RESUMEN
Whole blood transcriptome analysis is a valuable approachin medical research, primarily due to the ease of sample collection and the richness of the information obtained. Since the expression profile of individual genes in the analysis is influenced by medical traits and demographic attributes such as age and gender, there has been a growing demand for a comprehensive database for blood transcriptome analysis. Here, we performed whole blood RNA sequencing (RNA-seq) analysis on 576 participants stratified by age (20-30s and 60-70s) and gender from cohorts of the Tohoku Medical Megabank (TMM). A part of female segment included pregnant women. We did not exclude the globin gene family in our RNA-seq study, which enabled us to identify instances of hereditary persistence of fetal hemoglobin based on the HBG1 and HBG2 expression information. Comparing stratified populations allowed us to identify groups of genes associated with age-related changes and gender differences. We also found that the immune response status, particularly measured by neutrophil-to-lymphocyte ratio (NLR), strongly influences the diversity of individual gene expression profiles in whole blood transcriptome analysis. This stratification has resulted in a data set that will be highly beneficial for future whole blood transcriptome analysis in the Japanese population.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Humanos , Femenino , Masculino , Adulto , Persona de Mediana Edad , Perfilación de la Expresión Génica/métodos , Japón , Anciano , Adulto Joven , Factores de Edad , Factores Sexuales , Pueblo Asiatico/genética , Pueblos del Este de AsiaRESUMEN
In human cancer, activating mutations in the KEAP1-NRF2 pathway are frequently observed, and positively selected for, as they confer the cytoprotective functions of the transcription factor NRF2 on the cancer cells. This results in the development of aggressive tumours which are resistant to treatment with chemotherapeutic compounds. Recent clinical developments have also revealed that NRF2-activated cancers are similarly resistant to immune checkpoint inhibitor drugs. As the mechanism of action of these immune modulating therapies is tangential to the classical cytoprotective function of NRF2, it is unclear how aberrant NRF2 activity could impact the anti-cancer functionality of the immune system. In this context, we found that in human cancer, NRF2-activated cells are highly immunoedited, which allows the cancer cells to escape immune surveillance and develop into malignant tumours. This immunoediting takes the form of reduced antigen presentation by the MHC-I complex, coupled with reduced expression of activating ligands for NK cells. Together, these modifications to the immunogenicity of NRF2-activated cancers inhibit immune effector cell infiltration and engagement, and contribute to the formation of the immunologically cold tumour microenvironment which is a characteristic feature of NRF2-activated cancers.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Neoplasias , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Mutación , Estrés Oxidativo , Microambiente Tumoral/genéticaRESUMEN
While it is well established that the KEAP1-NRF2 pathway regulates the main inducible cellular response to oxidative stress, this cytoprotective function of NRF2 could become deleterious to the host if it confers survival onto irreparably damaged cells. In this regard, we have found that in diseased states, NRF2 promotes the transcriptional activation of a specific subset of the senescence-associated secretory phenotype (SASP) gene program, which we have named the NRF2-induced secretory phenotype (NISP). In two models of hepatic disease using Pten::Keap1 and Keap1::Atg7 double knockout mice, we found that the NISP functions in the liver to recruit CCR2 expressing monocytes, which function as immune system effector cells to directly remove the damaged cells. Through activation of this immune surveillance pathway, in non-transformed cells, NRF2 functions as a tumour suppressor to mitigate the long-term survival of damaged cells which otherwise would be detrimental for host survival. This pathway represents the final stage of the oxidative stress response, as it allows cells to be safely removed if the macromolecular damage caused by the original stressor is so extensive that it is beyond the repair capacity of the cell.
