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1.
Anticancer Res ; 39(9): 4795-4803, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519581

RESUMO

BACKGROUND/AIM: To determine the mechanism of vitamin D3-induced modulation of antioxidant-related factors in endometrial cancer, we investigated their role in apoptosis of human endometrial cancer cells exposed to vitamin D3 Materials and Methods: The survival rate of human endometrial cancer cells was estimated after treatment with activated vitamin D3 Reactive oxygen species (ROS) levels were measured using flow cytometry. The levels of VDR, Trx, TXNIP and apoptosis-related proteins were investigated using western blotting and immunocytochemistry in human tissues. RESULTS: Treatment with D3 induced apoptotic cell death and cell-cycle arrest by increasing ROS concentration. Vitamin D3 inhibited proliferation of human endometrial cancer cells. It regulated intracellular ROS concentration in endometrial cancer cells via increased TXNIP expression. CONCLUSION: Antioxidant regulation via TXNIP is an important cell death mechanism in human endometrial cancer, and occurs via induction by vitamin D3.


Assuntos
Antioxidantes/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias do Endométrio/metabolismo , Tiorredoxinas/metabolismo , Vitamina D/análogos & derivados , Apoptose/efeitos dos fármacos , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Vitamina D/farmacologia
2.
Toxicol Lett ; 314: 43-52, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31310794

RESUMO

Thioredoxin is an evolutionarily conserved antioxidant protein that plays a crucial role for fundamental cellular processes and embryonic development. Growing evidence support that Thioredoxin influences cellular response to chemicals insults, particularly those accompanying oxidative stress. The mechanisms underlying the functions of Thioredoxin1 in the embryonic development under the environmental toxicant exposure remain, however, largely unexplored. We report here that thioredoxin1 becomes differentially expressed in zebrafish embryos after exposure to 9 out of 11 environmental chemicals. In situ gene expression analysis show that thioredoxin1 is expressed in neurons, olfactory epithelia, liver and swim bladder under normal conditions. After MeHg exposure, however, thioredoxin1 is ectopically induced in the hair cells of the lateral line and in epithelia cells of the pharynx. Knockdown of Thioredoxin1 induces hydrocephalus and increases cell apoptosis in the brain ventricular epithelia cells. In comparison with 5% malformation in embryos injected with control morpholino, MeHg induces more than 77% defects in Thioredoxin1 knockdown embryos. Our data suggest that there is an association between hydrocephalus and Thioredoxin1 malfunction in embryonic development, and provide valuable information to elucidate the protective role of Thioredoxin1 against chemicals disruption.


Assuntos
Encéfalo/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Hidrocefalia/induzido quimicamente , Tiorredoxinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Hidrocefalia/embriologia , Hidrocefalia/genética , Hidrocefalia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxinas/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
3.
DNA Cell Biol ; 38(8): 874-879, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215797

RESUMO

Microglia-mediated neuroinflammation plays an important role in Alzheimer's disease development. Resveratrol, a natural polyphenol from the Japanese knotweed (Polygonum cuspidatumand), is known to protect against neuroinflammation, but the mechanism remains unclear. To begin to explore potential mechanisms, we created a model of inflammatory injury in BV-2 murine microglial cells based on the induction of amyloid-ß. We found that resveratrol (10 and 50 nM) significantly inhibited Aß-induced proliferation and activation of BV-2 cells, as well as their release of the proinflammatory cytokines, IL-6 and TNF-α. Resveratrol also suppressed the overexpression of cleaved caspase-1 and IL-1ß, and decreased Aß-stimulated degradation of IkBα and phosphorylation of NF-κB phosphorylation. Western blot analysis showed that Aß upregulated the TXNIP/TRX/NLRP3 pathway, while resveratrol treatment inhibited it. We conclude that resveratrol protects microglia from Aß-stimulated inflammation by suppressing the inflammatory response, at least in part by inhibiting the TXNIP/TRX/NLRP3 signaling pathway.


