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Suppressed ubiquitination of Nrf2 by p47 contributes to Nrf2 activation.
Ha Kim K, Sadikot RT, Yeon Lee J, Jeong HS, Oh YK, Blackwell TS, Joo M
(2017) Free Radic Biol Med 113: 48-58
MeSH Terms: Animals, Disease Models, Animal, HEK293 Cells, Humans, Kelch-Like ECH-Associated Protein 1, Lipopolysaccharides, Mice, NADPH Oxidases, NF-E2-Related Factor 2, Pneumonia, RAW 264.7 Cells, Reactive Oxygen Species, Signal Transduction, Ubiquitination
Show Abstract · Added March 21, 2018
Although critical in phagocytosis in innate immunity, reactive oxygen species (ROS) collaterally inflict damage to host phagocytes because they indiscriminate targets. Since Nrf2 increases the expression of anti-oxidant enzymes that nullifies ROS, ROS activating Nrf2 is a critical negative regulatory step for countering the deleterious effects of ROS. Here, we postulate whether, along with ROS activating Nrf2, NADPH oxidase components also participate in direct activation of Nrf2, contributing to protection from ROS. Our results show that the p47 of the NADPH oxidase, but not p65 or p40, physically binds to Nrf2, activating the Nrf2 function. p47 binding to Nrf2/Keap1 complex suppresses the ubiquitination of Nrf2, while p47 becomes ubiquitinated by Keap1. p47 increases the nuclear translocation of Nrf2 and the expression of Nrf2-dependent genes, whereas genetic ablation of p47 decreases the expression of those genes. In a lipopolysaccharide-induced acute lung inflammation mouse model, selective expression of p47 in mouse lungs induces the expression of Nrf2-dependent genes and is sufficient to suppress neutrophilic lung inflammation. Therefore, our findings suggest that p47 is a novel regulator of Nrf2 function.
Copyright © 2017 Elsevier Inc. All rights reserved.
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14 MeSH Terms
NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury.
Nlandu-Khodo S, Dissard R, Hasler U, Schäfer M, Pircher H, Jansen-Durr P, Krause KH, Martin PY, de Seigneux S
(2016) Sci Rep 6: 38598
MeSH Terms: Animals, Apoptosis, Cell Death, Creatinine, Disease Models, Animal, Gene Expression, Gene Expression Regulation, Genetic Predisposition to Disease, Glutathione, Kelch-Like ECH-Associated Protein 1, Kidney Diseases, Kidney Tubules, Mice, Mice, Knockout, Mitochondria, NADPH Oxidase 4, NF-E2-Related Factor 2, Oxidation-Reduction, Proto-Oncogene Proteins c-bcl-2, Reperfusion Injury
Show Abstract · Added December 26, 2018
NADPH oxidase 4 (NOX4) is highly expressed in kidney proximal tubular cells. NOX4 constitutively produces hydrogen peroxide, which may regulate important pro-survival pathways. Renal ischemia reperfusion injury (IRI) is a classical model mimicking human ischemic acute tubular necrosis. We hypothesized that NOX4 plays a protective role in kidney IRI. In wild type (WT) animals subjected to IRI, NOX4 protein expression increased after 24 hours. NOX4 KO (knock-out) and WT littermates mice were subjected to IRI. NOX4 KO mice displayed decreased renal function and more severe tubular apoptosis, decreased Bcl-2 expression and higher histologic damage scores compared to WT. Activation of NRF2 was decreased in NOX4 KO mice in response to IRI. This was related to decreased KEAP1 oxidation leading to decreased NRF2 stabilization. This resulted in decreased glutathione levels. In vitro silencing of NOX4 in cells showed an enhanced propensity to apoptosis, with reduced expression of NRF2, glutathione content and Bcl-2 expression, similar to cells derived from NOX4 KO mice. Overexpression of a constitutively active form of NRF2 (caNRF2) in NOX4 depleted cells rescued most of this phenotype in cultured cells, implying that NRF2 regulation by ROS issued from NOX4 may play an important role in its anti-apoptotic property.
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LKB1 Loss induces characteristic patterns of gene expression in human tumors associated with NRF2 activation and attenuation of PI3K-AKT.
