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Masayuki Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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stress sensing mechanisms and the physiological roles of the KEAP1 nrf2 system during cellular stress
Journal of Biological Chemistry, 2017Co-Authors: Takafumi Suzuki, Masayuki YamamotoAbstract:Abstract Transcription factor Nrf2 (NF-E2-related factor 2) is a master regulator of cellular responses against environmental stresses. Nrf2 induces the expression of detoxification and antioxidant enzymes and suppresses the induction of pro-inflammatory cytokine genes. KEAP1 (Kelch-like ECH-associated protein 1) is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase. KEAP1 regulates the activity of Nrf2 and acts as a sensor for oxidative and electrophilic stresses. In this review, we discuss the molecular mechanisms by which the KEAP1–Nrf2 system senses and regulates the cellular response to environmental stresses. In particular, we focus on the multiple stress-sensing mechanisms of KEAP1 and novel regulatory functions of Nrf2.
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Overview of redox regulation by KEAP1–Nrf2 system in toxicology and cancer
Current Opinion in Toxicology, 2016Co-Authors: Mikiko Suzuki, Akihito Otsuki, Nadine Keleku-lukwete, Masayuki YamamotoAbstract:Abstract The KEAP1–Nrf2 pathway is a cellular defense system against oxidative and xenobiotic stresses derived from reactive oxygen species (ROS) and electrophiles, respectively. Nrf2 is a key transcription factor that activates a set of cytoprotective genes, including those encoding antioxidative and detoxifying enzymes. KEAP1 is an adaptor protein of Cullin3-based E3 ligase, which regulates Nrf2 activity in response to these stresses. Under unstressed conditions, KEAP1 constitutively degrades Nrf2 via the proteasome pathway. KEAP1 interacts with Nrf2 through DLGex and ETGE sites in Nrf2 Neh2 domain, which is critical for regulation of Nrf2 degradation. ROS and electrophiles modify cysteine residues of KEAP1 to inactivate the ubiquitin E3 ligase activity of KEAP1, so that Nrf2 escapes from the KEAP1-mediated repression, migrates into the nucleus, and activates expression of its target genes. As oxidative stresses give rise to many diseases, Nrf2 inducers that interact with KEAP1 cysteine residues or KEAP1–Nrf2 binding surface are expected as drugs against these diseases. On the other hand, several lines of evidence have showed that cancer cells hijack the KEAP1–Nrf2 system to obtain resistance of chemo- and radiotherapies. Somatic mutations in human KEAP1 and NRF2 genes are observed in a number of cancers, resulting in constitutive activation of NRF2 and poor prognosis. In this review, we describe molecular basis underlying the KEAP1–Nrf2 function and drug discovery.
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Development of Novel Inhibitors for KEAP1-Nrf2 and KEAP1-P62 Protein-Protein Interaction
Free Radical Biology and Medicine, 2016Co-Authors: Daisuke Yasuda, Masaaki Komatsu, Mao Nakajima, Akihiro Yuasa, Rika Obata, Kyoko Takahashi, Yoshinobu Ichimura, Taketo Yoshida, Masayuki YamamotoAbstract:Nrf2-KEAP1 system is a biological defense mechanism in response to various stressors. An inhibitor for KEAP1-Nrf2 interaction to activate Nrf2 is expected as a novel target for drug discovery in oxidative stress-related disorders. However, Nrf2 is accumulated in many types of cancer and its expression is induced during the course of drug resistance, which is mediated by phosphorylated p62 (p-p62), an autophagy-related protein that forms a complex with KEAP1. We performed a high-throughput screening for KEAP1-Nrf2 inhibitory activity toward around 160,000 compounds, and identified a hit compound #1 that has a benzoindole skeleton. #1 inhibited KEAP1-Nrf2 interaction with a submicromolar IC50 value, suggesting that #1 would be a novel lead-compound for Nrf2 activator. Moreover, we also found #2, a unique compound that more strongly inhibits KEAP1-p-p62 interaction than KEAP1-Nrf2 interaction. #2 has an acetonyl side-chain on the naphthalene skeleton and the treatment of #2 suppressed proliferation and reduced tolerance to cisplatin or sorafenib in HCC. This result showed that #2 was expected as an Nrf2 suppressor which should be developed for the treatment of malignancy progression and chemoresistance.
