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1.
Nat Commun ; 10(1): 2745, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227705

RESUMO

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.


Assuntos
Descoberta de Drogas/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Sondas Moleculares/química , Tiorredoxina Redutase 1/metabolismo , Animais , Produtos Biológicos/farmacologia , Misturas Complexas , Dissulfetos/química , Corantes Fluorescentes/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/metabolismo , Oxirredução , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/genética
2.
Nutrients ; 11(4)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925798

RESUMO

The application of plant extracts for therapeutic purposes has been used in traditional medicine since the plants are a source of a great variety of chemical compounds that possess biological activity. Actually, the effect of these extracts on diseases such as cancer is being widely studied. Colorectal adenocarcinoma is one of the main causes of cancer related to death and the second most prevalent carcinoma in Western countries. The aim of this work is to study the possible effect of two fenugreek (Trigonella foenum graecum) protein hydrolysates on treatment and progression of colorectal cancer. Fenugreek proteins from seeds were hydrolysed by using two enzymes separately, which are named Purafect and Esperase, and were then tested on differentiated and undifferentiated human colonic adenocarcinoma Caco2/TC7 cells. Both hydrolysates did not affect the growth of differentiated cells, while they caused a decrease in undifferentiated cell proliferation by early apoptosis and cell cycle arrest in phase G1. This was triggered by a mitochondrial membrane permeabilization, cytochrome C release to cytoplasm, and caspase-3 activation. In addition, the hydrolysates of fenugreek proteins displayed antioxidant activity since they reduce the intracellular levels of ROS. These findings suggest that fenugreek protein hydrolysates could be used as nutraceutical molecules in colorectal cancer treatment.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Suplementos Nutricionais , Proteínas de Plantas/química , Hidrolisados de Proteína/farmacologia , Trigonella/química , Apoptose/efeitos dos fármacos , Células CACO-2 , Caspase 3 , Neoplasias do Colo/metabolismo , Citocromos c , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrolisados de Proteína/química , Espécies Reativas de Oxigênio , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo
3.
J Immunol Res ; 2018: 6140320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30225271

RESUMO

Thioredoxin reductase (TrxR), an antioxidant enzyme dependent on nicotinamide adenine dinucleotide phosphate, plays a vital role in defense against oxidative stress. However, the role of microRNAs targeting TrxR under oxidative stress has not yet been determined. In this study, we tested the involvement of miRNA-mediated posttranscriptional regulation in H2O2-induced TrxR1 expression in endothelial cells. Dual luciferase assay combined with expression analysis confirmed that miR-125a suppressed TrxR1 expression by targeting its 3'-UTR. Furthermore, H2O2 induced TrxR1 expression partly through downregulation of miR-125a. These findings indicate that miRNA-mediated posttranscriptional mechanism is involved in H2O2-induced TrxR1 expression in endothelial cells, suggesting an important role of miRNAs in the response to oxidative stress.


Assuntos
Endotélio Vascular/fisiologia , MicroRNAs/genética , Tiorredoxina Redutase 1/genética , Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Peróxido de Hidrogênio/metabolismo , NADP/metabolismo , Estresse Oxidativo , Processamento Pós-Transcricional do RNA , Tiorredoxina Redutase 1/metabolismo
4.
Biol Trace Elem Res ; 185(1): 11-19, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29247444

