Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.
The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
To maintain intracellular redox homeostasis, genes encoding many antioxidants and detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis antioxidant responsive elements (AREs) in their promoter regions. Nrf2 is the critical transcription factor responsible for ARE-dependent transcription. We and others have previously demonstrated that Nrf2 is targeted for ubiquitin-mediated degradation by Keap1 in a redox-sensitive manner through modifications of distinct cysteine residues of Keap1. Here, we report that p300/CBP directly acetylates Nrf2 in response to arsenite-induced stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.
Nrf2 is the key transcription factor regulating the antioxidant response. Nrf2 signaling is repressed by Keap1 at basal condition and induced by oxidative stress. Keap1 is recently identified as a Cullin 3-dependent substrate adaptor protein. A two-sites binding “hinge & latch” model vividly depicts how Keap1 can efficiently present Nrf2 as substrate for ubiquitination. Oxidative perturbation can impede Keap1-mediated Nrf2 ubiquitination but fail to disrupt Nrf2/Keap1 binding. Nrf2 per se is a redox-sensitive transcripon factor. A new Nrf2-mediated redox signaling model is proposed based on these new discoveries. Free floating Nrf2 protein functions as a redox-sensitive probe. Keap1 instead functions as a gate keeper to control the availability of Nrf2 probes and thus regulates the overall sensitivity of the redox signaling.
Nrf2; Keap1; redox
Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosporylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3β that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-α mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An autoregulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an autoregulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.
Nrf; INrf2 (keap1); Oxidative/electrophilic stress; Defensive gene expression; Cell signaling; Cell survival
Activation of the antioxidant response element (ARE) up-regulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein, Keap1, and the transcription factor, Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small molecule inhibitors of Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance (SPR) assay. The FP assay exhibits considerable tolerance towards DMSO and produced a Z'-factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high throughput screening (HTS) assay to discover small molecule inhibitors of Keap1-Nrf2 interaction.
Nrf2; Keap1; ARE; fluorescence polarization; high throughput screening; oxidative response
Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds antioxidant response element (ARE) that leads to coordinated activation of gene expression. It takes less than fifteen minutes from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls switching off of Nrf2 activation of gene expression. GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to nuclear localization of Fyn. Fyn phosphorylates Nrf2tyrosine568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2. The switching on and off of Nrf2 protect cells against free radical damage, prevents apoptosis and promotes cell survival.
Nrf2 is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the antioxidant response element (ARE). Nrf2 is repressed by the cysteine-rich Keap1 protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3-mediated ubiquitination complex. We find that modification of C151 of human Keap1 by mutation to a tryptophan relieves the repression by Keap1 and allows activation of the ARE by Nrf2. Keap1 C151W has a decreased affinity for Cul3, and can no longer serve to target Nrf2 for ubiquitination, though it retains its affinity for Nrf2. A series of 12 mutant Keap1 proteins, each containing a different residue at position 151, was constructed to explore the chemistry required for the effect. The series reveals that the extent to which Keap1 loses the ability to target Nrf2 for degradation, and hence the ability to repress ARE activation, correlates well with the partial molar volume of the residue. Other physico-chemical properties do not appear to contribute significantly to the effect. Based on this finding, a structural model is proposed whereby large residues at position 151 cause steric clashes that lead to alteration of the Keap1-Cul3 interaction. This model has significant implications for how electrophiles, which modify C151, disrupt the repressive function of Keap1.
Keap1; Nrf2; partial molar volume; antioxidant response element; cysteine; tryptophan
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element, a cis-acting regulatory element that increases expression of detoxifying enzymes and antioxidant proteins. Kelch-like ECH associating protein 1 (Keap1) protein is a negative regulator of Nrf2. Previous work has shown that genetic overexpression of Nrf2 is protective in vitro and in vivo. To modulate the Nrf2-ARE system without overexpressing Nrf2, we used short interfering RNA (siRNA) directed against Keap1. Keap1 siRNA administration in primary astrocytes increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage. These data indicate that activation of endogenous intracellular levels of Nrf2 is sufficient to protect in models of oxidative stress and Parkinson's disease.