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Synthesis of KEAP1-phosphorylated p62 and KEAP1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity
Bioorganic & Medicinal Chemistry Letters, 2016Co-Authors: Daisuke Yasuda, Masayuki Yamamoto, Masaaki Komatsu, Mao Nakajima, Akihiro Yuasa, Rika Obata, Kyoko Takahashi, Yoshinobu Ichimura, Riyo ImamuraAbstract:Abstract The KEAP1-Nrf2 system is involved not only in biological defense but also in malignancy progression and chemoresistance. The ubiquitin-binding protein p62/Sqstm1 (p62), which is highly expressed in several cancers, competes with Nrf2 for KEAP1 binding, leading to activation of Nrf2-mediated gene expression and survival of cancer cells. We had previously identified an inhibitor for the KEAP1-phosphorylated-p62 ( p -p62) protein-protein interaction (PPI), the acetonyl naphthalene derivative K67. In this study, we established facile synthetic routes for K67 and derivatives with various side chains on the C-2 position of naphthalene ring. K67 possessed high selectivity in the inhibition of KEAP1- p -p62. Other derivatives showed potent KEAP1-Nrf2 and KEAP1- p -p62 PPI inhibitory activities, though the selectivity between the two activities was lower than K67.
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the KEAP1 nrf2 system and diabetes mellitus
Archives of Biochemistry and Biophysics, 2015Co-Authors: Akira Uruno, Yoko Yagishita, Masayuki YamamotoAbstract:Abstract Nrf2 (NF-E2-related factor 2) plays a key role in the protection of vertebrates against environmental stress by contributing to the inducible expression of detoxification and antioxidant enzymes. KEAP1 (Kelch-like ECH-associated protein 1) is a sensor for oxidative and electrophilic stresses. KEAP1 also acts as an E3 ubiquitin ligase substrate-recognition subunit that specifically targets Nrf2. KEAP1 causes Nrf2 to be degraded through the ubiquitin–proteasome pathway and thus ensures that Nrf2 is constitutively suppressed under unstressed conditions. Upon exposure to oxidative or electrophilic stress, KEAP1 loses its ability to ubiquitinate Nrf2. Many lines of evidence have recently clarified that the KEAP1–Nrf2 system also plays critical roles in the maintenance of cellular homeostasis. One of the most salient examples is the contribution of KEAP1–Nrf2 to metabolic and energy-balance regulation. In particular, how the KEAP1–Nrf2 system protects the body against diabetes mellitus and how perturbations in this system provoke the disease condition are now under intense investigation. This review will summarize the recent progress made in this area.
Mark Hannink - One of the best experts on this subject based on the ideXlab platform.
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pgam5 tethers a ternary complex containing KEAP1 and nrf2 to mitochondria
Experimental Cell Research, 2008Co-Authors: Shih Ching Lo, Mark HanninkAbstract: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).
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structure of the KEAP1 nrf2 interface provides mechanistic insight into nrf2 signaling
The EMBO Journal, 2006Co-Authors: Shih Ching Lo, Lesa J. Beamer, Xuchu Li, Michael T Henzl, Mark HanninkAbstract:KEAP1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of KEAP1 bound to a 16-mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel β-strands connected by two overlapping type I β-turns stabilized by the aspartate and threonine residues. The β-turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in KEAP1. Mutagenesis experiments confirmed the role of individual amino acids for binding of Nrf2 to KEAP1 and for KEAP1-mediated repression of Nrf2-dependent gene expression. Our results provide a detailed picture of how a BTB-Kelch substrate adaptor protein binds to its cognate substrate and will enable the rational design of novel chemopreventive agents.