RESUMO

Arsenic (As) is a known toxic element and carcinogen. Transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) controls cellular adaptation to oxidants and electrophiles by inducing antioxidant genes in response to redox stress. To explore associations between As level and NRF2-regulated cytoprotective genes expression, an observational study was conducted in a population of 61 occupationally exposed men with median (Me) age 50 years (interquartile range (IQR) 42-54) and in a control group of 52 men aged 40 (IQR 31-51.5) without occupational exposure. NRF2, KEAP1, GSTP1, HMOX1, NQO1, PRDX1, and TXNRD1 transcript levels were determined by means of quantitative real-time PCR along with the gene expression, methylation of NRF2 and KEAP1, as well as global DNA methylation were assessed. The median urine As tot. level in the exposed and control group was found to be 21.8 µg/g creat. (IQR 15.5-39.8 µg/g creat.) and 3.8 µg/g creat. (IQR 2.5-9.3) (p < 0.001). Global DNA methylation was significantly higher in occupationally exposed workers than in controls (Me 14.1 (IQR 9.5-18.1) vs Me 8.5 (IQR 5.9-12.6) p < 0.0001). NRF2 mRNA level was positively correlated with expression of all investigated NRF2-target genes in both groups (0.37 > R < 0.76, all p values < 0.0001). The multivariate linear regression adjusting for global methylation showed that As(III) level was significantly associated with expression of TXNRD1, GSTP1, HMOX1, and PRDX1. The results of this study indicate that arsenic occupational exposure is positively associated with global DNA methylation. The findings provide evidence for rather inactivation of NRF2-KEAP1 pathway in response to chronic arsenic exposure.


Assuntos
Arsênico/toxicidade , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Adulto , Arsênico/urina , Metilação de DNA/genética , Metilação de DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glutationa S-Transferase pi/genética , Heme Oxigenase-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/genética , Exposição Ocupacional/efeitos adversos , Peroxirredoxinas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiorredoxina Redutase 1/genética
5.
PLoS One ; 12(11): e0187934, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29121093

RESUMO

OBJECTIVE: Omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), likely prevent cardiovascular disease, however their mechanisms remain unclear. Recently, the role of DNA damage in atherogenesis has been receiving considerable attention. Here, we investigated the effects of EPA and DHA on DNA damage in vascular endothelial cells to clarify their antiatherogenic mechanisms. METHODS AND RESULTS: We determined the effect of EPA and DHA on H2O2-induced DNA damage response in human aortic endothelial cells. Immunofluorescence staining showed that γ-H2AX foci formation, a prominent marker of DNA damage, was significantly reduced in the cells treated with EPA and DHA (by 47% and 48%, respectively). H2O2-induced activation of ATM, a major kinase orchestrating DNA damage response, was significantly reduced with EPA and DHA treatment (by 31% and 33%, respectively). These results indicated EPA and DHA attenuated DNA damage independently of the DNA damage response. Thus the effects of EPA and DHA on a source of DNA damage were examined. EPA and DHA significantly reduced intracellular reactive oxygen species under both basal condition and H2O2 stimulation. In addition, the mRNA levels of antioxidant molecules, such as heme oxygenase-1, thioredoxin reductase 1, ferritin light chain, ferritin heavy chain and manganese superoxide dismutase, were significantly increased with EPA and DHA. Silencing nuclear factor erythroid 2-related factor 2 (NRF2) remarkably abrogated the increases in mRNA levels of antioxidant molecules and the decrease in intracellular reactive oxygen species. Furthermore, EPA and DHA significantly reduced H2O2-induced senescence-associated ß-galactosidase activity in the cells (by 31% and 22%, respectively), which was revoked by NRF2 silencing. CONCLUSIONS: Our results suggested that EPA and DHA attenuate oxidative stress-induced DNA damage in vascular endothelial cells through upregulation of NRF2-mediated antioxidant response. Therefore omega-3 fatty acids likely help prevent cardiovascular disease, at least in part, by their genome protective properties.


Assuntos
Células Endoteliais/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Óleos de Peixe/farmacologia , Peróxido de Hidrogênio/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Humanos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Tiorredoxina Redutase 1/genética
6.
J Infect Dis ; 216(4): 477-488, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28931237

