RESUMO
GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.
Assuntos
Proteínas Serina-Treonina Quinases , Animais , Humanos , Camundongos , Aminoácidos/metabolismo , Homeostase , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismoRESUMO
Sulforaphane (SFN) is a potent activator of the transcriptional factor, Nuclear Factor Erythroid 2 (NF-E2)-Related factor 2 (NRF2). SFN and its precursor, glucoraphanin (sulforaphane glucosinolate, SGS), have been shown to ameliorate cognitive function in clinical trials and in vivo studies. However, the effects of SGS on age-related cognitive decline in Senescence-Accelerated Mouse Prone 8 (SAMP8) is unknown. In this study, we determined the preventive potential of SGS on age-related cognitive decline. One-month old SAMP8 mice or control SAM resistance 1 (SAMR1) mice were fed an ad libitum diet with or without SGS-containing broccoli sprout powder (0.3% w/w SGS in diet) until 13 months of age. SGS significantly improved long-term memory in SAMP8 at 12 months of age. Interestingly, SGS increased hippocampal mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α) and mitochondrial transcription factor A (TFAM), which are master regulators of mitochondrial biogenesis, both in SAMR1 and SAMP8 at 13 months of age. Furthermore, mRNAs for nuclear respiratory factor-1 (NRF-1) and mitochondrial DNA-encoded respiratory complex enzymes, but not mitochondrial DNA itself, were increased by SGS in SAMP8 mice. These results suggest that SGS prevents age-related cognitive decline by maintaining mitochondrial function in senescence-accelerated mice.
Assuntos
Disfunção Cognitiva , Biogênese de Organelas , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , DNA/metabolismo , Expressão Gênica , Hipocampo/metabolismo , Isotiocianatos , Camundongos , SulfóxidosRESUMO
GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial ß-oxidation that accompanied increases of peroxisomal ß-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.
Assuntos
Proteínas de Saccharomyces cerevisiae , Animais , Lipídeos , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Knockout , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases , Proteômica , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tamoxifeno/efeitos adversos , Tamoxifeno/metabolismo , Transativadores/metabolismo , Redução de PesoRESUMO
Jun dimerization protein 2 (JDP2) is a bZip-type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4), enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Repressoras/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Repressoras/genética , Células Tumorais CultivadasRESUMO
PURPOSE: Pulse wave velocity (PWV), an indicator of vascular stiffness, increases with age and is increasingly recognized as an independent risk factor for cardiovascular disease (CVD). Although many mechanical and chemical factors underlie the stiffness of the elastic artery, genetic risk factors related to age-dependent increases in PWV in apparently healthy people are largely unknown. The transcription factor nuclear factor E2 (NF-E2)-related factor 2 (Nrf2), which is activated by unidirectional vascular pulsatile shear stress or oxidative stress, regulates vascular redox homeostasis. Previous reports have shown that a SNP in the NRF2 gene regulatory region (-617C>A; hereafter called SNP-617) affects NRF2 gene expression such that the minor A allele confers lower gene expression compared to the C allele, and it is associated with various diseases, including CVD. We aimed to investigate whether SNP-617 affects vascular stiffness with aging in apparently healthy people. METHODS: Analyzing wide-ranging data obtained from a public health survey performed in Japan, we evaluated whether SNP-617 affected brachial-ankle PWV (baPWV) in never-smoking healthy subjects (n = 642). We also evaluated the effects of SNP-617 on other cardiovascular and blood test measurements. RESULTS: We have shown that not only AA carriers (n = 55) but also CA carriers (n = 247) show arterial stiffness compared to CC carriers (n = 340). Furthermore, SNP-617 also affected blood pressure indexes such as systolic blood pressure and mean arterial pressure but not the ankle brachial pressure index, an indicator of atherosclerosis. Multivariate analysis showed that SNP-617 accelerates the incremental ratio of baPWV with age. CONCLUSIONS: This study is the first to show that SNP-617 affects the age-dependent increase in vascular stiffness. Our results indicate that low NRF2 activity induces premature vascular aging and could be targeted for the prevention of cardiovascular diseases associated with aging.
