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1.
Nat Genet ; 53(3): 322-331, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33649593

RESUMO

The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 and leverage our multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.


Assuntos
Antígeno B7-H1/genética , Análise de Célula Única/métodos , Antígeno B7-2/metabolismo , Antígeno B7-H1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Culina/genética , Proteínas Culina/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptores de Interferon/genética , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Zhongguo Zhong Yao Za Zhi ; 46(1): 24-32, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-33645047

RESUMO

Nrf2 is the key transcription factor mainly for regulating oxidative homeostasis and cytoprotective responses against oxidative stress. Nrf2/Keap1 pathway is one of the most important cellular defense mechanisms against endogenous or exogenous oxidative stress. With its activation, a wide range of stress-related genes is transactivated to restore the cellular homeostasis. Recent studies revealed that the aberrant activation of Nrf2 is related to the malignant progression, chemotherapeutic drug resistance and poor prognosis. Nrf2 plays a crucial role in cancer malignancy and chemotherapeutic resistance by controlling the intracellular redox homeostasis through the activation of cytoprotective antioxidant genes. Nrf2 inhibitor containing many natural products has been deemed as a novel therapeutic strategy for human malignancies. This article reviews the progress of studies of the Nrf2/Keap1 pathway, and its biological impact in solid malignancies and molecular mechanisms for causing Nrf2 hyperactivation in cancer cells. In conclusion, we summarized the deve-lopment of Nrf2 inhibitors in recent years, in the expectation of providing reference for further drug development and clinical studies.


Assuntos
Fator 2 Relacionado a NF-E2 , Neoplasias , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oxirredução , Estresse Oxidativo
3.
Ecotoxicol Environ Saf ; 208: 111614, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396134

RESUMO

A novel gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀, PGGG cell line) was established, its application in cadmium (Cd) toxicology was demonstrated in this study. Primary cultures and PGGG subcultures were carried out at 25 °C in Dulbecco's Modified Eagle medium/F12 medium (1:1; pH 7.2) supplemented with 15% fetal bovine serum (FBS). Primary PGGG cells were spindle-shaped, proliferated into a confluent monolayer within two weeks and were continuously subcultured over passage 60. The growth of cells at passages 20, 40, and 60 was examined. Chromosome analysis revealed that the chromosomal number of normal PGGG cells was 48, but the number of cells with the normal chromosomes number decreased during the passaging process. Cadmium is one of the most toxic metals in aquatic systems and has been associated with multiple animal and human health problems. To interpret the cytotoxicity and related mechanisms of cadmium, PGGG cells were used as an in vitro model. After treatment with cadmium at concentrations ranging from 1 µM to 500 µM, PGGG cells demonstrated dose- and time-dependent cytotoxicity, manifested as morphological abnormalities and a viability decline. Further, it was found that the reactive oxygen species (ROS) and malondialdehyde (MDA) levels were elevated following cadmium exposure, and related genes involved in the antioxidant system, including those encoding catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and Kelch-like- ECH-associated protein 1 (Keap1), were regulated differently. In addition, PGGG cells treated with cadmium had the typical features associated with apoptosis, including phosphatidylserine (PS) externalization; upregulated expression of caspase-3, -8, and -9; and apoptotic body formation. In general, the PGGG cell line may serve as a useful tool for studying the toxic mechanisms of cadmium or other toxicants or for toxicity testing and environment monitoring.


Assuntos
Apoptose/efeitos dos fármacos , Bass , Cádmio/toxicidade , Expressão Gênica/efeitos dos fármacos , Brânquias , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Catalase/genética , Catalase/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Brânquias/citologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
4.
Mol Cell Biol ; 40(22)2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-32868290

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Antineoplásicos/química , Benzoquinonas/química , Benzoquinonas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Lactamas Macrocíclicas/química , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Oncogênica v-akt/antagonistas & inibidores , Oxirredução , Paclitaxel/farmacologia
5.
Ecotoxicol Environ Saf ; 202: 110914, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800249

RESUMO

Bombyx mori(Linnaeus, 1758) is an important economical insect, and the sericulture is a flourishing industry in many developing countries. Pyriproxyfen, a juvenile hormone pesticide, is often applied to cultivations widely in the world, and its exposure often resulted in silk yield reduction and non-cocooning. However, the effect of pyriproxyfen exposure on cocooning and gene expression level in the silk gland of B. mori has not been studied yet, and this study focused on the above issues. The result indicated that pyriproxyfen exposure can lead to silk gland injury, reduction of silk yield and cocooning rate. Furthermore, the expression levels of silk protein synthesis related genes were down regulated significantly. The same change trends were shown between PI3K/Akt and CncC/Keap1 pathway, which is the expressions of key genes can be elevated by pyriproxyfen exposure. In addition, the activity of detoxification enzymes (P450, GST and CarE) and the expression levels of detoxification genes were elevated after pyriproxyfen exposure, suggesting that detoxification enzymes may play an important role in detoxification of pyriproxyfen in silk gland. These results provided possible clues to the silk gland injury and gene transcriptional level changes in silkworm after pyriproxyfen exposure.