Assuntos
Proteínas de Transporte/metabolismo , Microglia/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Resveratrol/farmacologia , Tiorredoxinas/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Caspase 1/metabolismo , Linhagem Celular , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Microglia/fisiologia , NF-kappa B/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
4.
Nat Commun ; 10(1): 2629, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201355

RESUMO

Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fitocromo/metabolismo , Tiorredoxinas/metabolismo , Transcrição Genética/fisiologia , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Transcrição Genética/efeitos da radiação
5.
Oxid Med Cell Longev ; 2019: 2715810, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049129

RESUMO

Background: Hyperosmotic stress is an important pathophysiologic condition in diabetes, severe trauma, dehydration, infection, and ischemia. Furthermore, brain neuronal cells face hyperosmotic stress in ageing and Alzheimer's disease. Despite the enormous importance of knowing the homeostatic mechanisms underlying the responses of nerve cells to hyperosmotic stress, this topic has been underrepresented in the literature. Recent evidence points to autophagy induction as a hallmark of hyperosmotic stress, which has been proposed to be controlled by mTOR inhibition as a consequence of AMPK activation. We previously showed that methylglyoxal induced a decrease in the antioxidant proteins thioredoxin 1 (Trx1) and glyoxalase 2 (Glo2), which was mediated by AMPK-dependent autophagy. Thus, we hypothesized that hyperosmotic stress would have the same effect. Methods: HT22 hippocampal nerve cells were treated with NaCl (37, 75, or 150 mM), and the activation of the AMPK/mTOR pathway was investigated, as well as the levels of Trx1 and Glo2. To determine if autophagy was involved, the inhibitors bafilomycin (Baf) and chloroquine (CQ), as well as ATG5 siRNA, were used. To test for AMPK involvement, AMPK-deficient mouse embryonic fibroblasts (MEFs) were used. Results: Hyperosmotic stress induced a clear increase in autophagy, which was demonstrated by a decrease in p62 and an increase in LC3 lipidation. AMPK phosphorylation, linked to a decrease in mTOR and S6 ribosomal protein phosphorylation, was also observed. Deletion of AMPK in MEFs did not prevent autophagy induction by hyperosmotic stress, as detected by decreased p62 and increased LC3 II, or mTOR inhibition, inferred by decreased phosphorylation of P70 S6 kinase and S6 ribosomal protein. These data indicating that AMPK was not involved in autophagy activation by hyperosmotic stress were supported by a decrease in pS555-ULK1, an AMPK phosphorylation site. Trx1 and Glo2 levels were decreased at 6 and 18 h after treatment with 150 mM NaCl. However, this decrease in Trx1 and Glo2 in HT22 cells was not prevented by autophagy inhibition by Baf, CQ, or ATG5 siRNA. AMPK-deficient MEFs under hyperosmotic stress presented the same Trx1 and Glo2 decrease as wild-type cells. Conclusion: Hyperosmotic stress induced AMPK activation, but this was not responsible for its effects on mTOR activity or autophagy induction. Moreover, the decrease in Trx1 and Glo2 induced by hyperosmotic stress was independent of both autophagy and AMPK activation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Neurônios/metabolismo , Pressão Osmótica , Transdução de Sinais , Tioléster Hidrolases/metabolismo , Tiorredoxinas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Linhagem Celular Transformada , Ativação Enzimática , Camundongos , Neurônios/citologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Tioléster Hidrolases/genética , Tiorredoxinas/genética
6.
J Agric Food Chem ; 67(22): 6432-6444, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31095381

RESUMO

Liquid feeding strategies have been devised with the aim of enhancing grain nutrient availability for livestock. It is characterized by a steeping/soaking period that softens the grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples supplemented with either protease inhibitors or NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in the water-extractable fraction of feed containing protease inhibitors, illustrating significant inhibition of proteolytic activities arising during the steeping period. Application of functional NTR/Trx to liquid feed reductively increased the solubility of known and potentially new Trx-target proteins, e.g., outer membrane protein X, and their susceptibility to proteolysis. Thus, the NTR/Trx system exhibits important potential as a feed additive to enhance nutrient digestibility in monogastric animals.


Assuntos
Ração Animal/análise , Hordeum/enzimologia , Proteínas de Plantas/química , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/química , Eletroforese em Gel Bidimensional , Manipulação de Alimentos , Hordeum/química , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Proteômica , Sementes/química , Sementes/enzimologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
7.
Parasitol Res ; 118(6): 1785-1797, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31062084

RESUMO

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.