RESUMO

NQO1 and TRXR1 are important host reductases implicated in the regulation of inflammation and apoptosis. Although the transcriptional machinery governing these processes have been extensively investigated, the associated epigenetic regulatory events remain unclear. Here, we report that SET8, a histone H4 lysine 20 monomethylase (H4K20me1), is highly induced during Mycobacterium tuberculosis infection that orchestrates immune evasion strategies through the induction of NQO1 and TRXR1 in vivo. SET8, along with FoxO3a, mediates an active NQO1-PGC1-α complex, which promotes the anti-inflammatory M2 macrophage phenotype, and assists TRXR1-regulated arrest of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Strikingly, the loss-of-function analysis in an in vivo mouse tuberculosis model further corroborated the pivotal role of SET8-responsive NQO1 and TRXR1 in mycobacterial survival. Thus, augmenting host immune responses against Mycobacterium tuberculosis by harnessing the SET8-NQO1/TRXR1 axis with its specific and potent inhibitors could lead to promising host-directed therapeutic adjuvants for tuberculosis treatment.


Assuntos
Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/imunologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Humanos , Evasão da Resposta Imune , Leucócitos Mononucleares/microbiologia , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Células RAW 264.7 , Reprodutibilidade dos Testes , Transdução de Sinais , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Tuberculose/microbiologia
7.
Free Radic Biol Med ; 112: 277-286, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28774816

RESUMO

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.


Assuntos
Derivados de Benzeno/farmacologia , Carcinógenos/farmacologia , Fibroblastos/efeitos dos fármacos , PTEN Fosfo-Hidrolase/química , Tiorredoxina Redutase 1/metabolismo , Tiorredoxinas/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , NADP/metabolismo , Oxirredução , Estresse Oxidativo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxina Redutase 1/genética , Tiorredoxinas/genética
8.
J Biol Chem ; 292(35): 14371-14380, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28684416

RESUMO

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H2O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2O2 Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways.


Assuntos
NADP/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Tiorredoxina Redutase 1/metabolismo , Animais , Auranofina/farmacologia , Domínio Catalítico , Células Cultivadas , Dimerização , Embrião de Mamíferos/citologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Oxidantes/farmacologia , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Ratos , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Selenocisteína/química , Selenocisteína/metabolismo , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/química , Tiorredoxina Redutase 1/genética , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
9.
Biomed Res Int ; 2017: 4698167, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28536696

RESUMO

Thioredoxin reductase 1 (TXNRD1) which is a selenocysteine-containing protein is overexpressed in many malignancies. Its role in the hepatocellular carcinoma (HCC) prognosis has not been investigated. In this study, we investigated whether TXNRD1 functions as an independent prognostic factor for HCC patients. We found TXNRD1 was overexpressed in HCC tissues and cells, immunohistochemical analysis suggested TXNRD1 was elevated in 57 of 120 (47.5%) clinical samples, and its level was increased with the increasing clinical stage. In addition, TXNRD1 expression was positively correlated with clinical stage (p = 3.5e - 5), N classification (p = 4.4e - 4), and M classification (p = 0.037) of HCC patients. Kaplan-Meier analysis revealed that patients with high TXNRD1 expression had significantly shorter survival time than patients with low TXNRD1 expression. Multivariate analysis found TXNRD1 was an independent prognostic factor for HCC patients. In conclusion, our data suggested that TXNRD1 was a biomarker for the prognosis of patients with HCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Tiorredoxina Redutase 1/genética , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico
10.
Aging Cell ; 16(4): 683-692, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28474396

RESUMO

In a survey of enzymes related to protein oxidation and cellular redox state, we found activity of the redox enzyme thioredoxin reductase (TXNRD) to be elevated in cells from long-lived species of rodents, primates, and birds. Elevated TXNRD activity in long-lived species reflected increases in the mitochondrial form, TXNRD2, rather than the cytosolic forms TXNRD1 and TXNRD3. Analysis of published RNA-Seq data showed elevated TXNRD2 mRNA in multiple organs of longer-lived primates, suggesting that the phenomenon is not limited to skin-derived fibroblasts. Elevation of TXNRD2 activity and protein levels was also noted in liver of three different long-lived mutant mice, and in normal male mice treated with a drug that extends lifespan in males. Overexpression of mitochondrial TXNRD2 in Drosophila melanogaster extended median (but not maximum) lifespan in female flies with a small lifespan extension in males; in contrast, overexpression of the cytosolic form, TXNRD1, did not produce a lifespan extension.