Assuntos
Bombyx/fisiologia , Inseticidas/toxicidade , Piridinas/toxicidade , Animais , Bombyx/efeitos dos fármacos , Bombyx/genética , Regulação para Baixo , Proteínas de Insetos/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Larva/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Biossíntese de Proteínas , Seda/biossíntese , Seda/genética , Seda/metabolismo
6.
Chem Biol Interact ; 329: 109220, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32763245

RESUMO

The sepsis is considered as serious clinic-pathological condition related with high rate of morbidity and mortality in critical care settings. In the proposed study, the hydrazides derivatives N-(benzylidene)-2-((2-hydroxynaphthalen-1-yl)diazenyl)benzohydrazides (1-2) (NCHDH and NTHDH) were investigated against the LPS-induced sepsis in rodents. The NCHDH and NTHDH markedly improved the physiological sign and symptoms associated with the sepsis such as mortality, temperature, and clinical scoring compared to negative control group, which received only LPS (i.p.). The NCHDH and NTHDH also inhibited the production of the NO and MPO compared to the negative control. Furthermore, the treatment control improved the histological changes markedly of all the vital organs. Additionally, the Masson's trichrome and PAS (Periodic Acid Schiff) staining also showed improvement in the NCHDH and NTHDH treated group in contrast to LPS-induced group. The antioxidants were enhanced by the intervention of the NCHDH and NTHDH and the level of the MDA and POD were attenuated marginally compared to the LPS-induced group. The hematology study showed marked improvement and the reversal of the LPS-induced changes in blood composition compared to the negative control. The synthetic function of the liver and kidney were preserved in the NCHDH and NTHDH treated group compared to the LPS-induced group. The NCHDH and NTHDH markedly enhanced the Nrf2, HO-1 (Heme oxygenase-1), while attenuated the Keap1 and TRPV1 expression level as compared to LPS treated group. Furthermore, the NCHDH and NTHDH treatment showed marked increased in the mRNA expression level of the HSP70/90 proteins compared to the negative control.


Assuntos
Hidrazinas/farmacologia , Insuficiência de Múltiplos Órgãos/etiologia , Sepse/etiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Heme Oxigenase-1/metabolismo , Hidrazinas/química , Hidrazinas/uso terapêutico , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Insuficiência de Múltiplos Órgãos/mortalidade , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Sepse/tratamento farmacológico , Sepse/mortalidade , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
7.
Am J Physiol Renal Physiol ; 319(4): F686-F696, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32830535

RESUMO

Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis as well as the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI-specific siRNA in the absence and presence of an antioxidant, tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in PDI-inhibited cells. Also, PDI inhibition caused nuclear factor erythroid 2-related factor 2 (Nrf2; a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase and glutathione-S-transferase activities, and increased oxidative stress. In PDI-inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, whereas Kelch-like ECH-associated protein 1 (an Nrf2 regulatory protein) silencing protected cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation, causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. This study suggests an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.


Assuntos
Apoptose , Células Epiteliais/enzimologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Túbulos Renais Proximais/enzimologia , Mitocôndrias/enzimologia , Fator 2 Relacionado a NF-E2/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Transporte Ativo do Núcleo Celular , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Metabolismo Energético , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/genética , Interferência de RNA , Transdução de Sinais , Marcadores de Spin
8.
Nat Commun ; 11(1): 4225, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839463

RESUMO

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.