Kaufman JM, Amann JM, Park K, Arasada RR, Li H, Shyr Y, Carbone DP
(2014) J Thorac Oncol 9: 794-804
MeSH Terms: AMP-Activated Protein Kinases, Adaptor Proteins, Signal Transducing, Adenocarcinoma, Animals, Ataxia Telangiectasia Mutated Proteins, Carcinoma, Non-Small-Cell Lung, Cell Line, Tumor, Cytoskeletal Proteins, ErbB Receptors, GA-Binding Protein Transcription Factor, Humans, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Lung Neoplasms, Mice, Mitochondria, Multigene Family, Mutation, NF-E2-Related Factor 2, Phosphatidylinositol 3-Kinase, Phosphorylation, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins p21(ras), RNA, Messenger, Signal Transduction, Transcriptome, Tumor Suppressor Protein p53, ras Proteins
Show Abstract · Added February 19, 2015
INTRODUCTION - Inactivation of serine/threonine kinase 11 (STK11 or LKB1) is common in lung cancer, and understanding the pathways and phenotypes altered as a consequence will aid the development of targeted therapeutic strategies. Gene and protein expressions in a murine model of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (Kras)-mutant lung cancer have been studied to gain insight into the biology of these tumors. However, the molecular consequences of LKB1 loss in human lung cancer have not been fully characterized.
METHODS - We studied gene expression profiles associated with LKB1 loss in resected lung adenocarcinomas, non-small-cell lung cancer cell lines, and murine tumors. The biological significance of dysregulated genes was interpreted using gene set enrichment and transcription factor analyses and also by integration with somatic mutations and proteomic data.
RESULTS - Loss of LKB1 is associated with consistent gene expression changes in resected human lung cancers and cell lines that differ substantially from the mouse model. Our analysis implicates novel biological features associated with LKB1 loss, including altered mitochondrial metabolism, activation of the nuclear respiratory factor 2 (NRF2) transcription factor by kelch-like ECH-associated protein 1 (KEAP1) mutations, and attenuation of the phosphatidylinositiol 3-kinase and v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway. Furthermore, we derived a 16-gene classifier that accurately predicts LKB1 mutations and loss by nonmutational mechanisms. In vitro, transduction of LKB1 into LKB1-mutant cell lines results in attenuation of this signature.
CONCLUSION - Loss of LKB1 defines a subset of lung adenocarcinomas associated with characteristic molecular phenotypes and distinctive gene expression features. Studying these effects may improve our understanding of the biology of these tumors and lead to the identification of targeted treatment strategies.
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30 MeSH Terms
Cysteine-based regulation of the CUL3 adaptor protein Keap1.
Sekhar KR, Rachakonda G, Freeman ML
(2010) Toxicol Appl Pharmacol 244: 21-6
MeSH Terms: Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Animals, Cullin Proteins, Cysteine, Cytoskeletal Proteins, Gene Expression Regulation, Humans, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, NF-E2-Related Factor 2, Oxidation-Reduction, Proteasome Endopeptidase Complex, Protein Processing, Post-Translational, Protein Structure, Tertiary, Structure-Activity Relationship, Sulfhydryl Compounds, Transcriptional Activation, Ubiquitination
Show Abstract · Added March 5, 2014
Nrf2 (NF-E2-related factor 2) is a master transcription factor containing a powerful acidic transcriptional activation domain. Nrf2-dependent gene expression impacts cancer chemoprevention strategies, inflammatory responses, and progression of neurodegenerative diseases. Under basal conditions, association of Nrf2 with the CUL3 adaptor protein Keap1 results in the rapid Nrf2 ubiquitylation and proteasome-dependent degradation. Inhibition of Keap1 function blocks ubiquitylation of Nrf2, allowing newly synthesized Nrf2 to translocate into the nucleus, bind to ARE sites and direct target gene expression. Site-directed mutagenesis experiments coupled with proteomic analysis support a model in which Keap1 contains at least 2 distinct cysteine motifs. The first is located at Cys 151 in the BTB domain. The second is located in the intervening domain and centers around Cys 273 and 288. Adduction or oxidation at Cys151 has been shown to produce a conformational change in Keap1 that results in dissociation of Keap1 from CUL3, thereby inhibiting Nrf2 ubiquitylation. Thus, adduction captures specific chemical information and translates it into biochemical information via changes in structural conformation.
2009 Elsevier Inc. All rights reserved.
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19 MeSH Terms
Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.
Rachakonda G, Xiong Y, Sekhar KR, Stamer SL, Liebler DC, Freeman ML
(2008) Chem Res Toxicol 21: 705-10
MeSH Terms: Animals, COS Cells, Cell Cycle Proteins, Cell Line, Tumor, Cells, Cultured, Chlorocebus aethiops, Circular Dichroism, Cullin Proteins, Cysteine, DNA, Recombinant, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Kinetics, Mass Spectrometry, Recombinant Proteins
Show Abstract · Added March 5, 2014
The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl- N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in vivo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.
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17 MeSH Terms
Novel n-3 fatty acid oxidation products activate Nrf2 by destabilizing the association between Keap1 and Cullin3.