Assuntos
Citosol/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Mitocôndrias/metabolismo , Taenia solium/genética , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cysticercus/genética , Cysticercus/isolamento & purificação , Cysticercus/metabolismo , Citosol/química , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Fases de Leitura Aberta , Coelhos , Taenia solium/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo
8.
Gene ; 706: 32-42, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31028868

RESUMO

The chloroplastic thioredoxins (Trxs), a family of thiol-disulphide oxidoreductases, are reduced by either ferredoxin (Fd)-dependent Trx reductase (FTR) or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Trx reductase (NTR). Two Trx systems are present in chloroplasts including Trxs, Trx-like proteins, and reductase FTR and NTRC. FTR is the main reductant for Trxs in chloroplasts, while the flavoprotein NTRC integrates NTR and Trx activity, and plays multiple roles in the Calvin cycle, the oxidative pentose phosphate pathway (OPPP), anti-peroxidation, tetrapyrrole metabolism, ATP and starch synthesis, and photoperiodic regulation. In addition, not only there exists a reduction potential transfer pathway across the thylakoid membrane, but also FTR and NTRC coordinate with each other to regulate chloroplast redox homeostasis. Herein, we summarise the physiological functions of these two Trx reduction systems, discuss how both regulate redox homeostasis in plant plastids, and emphasize the significance of chloroplast thioredoxin systems in maintaining photosynthetic efficiency in plants.


Assuntos
Cloroplastos/metabolismo , Tiorredoxina Dissulfeto Redutase/fisiologia , Tiorredoxinas/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/fisiologia , Ferredoxinas/metabolismo , Proteínas com Ferro-Enxofre , Oxirredução , Oxirredutases/metabolismo , Peroxirredoxinas/metabolismo , Fotossíntese/fisiologia , Plastídeos/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
9.
Genesis ; 57(5): e23293, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30912273

RESUMO

The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.


Assuntos
Arginase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regeneração/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Transporte/metabolismo , Desenvolvimento Embrionário , Extremidades/embriologia , Larva/genética , Larva/metabolismo , Organogênese , Ligação Proteica/fisiologia , Regeneração/genética , Tiorredoxinas/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo
10.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897830

RESUMO

Plants are constantly subjected to a variety of environmental stresses and have evolved regulatory responses to overcome unfavorable conditions that might reduce or adversely change a plant's growth or development. Among these, the regulated production of reactive oxygen species (ROS) as a signaling molecule occurs during plant development and pathogen defense. This study demonstrates the possible antifungal activity of Oryza sativa Tetratricopeptide Domain-containing thioredoxin (OsTDX) protein against various fungal pathogens. The transcription of OsTDX was induced by various environmental stresses known to elicit the generation of ROS in plant cells. OsTDX protein showed potent antifungal activity, with minimum inhibitory concentrations (MICs) against yeast and filamentous fungi ranging between 1.56 and 6.25 and 50 and 100 µg/mL, respectively. The uptake of SYTOX-Green into fungal cells and efflux of calcein from artificial fungus-like liposomes suggest that its killing mechanism involves membrane permeabilization and damage. In addition, irregular blebs and holes apparent on the surfaces of OsTDX-treated fungal cells indicate the membranolytic action of this protein. Our results suggest that the OsTDX protein represents a potentially useful lead for the development of pathogen-resistant plants.


Assuntos
Anti-Infecciosos/farmacologia , Oryza/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Antifúngicos/farmacologia , Oryza/genética , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo
11.
Oxid Med Cell Longev ; 2019: 9291683, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911354