Asunto(s)
Hígado , Factor 2 Relacionado con NF-E2 , Animales , Ratones , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Transporte Biológico , Activación Transcripcional , Ratones NoqueadosRESUMEN
The KEAP1-NRF2 pathway regulates the main inducible cellular response to oxidative and electrophilic stresses. Activating mutations in the KEAP1-NRF2 pathway occur commonly in human cancer, where they contribute to the formation of aggressive tumours that are associated with a poor prognosis for patients. An important clinical feature of these tumours is their defiance to all current anti-cancer treatment regimens, highlighting the need for the development of new therapeutic strategies to target NRF2-activated cancers. In this review, we discuss the mechanisms through which acquired NRF2 hyperactivation can result in resistance of tumours to immune checkpoint inhibitor therapies in addition to classical chemotherapeutics, and propose with examples that using a synthetic lethal strategy mediated by NRF2-target gene-dependent bioactivation of prodrugs represents a promising strategy to specifically enhance toxicity to heretofore untreatable NRF2-hyperactivated human tumours.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Neoplasias , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Mutaciones Letales Sintéticas , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oxidación-Reducción , Estrés OxidativoRESUMEN
The Keap1-Nrf2 system is the master regulator of the cellular response against oxidative and xenobiotic stresses. Constitutive activation of Nrf2 is frequently observed in various types of cancers. Nrf2 hyperactivation induces metabolic reprogramming in cancer cells, which supports the increased energy demand required for rapid proliferation and confers high-level resistance against anticancer radio/chemotherapy. Hence, Nrf2 inhibition has emerged as an attractive therapeutic strategy to counter such acquired resistance in Nrf2-activated tumors. We previously identified Halofuginone (HF) as a promising Nrf2 inhibitor. In this study, we pursued preclinical characterization of HF and found that while HF markedly reduced the viability of cancer cells, it also caused severe hematopoietic and immune cell suppression in a dose-dependent manner. Hence, to overcome this toxicity, we decided to employ a nanomedicine approach to HF. We found that encapsulation of HF into a polymeric micelle (HF micelle; HFm) largely relieved the systemic toxicity exhibited by free HF while maintaining the tumor-suppressive properties of HF. LC-MS/MS analysis revealed that the reduction in the magnitude of adverse effects was the result of the ability to release HF from the HFm core in a slow and sustained manner. These results thus support the contention that HFm will potentially counteract Nrf2-activated cancers in the clinical settings.
Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Nanopartículas , Piperidinas , Quinazolinonas , Humanos , Adenocarcinoma del Pulmón/metabolismo , Cromatografía Liquida , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/patología , Micelas , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Piperidinas/farmacología , Quinazolinonas/farmacología , Espectrometría de Masas en TándemRESUMEN
Activating mutations in the KEAP1-NRF2 pathway are found in approximately 25% of lung tumors, where the hijacking of NRF2's cytoprotective functions results in aggressive tumor growth, chemoresistance, and a poor prognosis for patients. There are currently no approved drugs which target aberrant NRF2 activation, which means that there is an urgent clinical need to target this orphan oncogenic pathway in human tumors. In this study, we used an isogenic pair of wild-type and Keap1 knockout cells to screen a range of chemotherapeutic and pathway-targeted anticancer drugs in order to identify compounds which display enhanced toxicity toward cells with high levels of Nrf2 activity. Through this approach, complemented by validation across a panel of eight human cancer cell lines from a range of different tissues, we identified the DNA-damaging agent mitomycin C to be significantly more toxic in cells with aberrant Nrf2 activation. Mechanistically, we found that the NRF2 target genes for cytochrome P450 reductase, NQO1, and enzymes in the pentose phosphate pathway are all responsible for the NRF2-dependent enhanced bioactivation of mitomycin C. As mitomycin C is already approved for clinical use, it represents as excellent drug repositioning candidate to target the currently untreatable NRF2 activation in human tumors.