Assuntos
Drosophila melanogaster/enzimologia , Fibroblastos/enzimologia , Longevidade/genética , Mitocôndrias/enzimologia , Primatas/metabolismo , Tiorredoxina Redutase 2/genética , Animais , Citosol/efeitos dos fármacos , Citosol/enzimologia , Drosophila melanogaster/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Humanos , Longevidade/efeitos dos fármacos , Masculino , Masoprocol/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Especificidade de Órgãos , Cultura Primária de Células , Primatas/genética , Fatores Sexuais , Pele/citologia , Pele/efeitos dos fármacos , Pele/enzimologia , Especificidade da Espécie , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Tiorredoxina Redutase 2/metabolismo
11.
Free Radic Biol Med ; 106: 270-277, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28232204

RESUMO

Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family - all siblings of the index patient were homozygous and the parents heterozygous - were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by 75Se labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34±3% and 16±8% of controls, respectively). In fibroblasts, we detected reduced 75Se-labeling of the enzyme (41±3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30-40% lowered kcat/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and 75Se labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy.


Assuntos
Epilepsia Generalizada/genética , Predisposição Genética para Doença , Estresse Oxidativo/genética , Tiorredoxina Redutase 1/genética , Adolescente , Adulto , Criança , Pré-Escolar , Epilepsia Generalizada/fisiopatologia , Feminino , Glutationa/metabolismo , Homozigoto , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Espécies Reativas de Oxigênio/metabolismo , Sequenciamento Completo do Exoma
12.
Biochim Biophys Acta Gene Regul Mech ; 1860(2): 196-204, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27932267

RESUMO

The Polycomb/Trithorax Responsive Elements (PRE/TREs) are the cis-regulatory sequences that interact with both repressive (PcG) as well as activating (TrxG) complexes. However, most of the mammalian PREs are demonstrated to interact with the repressive polycomb (PcG) complexes only. We have carried out an unbiased search for proteins interacting with human PRE-PIK3C2B (hPRE-PIK3C2B) based on DNA affinity purification followed by mass spectrometry and identified MLL, MLL4 and WDR87 among other proteins in three biological replicates in HEK, U87 and HeLa cell lines. The hPRE-PIK3C2B interacts with the members of multiple activating complexes (COMPASS-like). The increase in the interaction of MLL and MLL4 on depletion of YY1 and the increase in the enrichment of YY1 and EZH2 upon MLL knockdown at the hPRE-PIK3C2B indicate the dual occupancy and suggest a concentration dependent enrichment of the activator or the repressor complex at hPRE-PIK3C2B. Further, we show that the hPRE-PIK3C2B interacts with the Drosophila homologues of PcG and TrxG proteins in transgenic flies. Here, we found that there is an increased enrichment of Pc (Polycomb) in comparison to Trx (TrxG protein) at hPRE-PIK3C2B in the Drosophila transgenic flies and this seems to be the default state while the balance is tipped towards the trithorax complex in PcG mutants. To the best of our knowledge, this is one of the early demonstrations of human PRE acting as a TRE without any sequence alteration.


Assuntos
Classe II de Fosfatidilinositol 3-Quinases/genética , Repressão Epigenética/genética , Íntrons/genética , Proteínas do Grupo Polycomb/genética , Ativação Transcricional/genética , Animais , Animais Geneticamente Modificados/genética , Linhagem Celular , Linhagem Celular Tumoral , Drosophila melanogaster/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Células HEK293 , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Tiorredoxina Redutase 1/genética , Fator de Transcrição YY1/genética
13.
Free Radic Biol Med ; 109: 125-131, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27993729