The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.
PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.
Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders. The overexpression of Nrf2 has become a potential therapeutic avenue for various neurodegenerative disorders such as Parkinson, Amyotrophic lateral sclerosis, and Alzheimer’s disease. The expression of phase II detoxification enzymes is governed by the cis-acting regulatory element known as antioxidant response element (ARE). The transcription factor Nrf2 binds to ARE thereby transcribing multitude of antioxidant genes. Keap1, a culin 3-based E3 ligase that targets Nrf2 for degradation, sequesters Nrf2 in cytoplasm. Disruption of Keap1-Nrf2 interaction or genetic overexpression of Nrf2 can increase the endogenous antioxidant capacity of the brain thereby rendering protection against oxidative stress in neurodegenerative disorders. This review primarily focuses on targeted Nrf2 overexpression as a promising therapeutic strategy for the treatment of neurodegenerative disorders.
A common feature of diverse chemopreventive agents is the ability to activate expression of a genetic program that protects cells from reactive chemical species that, if left unchecked, would cause mutagenic DNA damage. The bZIP transcription factor Nrf2 has emerged as a key regulator of this cancer-preventive genetic program. Nrf2 is normally sequestered in the cytoplasm by a protein known as Keap1. Chemopreventive agents allow Nrf2 to escape from Keap1-mediated repression, although the molecular mechanism(s) responsible for activation of Nrf2 is not understood. In this report, we demonstrate that Keap1 does not passively sequester Nrf2 in the cytoplasm but actively targets Nrf2 for ubiquitination and degradation by the proteosome under basal culture conditions. We have identified two critical cysteine residues in Keap1, C273 and C288, that are required for Keap1-dependent ubiquitination of Nrf2. Both sulforaphane, a chemopreventive isothiocyanate, and oxidative stress enable Nrf2 to escape Keap1-dependent degradation, leading to stabilization of Nrf2, increased nuclear localization of Nrf2, and activation of Nrf2-dependent cancer-protective genes. We have identified a third cysteine residue in Keap1, C151, that is uniquely required for inhibition of Keap1-dependent degradation of Nrf2 by sulforaphane and oxidative stress. This cysteine residue is also required for a novel posttranslational modification to Keap1 that is induced by oxidative stress. We propose that Keap1 is a component of a novel E3 ubiquitin ligase complex that is specifically targeted for inhibition by both chemopreventive agents and oxidative stress.
The Keap1-Nrf2 system protects animals from oxidative and electrophilic stresses. Nrf2 is a transcription factor that induces the expression of genes essential for detoxifying reactive oxygen species (ROS) and cytotoxic electrophiles. Keap1 is a stress sensor protein that binds to and ubiquitinates Nrf2 under unstressed conditions, leading to the rapid proteasomal degradation of Nrf2. Upon exposure to stress, Keap1 is modified and inactivated, which allows Nrf2 to accumulate and activate the transcription of a battery of cytoprotective genes. Antioxidative and detoxification activities are important for many types of cells to avoid DNA damage and cell death. Accumulating lines of recent evidence suggest that Nrf2 is also required for the primary functions of myeloid cells, which include phagocytosis, inflammation regulation, and ROS generation for bactericidal activities. In fact, results from several mouse models have shown that Nrf2 expression in myeloid cells is required for the proper regulation of inflammation, antitumor immunity, and atherosclerosis. Moreover, several molecules generated upon inflammation activate Nrf2. Although ROS detoxification mediated by Nrf2 is assumed to be required for anti-inflammation, the entire picture of the Nrf2-mediated regulation of myeloid cell primary functions has yet to be elucidated. In this review, we describe the Nrf2 inducers characteristic of myeloid cells and the contributions of Nrf2 to diseases.