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Proteína 1 Asociada A ECH Tipo Kelch/genética , Mitomicina/farmacología , NADP/metabolismo , Factor 2 Relacionado con NF-E2/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/deficiencia , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , Factor 2 Relacionado con NF-E2/deficiencia , Estrés Oxidativo , Paclitaxel/farmacología , Vía de Pentosa Fosfato/efectos de los fármacos , Vía de Pentosa Fosfato/genética , Transducción de Señal , Proteína Fluorescente RojaRESUMEN
Activating mutations in KEAP1-NRF2 are frequently found in tumors of the lung, esophagus, and liver, where they are associated with aggressive growth, resistance to cancer therapies, and low overall survival. Despite the fact that NRF2 is a validated driver of tumorigenesis and chemotherapeutic resistance, there are currently no approved drugs which can inhibit its activity. Therefore, there is an urgent clinical need to identify NRF2-selective cancer therapies. To this end, we developed a novel synthetic lethal assay, based on fluorescently labeled isogenic wild-type and Keap1 knockout cell lines, in order to screen for compounds which selectively kill cells in an NRF2-dependent manner. Through this approach, we identified three compounds based on the geldanamycin scaffold which display synthetic lethality with NRF2. Mechanistically, we show that products of NRF2 target genes metabolize the quinone-containing geldanamycin compounds into more potent HSP90 inhibitors, which enhances their cytotoxicity while simultaneously restricting the synthetic lethal effect to cells with aberrant NRF2 activity. As all three of the geldanamycin-derived compounds have been used in clinical trials, they represent ideal candidates for drug repositioning to target the currently untreatable NRF2 activity in cancer.
Asunto(s)
Antineoplásicos/farmacología , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Antineoplásicos/química , Benzoquinonas/química , Benzoquinonas/metabolismo , Muerte Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Lactamas Macrocíclicas/química , Ratones , Ratones Endogámicos BALB C , Mutación , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína Oncogénica v-akt/antagonistas & inhibidores , Oxidación-Reducción , Paclitaxel/farmacologíaRESUMEN
The KEAP1-NRF2 pathway is the principal protective response to oxidative and electrophilic stresses. Under homeostatic conditions, KEAP1 forms part of an E3 ubiquitin ligase, which tightly regulates the activity of the transcription factor NRF2 by targeting it for ubiquitination and proteasome-dependent degradation. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program. Recent advances have revealed that KEAP1 contains multiple stress sensors and inactivation modalities, which together allow diverse cellular inputs, from oxidative stress and cellular metabolites to dysregulated autophagy, to regulate NRF2 activity. This integration of the KEAP1-NRF2 system into multiple cellular signaling and metabolic pathways places NRF2 activation as a critical regulatory node in many disease phenotypes and suggests that the pharmaceutical modulation of NRF2's cytoprotective activity will be beneficial for human health in a broad range of noncommunicable diseases.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , Animales , Autofagia , Humanos , Redes y Vías Metabólicas , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.
Asunto(s)
Cisteína/metabolismo , Peróxido de Hidrógeno/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Estrés Oxidativo/genética , Animales , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Factor de Transcripción NF-E2/genética , Factor de Transcripción NF-E2/metabolismo , Estrés Oxidativo/fisiologíaRESUMEN
NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2.