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ubiquitination of KEAP1 a btb kelch substrate adaptor protein for cul3 targets KEAP1 for degradation by a proteasome independent pathway
Journal of Biological Chemistry, 2005Co-Authors: Donna D. Zhang, Shih Ching Lo, Geetha M Habib, Michael W Lieberman, Mark HanninkAbstract:KEAP1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. KEAP1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of KEAP1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that KEAP1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of KEAP1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of KEAP1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of KEAP1, particularly in cells that are unable to synthesize glutathione. Degradation of KEAP1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of KEAP1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.
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KEAP1 is a redox regulated substrate adaptor protein for a cul3 dependent ubiquitin ligase complex
Molecular and Cellular Biology, 2004Co-Authors: Donna D. Zhang, Shih Ching Lo, Janet V Cross, Dennis J Templeton, Mark HanninkAbstract: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.
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distinct cysteine residues in KEAP1 are required for KEAP1 dependent ubiquitination of nrf2 and for stabilization of nrf2 by chemopreventive agents and oxidative stress
Molecular and Cellular Biology, 2003Co-Authors: Donna D. Zhang, Mark HanninkAbstract: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.
Longqin Hu - One of the best experts on this subject based on the ideXlab platform.
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Nrf2 activation through the inhibition of KEAP1–Nrf2 protein–protein interaction
Medicinal Chemistry Research, 2020Co-Authors: Longqin HuAbstract:Activation of the transcription factor Nrf2 via the KEAP1–Nrf2–ARE signaling system regulates the transcription and subsequent expression of cellular cytoprotective proteins and plays a crucial role in preventing pathological conditions exacerbated by the overproduction of oxidative stress. In addition to electrophilic modulators, direct noncovalent inhibitors that interrupt the KEAP1–Nrf2 protein–protein interaction (PPI) leading to Nrf2 activation have attracted a great deal of attention as potential preventive and therapeutic agents for oxidative stress-related diseases. Structural studies of KEAP1-binding ligands, development of biochemical and cellular assays, and new structure-based design approaches have facilitated the discovery of small molecule PPI inhibitors. This perspective reviews the KEAP1–Nrf2–ARE system, its physiological functions, and the recent progress in the discovery and the potential applications of direct inhibitors of KEAP1–Nrf2 PPI.
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discovery of direct inhibitors of KEAP1 nrf2 protein protein interaction as potential therapeutic and preventive agents
Acta Pharmaceutica Sinica B, 2015Co-Authors: Dhulfiqar Ali Abed, Longqin Hu, Melanie Goldstein, Haifa AlbanyanAbstract:The KEAP1–Nrf2–ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the KEAP1–Nrf2 protein–protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of KEAP1–Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of KEAP1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of KEAP1–Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target KEAP1 Kelch domain protein. These non-covalent direct inhibitors of KEAP1–Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.
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small molecule modulators of KEAP1 nrf2 are pathway as potential preventive and therapeutic agents
Medicinal Research Reviews, 2012Co-Authors: Sadagopan Magesh, Yu Chen, Longqin HuAbstract:KEAP1-Nrf2-ARE pathway represents one of the most important cellular defense mechanisms against oxidative stress and xenobiotic damage. Activation of Nrf2 signaling induces the transcriptional regulation of ARE-dependent expression of various detoxifying and antioxidant defense enzymes and proteins. KEAP1-Nrf2-ARE signaling has become an attractive target for the prevention and treatment of oxidative stress-related diseases and conditions including cancer, neurodegenerative, cardiovascular, metabolic and inflammatory diseases. Over the last few decades, numerous Nrf2 inducers have been developed and some of them are currently undergoing clinical trials. Recently, over-activation of Nrf2 has been implicated in cancer progression as well as in drug resistance to cancer chemotherapy. Thus, Nrf2 inhibitors could potentially be used to improve the effectiveness of cancer therapy. Herein, we review the signaling mechanism of KEAP1-Nrf2-ARE pathway, its disease relevance, and currently known classes of small molecule modulators. We also discuss several aspects of KEAP1-Nrf2 interaction, Nrf2-based peptide inhibitor design, and the screening assays currently used for the discovery of direct inhibitors of KEAP1-Nrf2 interaction.
