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1.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Background
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
Conclusions
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Background.
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030420
PMCID: PMC1584412  PMID: 17020408
2.  Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2▿  
Molecular and Cellular Biology  2007;27(18):6334-6349.
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.
doi:10.1128/MCB.00630-07
PMCID: PMC2099624  PMID: 17636022
3.  Regulation of Nrf2 – An update 
Free radical biology & medicine  2013;66:10.1016/j.freeradbiomed.2013.02.008.
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes which encode detoxifying enzymes, drug transporters (MRPs), anti-apoptotic proteins and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase mediated degradation of Nrf2. Chemicals including antioxidants, tocopherols including α-tocopherol (vitamin E), phytochemicals and radiations antagonize the Nrf2:INrf2 interaction and leads to the stabilization and activation of Nrf2. The signaling events involve pre-induction, induction and post-induction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinase(s) in a pre-induction response which phosphorylates specific residues on Nrf2 negative-regulators, INrf2, Fyn and Bach1, leading to their nuclear export, ubiquitination and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2cysteine151 followed by PKC phosphorylation of Nrf2serine40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus resulting in a coordinated activation of gene expression. This is followed by a post-induction response that controls the ‘switching off’ of Nrf2-activated gene expression. GSK3β under the control of AKT and PI3K, phosphorylates Fyn leading to Fyn nuclear localization. Fyn phosphorylates Nrf2Y568 resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provides protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
doi:10.1016/j.freeradbiomed.2013.02.008
PMCID: PMC3773280  PMID: 23434765
Nrf2; INrf2(Keap1); Antioxidants; Vitamins; Phytochemicals; ROS; Signaling; Regulation; Chemoprotection; Oncogenesis
4.  Cross-Regulations among NRFs and KEAP1 and Effects of their Silencing on Arsenic-Induced Antioxidant Response and Cytotoxicity in Human Keratinocytes 
Environmental Health Perspectives  2012;120(4):583-589.
Background: Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.
Objective: We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3–adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.
Results: In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs3+). In contrast, silencing KEAP1 led to a dramatic resistance to iAs3+-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs3+ toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs3+. In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs3+-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs3+-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.
Conclusions: Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs3+-induced oxidative stress.
doi:10.1289/ehp.1104580
PMCID: PMC3339469  PMID: 22476201
antioxidant response; arsenic; cytotoxicity; KEAP1; keratinocyte; NRF1; NRF2
5.  Promoter DNA demethylation of Keap1 gene in diabetic cardiomyopathy 
Researches have shown that the onset of diabetes is closely associated with oxidative stress and the chronic exposure leads to the development of complications such as diabetic cardiomyopathy. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. The aim of the present study was to investigate the status of Nrf2 mediated antioxidant system in myocardial biopsies of non-diabetic (NDM) and type-2 diabetic (DM-T2) cardiomyopathy patients. The western blot analysis of antioxidant proteins, real-time PCR analysis of Nrf2/Keap1 gene and bisulphate DNA sequencing analysis to study the methylation status of the CpG islands of Keap1 promoter DNA were performed. The immunoblot analysis showed the decreased level of antioxidant proteins other than Keap1 in the diabetic cardiopathy patients. Similarly, mRNA levels of Keap1 showed 5-fold increase in diabetic patients. Further analysis on promoter region of Keap1 gene revealed 80% demethylation in diabetic patients. Altogether, our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. Decreased Nrf2 activity represses the transcription of many antioxidant enzyme genes and alters the redox-balance up on diabetes. Thus, our study clearly demonstrates the failure of Nrf2 mediated antioxidant system revealed in biopsies of diabetic cardiomyopathy.
PMCID: PMC4313971
Nrf2; antioxidant system; CpG islands; bisulphate sequencing
6.  Regulatory Role of KEAP1 and NRF2 in PPARγ Expression and Chemoresistance in Human Non-small Cell Lung Carcinoma Cells 
Free radical biology & medicine  2012;53(4):758-768.
