RESUMO
Cap'n'Collar (CNC) proteins heterodimerize with small Maf proteins and regulate the transcription of various genes. Small Maf-deficient mice develop severe neurodegeneration, and it remains unclear whether CNC proteins are involved in this process. In this study, we examined the contribution of Nrf1, one of the CNC proteins, to neuronal homeostasis in vivo. As Nrf1 gene knockout mice are embryonic lethal, we developed a central nervous system (CNS)-specific Nrf1 knockout (CKO) mouse line using mice bearing an Nrf1(flox) allele and Nestin-Cre allele. At birth, the CKO mice appeared indistinguishable from control mice, but thereafter they showed progressive motor ataxia and severe weight loss. All Nrf1 CKO mice died within 3 weeks. These phenotypes are similar to those reported in small Maf-deficient mice, suggesting the presence of collaboration between Nrf1 and small Maf proteins. We also found aberrant accumulation of polyubiquitinated proteins in various CNS regions and apparent neuronal loss in the hippocampus of Nrf1 CKO mice. An oxidative stress marker was accumulated in the spinal cords of the mice, but the expression patterns of oxidative stress response genes regulated by Nrf2 did not change substantially. These results show that Nrf1 sustains the CNS homeostasis through regulating target genes distinct from those regulated by Nrf2.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Deleção de Genes , Neurônios Motores/metabolismo , Degeneração Neural/genética , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Animais , Ataxia/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Neurônios Motores/patologia , Degeneração Neural/patologia , Estresse Oxidativo/genética , Fenótipo , Proteínas Ubiquitinadas/metabolismoRESUMO
Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Estresse Oxidativo , Proteínas/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas Culina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína 1 Associada a ECH Semelhante a Kelch , Substâncias Macromoleculares , Camundongos , Fator 2 Relacionado a NF-E2 , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Nrf1 is a member of the vertebrate Cap'n'Collar (CNC) transcription factor family that commonly contains a unique basic-leucine zipper domain. Among CNC family members, Nrf2 is known to regulate a battery of antioxidant and xenobiotic-metabolizing enzyme genes through the antioxidant response element (ARE). Although Nrf1 has also been shown to bind the ARE, it is unclear whether it plays a distinct role from Nrf2 in regulating genes with this element. To address this issue in vivo, we generated mice bearing a hepatocyte-specific disruption of the Nrf1 gene. AlthoughNrf2 knock-out mice did not exhibit liver damage when they were maintained in an unstressed condition, hepatocyte-specific deletion of Nrf1 caused liver damage resembling the human disease non-alcoholic steatohepatitis. Gene expression analysis revealed that the disruption of Nrf1 causes stress that activates a number of ARE-driven genes in an Nrf2-dependent manner, indicating that Nrf2 cannot compensate completely for loss of Nrf1 function in the liver. In contrast, expression of metallothionein-1 and -2 (MT1 and MT2) genes, each of which harbors at least one ARE in its regulatory region, was decreased in the Nrf1-null mutant mice. Whereas Nrf1 and Nrf2 bound the MT1 ARE with comparable affinity, Nrf1 preferentially activated the reporter gene expression through the MT1 ARE. This study has, thus, identified the first ARE-dependent gene that relies exclusively on Nrf1, suggesting that it plays a distinct functional role in regulating ARE-driven genes.
Assuntos
Regulação da Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Metalotioneína/biossíntese , Fator 2 Relacionado a NF-E2/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Elementos de Resposta/fisiologia , Animais , Antioxidantes/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Técnicas de Silenciamento de Genes , Hepatite/genética , Hepatite/metabolismo , Zíper de Leucina/fisiologia , Metalotioneína/genética , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 1 Nuclear Respiratório/genética , Estrutura Terciária de Proteína/fisiologiaRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Estresse OxidativoRESUMO
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 beta 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 beta 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.