The structure of mouse Keap1-DC complexed with the DLG motif peptide of Nrf2 transcription factor was determined at 1.9 Å resolution. The structure showed that the peptide binds to Keap1-DC at the bottom region of the β-propeller domain.
The cytoplasmic repressor Keap1 regulates the function of transcription factor Nrf2 which plays critical roles in oxidative and xenobiotic stresses. The Neh2 domain of Nrf2 interacts with Keap1 at the bottom region of the Kelch/β-propeller domain which is formed by double-glycine repeat and C-terminal region domains (Keap1-DC). The structure of Keap1-DC complexed with an Nrf2 peptide containing a conserved DLG motif has been determined at 1.9 Å resolution. The Keap1-bound DLG peptide possesses a hairpin conformation, and it binds to the Keap1 protein at the bottom region of the β-propeller domain. The intermolecular interaction occurs through their complementary electrostatic interactions. Comparison of the present structure with the recently reported Keap1-DC complex structure suggests that the DLG and ETGE motifs of Neh2 in Nrf2 bind to Keap1 in a similar manner but with different binding potencies.
oxidative stress; Nrf2 transcription factor; Keap1; β-propeller domain; structure of the complex
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 α-helix followed by a mini antiparallel β-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 β-propeller structure of Keap1. In contrast, the central α-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 β-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 α-helix in a correct position to benefit ubiquitin signaling.
The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2. Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to repress steady-state levels of Nrf2 and Nrf2-dependent transcription. Cullin-dependent ubiquitin ligase complexes have been proposed to undergo dynamic cycles of assembly and disassembly that enable substrate adaptor exchange or recycling. In this report, we have characterized the importance of substrate adaptor recycling for regulation of Keap1-mediated repression of Nrf2. Association of Keap1 with Cul3 was decreased by ectopic expression of CAND1 and was increased by small interfering RNA (siRNA)-mediated knockdown of CAND1. However, both ectopic overexpression and siRNA-mediated knockdown of CAND1 decreased the ability of Keap1 to target Nrf2 for ubiquitin-dependent degradation, resulting in stabilization of Nrf2 and activation of Nrf2-dependent gene expression. Neddylation of Cul3 on Lys 712 is required for Keap1-dependent ubiquitination of Nrf2 in vivo. However, the K712R mutant Cul3 molecule, which is not neddylated, can still assemble with Keap1 into a functional ubiquitin ligase complex in vitro. These results provide support for a model in which substrate adaptor recycling is required for efficient substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes.
Keap1 is a key regulator of the Nrf2 transcription factor which transactivates the Antioxidant Response Element (ARE) and upregulates numerous proteins involved in antioxidant defense. Under basal conditions, Keap1 targets Nrf2 for ubiquitination and proteolytic degradation and as such is responsible for the rapid turnover of Nrf2. In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1 leading to loss of its ability to negatively regulate Nrf2. We have previously shown that nitric oxide and S-nitrosothiols cause nuclear accumulation of Nrf2 and upregulation of the ARE-regulated gene HO-1. Here we show that nitric oxide and S-nitrosocysteine (CSNO) cause time and dose-dependent Keap1 thiol modification. These studies were carried out in HEK293 and in HEK293 cells overexpressing hemagglutinin-tagged Keap1. Furthermore we demonstrate that in response to CSNO Keap1 accumulates in the nucleus with a time course similar to that of Nrf2.
Nitric Oxide; S-Nitrosocysteine; S-Nitrosylation; Oxidation; Keap1; Nrf2; Nuclear localization
In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.