The nuclear factor-E2-related factor 2 (NRF2) serves as a master regulator in cellular defense against oxidative stress and chemical detoxification. However, persistent activation of NRF2 resulting from mutations of NRF2 and/or downregulation or mutations of its suppressor Kelch-like ECH-associated protein 1 (KEAP1) are associated with tumorigenicity and chemoresistance of non-small-cell lung carcinomas (NSCLCs). Thus, inhibiting NRF2-mediated adaptive antioxidant response is widely considered a promising strategy to prevent tumor growth and reverse chemoresistance in NSCLCs. Unexpectedly, stable knockdown of KEAP1 by lentiviral shRNA sensitized three independent NSCLC cell lines (A549, HTB-178 and HTB-182) to multiple chemotherapeutic agents, including arsenic trioxide (As2O3), etoposide and doxorubicin, despite moderately increased NRF2 levels. In lung adenocarcinoma epithelial A549 cells, silencing of KEAP1 augmented the expression of peroxisome proliferator-activated receptor γ (PPARγ) and genes associated with cell differentiation, including E-Cadherin and Gelsolin. In addition, KEAP1-knockdown A549 cells displayed attenuated expression of proto-oncogene Cyclin D1 and markers for cancer stem cells (CSCs), and reduced non-adherent sphere formation. Moreover, deficiency of KEAP1 led to elevated induction of PPARγ in response to As2O3. Pretreatment of A549 cells with PPARγ agonists activated PPARγ and augmented the cytotoxicity of As2O3. A mathematical model was formulated to advance a hypothesis that differential regulation of PPARγ and detoxification enzymes by KEAP1 and NRF2 may underpin the observed landscape changes in chemo-sensitivity. Collectively, suppression of KEAP1 expression in human NSCLC cells resulted in sensitization to chemotherapeutic agents, which may be attributed to activation of PPARγ and subsequent alterations in cell differentiation and CSC abundance.
doi:10.1016/j.freeradbiomed.2012.05.041
PMCID: PMC3418425  PMID: 22684020
7.  Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy 
Purpose.
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Methods.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Results.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Conclusions.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
doi:10.1167/iovs.13-11598
PMCID: PMC3676188  PMID: 23633659
antioxidant defense; diabetic retinopathy; Nrf2
8.  The Keap1–Nrf2 system in cancers: stress response and anabolic metabolism 
Frontiers in Oncology  2012;2:200.
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.
doi:10.3389/fonc.2012.00200
PMCID: PMC3530133  PMID: 23272301
stress response; redox homeostasis; transcription; purine nucleotide; glutathione
9.  Nrf2:INrf2(Keap1) Signaling in Oxidative Stress 
Free radical biology & medicine  2009;47(9):1304-1309.
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.
doi:10.1016/j.freeradbiomed.2009.07.035
PMCID: PMC2763938  PMID: 19666107
10.  Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex 
Molecular and Cellular Biology  2004;24(24):10941-10953.
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.
doi:10.1128/MCB.24.24.10941-10953.2004
PMCID: PMC533977  PMID: 15572695
11.  Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein β during adipogenesis 
Free radical biology & medicine  2011;52(2):462-472.
Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper transcription factor that is involved in the cellular adaptive response to oxidative stress. Our previous study reported that targeted disruption of the Nrf2 gene in mice decreases adipose tissue mass and protects against obesity induced by a high-fat diet. Deficiency of Nrf2 in preadipocytes and mouse embryonic fibroblasts led to impaired adipogenesis. Consistent with these findings, the current study found that lack of Nrf2 in primary cultured mouse preadipocytes and 3T3-L1 cells hampered adipogenic differentiation induced by hormonal cocktails. Stable knockdown of Nrf2 in 3T3-L1 cells blocked the enhanced adipogenesis caused by deficiency of kelch-like ECH-associated protein 1 (Keap1), a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex. In addition, increased production of reactive oxygen species (ROS) and activation of Nrf2 occurred at the very early stage upon adipogenic hormonal challenge in 3T3-L1 cells, followed by an immediate induction of CCAAT/enhancer-binding protein β (C/EBPβ). Knockdown of Nrf2 led to reduced expression of C/EBPβ induced by adipogenic hormonal cocktails, chemical Nrf2 activators or Keap1 silencing. Cebpβ promoter-driven reporter assays and chromatin immunoprecipitation suggested that Nrf2 associates with a consensus antioxidant response element (ARE) binding site in the promoter of the Cebpβ gene during adipogenesis and upregulates its expression. These findings demonstrate a novel role of Nrf2 beyond xenobiotic detoxification and antioxidant response, and suggest that Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.
doi:10.1016/j.freeradbiomed.2011.10.453
PMCID: PMC3307524  PMID: 22138520
Nrf2; C/EBPβ; Adipogenesis
12.  Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level 
PLoS ONE  2009;4(5):e5492.