Assuntos
Proteínas de Ligação a DNA/genética , Mutação da Fase de Leitura , Neoplasias da Vesícula Biliar/genética , Predisposição Genética para Doença/genética , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Chile , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Vesícula Biliar/diagnóstico , Neoplasias da Vesícula Biliar/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Índia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas c-ets/imunologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , República da Coreia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
9.
Medicine (Baltimore) ; 99(26): e20433, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32590729

RESUMO

Bronchopulmonary dysplasia (BPD) is a chronic lung disease common in premature infants and is one of the leading causes of disability and death in newborns. The Keap-1/Nrf2 signaling pathway plays an important role in antioxidant and anti-inflammatory.Ten clean-grade, healthy pregnant Sprague-Dawley rats (purchased from Experimental Animal Center of Peking university, China) naturally gave birth to 55 neonatal rats from which 40 were selected and randomly divided into a hyperoxia group and a control group (N = 20, each). Thirty-two BPD patient samples are from Neonatal Department of the second Hospital of Jilin University from November 30, 2016 to May 1 2019.In present study, we observed that lung tissues of the control group did not undergo obvious pathological changes, whereas in the hyperoxia group, lung tissues had disordered structures. With increased time of hyperoxia exposure, the alveolar wall became attenuated. Under hypoxia conditions, the activity of oxidative stress-related enzymes (CAT, GSH-Px, SOD) in lung samples was significantly lower than that before treatment. The expression level of Keap1 mRNA and protein in the hyperoxia group was slightly lower than that of control group. The expression of Nrf2 and HO-1 mRNA and protein in the hyperoxia group was significantly higher than that of control group. For the infants with BPD, we found that the activity of SOD, GSH-Px, and CAT was significantly different from those of control group.We constructed a premature BPD animal model and found the abnormal of oxidative stress in different groups and the expression levels of Keap1/Nrf2 signaling pathway-related molecules, and we validated the results in premature infants with BPD.


Assuntos
Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Pulmão/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Animais , Animais Recém-Nascidos , Estudos de Casos e Controles , Catalase/metabolismo , Modelos Animais de Doenças , Feminino , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Hiperóxia , Hipóxia , Recém-Nascido Prematuro , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Pulmão/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/genética , Gravidez , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Superóxido Dismutase/metabolismo
10.
Aquat Toxicol ; 224: 105516, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32485495

RESUMO

Summer outbreaks of the hepatobiliary syndrome in fish impose a heavy burden on aquaculture in China. Curcumin is a polyphenol with antioxidant activity that has been used to protect the health of fish livers, but the mechanism underlying its protective effect is unclear. In this study, an in vitro model of hepatocyte oxidative damage in Oreochromis niloticus was established using H2O2. Treatment with 5 mM H2O2 for 2.5 h markedly reduced cell viability and antioxidant activity and elevated lactate dehydrogenase (LDH) activity, indicating conditions that can be used to establish an oxidative stress model. Under H2O2 stress, curcumin pretreatment significantly maintained cell viability, reduced malondialdehyde (MDA) levels, and increased superoxide dismutase (SOD) activity. RNA-seq results showed that acute H2O2 treatment resulted in minor changes in gene expression, whereas curcumin changed the expression profile and affected cytochrome P450 (Cyp 450), glutathione (GSH) metabolism, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Several critical antioxidant defense signaling pathways were identified, and altered expression was confirmed by q-PCR. These results indicate that curcumin might upregulate PPAR expression by increasing Cyp2J2 expression. Further experiments showed that curcumin can upregulate the Nrf2-Keap1 signaling pathway at the transcriptional level, and this upregulation can induce downstream defense genes, including glutamate cysteine ligase catalytic subunit(GCLC) and glutamate cysteine ligase modifier subunit (GCLM), and thereby promote GSH synthesis and the expression of related antioxidases. This study might shed light on the effects of curcumin on the prevention and alleviation of liver diseases in fish.


Assuntos
Antioxidantes/farmacologia , Curcumina/farmacologia , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Tilápia/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Células Cultivadas , Curcumina/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Hepatócitos/metabolismo , Peróxido de Hidrogênio/toxicidade , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tilápia/genética , Poluentes Químicos da Água/toxicidade
11.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376626