Assuntos
Envelhecimento , Fator 2 Relacionado a NF-E2/genética , Rigidez Vascular/fisiologia , Adulto , Alelos , Índice Tornozelo-Braço , Aterosclerose/genética , Aterosclerose/patologia , Pressão Sanguínea , Frequência do Gene , Genótipo , Inquéritos Epidemiológicos , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Análise de Onda de Pulso , FumarRESUMO
Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.
Assuntos
Ciclo Celular , Proliferação de Células , Desenvolvimento Fetal , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico , Transativadores/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Ciclina B1/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Transativadores/genéticaRESUMO
Reactive oxygen species (ROS) are byproducts of aerobic respiration and signaling molecules that control various cellular functions. Nrf2 governs the gene expression of endogenous antioxidant synthesis and ROS-eliminating enzymes in response to various electrophilic compounds that inactivate the negative regulator Keap1. Accumulating evidence has shown that mitochondrial ROS (mtROS) activate Nrf2, often mediated by certain protein kinases, and induce the expression of antioxidant genes and genes involved in mitochondrial quality/quantity control. Mild physiological stress, such as caloric restriction and exercise, elicits beneficial effects through a process known as "mitohormesis." Exercise induces NOX4 expression in the heart, which activates Nrf2 and increases endurance capacity. Mice transiently depleted of SOD2 or overexpressing skeletal muscle-specific UCP1 exhibit Nrf2-mediated antioxidant gene expression and PGC1α-mediated mitochondrial biogenesis. ATF4 activation may induce a transcriptional program that enhances NADPH synthesis in the mitochondria and might cooperate with the Nrf2 antioxidant system. In response to severe oxidative stress, Nrf2 induces Klf9 expression, which represses mtROS-eliminating enzymes to enhance cell death. Nrf2 is inactivated in certain pathological conditions, such as diabetes, but Keap1 down-regulation or mtROS elimination rescues Nrf2 expression and improves the pathology. These reports aid us in understanding the roles of Nrf2 in pathophysiological alterations involving mtROS.
Assuntos
Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/genética , Ativação TranscricionalRESUMO
: Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 µM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert-butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.
Assuntos
Abietanos/farmacologia , Fator 4 Ativador da Transcrição/genética , Citoproteção/genética , Regulação da Expressão Gênica , Fator 2 Relacionado a NF-E2/genética , Fator 4 Ativador da Transcrição/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroquinonas/farmacologia , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Tunicamicina/farmacologiaRESUMO
OBJECTIVES: Desmoplastic changes of extracellular matrix (ECM) containing large amounts of hyaluronan (HA) are of interest in chemo- and immunoresistance of pancreatic ductal adenocarcinoma (PDAC). The goal of this study was to evaluate the effects of 4-methylumbelliferone (MU), a selective inhibitor of HA, on ECM and to examine how MU affects adoptive immunotherapy. METHODS: The effect of MU on cell proliferation, HA synthesis and formation of ECM were investigated in four PDAC cell lines. In addition, the cytotoxicity of γδ T-cell-rich peripheral blood mononuclear cells (PBMCs) collected from healthy donors and stimulated with zoledronate and interleukin-2 was examined in the presence of MU. The amount of HA and tumor-infiltrating lymphocytes were also investigated in mice xenograft models. RESULTS: In vitro, 1.0 mM MU inhibited cell proliferation by 45-70% and HA synthesis by 55-80% in all four PDAC cell lines, and enhanced γδ T-cell-rich PBMC-mediated cytotoxicity against PDAC cells. In vivo, MU reduced intratumoral HA and promoted infiltration of inoculated γδ T-cells into tumor tissue, and consequently suppressed tumor growth. CONCLUSIONS: 4-methylumbelliferone may be an effective immunosensitizer against PDAC through induction of structural changes in the ECM.