The transcription factor Nrf2 is the master regulator of a cellular defense mechanism against environmental insults. The Nrf2-mediated antioxidant response is accomplished by the transcription of a battery of genes that encode phase II detoxifying enzymes, xenobiotic transporters, and antioxidants. Coordinated expression of these genes is critical in protecting cells from toxic and carcinogenic insults and in maintaining cellular redox homeostasis. Activation of the Nrf2 pathway is primarily controlled by Kelch-like ECH-associated protein 1 (Keap1), which is a molecular switch that turns on or off the Nrf2 signaling pathway according to intracellular redox conditions. Here we report our finding of a novel Nrf2 suppressor ectodermal-neural cortex 1 (ENC1), which is a BTB-Kelch protein and belongs to the same family as Keap1. Transient expression of ENC1 reduced steady-state levels of Nrf2 and its downstream gene expression. Although ENC1 interacted with Keap1 indirectly, the ENC1-mediated down-regulation of Nrf2 was independent of Keap1. The negative effect of ENC1 on Nrf2 was not due to a change in the stability of Nrf2 because neither proteasomal nor lysosomal inhibitors had any effects. Overexpression of ENC1 did not result in a change in the level of Nrf2 mRNA, rather, it caused a decrease in the rate of Nrf2 protein synthesis. These results demonstrate that ENC1 functions as a negative regulator of Nrf2 through suppressing Nrf2 protein translation, which adds another level of complexity in controlling the Nrf2 signaling pathway.
doi:10.1371/journal.pone.0005492
PMCID: PMC2675063  PMID: 19424503
13.  The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase 
Molecular and Cellular Biology  2004;24(19):8477-8486.
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.
doi:10.1128/MCB.24.19.8477-8486.2004
PMCID: PMC516753  PMID: 15367669
14.  Structural analysis of the complex of Keap1 with a prothymosin α peptide 
The crystal structure of the complex of mouse Keap1-DC with a fragment of the nuclear protein prothymosin α was determined and refined to 1.9 Å resolution and revealed that the peptide binds to the bottom region of the β-propeller domain of Keap1-DC.
The Nrf2 transcription factor, which plays important roles in oxidative and xenobiotic stress, is negatively regulated by the cytoplasmic repressor Keap1. The β-propeller/Kelch domain of Keap1, which is formed by the double-glycine repeat and C-terminal region domains (Keap1-DC), interacts directly with the Neh2 domain of Nrf2. The nuclear oncoprotein prothymosin α (ProTα) also interacts directly with Keap1 and may play a role in the dissociation of the Keap1–Nrf2 complex. The structure of Keap1-DC complexed with a ProTα peptide (amino acids 39–54) has been determined at 1.9 Å resolution. The Keap1-bound ProTα peptide possesses a hairpin conformation and binds to the Keap1 protein at the bottom region of the β-propeller domain. Complex formation occurs as a consequence of their complementary electrostatic interactions. A comparison of the present structure with recently reported Keap1-DC complex structures revealed that the DLG and ETGE motifs of the Neh2 domain of Nrf2 and the ProTα peptide bind to Keap1 in a similar manner but with different binding potencies.
doi:10.1107/S1744309108004995
PMCID: PMC2374262  PMID: 18391415
oxidative stress; Nrf2 transcription factor; prothymosin α; Keap1; β-propeller domain
15.  Increased Nrf2 Activation in Livers from Keap1-Knockdown Mice Increases Expression of Cytoprotective Genes that Detoxify Electrophiles more than those that Detoxify Reactive Oxygen Species 
Toxicological Sciences  2009;108(1):35-47.
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance–associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species.
doi:10.1093/toxsci/kfn267
PMCID: PMC2644398  PMID: 19129213
Nrf2; electrophilic stress; oxidative stress; cytoprotection
16.  Fetal Alz-50 Clone 1 Interacts with the Human Orthologue of the Kelch-like Ech-Associated Protein† 
Biochemistry  2004;43(38):12113-12122.