RESUMO

Expression of viral genes and activation of innate antiviral responses during infection result in an increase in reactive oxygen species (ROS) and toxic by-products of energy metabolism which can lead to cell death. The mitochondrion and its associated proteins are crucial regulators of these responses and related pathways such as autophagy and apoptosis. Through a mass spectrometry approach, we have shown that the herpes simplex virus 1 (HSV-1) neurovirulence- and autophagy-modulating protein ICP34.5 interacts with numerous mitochondrion-associated factors. Specifically, we showed that amino acids 68 to 87 of ICP34.5, the domain that binds beclin1 and controls neurovirulence, are necessary for interactions with PGAM5, KEAP1, and other regulators of the antioxidant response, mitochondrial trafficking, and programmed cell death. We further show that while this domain interacts with multiple cellular stress response factors, it does not alter apoptosis or antioxidant gene expression. That said, the attenuated replication of a recombinant virus lacking residues 68 to 87 (termed Δ68-87) in primary human fibroblasts was restored by addition of ferric nitrate. Furthermore, in primary mouse neurons, the perinuclear localization of mitochondria that follows infection with HSV-1 was notably absent following Δ68-87 infection. Through this 20-amino-acid domain, ICP34.5 significantly reduces mitochondrial motility in axons of neurons. We propose the hypothesis that ICP34.5 promotes perinuclear mitochondrial localization by modulating transport of mitochondria through interaction with PGAM5. These data expand upon previous observations of altered mitochondrial dynamics following alphaherpesvirus infections and identify a key determinant of this activity during HSV-1 infections.IMPORTANCE Herpes simplex virus persists lifelong in neurons and can reactivate to cause recurrent lesions in mucosal tissues. A key determinant of virulence is the viral protein ICP34.5, of which residues 68 to 87 significantly contribute to neurovirulence through an unknown mechanism. Our report provides evidence that residues 68 to 87 of ICP34.5 are required for binding mitochondrion-associated factors. These interactions alter mitochondrial dynamics in neurons, thereby facilitating viral replication and pathogenesis.


Assuntos
Axônios/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Mitocôndrias/metabolismo , Proteínas Virais/metabolismo , Axônios/patologia , Axônios/virologia , Células HEK293 , Herpes Simples/patologia , Herpesvirus Humano 1/genética , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Domínios Proteicos , Transporte Proteico , Proteínas Virais/genética
12.
Phytomedicine ; 71: 153241, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32454347

RESUMO

BACKGROUND: Oxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF). AIMS: To investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation. MATERIALS AND METHODS: In vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2-/- were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection. RESULTS: Our results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2-/- HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2-/- mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2-/- mice. CONCLUSIONS: These investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Inflamassomos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Umbeliferonas/farmacologia , Acetaminofen/efeitos adversos , Animais , Proteínas de Transporte/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Hep G2 , Humanos , Inflamassomos/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo/fisiologia , Substâncias Protetoras/farmacologia
15.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32161178

RESUMO

The rabbit hemorrhagic disease virus (RHDV), which belongs to the family Caliciviridae and the genus Lagovirus, causes lethal fulminant hepatitis in rabbits. RHDV decreases the activity of antioxidant enzymes regulated by Nrf2 in the liver. Antioxidants are important for the maintenance of cellular integrity and cytoprotection. However, the mechanism underlying the regulation of the Nrf2-antioxidant response element (ARE) signaling pathway by RHDV remains unclear. Using isobaric tags for relative and absolute quantification (iTRAQ) technology, the current study demonstrated that RHDV inhibits the induction of ARE-regulated genes and increases the expression of the p50 subunit of the NF-κB transcription factor. We showed that RHDV replication causes a remarkable increase in reactive oxygen species (ROS), which is simultaneously accompanied by a significant decrease in Nrf2. It was found that nuclear translocation of Keap1 plays a key role in the nuclear export of Nrf2, leading to the inhibition of Nrf2 transcriptional activity. The p50 protein partners with Keap1 to form the Keap1-p50/p65 complex, which is involved in the nuclear translocation of Keap1. Moreover, upregulation of Nrf2 protein levels in liver cell nuclei by tert-butylhydroquinone (tBHQ) delayed rabbit deaths due to RHDV infection. Considered together, our findings suggest that RHDV inhibits the Nrf2-dependent antioxidant response via nuclear translocation of Keap1-NF-κB complex and nuclear export of Nrf2 and provide new insight into the importance of oxidative stress during RHDV infection.IMPORTANCE Recent studies have reported that rabbit hemorrhagic disease virus (RHDV) infection reduced Nrf2-related antioxidant function. However, the regulatory mechanisms underlying this process remain unclear. The current study showed that the NF-κB p50 subunit partners with Keap1 to form the Keap1-NF-κB complex, which plays a key role in the inhibition of Nrf2 transcriptional activity. More importantly, upregulated Nrf2 activity delayed the death of RHDV-infected rabbits, strongly indicating the importance of oxidative damage during RHDV infection. These findings may provide novel insights into the pathogenesis of RHDV.