Targeting Nrf2 signaling appears to be an attractive approach for the treatment of maladaptive cardiac remodeling and dysfunction; however, pharmacological modulation of the Nrf2 pathway in the cardiovascular system remains to be established. Herein, we report that a novel synthetic triterpenoid derivative, dihydro-CDDO-trifluoroethyl amide (dh404), activates Nrf2 and suppresses oxidative stress in cardiomyocytes. Dh404 interrupted the Keap1-Cul3-Rbx1 E3 ligase complex-mediated Nrf2 ubiquitination and subsequent degradation saturating the binding capacity of Keap1 to Nrf2, thereby rendering more Nrf2 to be translocated into the nuclei to activate Nrf2-driven gene transcription. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 was resistant to dh404-induced stabilization of Nrf2 protein. In addition, dh404 did not dissociate the interaction of Nrf2 with the Keap1-Cul3-Rbx1 E3 ligase complex. Thus, it is likely that dh404 inhibits the ability of Keap1-Cul3-Rbx1 E3 ligase complex to target Nrf2 for ubiquitination and degradation via modifying Cys-151 of Keap1 to change the conformation of the complex. Moreover, dh404 was able to stabilize Nrf2 protein, to enhance Nrf2 nuclear translocation, to activate Nrf2-driven transcription, and to suppress angiotensin II (Ang II)-induced oxidative stress in cardiomyocytes. Knockdown of Nrf2 almost blocked the anti-oxidative effect of dh404. Dh404 activated Nrf2 signaling in the heart. Taken together, dh404 appears to be a novel Nrf2 activator with a therapeutic potential for cardiac diseases via suppressing oxidative stress.
Eukaryote cells balance production of reactive oxygen species (ROS) with levels of anti-oxidant enzyme activity to maintain cellular redox homeostasis. Mitochondria are a major source of ROS, while many anti-oxidant genes are regulated by the Nrf2 transcription factor. Keap1, a redox-regulated substrate adaptor for a cullin-based ubiquitin ligase, targets Nrf2 for proteosome-mediated degradation and represses Nrf2-dependent gene expression. We have previously identified a member of the phosphoglycerate mutase family, PGAM5, as a Keap1-binding protein. In this report, we demonstrate that PGAM5 is targeted to the outer membrane of mitochondria by an N-terminal mitochondrial-localization sequence. Furthermore, we provide evidence that PGAM5 forms a ternary complex containing both Keap1 and Nrf2, in which the dimeric Keap1 protein simultaneously binds both PGAM5 and Nrf2 through their conserved E(S/T)GE motifs. Knockdown of either Keap1 or PGAM5 activates Nrf2-dependent gene expression. We suggest that this ternary complex provides a molecular framework for understanding how nuclear anti-oxidant gene expression is regulated in response to changes in mitochondrial function(s).
Mitochondrial proteins; Anti-oxidant gene expression; Oxidative stress; ubiquitin ligases; chemoprevention
The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.
The Keap1–Nrf2 [Kelch-like ECH-associated protein 1–nuclear factor (erythroid-derived 2)-like 2] pathway plays a central role in the protection of cells against oxidative and xenobiotic stresses. Nrf2 is a potent transcription activator that recognizes a unique DNA sequence known as the antioxidant response element (ARE). Under normal conditions, Nrf2 binds to Keap1 in the cytoplasm, resulting in proteasomal degradation. Following exposure to electrophiles or reactive oxygen species, Nrf2 becomes stabilized, translocates into the nucleus, and activates the transcription of various cytoprotective genes. Increasing attention has been paid to the role of Nrf2 in cancer cells because the constitutive stabilization of Nrf2 has been observed in many human cancers with poor prognosis. Recent studies have shown that the antioxidant and detoxification activities of Nrf2 confer chemo- and radio-resistance to cancer cells. In this review, we provide an overview of the Keap1–Nrf2 system and discuss its role under physiological and pathological conditions, including cancers. We also introduce the results of our recent study describing Nrf2 function in the metabolism of cancer cells. Nrf2 likely confers a growth advantage to cancer cells through enhancing cytoprotection and anabolism. Finally, we discuss the possible impact of Nrf2 inhibitors on cancer therapy.
stress response; redox homeostasis; transcription; purine nucleotide; glutathione
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.