The fetal Alz-50 reactive clone 1 (FAC1) protein exhibits altered expression and subcellular localization during neuronal development and neurodegenerative diseases such as Alzheimer's disease. Using the yeast two-hybrid screen, the human orthologue of Keap1 (hKeap1) was identified as a FAC1 interacting protein. Keap1 is an important regulator of the oxidative stress response pathway through its interaction with the Nrf family of transcription factors. An interaction between full-length FAC1 and hKeap1 proteins has been demonstrated, and the FAC1 binding domain of hKeap1 has been identified as the Kelch repeats. In addition, FAC1 colocalizes with endogenous Keap1 within the cytoplasm of PT67 cells. Exogenously introduced eGFP:hKeap1 fusion protein redistributed FAC1 to colocalize with eGFP: hKeap1 in perinuclear, spherical structures. The interaction between FAC1 and hKeap1 is reduced by competition with the Nrf2 protein. However, competition by Nrf2 for hKeap1 is reduced by diethylmaleate (DEM), a known disrupter of the Nrf2:Keap1 interaction. DEM does not affect the ability of FAC1 to bind hKeap1 in our assay. These results suggest that hKeap1 regulates FAC1 in addition to its known role in control of Nrf2. Furthermore, the observed competition between FAC1 and Nrf2 for binding hKeap1 indicates that the interplay between these three proteins has important implications for neuronal response to oxidative stress.
doi:10.1021/bi0494166
PMCID: PMC3670950  PMID: 15379550
17.  Enhanced 4-Hydroxynonenal Resistance in KEAP1 Silenced Human Colon Cancer Cells 
Nuclear factor erythroid 2-related factor 2 (NRF2) is the transcription factor that regulates an array of antioxidant/detoxifying genes for cellular defense. The conformational changes of Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor protein of NRF2, by various stimuli result in NRF2 liberation and accumulation in the nucleus. In the present study, we aimed to investigate the effect of KEAP1 knockdown on NRF2 target gene expression and its toxicological implication using human colon cancer cells. The stable KEAP1-knockdown HT29 cells exhibit elevated levels of NRF2 and its target gene expressions. In particular, the mRNA levels of aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, and 1B10) were substantially increased in KEAP1 silenced HT29 cells. These differential AKRs expressions appear to contribute to protection against oxidative stress. The KEAP1-knockdown cells were relatively more resistant to hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) compared to the control cells. Accordantly, we observed accumulation of 4HNE protein adducts in H2O2- or 4HNE-treated control cells, whereas KEAP1-knockdown cells did not increase adduct formation. The treatment of KEAP1-silenced cells with AKR1C inhibitor flufenamic acid increased 4HNE-induced cellular toxicity and protein adduct formation. Taken together, these results indicate that AKRs, which are NRF2-dependent highly inducible gene clusters, play a role in NRF2-mediated cytoprotection against lipid peroxide toxicity.
doi:10.1155/2013/423965
PMCID: PMC3674683  PMID: 23766854
18.  Keap1 Regulates the Oxidation-Sensitive Shuttling of Nrf2 into and out of the Nucleus via a Crm1-Dependent Nuclear Export Mechanism†  
Molecular and Cellular Biology  2005;25(11):4501-4513.
Keap1 is a negative regulator of Nrf2, a transcription factor essential for antioxidant response element (ARE)-mediated gene expression. We find that Keap1 sequesters Nrf2 in the cytoplasm, not by docking it to the actin cytoskeleton but instead through an active Crm1/exportin-dependent nuclear export mechanism. Deletion and mutagenesis studies identified a nuclear export signal (NES) in the intervening region of Keap1 comprised of hydrophobic leucine and isoleucine residues in agreement with a traditional NES consensus sequence. Mutation of the hydrophobic amino acids resulted in nuclear accumulation of both Keap1 and Nrf2, as did treatment with the drug leptomycin B, which inactivates Crm1/exportin. ARE genes were partially activated under these conditions, suggesting that additional oxidation-sensitive elements are required for full activation of the antioxidant response. Based on these data, we propose a new model for regulation of Nrf2 by Keap1. Under normal conditions, Keap1 and Nrf2 are complexed in the cytoplasm where they are targeted for degradation. Oxidative stress inactivates Keap1's NES, allowing entry of both Keap1 and Nrf2 into the nucleus and transcriptional transactivation of ARE genes.
doi:10.1128/MCB.25.11.4501-4513.2005
PMCID: PMC1140621  PMID: 15899855
19.  Identification of Novel microRNAs in Post-Transcriptional Control of Nrf2 Expression and Redox Homeostasis in Neuronal, SH-SY5Y Cells 
PLoS ONE  2012;7(12):e51111.
