the NRF2 pathway in response to reactive oxygen species.
The interaction of NRP/B and NRF2 was essential for the activation of NRF2 target genes (Fig. 3C). We examined which structural domain of NRF2 was responsible for induction of NRF2 target genes. Several truncation mutants of NRP/B were analyzed for their ability to interact with NRF2. The NRP/B BTB and BTB-IVS regions strongly interacted with NRF2, whereas the IVS region displayed a much weaker association (Fig. 4D). The mutations D47A, H60A, and R61D in the BTB domain (NRP/B Mut1), which we predicted from homology modeling to destabilize the BTB fold, reduced the level of NRP/B interaction with NRF2 (Fig. 5F). Furthermore, wild-type NRP/B, but not NRP/B Mut1, induced ARE-driven transcriptional activity of the phase II detoxifying and antioxidant enzymes, HO1 and NQO1 (Fig. 5, C and D). The NRB/B BTB domain may, therefore, significantly contribute to the activation of NRF2 target genes. NRF2 contains 6 domains, Neh2, Neh4, and Neh5 at the N-terminal, and Neh6, Neh1, and Neh3 at the C-terminal (22). KEAP1 interacts with Neh2 of NRF2 to inhibit transcriptional activity (8). Neh4 and Neh5 are conserved in several species and interact with the co-activator CREB-binding protein to synergistically activate the transcription of NRF2 target genes (22). To determine which domain(s) of NRF2 is associated with NRP/B, we adopted a deletion mapping approach, which revealed that the N-terminal region of NRF2 spanning amino acids 1-339 containing Neh2, Neh4, and Neh5 interacted specifically with NRP/B (Fig. 4). Furthermore, the NRF2 region spanning amino acids 100 to 150 containing Neh4 strongly associated with NRP/B (Fig. 4C) (22). It was observed that NRP/B also interacted with the Neh5 activation domain of NRF2, although the affinity of the association was not as strong as the Neh4 domain. These data reveal the core domains of NRP/B and NRF2 that are critical for activation of NRF2 target genes HO1 and NQO1.
In conclusion, we elucidated the molecular mechanism by which NRP/B activates NRF2-mediated ARE-driven transcription of phase II detoxifying and antioxidant enzymes NQO1 and HO1. The association of the NRP/B BTB domain with the NRF2 Neh4/Neh5 domains is central to NRF2-mediated cellular protection against damage caused by oxidative stress. NQO1 is an activating enzyme for some anti-cancer drugs and has been implicated in cancer prevention (36 -38). HO1 exerts antioxidant effects, including inhibition of the proliferation of vascular and airway smooth muscle cells (39,40), and has been implicated in the endogenous defense against oxidative stress (41). Understanding the molecular mechanism by which the NRF2-NRP/B interaction mediates enzyme activation may have potential therapeutic consequences for the treatment of cancer and neurodegenerative disorders.