Assuntos
Carcinoma Ductal Pancreático/imunologia , Linfócitos Intraepiteliais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/imunologia , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Himecromona/farmacologia , Interleucina-2/farmacologia , Linfócitos Intraepiteliais/efeitos dos fármacos , Linfócitos Intraepiteliais/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Ácido Zoledrônico/farmacologiaRESUMO
Iron has played an important role in energy production since the beginning of life, as iron-catalyzed redox reactions are required for energy production. Oxygen, a highly efficient electron acceptor with high reduction potential, facilitates highly efficient energy production in eukaryotic cells. However, the increasing atmospheric oxygen concentration produces new threats to the organism, as oxygen reacts with iron and produces reactive oxygen species unless its levels are strictly regulated. As the size of multicellular organisms increases, these organisms must transport oxygen to the peripheral tissues and begin to employ red blood cells containing hemoglobin. This system is potentially a double-edged sword, as hemoglobin autoxidation occurs at a certain speed and releases free iron into the cytoplasm. Nrf2 belongs to the CNC transcription factor family, in which NF-E2p45 is the founding member. NF-E2p45 was first identified as a transcription factor that binds to the erythroid gene regulatory element NF-E2 located in the promoter region of the heme biosynthetic porphobilinogen deaminase gene. Human Nrf2 was also identified as a transcription factor that binds to the regulatory region of the ß-globin gene. Despite these original findings, NF-E2p45 and Nrf2 knockout mice exhibit few erythroid phenotypes. Nrf2 regulates the expression of a wide range of antioxidant and detoxification enzymes. In this review article, we describe and discuss the roles of Nrf2 in various iron-mediated bioreactions and its possible coevolution with iron and oxygen.
RESUMO
Abnormal α-synuclein is deposited in neuronal cytoplasmic inclusions and presynapses in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NUB1 is accumulated in these specific regions together with abnormal α-synuclein and that NUB1 is able to inhibit α-synuclein aggregation in cultured cells. We therefore created transgenic (Tg) mice expressing both NUB1 and abnormal α-synuclein to investigate the role of NUB1 on degradation of abnormal α-synuclein in vivo. Immunohistochemical and biochemical studies confirmed that NUB1 was over-expressed in neurons of mice expressing NUB1 (NUB1 Tg), and both NUB1 and abnormal α-synuclein (double Tg). NUB1 levels were increased by 4.7-fold in NUB1 Tg mice compared with wild type mice. Unexpectedly, normal and abnormal α-synuclein levels were unchanged between abnormal α-synuclein Tg mice (Lewy body disease model mice) and double Tg mice, and pathological observations were almost similar between them. Finally, we found that the levels of insoluble α-synuclein were lower and those of some chaperone molecules were higher in double Tg mice compared with abnormal α-synuclein Tg mice. These results suggest that increased levels of NUB1 play a potential role in degradation of detergent-insoluble α-synuclein in vivo, although it is insufficient to degrade abnormal α-synuclein in Lewy body disease model mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Doença por Corpos de Lewy/metabolismo , alfa-Sinucleína/metabolismo , Animais , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The accumulation of mis-folded and/or abnormally modified proteins is a major characteristic of many neurodegenerative diseases. In Lewy body disease (LBD), which includes Parkinson's disease and dementia with Lewy bodies, insoluble α-synuclein is widely deposited in the presynaptic terminals as well as in the neuronal cytoplasm in distinct brain regions. It is well known that the autophagy-lysosome system serves as an efficient degradation pathway for abnormal molecules within cells. To test the possibility that activated autophagy can degrade abnormal molecules, we investigated the effect of trehalose on abnormal aggregation of α-synuclein in a model of LBD. Trehalose is a natural disaccharide composed of two glucose units and functions as an autophagy inducer. Consistent with previous studies, trehalose increased level of the autophagosomal protein LC3, especially a lipidated form LC3-II in cultured cells and mice brain. Also, trehalose increased levels of several chaperon molecules, such as HSP90 and SigmaR1, in the brains of LBD model mice. Further studies revealed that level of detergent-insoluble α-synuclein was suppressed in mice following oral administration of trehalose, despite an apparent alteration was not observed regarding abnormal aggregation of α-synuclein. These results suggest that the oral intake of trehalose modulates propensity of molecules prior to aggregation formation.