Gao L, Wang J, Sekhar KR, Yin H, Yared NF, Schneider SN, Sasi S, Dalton TP, Anderson ME, Chan JY, Morrow JD, Freeman ML
(2007) J Biol Chem 282: 2529-37
MeSH Terms: Animals, Cell Cycle Proteins, Cell Line, Cullin Proteins, Fatty Acids, Omega-3, Gene Expression Regulation, Genes, Reporter, Humans, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Mice, Molecular Structure, NF-E2-Related Factor 2, Oxidation-Reduction, Oxidative Stress, Transcriptional Activation
Show Abstract · Added March 5, 2014
Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.
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16 MeSH Terms
Increased expression of nuclear factor E2 p45-related factor 2 (NRF2) in head and neck squamous cell carcinomas.
Stacy DR, Ely K, Massion PP, Yarbrough WG, Hallahan DE, Sekhar KR, Freeman ML
(2006) Head Neck 28: 813-8
MeSH Terms: Adult, Aged, Biomarkers, Tumor, Carcinoma, Squamous Cell, Female, Gene Expression Regulation, Neoplastic, Head and Neck Neoplasms, Humans, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Male, Middle Aged, Mouth Mucosa, NF-E2-Related Factor 2
Show Abstract · Added March 5, 2014
BACKGROUND - Head and neck squamous cell carcinoma (HNSCC) continues to cause significant morbidity and mortality. Overexpression of specific phase II gene products may represent an important biomarker. One regulator of phase II gene expression is the transcription factor nuclear factor E2 p45-related factor 2 (Nrf2). Nrf2 expression was evaluated in HNSCC, to determine whether it might serve as a biomarker for early detection of disease.
METHODS - A tissue microarray was constructed of 141 HNSCC biopsy cores from 47 HNSCCs. In addition, histologically normal squamous mucosa was obtained from 7 patients. Protein expression was evaluated by immunohistochemistry.
RESULTS - Nrf2 expression was increased in 91.5% of tumors. Expression of thioredoxin, a Nrf2-reguated gene product, was elevated in 75% of tumors. Keap1, which regulates the rate of Nrf2 ubiquitination and degradation, was relatively overexpressed in HNSCC compared with normal mucosa.
CONCLUSIONS - Nrf2 expression may be a possible HNSCC candidate biomarker.
(c) 2006 Wiley Periodicals, Inc.
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Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane.
Hong F, Freeman ML, Liebler DC
(2005) Chem Res Toxicol 18: 1917-26
MeSH Terms: Amino Acid Sequence, Antineoplastic Agents, Cells, Cultured, Cysteine, DNA Adducts, Gene Expression Regulation, Neoplastic, Humans, Intracellular Signaling Peptides and Proteins, Isothiocyanates, Kelch-Like ECH-Associated Protein 1, Mass Spectrometry, Molecular Sequence Data, NF-E2-Related Factor 2, Proteins, Thiocyanates
Show Abstract · Added March 20, 2014
The chemopreventive agent sulforaphane is an isothiocyanate derived from cruciferous vegetables. Sulforaphane exerts cancer chemopreventive effects by inducing antioxidant/electrophile response element (ARE)-regulated phase 2 enzyme and antioxidant genes through activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which is regulated by the thiol-rich sensor protein Kelch-like ECH-associated protein 1 (Keap1). Sulforaphane is an electrophile that can react with protein thiols to form thionoacyl adducts. We hypothesized that, like other electrophilic Nrf2 activators, sulforaphane activates this system through specific modifications of the Keap1 protein. However, thionoacyl adducts are labile to hydrolysis and transacylation reactions, which complicate the identification of the sulforaphane adduct sites on Keap1. In this study, we characterized the stability of sulforaphane thionoacyl adducts and developed a liquid chromatography-tandem mass spectrometry method to map labile sulforaphane adduct sites formed on Keap1 in vitro. Sulforaphane displays a distinctly different pattern of Keap1 modification than previously studied ARE inducers that modify Keap1 by alkylation. Sulforaphane modified Keap1 most readily in the Kelch domain, rather than in the central linker domain, which is targeted by previously characterized ARE inducers. Also, in contrast to previously studied ARE inducers and as reported recently [Zhang, et al. (2005) J. Biol. Chem. 280, 30091-30099], sulforaphane treatment in vivo does not lead to the accumulation of ubiquitinated Keap1. Our observations suggest a novel mechanism for Nrf2 stabilization by sulforaphane-Keap1 thionoacyl adduct formation.
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15 MeSH Terms
Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2 activation.