RESUMO

Myocardial ischemia/reperfusion and heart failure are the major cardiac conditions in which an imbalance between oxidative stress and anti-oxidant mechanisms is observed. The myocardium has endogenous reducing mechanisms, including the thioredoxin (Trx) and glutathione systems, that act to scavenge reactive oxygen species (ROS) and reduce oxidized proteins. The Trx system consists of Trx, Trx reductase (TrxR), and an electron donor, NADPH, where Trx is maintained in a reduced state in the presence of TrxR and NADPH. Trx1, a major isoform of Trx, is abundantly expressed in the heart and exerts its oxidoreductase activity through conserved Cys32 and Cys35, reducing oxidized proteins through thiol disulfide exchange reactions. In this review, we will focus on molecular targets of Trx1 in the heart, including transcription factors, microRNAs, histone deactylases, and protein kinases. We will then discuss how Trx1 regulates the functions of its targets, thereby affecting the extent of myocardial injury caused by myocardial ischemia/reperfusion and the progression of heart failure.


Assuntos
Insuficiência Cardíaca/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , NADP/metabolismo , Oxirredução , Estresse Oxidativo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Tiorredoxinas/genética
14.
J Pathol ; 241(4): 475-487, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27873306

RESUMO

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early-stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95% CI 1.01-17.76). This difference was more significant in patients with early-stage moderately-poorly differentiated tumours (HR 9.259; 95% CI 2.12-53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early-stage disease (HR 6.027; 95% CI 1.41-25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM-mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early-stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Antígenos CD/genética , Biomarcadores Tumorais/genética , Carcinoma Endometrioide/genética , Moléculas de Adesão Celular Neuronais/genética , Neoplasias do Endométrio/genética , Proteínas Fetais/genética , Regulação Neoplásica da Expressão Gênica , Idoso , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Endometrioide/diagnóstico , Carcinoma Endometrioide/patologia , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/patologia , Feminino , Proteínas Fetais/metabolismo , Filaminas/genética , Filaminas/metabolismo , Humanos , Laminina/genética , Laminina/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Estudos Retrospectivos , Transdução de Sinais , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo
15.
Free Radic Biol Med ; 101: 53-70, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27693380

RESUMO

Oxidative damage and aggregation of cellular proteins is a hallmark of neuronal cell death after neurotrauma and chronic neurodegenerative conditions. Autophagy and ubiquitin protease system are involved in degradation of protein aggregates, and interruption of their function is linked to apoptotic cell death in these diseases. Oxidative modification of cysteine groups in key molecular proteins has been linked to modification of cellular systems and cell death in these conditions. Glutathione and thioredoxin systems provide reducing protons that can effectively reverse protein modifications and promote cell survival. The central role of Thioredoxin in inhibition of apoptosis is well identified. Additionally, its involvement in initiation of autophagy has been suggested recently. We therefore aimed to investigate the involvement of Thioredoxin system in autophagy-apoptosis processes. A model of serum deprivation in SH-SY5Y was used that is associated with autophagy and apoptosis. Using pharmacological and RNA-editing technology we show that Thioredoxin reductase deficiency in this model enhances oxidative stress and interrupts the early protective autophagy and promotes apoptosis. This was associated with decreased protein-degradation in lysosomes due to altered lysosomal acidification and accumulation of autophagosomes as well as impairment in proteasome pathway. We further confirmed that the extent of oxidative stress is a determining factor in autophagy- apoptosis interplay, as upregulation of cellular reducing capacity by N-acetylcysteine prevented impairment in autophagy and proteasome systems thus promoted cell viability. Our study provides evidence that excessive oxidative stress inhibits protein degradation systems and affects the final stages of autophagy by inhibiting autolysosome maturation: a novel mechanistic link between protein aggregation and conversion of autophagy to apoptosis that can be applicable to neurodegenerative diseases.