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Kinetic Analyses of KEAP1–Nrf2 Interaction and Determination of the Minimal Nrf2 Peptide Sequence Required for KEAP1 Binding Using Surface Plasmon Resonance
Chemical Biology & Drug Design, 2011Co-Authors: Yu Chen, Ah-ng Tony Kong, Daigo Inoyama, Lesa J. Beamer, Longqin HuAbstract:The KEAP1-Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for KEAP1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of KEAP1 in solution using a surface plasmon resonance (SPR)-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and KEAP1 Kelch domain in solution (KDsolution) was found to be 23.9 nM, which is 10× lower than the surface binding constant (KDsurface) of 252 nM obtained for the direct binding of KEAP1 Kelch domain to the immobilized 16mer Nrf2 peptide on a SPR sensor chip surface. The binding affinity of Nrf2 peptides to KEAP1 Kelch domain was not lost until after deletion of 8 residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a KDsolution of 352 nM and the affinity was increased 15× upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for KEAP1 binding is the 9mer sequence of LDEETGEFL.
Donna D. Zhang - One of the best experts on this subject based on the ideXlab platform.
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USP15 negatively regulates Nrf2 through deubiquitination of KEAP1
Molecular Cell, 2013Co-Authors: Nicole Villeneuve, Wang Tian, Tongde Wu, Eli Chapman, Deyu Fang, Donna D. ZhangAbstract:Summary Nrf2 is a master regulator of the antioxidant response. Under basal conditions, Nrf2 is polyubiquitinated by the KEAP1-Cul3 E3 ligase and degraded by the 26S proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to KEAP1. Currently, regulation of the Nrf2-KEAP1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or KEAP1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates KEAP1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated KEAP1 incorporates into the KEAP1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-KEAP1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.
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a noncanonical mechanism of nrf2 activation by autophagy deficiency direct interaction between KEAP1 and p62
Molecular and Cellular Biology, 2010Co-Authors: Xiaojun Wang, Nicole Villeneuve, Tongde Wu, Fei Zhao, Tao Jiang, Eileen White, Donna D. ZhangAbstract: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.
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KEAP1 controls postinduction repression of the nrf2 mediated antioxidant response by escorting nuclear export of nrf2
Molecular and Cellular Biology, 2007Co-Authors: Shirley L Zhang, Jefferson Y Chan, Donna D. ZhangAbstract: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.
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ubiquitination of KEAP1 a btb kelch substrate adaptor protein for cul3 targets KEAP1 for degradation by a proteasome independent pathway
Journal of Biological Chemistry, 2005Co-Authors: Donna D. Zhang, Shih Ching Lo, Geetha M Habib, Michael W Lieberman, Mark HanninkAbstract:KEAP1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. KEAP1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of KEAP1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that KEAP1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of KEAP1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of KEAP1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of KEAP1, particularly in cells that are unable to synthesize glutathione. Degradation of KEAP1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of KEAP1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.
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KEAP1 is a redox regulated substrate adaptor protein for a cul3 dependent ubiquitin ligase complex
Molecular and Cellular Biology, 2004Co-Authors: Donna D. Zhang, Shih Ching Lo, Janet V Cross, Dennis J Templeton, Mark HanninkAbstract: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.
Akira Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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physiological significance of reactive cysteine residues of KEAP1 in determining nrf2 activity
Molecular and Cellular Biology, 2008Co-Authors: Tae Yamamoto, Takafumi Suzuki, Hozumi Motohashi, Akira Kobayashi, Junko Wakabayashi, Jon Maher, Masayuki YamamotoAbstract:KEAP1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. KEAP1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each KEAP1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant KEAP1(C273A) and/or KEAP1(C288A) protein in KEAP1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for KEAP1 to repress Nrf2 activity in vivo. In contrast, KEAP1(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from KEAP1(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining KEAP1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.