Hong F, Sekhar KR, Freeman ML, Liebler DC
(2005) J Biol Chem 280: 31768-75
MeSH Terms: Biotin, Cell Cycle Proteins, Cell Line, Cullin Proteins, Cysteine, DNA-Binding Proteins, Electrophoresis, Polyacrylamide Gel, Humans, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Mass Spectrometry, NF-E2-Related Factor 2, Peptides, Proteins, Trans-Activators, Transfection, Ubiquitin
Show Abstract · Added March 5, 2014
Activation of the transcription factor Nrf2 regulates expression of phase II enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2 concentrations are regulated by the thiol-rich sensor protein Keap1, which is an adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2. However, the links between site specificity of Keap1 modification by electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (tBHQ) and two biotin-tagged, thiol-reactive electrophiles, N-iodoacetyl-N-biotinylhexylenediamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane (BMCC). Both IAB and tBHQ induced antioxidant response element (ARE)-directed green fluorescent protein (GFP) expression in ARE/thymidine kinase GFP HepG2 cells, and both initiated nuclear Nrf2 accumulation and induction of heme oxygenase 1 in HEK293 cells. In contrast, BMCC produced none of these effects. Liquid chromatography tandem mass spectrometry (MS-MS) analysis of human Keap1 modified by IAB or BMCC in vitro indicated that IAB adduction occurred primarily in the central linker domain, whereas BMCC modified other Keap1 domains. Treatment of FLAG-Keap1-transfected HEK293 with the Nrf2-activating compounds IAB and tBHQ generated high molecular weight Keap1 forms, which were identified as K-48-linked polyubiquitin conjugates by immunoblotting and liquid chromatography MS-MS. Keap1 polyubiquitination coincided with Nrf2 stabilization and nuclear accumulation. In contrast, BMCC did not induce Keap1 polyubiquitination. Our results suggest that Nrf2 activation is regulated through the polyubiquitination of Keap1, which in turn is triggered by specific patterns of electrophile modification of the Keap1 central linker domain. These results suggest that Keap1 adduction triggers a switching of Cul3-dependent ubiquitination from Nrf2 to Keap1, leading to Nrf2 activation.
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17 MeSH Terms
NADPH oxidase activity is essential for Keap1/Nrf2-mediated induction of GCLC in response to 2-indol-3-yl-methylenequinuclidin-3-ols.
Sekhar KR, Crooks PA, Sonar VN, Friedman DB, Chan JY, Meredith MJ, Starnes JH, Kelton KR, Summar SR, Sasi S, Freeman ML
(2003) Cancer Res 63: 5636-45
MeSH Terms: Animals, Anticarcinogenic Agents, Gene Expression Regulation, Enzymologic, Glutamate-Cysteine Ligase, Humans, Indoles, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Mice, NADPH Oxidases, Proteins, Quinuclidines, Structure-Activity Relationship, Tumor Cells, Cultured
Show Abstract · Added March 5, 2014
Glutamate cysteine ligase, the rate-limiting enzyme for the synthesis of glutathione, represents an important component of chemoprevention paradigms. GCLC and GCLM, the genes encoding glutamate cysteine ligase subunits, are induced by indoles, such as indomethacin. Novel functionalized indole analogues and other structurally related compounds were synthesized and used for a comparative structure analysis of GCLC induction. Use of mouse embryo fibroblasts null for Nrf2 (nuclear factor-erythroid 2p45-related transcription factor) and HepG2 cells overexpressing Keap1 demonstrated that indole analogue-mediated GCLC expression was regulated by Nrf2-Keap1 interactions. Indole analogues capable of inducing GCLC were found to increase NADPH oxidase activity. Indole analogues unable to induce GCLC did not increase oxidase activity. HepG2 cells transfected with FLAG/Keap1 were exposed to indomethacin, and the redox state of Keap1 cysteine residues was assessed. The data indicated that Keap1 exhibited several oxidation states that were sensitive to indomethacin treatment. These indomethacin-mediated changes in thiol oxidation states were suppressed by diphenyleneiodonium, a NADPH oxidase inhibitor. Diphenyleneiodonium also suppressed indole analogue-mediated increases in GCLC mRNA. In summary, the use of the indole analogues identified NADPH oxidase activity as a novel upstream activity regulating Nrf2/Keap1 signaling of GCLC, provided data supporting the hypothesis that Keap1 is a downstream effector for oxidase activity, and afforded in vivo data to support the hypothesis that Keap1 thiols can act as molecular sensors of reactive oxygen species. Finally, the comparative structure analysis suggests that 2-indol-3-yl-methylenequinuclidin-3-ols may represent a prototype for the development of novel chemopreventative agents able to activate Keap1/Nrf2 signaling.
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14 MeSH Terms