Assuntos
Proteína 7 Relacionada à Autofagia/genética , Autofagia/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Tiorredoxina Redutase 1/genética , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Proteína 7 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/farmacologia , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Lisossomos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oxirredução , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/metabolismo
16.
Genet Mol Res ; 15(3)2016 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-27706680

RESUMO

Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse reaction to anti-tuberculosis (TB) treatment. Thioredoxin reductase 1 (TXNRD1), encoded by the TXNRD1 gene, is an important enzyme involved in oxidant challenge. TXNRD1 plays a key role in regulating cell growth and transformation, and protects cells against oxidative damage. We investigated the association between TXNRD1 polymorphisms and ATDH susceptibility. In this prospective study, 280 newly diagnosed TB patients were followed-up for 3 months after beginning anti-TB therapy. Tag single-nucleotide polymorphisms (tag-SNPs) of TXNRD1 were selected using Haploview 4.2 based on the HapMap database of the Chinese Han in Beijing (CHB) panel. Genotyping was performed using the MassARRAY platform. Of the 280 patients enrolled in this study, 33 were lost to follow-up, 24 had ATDH, and 223 were free from ATDH. After adjusting for sex, age, smoking status, and body mass index, there were no significant differences in the allele and genotype frequency distributions of TXNRD1 SNPs between the ATDH and non-ATDH groups (all P > 0.05). The haplotype analysis showed that haplotype TCAGCC was associated with an increased risk of ATDH susceptibility [P = 0.024, OR (95%CI) = 6.273 (1.023-38.485)]. Further stratified analyses showed that the haplotype TCAGCC was associated with ATDH susceptibility in female subjects [P = 0.036, OR (95%CI) = 5.711 (0.917-35.560)] and non-smokers [P = 0.029, OR (95%CI) = 6.008 (0.971-37.158)]. Our results suggest that TXNRD1 variants may favor ATDH susceptibility in females and non-smokers. Further studies are required to verify this association.


Assuntos
Antituberculosos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/genética , Polimorfismo de Nucleotídeo Único , Tiorredoxina Redutase 1/genética , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Fatores Etários , Alelos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Etambutol/efeitos adversos , Feminino , Expressão Gênica , Frequência do Gene , Haplótipos , Humanos , Isoniazida/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estudos Prospectivos , Pirazinamida/efeitos adversos , Rifampina/efeitos adversos , Fatores de Risco , Fatores Sexuais , Tuberculose Pulmonar/microbiologia
17.
Glia ; 64(12): 2219-2230, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27641725

RESUMO

Oxidative stress critically contributes to the pathogenesis of a variety of neurodegenerative diseases such as multiple sclerosis. Astrocytes are the main regulators of oxidative homeostasis in the brain and dysregulation of these cells likely contributes to the accumulation of oxidative damage. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of the anti-oxidant stress defense. In this study, we elucidate the effects of astrocytic Nrf2-activation on brain-intrinsic inflammation and lesion development. Cells deficient for the Nrf2 repressor kelch-like ECH-associated protein 1 (Keap1) are characterized by hyperactivation of Nrf2-signaling. Therefore, wild type mice and mice with a GFAP-specific Keap1-deletion were fed with 0.25% cuprizone for 1 or 3 weeks. Cuprizone intoxication induced pronounced oligodendrocyte loss, demyelination and reactive gliosis in wild type animals. In contrast, astrocyte-specific Nrf2-activation was sufficient to prevent oligodendrocyte loss and demyelination, to ameliorate brain intrinsic inflammation and to counteract axonal damage. Our results highlight the potential of the Nrf2/ARE system for the treatment of neuroinflammation in general and of multiple sclerosis in particular. © GLIA 2016;64:2219-2230.