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loss of KEAP1 function activates nrf2 and provides advantages for lung cancer cell growth
Cancer Research, 2008Co-Authors: Tsutomu Ohta, Takafumi Suzuki, Akira Kobayashi, Makiko Ohtsuji, Kumiko Iijima, Mamiko Miyamoto, Izumi Nakahara, Hiroshi Tanaka, Jun Yokota, Tokuki SakiyamaAbstract:Oxidative and electrophilic stresses are sensed by KEAP1, which activates Nrf2 to achieve cytoprotection by regulating the expression of drug-metabolizing and antioxidative stress enzymes/proteins. Because oxidative and electrophilic stresses cause many diseases, including cancer, we hypothesized that an abnormality in the Nrf2-KEAP1 system may facilitate the growth of cancer cells. We sequenced the KEAP1 gene of 65 Japanese patients with lung cancer and identified five nonsynonymous somatic mutations at a frequency of 8%. We also identified two nonsynonymous somatic KEAP1 gene mutations and two lung cancer cell lines expressing KEAP1 at reduced levels. In lung cancer cells, low KEAP1 activity (due to mutations or low-level expression) led to nuclear localization and constitutive activation of Nrf2. The latter resulted in constitutive expression of cytoprotective genes encoding multidrug resistance pumps, phase II detoxifying enzymes, and antioxidative stress enzymes/proteins. Up-regulation of these target genes in lung cancer cells led to cisplatin resistance. Nrf2 activation also stimulated growth of lung cancer–derived cell lines expressing KEAP1 at low levels and in mutant cell lines and in KEAP1 -null mouse embryonic fibroblasts under homeostatic conditions. Thus, inhibition of NRF2 may provide new therapeutic approaches in lung cancers with activation of Nrf2. [Cancer Res 2008;68(5):1303–9]
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Subcellular localization and cytoplasmic complex status of endogenous KEAP1.
Genes to Cells, 2007Co-Authors: Yoriko Watai, Akira Kobayashi, Ken Itoh, Hiroko Nagase, Mio Mizukami, Justina Dolorita Mcevoy, Jeffrey D. Singer, Masayuki YamamotoAbstract:KEAP1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous KEAP1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous KEAP1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, KEAP1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of KEAP1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that KEAP1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous KEAP1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of KEAP1.
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structural basis for defects of KEAP1 activity provoked by its point mutations in lung cancer
Molecular Cell, 2006Co-Authors: Balasundaram Padmanabhan, Yoshihiro Nakamura, Shigeyuki Yokoyama, Akira Kobayashi, Moonil Kang, Kit I Tong, Tsutomu Ohta, Maria Scharlock, Makiko Ohtsuji, Masayuki YamamotoAbstract:Nrf2 regulates the cellular oxidative stress response, whereas KEAP1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the KEAP1 and Nrf2 interaction, we resolved the six-bladed β propeller crystal structure of the Kelch/DGR and CTR domains of mouse KEAP1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of KEAP1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the β propeller of KEAP1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce KEAP1's affinity for Nrf2. These results provide a structural basis for the loss of KEAP1 function and gain of Nrf2 function.
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oxidative and electrophilic stresses activate nrf2 through inhibition of ubiquitination activity of KEAP1
Molecular and Cellular Biology, 2006Co-Authors: Akira Kobayashi, Moonil Kang, Yoriko Watai, Kit I Tong, Takahiro Shibata, Koji Uchida, Masayuki YamamotoAbstract:The KEAP1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. KEAP1 acts as a stress sensor protein in this system. While KEAP1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of KEAP1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from KEAP1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the KEAP1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of KEAP1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of KEAP1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the KEAP1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the KEAP1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.