Nuclear factor-erythroid 2-related factor 2 (Nrf2/NFE2L2), a redox-sensitive transcription factor plays a critical role in adaptation to cellular stress and affords cellular defense by initiating transcription of antioxidative and detoxification genes. While a protein can be regulated at multiple levels, control of Nrf2 has been largely studied at post-translational regulation points by Keap1. Importantly, post-transcriptional/translational based regulation of Nrf2 is less understood and to date there are no reports on such mechanisms in neuronal systems. In this context, studies involving the role of microRNAs (miRs) which are normally considered as fine tuning regulators of protein production through translation repression and/or post-transcriptional alterations, are in place. In the current study, based on in-silico analysis followed by immunoblotting and real time analysis, we have identified and validated for the first time that human NFE2L2 could be targeted by miR153/miR27a/miR142-5p/miR144 in neuronal, SH-SY5Y cells. Co-transfection studies with individual miR mimics along with either WT 3′ UTR of human Nrf2 or mutated miRNA targeting seed sequence within Nrf2 3′ UTR, demonstrated that Nrf2 is a direct regulatory target of these miRs. In addition, ectopic expression of miR153/miR27a/miR142-5p/miR144 affected Nrf2 mRNA abundance and nucleo-cytoplasmic concentration of Nrf2 in a Keap1 independent manner resulting in inefficient transactivating ability of Nrf2. Furthermore, forced expression of miRs diminished GCLC and GSR expression resulting in alteration of Nrf2 dependent redox homeostasis. Finally, bioinformatics based miRNA-disease network analysis (MDN) along with extended computational network analysis of Nrf2 associated pathologic processes suggests that if in a particular cellular scenario where any of these miR153/miR27a/miR142-5p/miR144 either individually or as a group is altered, it could affect Nrf2 thus triggering and/or determining the fate of wide range of disease outcomes.
doi:10.1371/journal.pone.0051111
PMCID: PMC3517581  PMID: 23236440
20.  Acetylation of Nrf2 by p300/CBP Augments Promoter-Specific DNA Binding of Nrf2 during the Antioxidant Response▿ †  
Molecular and Cellular Biology  2009;29(10):2658-2672.
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.
doi:10.1128/MCB.01639-08
PMCID: PMC2682049  PMID: 19273602
21.  Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors 
Antioxidants & Redox Signaling  2011;15(8):2335-2381.
Abstract
Convincing concepts of redox control of gene transcription have been worked out for prokaryotes and lower eukaryotes, whereas the knowledge on complex mammalian systems still resembles a patchwork of poorly connected findings. The article, therefore, reviews principles of redox regulation with special emphasis on chemical feasibility, kinetic requirements, specificity, and physiological context, taking well investigated mammalian transcription factor systems, nuclear transcription factor of bone marrow-derived lymphocytes (NF-κB), and kelch-like ECH-associated protein-1 (Keap1)/Nrf2, as paradigms. Major conclusions are that (i) direct signaling by free radicals is restricted to O2•− and •NO and can be excluded for fast reacting radicals such as •OH, •OR, or Cl•; (ii) oxidant signals are H2O2, enzymatically generated lipid hydroperoxides, and peroxynitrite; (iii) free radical damage is sensed via generation of Michael acceptors; (iv) protein thiol oxidation/alkylation is the prominent mechanism to modulate function; (v) redox sensors must be thiol peroxidases by themselves or proteins with similarly reactive cysteine or selenocysteine (Sec) residues to kinetically compete with glutathione peroxidase (GPx)- and peroxiredoxin (Prx)-type peroxidases or glutathione-S-transferases, respectively, a postulate that still has to be verified for putative mammalian sensors. S-transferases and Prxs are considered for system complementation. The impact of NF-κB and Nrf2 on hormesis, management of inflammatory diseases, and cancer prevention is critically discussed. Antioxid. Redox Signal. 15, 2335–2381.