Assuntos
Astrócitos/metabolismo , Doenças Desmielinizantes/etiologia , Regulação da Expressão Gênica/fisiologia , Esclerose Múltipla/complicações , Esclerose Múltipla/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Cuprizona/toxicidade , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Esclerose Múltipla/induzido quimicamente , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo
18.
Appl Microbiol Biotechnol ; 100(24): 10363-10374, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27344592

RESUMO

Two thioredoxin (Trx) reductases (Trr1/2) are known to play overlapping roles in the yeast Trx-Trr redox system but are generally unexplored in filamentous fungi, which possess multiple Trx homologues. This study seeks to characterize the functions of Trr1 and Trr2 in Beauveria bassiana, a filamentous fungal insect pathogen, and to probe their Trx partners. Both Trr1 and Trr2 were evidently localized in the cytoplasm of B. bassiana, unlike the two yeast homologues that have been reported to localize in the cytoplasm and mitochondria, respectively. Most of the six trx genes were greatly upregulated at the transcriptional level in the absence of trr1 instead of trr2 in B. bassiana, in which the trr1/2 double deletion failed in many attempts. Deletion of trr1 resulted in increased Trx activity, severe cysteine auxotrophy, and drastically reduced activities of peroxidases and superoxide dismutases under normal or oxidative conditions despite little change in catalase activity. Such changes disappeared in the absence of trr2 and were completely restored by complementation of trr1/2 or overexpression of trx1/6 in the Δtrr1 mutant, but were not restored at all by overexpression of trx2/3/4/5 or trr2 in the same mutant. All of these mutants exhibited similar trends of changes in the antioxidant response, conidiation, germination, thermotolerance, UV-B resistance, and virulence. Taken together, the findings indicate that Trr1 could reduce Trx2-5 and hence dominate the intracellular redox state, profoundly affecting the potential of B. bassiana against arthropod pests. Trr2 could reduce Trx1/6 but function only in the absence of Trr1.


Assuntos
Artrópodes/microbiologia , Beauveria/enzimologia , Beauveria/crescimento & desenvolvimento , Cisteína/biossíntese , Tiorredoxina Redutase 1/metabolismo , Tiorredoxina Redutase 2/metabolismo , Fatores de Virulência/metabolismo , Animais , Beauveria/genética , Citoplasma/enzimologia , Deleção de Genes , Teste de Complementação Genética , Oxirredução , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 2/genética , Virulência , Fatores de Virulência/genética
19.
Sci Rep ; 6: 28080, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27346647

RESUMO

Recently thioredoxin reductase 1 (TrxR1), encoded by Txnrd1, was suggested to modulate glucose and lipid metabolism in mice. Here we discovered that TrxR1 suppresses insulin responsiveness, anabolic metabolism and adipocyte differentiation. Immortalized mouse embryonic fibroblasts (MEFs) lacking Txnrd1 (Txnrd1(-/-)) displayed increased metabolic flux, glycogen storage, lipogenesis and adipogenesis. This phenotype coincided with upregulated PPARγ expression, promotion of mitotic clonal expansion and downregulation of p27 and p53. Enhanced Akt activation also contributed to augmented adipogenesis and insulin sensitivity. Knockdown of TXNRD1 transcripts accelerated adipocyte differentiation also in human primary preadipocytes. Furthermore, TXNRD1 transcript levels in subcutaneous adipose tissue from 56 women were inversely associated with insulin sensitivity in vivo and lipogenesis in their isolated adipocytes. These results suggest that TrxR1 suppresses anabolic metabolism and adipogenesis by inhibition of intracellular signaling pathways downstream of insulin stimulation.


Assuntos
Adipócitos/enzimologia , Adipogenia , Diferenciação Celular , Resistência à Insulina , Tiorredoxina Redutase 1/metabolismo , Animais , Linhagem Celular Transformada , Feminino , Humanos , Camundongos , Camundongos Knockout , Tiorredoxina Redutase 1/genética
20.
Cell Microbiol ; 18(12): 1800-1814, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27235692

RESUMO

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis.


Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ricina/farmacocinética , Ricina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Fracionamento Celular , Citocromos c/genética , Citocromos c/metabolismo , Fragmentação do DNA , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/metabolismo , Endossomos/ultraestrutura , Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Fígado/citologia , Fígado/metabolismo , Masculino , Mitocôndrias/metabolismo , Multimerização Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Ricina/síntese química , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo
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