Asunto(s)
Diabetes Insípida Nefrogénica/patología , Hidronefrosis/patología , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/metabolismo , Poliuria/patología , Animales , Acuaporina 2/metabolismo , Diferenciación Celular/genética , Diabetes Insípida Nefrogénica/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Hidronefrosis/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/genética , Poliuria/genética , Reabsorción Renal/genéticaRESUMEN
Eukaryotic cells maintain protein homeostasis through the activity of multiple basal and inducible systems, which function in concert to allow cells to adapt to a wide range of environmental conditions. Although the transcriptional programs regulating individual pathways have been studied in detail, it is not known how the different pathways are transcriptionally integrated such that a deficiency in one pathway can be compensated by a change in an auxiliary response. One such pathway that plays an essential role in many proteostasis responses is the ubiquitin-proteasome system, which functions to degrade damaged, unfolded, or short half-life proteins. Transcriptional regulation of the proteasome is mediated by the transcription factor Nrf1. Using a conditional knockout mouse model, we found that Nrf1 regulates protein homeostasis in the endoplasmic reticulum (ER) through transcriptional regulation of the ER stress sensor ATF6. In Nrf1 conditional-knockout mice, a reduction in proteasome activity is accompanied by an ATF6-dependent downregulation of the endoplasmic reticulum-associated degradation machinery, which reduces the substrate burden on the proteasome. This indicates that Nrf1 regulates a homeostatic shift through which proteostasis in the endoplasmic reticulum and cytoplasm are coregulated based on a cell's ability to degrade proteins.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Degradación Asociada con el Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Homeostasis , Transcripción Genética , Factor de Transcripción Activador 6/metabolismo , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Regulación hacia Abajo/genética , Elementos de Facilitación Genéticos/genética , Homeostasis/genética , Masculino , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Modelos Biológicos , Factor Nuclear 1 de Respiración/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The transcription factor Nrf2 (NF-E2-related-factor 2) is essential for the oxidative and electrophilic stress responses. Keap1 (Kelch-like-ECH-associated-protein 1), an adaptor for a cullin-3 (Cul3)-based ubiquitin ligase, regulates Nrf2 activity through proteasomal degradation, and acts as a sensor for oxidative and electrophilic stresses. The Keap1-Cul3 complex is a critical regulator of the cellular Nrf2 level, and yet quantitative information regarding their endogenous intracellular concentrations in homeostatic conditions and during stress responses is unknown. We analyzed the absolute amounts of the Nrf2, Keap1, and Cul3 proteins in five murine cell lines by comparison with serial dilutions of purified recombinant protein standards in combination with quantitative immunoblot analyses. In the basal state, the amount of Nrf2 was maintained at lower levels than those of Keap1 and Cul3 proteins, whereas the electrophilic agent diethylmaleate dramatically increased Nrf2 to a level greater than that of Keap1 and Cul3, resulting in the accumulation of Nrf2 in the nucleus. In contrast, Keap1 and Cul3 did not display any changes in their abundance, subcellular localization, or interaction in response to electrophilic stimuli. Our results demonstrate that the regulation of the Nrf2 protein level during stress responses is mediated by the activity but not the composition of the Nrf2-Keap1-Cul3 complex.
Asunto(s)
Proteínas Cullin/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Complejos Multiproteicos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Maleatos/farmacología , Ratones , Estrés Oxidativo , Células RAW 264.7 , Proteínas Recombinantes/metabolismoRESUMEN
Nrf2 activation promotes resistance to chemical carcinogenesis in animal models, but activating mutations in Nrf2 also confer malignant characters to human cells by activating antioxidative/detoxifying enzymes and metabolic reprogramming. In this study, we examined how these contradictory activities of Nrf2, cancer chemoprevention and cancer cell growth enhancement, can be reconciled in an established mouse model of urethane-induced lung carcinogenesis. Using Keap1-knockdown (kd) mice, which express high levels of Nrf2, we found that urethane was rapidly excreted into the urine, consistent with an upregulation in the expression of urethane detoxification genes. Consequently, urethane-induced tumors were significantly smaller and less frequent in Keap1-kd mice than in wild-type mice. In contrast, tumor cells derived from Keap1-kd mice and transplanted into nude mice exhibited higher tumorigenicity compared with cells derived from wild-type mice. To identify the factors contributing to the tumor growth phenotype in the transplantation model, we performed a microarray analysis and found that many antioxidative stress genes were upregulated in the Keap1-kd-derived tumors. Therefore, we suggest that Nrf2 activation in cancer cells enhances their tumorigenicity, but global Nrf2 activation, as in Keap1-kd mice, simultaneously enhances anticancer immunity, thereby suppressing the growth potential of Keap1-kd tumors. Our findings provide relevant insight into the dual role of Nrf2 in cancer and warrant further studies of Nrf2 function during different stages of carcinogenesis. Cancer Res; 76(10); 3088-96. ©2016 AACR.