I. Introduction
II. Mechanistic Principles in Redox Regulation
A. Indispensable components of regulatory circuits
B. Radicals or hydroperoxides as oxidant signals in redox signaling?
C. Signals of free radical damage
D. Sensing and transducing proteins
1. Thiol peroxidases as sensors
2. Transcription factors as sensors for hydroperoxides
3. PPs and PKs as potential sensors
4. Redox sensing by cytosolic inhibitory complexes of transcription factors
5. Sensing by selenocysteine-containing proteins?
6. Oxygen sensing
E. Adjusting sensitivity by competing systems
F. The problems beyond signals and sensors
III. Orchestrating the Adaptive Response by the Keap1/Nrf2 System
A. Discovery
B. The physiological context of Nrf2-dependent gene activation
C. Mechanistic aspects of Nrf2 signaling
1. Basics of Nrf2 activation
2. Keap1 as primary redox sensor of Nrf2 signaling
3. Downstream signaling events
4. Nrf2-mediated transduction events
5. Shut-off mechanisms
6. Modulation of Nrf2 function by cross-talk with phosphorylation cascades
D. Synopsis of the Nrf2 system
IV. NF-κB, a Key Regulator of the Immune Response
A. Discovery and definitions
B. The biological context of NF-κB
C. Basics of NF-κB activation
D. Redox regulation of NF-κB activation
1. The source of the oxidants
2. Modulation of NF-κB activation via redox-sensitive phosphorylation
3. Regulation of NF-κB by redoxin systems
E. Termination of NF-κB signaling
F. Synopsis of the NF-κB system
V. Loose Ends and Perspectives
doi:10.1089/ars.2010.3534
PMCID: PMC3166203  PMID: 21194351
22.  Nuclear Oncoprotein Prothymosin α Is a Partner of Keap1: Implications for Expression of Oxidative Stress-Protecting Genes 
Molecular and Cellular Biology  2005;25(3):1089-1099.
Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin α. The in vivo and in vitro data indicated that the prothymosin α-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin α was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin α by using prothymosin α overproduction and mRNA interference approaches. Our data attribute to prothymosin α the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin α and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.
doi:10.1128/MCB.25.3.1089-1099.2005
PMCID: PMC544000  PMID: 15657435
23.  Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response 
PLoS ONE  2009;4(8):e6588.
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.
doi:10.1371/journal.pone.0006588
PMCID: PMC2719090  PMID: 19668370
24.  MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism 
NF-E2-related factor 2 (Nrf2) is an important transcription factor involved in antioxidant response. Nrf2 binds antioxidant response elements (ARE) within promoters of genes encoding detoxification enzymes (e.g., NAD (P) H-quinone oxidoreductase 1 (NQO1)) leading to their transcriptional activation. Nrf2 function is regulated post-translationally by its negative regulator Kelch-like ECH-associated protein 1 (Keap1) that binds Nrf2 and induces cytoplasmic Nrf2 degradation. Our present studies provide new evidence that Nrf2 expression can be regulated by a Keap1-independent mechanism. Here, we utilized breast epithelial cells to explore the impact of microRNA (miRNA) on Nrf2 expression. We found that Nrf2 mRNA levels are reversibly correlated with miR-28 expression and that ectopic expression of miR-28 alone reduces Nrf2 mRNA and protein levels. We further investigated the molecular mechanisms by which miR-28 inhibits Nrf2 mRNA expression. Initially, the ability of miR-28 to regulate the 3′ untranslated region (3′UTR) of Nrf2 mRNA was evaluated via luciferase reporter assay. We observed that miR-28 reduces wild-type Nrf2 3′UTR luciferase reporter activity and this repression is eliminated upon mutation of the miR-28 targeting seed sequence within the Nrf2 3′UTR. Moreover, over-expression of miR-28 decreased endogenous Nrf2 mRNA and protein expression. We also explored the impact of miR-28 on Keap1-Nrf2 interactions and found that miR-28 overexpression does not alter Keap1 protein levels and has no effect on the interaction of Keap1 and Nrf2. Our findings, that miR-28 targets the 3′UTR of Nrf2 mRNA and decreases Nrf2 expression, suggest that this miRNA is involved in the regulation of Nrf2 expression in breast epithelial cells.
doi:10.1007/s10549-011-1604-1
PMCID: PMC3752913  PMID: 21638050
Mammary epithelial cells; miR-28; Nrf2; Chemoprevention
25.  CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1 
Molecular and Cellular Biology  2006;26(4):1235-1244.
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.
doi:10.1128/MCB.26.4.1235-1244.2006
PMCID: PMC1367193  PMID: 16449638

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