Assuntos
Antioxidantes/metabolismo , Infecções por Caliciviridae/metabolismo , Vírus da Doença Hemorrágica de Coelhos/imunologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Animais , Elementos de Resposta Antioxidante , Antioxidantes/farmacologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/patologia , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Vírus da Doença Hemorrágica de Coelhos/patogenicidade , Humanos , Hidroquinonas , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Proteômica , Coelhos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA , Replicação Viral
16.
Environ Sci Pollut Res Int ; 27(13): 15663-15673, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32080815

RESUMO

Aspirin (ASA) is a widely used non-steroidal anti-inflammatory drug. Its high detection frequency in various waterborne and environmental residues has drawn wide attention. Limited information were provided for the effects of aspirin exposure on oxidative stress signaling pathway in fish, which is closely related to pathological and immunological process of fish. In this study, a small fish - Mugilogobius abei (M. abei) distributing widely in aquatic ecosystems in southern China, was employed as testing organism and the key genes of the detoxification metabolism were cloned for the first time. The responses of Nrf2/Keap1 signaling pathway were investigated under the environmentally relevant concentration aspirin exposure (0.5 µg L-1, 5 µg L-1, and 50 µg L-1) for 24 h, 72 h, and 168 h then. The transcriptional expression of the key genes (Nrf2, Keap1, GCLC, GPx, GST, SOD, CAT, Trx2, and TrxR) as well as the changes of the related enzymatic activities (GPx, GST, SOD, and CAT) and GSH and MDA content were also determined. Results showed that Nrf2 and Keap1 gene expression displayed a negative correlation to some extent under ASA exposure, the transcriptional expressions of the downstream related genes (GCLC, GST, SOD, CAT, Trx2, and TrxR) in Nrf2/Keap1 signaling pathway showed inhibition at 24 h but induction at 72 h and 168 h. At the protein level, ASA exposure can improve the antioxidant capacity by increasing GSH synthesis and enzymatic activity of GPx, GST, SOD, and CAT to reduce the degree of lipid peroxidation. We proposed that ASA exposure may interfere with the redox balance in M. abei at an early stage but sub-chronic ASA exposure can activate the Nrf2 signaling pathway to improve the antioxidant capacity of M. abei.


Assuntos
Aspirina , Fator 2 Relacionado a NF-E2 , Animais , Antioxidantes , Atenção , China , Ecossistema , Regulação da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Estresse Oxidativo , Transdução de Sinais
17.
Mycotoxin Res ; 36(3): 287-299, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32076947

RESUMO

Deoxynivalenol (DON) is a type B trichothecenes that is widely contaminating human and animal foods, leading to several toxicological implications if ingested. Induction of oxidative stress and production of lipid peroxides were suggested to be the reasons for DON-induced cytotoxicity. However, detailed and comprehensive profiling of DON-related lipid hydroperoxides was not identified. Furthermore, the mechanisms behind DON-induced cytotoxicity and oxidative stress have received less attention. Zinc (Zn) is an essential element that has antioxidant activities; however, the protective effects of Zn against DON-induced adverse effects were not examined. Therefore, this study was undertaken to investigate DON-induced cytotoxicity and oxidative damage to human HepG2 cell lines. Furthermore, a quantitative estimation for the formed lipid hydroperoxides was conducted using LC-MS/MS. In addition, DON-induced transcriptomic changes on the inflammatory markers and antioxidant enzymes were quantitatively examined using qPCR. The protective effects of Zn against DON-induced cytotoxicity and oxidative stress, the formation of lipid hydroperoxides (LPOOH), and antioxidant status in HepG2 cells were investigated. Finally, the effects of DON and Zn on the Nrf2-Keap1 pathway were further explored. The achieved results indicated that DON caused significant cytotoxicity in HepG2 cells accompanied by significant oxidative damage and induction of the inflammatory markers. Identification of DON-related LPOOH revealed the formation of 22 LPOOH species including 14 phosphatidylcholine hydroperoxides, 5 triacylglycerol hydroperoxides, and 3 cholesteryl ester hydroperoxides. DON caused significant downregulation of Nrf2-regulated antioxidant enzymes. Zn administration led to significant protection of HepG2 cells against DON-induced adverse effects, probably via activation of the Nrf2-Keap1 pathway.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Peróxidos Lipídicos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tricotecenos/toxicidade , Zinco/farmacologia , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Peróxidos Lipídicos/química , Fator 2 Relacionado a NF-E2/genética , Oxirredução/efeitos dos fármacos
18.
Biochem Biophys Res Commun ; 524(4): 895-902, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057361