Asunto(s)
Transformación Celular Neoplásica/patología , Neoplasias Pulmonares/patología , Mutación/genética , Factor 2 Relacionado con NF-E2/fisiología , Microambiente Tumoral , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Perfilación de la Expresión Génica , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Uretano/toxicidadRESUMEN
The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cisteína/metabolismo , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Cisteína/química , Cisteína/genética , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , TransfecciónRESUMEN
The Kelch-like ECH associated protein 1 (Keap1)-NF-E2 p45-related factor 2 (Nrf2) pathway regulates networks of proteins that protect against the cumulative damage of oxidants, electrophiles and misfolded proteins. The interaction between transcription factor Nrf2 and its main negative cytoplasmic regulator Keap1 follows a cycle whereby the protein complex sequentially adopts two conformations: 'open', in which Nrf2 binds to one monomer of Keap1, followed by 'closed', in which Nrf2 interacts with both members of the Keap1 dimer. Electrophiles and oxidants (inducers) are recognized by cysteine sensors within Keap1, disrupting its ability to target Nrf2 for ubiquitination and degradation. Consequently, the protein complex accumulates in the 'closed' conformation, free Keap1 is not regenerated and newly synthesized Nrf2 is stabilized to activate target-gene transcription. The prevailing view of the Keap1-Nrf2 pathway, for which there exists a wealth of experimental evidence, is that it lies at the heart of cellular defence, playing crucial roles in adaptation and survival under conditions of stress. More recently, the significance of Nrf2 in intermediary metabolism and mitochondrial physiology has also been recognized, adding another layer of cytoprotection to the repertoire of functions of Nrf2. One way by which Nrf2 influences mitochondrial activity is through increasing the availability of substrates (NADH and FADH2) for respiration. Another way is through accelerating fatty acid oxidation (FAO). These findings reinforce the reciprocal relationship between oxidative phosphorylation and the cellular redox state, and highlight the key role of Nrf2 in regulating this balance.
Asunto(s)
Metabolismo Energético , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteína 1 Asociada A ECH Tipo Kelch , Mitocondrias/metabolismo , Modelos Moleculares , Factor 2 Relacionado con NF-E2/química , Oxidación-Reducción , Transducción de SeñalRESUMEN
The transcription factor Nrf2 regulates the expression of a large network of cytoprotective and metabolic enzymes and proteins. Compounds that directly and reversibly inhibit the interaction between Nrf2 and its main negative regulator Keap1 are potential pharmacological agents for a range of disease types including neurodegenerative conditions and cancer. We describe the development of a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit the Nrf2-Keap1 protein-protein interaction (PPI) in vitro and in live cells and up-regulate the expression of Nrf2-dependent gene products.