Assuntos
Autofagia/efeitos dos fármacos , Doença por Corpos de Lewy/tratamento farmacológico , Chaperonas Moleculares/biossíntese , Trealose/administração & dosagem , Administração Oral , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Proteínas de Choque Térmico HSP90/biossíntese , Células HeLa , Humanos , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Maltose/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/biossíntese , Agregação Patológica de Proteínas/prevenção & controle , Receptores sigma/biossíntese , Solubilidade , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Receptor Sigma-1RESUMO
Atherosclerosis is a chronic inflammatory disease of the vascular arterial walls. A number of studies have revealed the biological and genetic bases of atherosclerosis, and over 100 genes influence atherosclerosis development. Nrf2 plays an important role in oxidative stress response and drug metabolism, but the Nrf2 signaling pathway is closely associated with atherosclerosis development. During atherosclerosis progression, Nrf2 signaling modulates many physiological and pathophysiological processes, such as lipid homeostasis regulation, foam cell formation, macrophage polarization, redox regulation and inflammation. Interestingly, Nrf2 exhibits both pro- and anti-atherogenic effects in experimental animal models. These observations make the Nrf2 pathway a promising target to prevent atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Aterosclerose/fisiopatologia , Humanos , Camundongos , Transdução de Sinais/fisiologiaRESUMO
In Lewy body disease (LBD) such as dementia with LBs and Parkinson's disease, several lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly involved with intracellular proteolysis and is a component of ubiquitin-positive inclusions in various neurodegenerative disorders. However, it is not clear whether p62 deficiency affects inclusion formation and abnormal protein accumulation. To answer this question, we used a mouse model of LBD that lacks p62, and found that LB-like inclusions were observed in transgenic mice that overexpressed α-synuclein (Tg mice) with or without the p62 protein. p62 deficiency enhanced α-synuclein pathology with regard to the number of inclusions and staining intensity compared with Tg mice that expressed p62. To further investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we assessed the mRNA and protein levels of several molecules, and found that the neighbor of the brca1 gene (NBr1), which is functionally and structurally similar to p62, is increased in Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative modulation of α-synuclein aggregation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Encéfalo/patologia , Proteínas de Choque Térmico/deficiência , Corpos de Inclusão/patologia , Doença por Corpos de Lewy/patologia , alfa-Sinucleína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Encéfalo/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Corpos de Inclusão/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Doença por Corpos de Lewy/metabolismo , Camundongos , Camundongos Transgênicos , Atividade Motora , Proteínas/metabolismo , Proteólise , Proteína Sequestossoma-1 , Estresse Fisiológico , alfa-Sinucleína/genéticaRESUMO
Amyloid-beta (Aß) peptides, Aß 1-42 (Aß42) and Aß43 in particular, cause neurotoxicity and cell death in the brain of Alzheimer's disease (AD) at higher concentrations. Carnosic acid (CA), a phenolic diterpene compound in the labiate herbs rosemary and sage, serves as an activator for neuroprotective and neurotrophic functions in brain cells. We investigated the effect of CA on apoptosis induced by Aß42 or Aß43 in cultured SH-SY5Y human neuroblastoma cells. Treatment of the cells with Aß42 or Aß43 (monomer, 10 µM each) induced apoptosis, which was confirmed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF). Concurrently, the Aß treatment induced the activation of caspase (Casp) cascades including an effector Casp (Casp3) and initiator Casps (Casp4, Casp8 and Casp9). Pretreatment of the cells with CA (10 µM) partially attenuated the apoptosis induced by Aß42 or Aß43. CA pretreatment also reduced the cellular oligomers of Aß42 and Aß43. These results suggest that CA suppressed the activation of Casp cascades by reducing the intracellular oligomerization of exogenous Aß42/43 monomer. The ingestion of an adequate amount of CA may have a potential in the prevention of Aß-mediated diseases, particularly AD.
Assuntos
Abietanos/farmacologia , Peptídeos beta-Amiloides/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Extratos Vegetais/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de TempoRESUMO
Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5' to 3') eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction.