RESUMO

The transcription factor NRF2 plays a key role in the protection against environmental stress and maintaining cellular homeostasis. The acetyltransferase p300 is a known component of the NRF2 transcriptional complex and promotes its transcriptional activity. In this study we describe a novel mechanism by which p300 facilitates NRF2 activity. p300 physically interacts with NRF2 and interferes with NRF2-KEAP1 complex formation. In particular, p300 increases NRF2 protein abundance and stability, thereby promoting NRF2 nuclear localization. Notably, the acetyltransferase activity of p300 was indispensable for the stabilizing effects towards NRF2. Furthermore, overexpression of p300 protected HEK293T cells from oxidative stress and increased viability. Together our study uncovers a link between p300 and control of NRF2-KEAP1 signaling via regulation of NRF2 stability and this may act as a novel checkpoint on the adaptation to oxidative stress.


Assuntos
Regulação da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fatores de Transcrição de p300-CBP/genética , Adaptação Fisiológica , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Transcrição Genética , Fatores de Transcrição de p300-CBP/deficiência
19.
J Endocrinol ; 245(1): 129-140, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32031966

RESUMO

Over a half of the diabetic individuals develop macrovascular complications that cause high mortality. Oxidative stress (OS) promotes endothelial dysfunction (ED) which is a critical early step toward diabetic macrovascular complications. Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular antioxidant defense system and combats diabetes-induced OS. Previously, we found that impaired NRF2 antioxidant signaling contributed to diabetes-induced endothelial OS and dysfunction in mice. The present study has investigated the effect of microRNA-200a (miR-200a) on NRF2 signaling and diabetic ED. In aortic endothelial cells (ECs) isolated from C57BL/6 wild-type (WT) mice, high glucose (HG) reduced miR-200a levels and increased the expression of kelch-like ECH-associated protein 1 (Keap1) - a target of miR-200a and a negative regulator of NRF2. This led to the inactivation of NRF2 signaling and exacerbation of OS and inflammation. miR-200a mimic (miR-200a-M) or inhibitor modulated KEAP1/NRF2 antioxidant signaling and manipulated OS and inflammation under HG conditions. These effects were completely abolished by knockdown of Keap1, indicating that Keap1 mRNA is a major target of miR-200a. Moreover, the protective effect of miR-200a-M was completely abrogated in aortic ECs isolated from C57BL/6 Nrf2 knockout (KO) mice, demonstrating that NRF2 is required for miR-200a's actions. In vivo, miR-200a-M inhibited aortic Keap1 expression, activated NRF2 signaling, and attenuated hyperglycemia-induced OS, inflammation and ED in the WT, but not Nrf2 KO, mice. Therefore, the present study has uncovered miR-200a/KEAP1/NRF2 signaling that controls aortic endothelial antioxidant capacity, which protects against diabetic ED.


Assuntos
Complicações do Diabetes/genética , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch/genética , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Animais , Antioxidantes , Complicações do Diabetes/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/fisiopatologia , Inflamação/complicações , Inflamação/genética , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Transdução de Sinais/genética
20.
Curr Diabetes Rev ; 16(8): 797-806, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32000646

RESUMO

Diabetes mellitus is associated with an increased risk of micro and macrovascular complications. During hyperglycemic conditions, endothelial cells and vascular smooth muscle cells are exquisitely sensitive to high glucose. This high glucose-induced sustained reactive oxygen species production leads to redox imbalance, which is associated with endothelial dysfunction and vascular wall remodeling. Nrf2, a redox-regulated transcription factor plays a key role in the antioxidant response element (ARE)-mediated expression of antioxidant genes. Although accumulating data indicate the molecular mechanisms underpinning the Nrf2 regulated redox balance, understanding the influence of the Nrf2/ARE axis during hyperglycemic condition on vascular cells is paramount. This review focuses on the context-dependent role of Nrf2/ARE signaling on vascular endothelial and smooth muscle cell function during hyperglycemic conditions. This review also highlights improving the Nrf2 system in vascular tissues, which could be a potential therapeutic strategy for vascular dysfunction.


Assuntos
Elementos de Resposta Antioxidante/genética , Diabetes Mellitus Tipo 2/genética , Células Endoteliais/metabolismo , Hiperglicemia/genética , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/genética , Animais , Elementos de Resposta Antioxidante/fisiologia , Antioxidantes/metabolismo , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Epigênese Genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Hiperglicemia/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Fosfotransferases/genética , Fosfotransferases/metabolismo , Espécies Reativas de Oxigênio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
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