Asunto(s)
Hemo-Oxigenasa 1/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , Factor 2 Relacionado con NF-E2/metabolismo , Triazoles/química , Línea Celular Tumoral , Química Clic , Simulación por Computador , Bases de Datos de Compuestos Químicos , Relación Dosis-Respuesta a Droga , Polarización de Fluorescencia , Células HEK293 , Hemo-Oxigenasa 1/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Isotiocianatos/química , Isotiocianatos/farmacología , Proteína 1 Asociada A ECH Tipo Kelch , Simulación del Acoplamiento Molecular , NAD(P)H Deshidrogenasa (Quinona)/genética , Factor 2 Relacionado con NF-E2/química , Unión Proteica , Relación Estructura-Actividad , Sulfóxidos , Triazoles/síntesis química , Triazoles/farmacologíaRESUMEN
NRF1 (NF-E2-p45-related factor 1) plays an important role in the regulation of genes encoding proteasome subunits, a cystine transporter, and lipid-metabolizing enzymes. Global and tissue-specific disruptions of the Nrf1 gene in mice result in embryonic lethality and spontaneous development of severe tissue defects, respectively, suggesting NRF1 plays a critical role in vivo. Mechanistically, the continuous degradation of the NRF1 protein by the proteasome is regarded as a major regulatory nexus of NRF1 activity. To develop NRF1-specific inducers that act to overcome the phenotypes related to the lack of NRF1 activity, we constructed a novel NRF1ΔC-Luc fusion protein reporter and developed cell lines that stably express the reporter in Hepa1c1c7 cells for use in high-throughput screening. In screening of a chemical library with this reporter system, we identified two hit compounds that significantly induced luciferase activity. Through an examination of a series of derivatives of one of the hit compounds, we identified T1-20, which induced a 70-fold increase in luciferase activity. T1-20 significantly increased the level of NRF1 protein in the mouse liver, indicating that the compound is also functional in vivo. Thus, these results show the successful identification of the first small chemical compounds which specifically and significantly induce NRF1.
Asunto(s)
Bases de Datos de Compuestos Químicos , Descubrimiento de Drogas , Factor Nuclear 1 de Respiración/química , Factor Nuclear 1 de Respiración/metabolismo , Compuestos Orgánicos/metabolismo , Animales , Línea Celular Tumoral , Vectores Genéticos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Hígado/metabolismo , RatonesRESUMEN
The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.
Asunto(s)
Genes Reporteros , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Secuencia de Bases , Hipoxia de la Célula/genética , Línea Celular , Humanos , Luciferasas/metabolismo , Ratones , Datos de Secuencia Molecular , Ratas , Elementos de Respuesta/genética , Estrés Fisiológico/genéticaRESUMEN
Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1(flox) allele and a rat CYP1A1-Cre transgene (Nrf1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Ácido Graso Desaturasas/genética , Regulación Enzimológica de la Expresión Génica , Metabolismo de los Lípidos , Factor Nuclear 1 de Respiración/fisiología , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animales , Línea Celular , Cistina/metabolismo , Citocromo P-450 CYP1A1/genética , Ácido Graso Desaturasas/metabolismo , Femenino , Glutatión/metabolismo , Hepatocitos/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Unión Proteica , Ratas , Receptores de Lipoproteína/genética , Receptores de Lipoproteína/metabolismo , Elementos de Respuesta , Activación Transcripcional , Triglicéridos/metabolismoRESUMEN
The transcription factor NF-E2 p45-related factor 2 (Nrf2) and its negative regulator Kelch-like ECH associated protein 1 (Keap1) control the expression of nearly 500 genes with diverse cytoprotective functions. Keap1, a substrate adaptor protein for Cullin3/Rbx1 ubiquitin ligase, normally continuously targets Nrf2 for degradation, but loses this ability in response to electrophiles and oxidants (termed inducers). Consequently, Nrf2 accumulates and activates transcription of its downstream target genes. Many inducers are phytochemicals, and cruciferous vegetables represent one of the richest sources of inducer activity among the most commonly used edible plants. Here we summarize the discovery of the isothiocyanate sulforaphane as a potent inducer which reacts with cysteine sensors of Keap1, leading to activation of Nrf2. We then describe the development of a quantitative Förster resonance energy transfer (FRET)-based methodology combined with multiphoton fluorescence lifetime imaging microscopy (FLIM) to investigate the interactions between Keap1 and Nrf2 in single live cells, and the effect of sulforaphane, and other cysteine-reactive inducers, on the dynamics of the Keap1-Nrf2 protein complex. We present the experimental evidence for the "cyclic sequential attachment and regeneration" or "conformation cycling" model of Keap1-mediated Nrf2 degradation. Finally, we discuss the implications of this mode of regulation of Nrf2 for achieving a fine balance under normal physiological conditions, and the consequences and mechanisms of disrupting this balance for tumor biology.