Assuntos
Elementos Facilitadores Genéticos , Heme Oxigenase-1/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/metabolismo , Ativação Transcricional , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células HeLa , Heme Oxigenase-1/biossíntese , Humanos , Maleatos/farmacologia , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , RNA não Traduzido/biossíntese , RNA não Traduzido/genéticaRESUMO
The ubiquitin-proteasome pathway degrades ubiquitinated proteins to remove damaged or misfolded protein and thus plays an important role in the maintenance of many important cellular processes. Because the pathway is also crucial for tumor cell growth and survival, proteasome inhibition by specific inhibitors exhibits potent antitumor effects in many cancer cells. xCT, a subunit of the cystine antiporter system xc (-), plays an important role in cellular cysteine and glutathione homeostasis. Several recent reports have revealed that xCT is involved in cancer cell survival; however, it was unknown whether xCT affects the cytotoxic effects of proteasome inhibitors. In this study, we found that two stress-inducible transcription factors, Nrf2 and ATF4, were upregulated by proteasome inhibition and cooperatively enhance human xCT gene expression upon proteasome inhibition. In addition, we demonstrated that the knockdown of xCT by small interfering RNA (siRNA) or pharmacological inhibition of xCT by sulfasalazine (SASP) or (S)-4-carboxyphenylglycine (CPG) significantly increased the sensitivity of T24 cells to proteasome inhibition. These results suggest that the simultaneous inhibition of both the proteasome and xCT could have therapeutic benefits in the treatment of bladder tumors.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Antineoplásicos/farmacologia , Cisteína/metabolismo , Glutationa/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glicina/análogos & derivados , Glicina/farmacologia , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Oligopeptídeos/farmacologia , Pirazinas/farmacologia , RNA Interferente Pequeno/farmacologia , Sulfassalazina/farmacologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Extensive research on p62 has established its role in oxidative stress, protein degradation and in several diseases such as Paget's disease of the bone, frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Importantly, previous studies showed that p62 binds directly to Keap1, which is a ubiquitin E3 ligase responsible for degrading Nrf2. Indeed, colocalisation of p62 and Keap1 occurs in tumorigenesis and neurodegeneration. A serine (S) residue in the Keap1-interacting region of p62 is phosphorylated in hepatocellular carcinoma, and this phosphorylation contributes to tumour growth through the higher affinity of p62 to Keap1. However, it remains largely unknown whether p62 is phosphorylated in the Keap1-interacting region under neurodegenerative conditions. RESULTS: To answer this question, we generated an antibody against phosphorylated S349 (P-S349) of p62 and showed that S349 is phosphorylated following disruption of protein degradation. In particular, the ratio of P-S349 to total p62 levels was significantly increased in the brains with Alzheimer's disease (AD) compared with controls. We also compared the reactivity of the P-S349 antibody with P-S403 of p62 and showed that these two phosphorylated sites on p62 cause different responses with proteasome inhibition and show distinct localisation patterns in AD brains. In addition to disruption of protein degradation systems, activation of oxidative stress can induce P-S349. CONCLUSION: These results support the hypothesis that disruption of protein degradation systems and sustained activation of the Keap1-Nrf2 system occur in the brains with AD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Serina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/farmacologia , Encéfalo/citologia , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Masculino , Pessoa de Meia-Idade , Neuroblastoma/patologia , Emaranhados Neurofibrilares/metabolismo , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Sequestossoma-1RESUMO
Amyloid beta (Aß) peptides are key molecules in the pathogenesis of Alzheimer's disease (AD). The sequential cleavage of amyloid precursor protein (APP) by the ß- and γ-secretases generates Aß peptides; however, the alternate cleavage of APP by the α- and γ-secretases decreases Aß production. We previously reported that carnosic acid (CA), a phenolic diterpene compound found in the labiate herbs rosemary and sage, suppresses Aß (1-40 and 1-42) production by activating α-secretase in cultured SH-SY5Y human neuroblastoma cells (Neurosci. Res. 2013; 75: 94-102). Here, we investigated the effect of CA on the production of Aß peptides (1-40, 1-42 and 1-43) in U373MG human astrocytoma cells. The treatment of cells with CA suppressed Aß40/42/43 release (55-71% decrease at 50µM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17); however, the ß-secretase BACE1 (ß-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα release enhanced by CA and partially recovered the CA-suppressed Aß40/42/43 release. These results suggest that CA reduces Aß production, at least partially, by activating TACE in human astroglial cells. The use of CA may have a potential in the prevention of Aß-mediated diseases.