RESUMO

Background: Thioredoxin-1 (Trx-1) is a small redox protein, which plays an important role in many biological processes. Although increased expression of Trx-1 in various solid tumors has been reported, the prognostic significance and function of Trx-1 in human gastric cancer (GC) are still unclear. Here, we investigated the clinical and prognostic significance of Trx-1 expression and the function and mechanism of Trx-1 in human GC. Methods: We analyzed Trx-1 mRNA expression from the GEO database and Trx-1 protein expression in 144 GC tissues using immunohistochemistry. Effects of Trx-1 on GC cell were assessed in vitro and in vivo through Trx-1 knockdown or overexpression. The antitumor effects of the Trx-1 inhibitor, PX-12, on GC cells were investigated. PTEN and p-AKT expressions were evaluated by Western blotting. Results: Increased Trx-1 expression was found in GC tissues and associated with poor prognosis and aggressive clinicopathological characteristics in patients with GC. High Trx-1 expression predicted poor prognosis, and its expression was an independent prognostic factor for overall survival of GC patients. Knockdown of Trx-1 expression inhibited GC cell growth, migration, and invasion in vitro and tumor growth and lung metastasis in vivo. Conversely, overexpression of Trx-1 promoted GC cell growth, migration, and invasion. We also found that PX-12 inhibited GC cell growth, migration, and invasion. Overexpression of Trx-1 caused a decrease in PTEN and increase in p-AKT levels whereas silencing Trx-1 caused an increase in PTEN and decrease in p-AKT levels in GC cells. Inhibition of AKT signaling pathway by MK2206 also inhibited GC cell growth, migration, and invasion. Conclusion: Our results indicate that Trx-1 may be a promising prognostic indicator and therapeutic target for GC patients.


Assuntos
Progressão da Doença , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Tiorredoxinas/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Movimento Celular , Proliferação de Células , Dissulfetos/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Lentivirus/metabolismo , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , PTEN Fosfo-Hidrolase/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
12.
Chem Pharm Bull (Tokyo) ; 67(3): 186-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30827998

RESUMO

Gold compounds have a long history of use in medicine. Auranofin was developed more than 30 years ago as an oral therapy for rheumatoid arthritis. Now, however, auranofin is rarely used in clinical practice despite its efficacy for treating rheumatoid arthritis because more novel antirheumatic medications are available. Although its use in clinical practice has decreased, studies on auranofin have continued and it shows promise for the treatment of several different diseases, including cancer and bacterial and parasitic infections. Several potential novel applications of auranofin for treating human disease have been proposed. Auranofin inhibits the activity of thioredoxin reductase (TrxR), an enzyme of the thioredoxin (Trx) system that is important for maintaining the intracellular redox state. Particularly in cancers, TrxR inhibition leads to an increase in cellular oxidative stress and induces apoptosis. TrxR overexpression is associated with aggressive tumor progression and poor survival in patients with breast, ovarian, and lung cancers. The Trx system may represent an attractive target for the development of new cancer treatments. Therefore, the TrxR inhibitor auranofin may be a potent anticancer agent. This review summarizes the current understanding of auranofin for cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Auranofina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
13.
Nat Nanotechnol ; 14(3): 252-259, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742135

RESUMO

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.


Assuntos
DNA/química , Nanopartículas/química , Organelas/enzimologia , Animais , Caenorhabditis elegans/metabolismo , Toxina Diftérica/metabolismo , Dissulfetos/metabolismo , Endossomos/metabolismo , Genes Reporter , Células HeLa , Humanos , Tiorredoxinas/metabolismo
14.
Biochimie ; 160: 1-13, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30710560

RESUMO

The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.


Assuntos
Dissulfetos/metabolismo , Conformação Proteica , Staphylococcus aureus/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dissulfetos/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , NADP/metabolismo , Oxirredução , Especificidade por Substrato , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/química
15.
J Exp Clin Cancer Res ; 38(1): 69, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755224

RESUMO

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death. METHODS: CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential. RESULTS: Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53's role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition. CONCLUSIONS: PRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.


Assuntos
Glutationa/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Quinuclidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteína Supressora de Tumor p53/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-30776436

RESUMO

Thioredoxin system plays an important role in antioxidative stress, thioredoxin 2 (Trx2) being one of the most important components in the thioredoxin system. The full-length cDNA sequence of thioredoxin 2 from Euphausia superba (EsTrx2) is 1276 bp and contain a 5' untranslated region (UTR) of 94 bp, a 3' UTR of 741 bp and an open reading frame (ORF) of 441 bp, encoding a putative protein of 146 amino acids. Multiple sequence alignments have indicated that EsTrx2 possesses a conserved (-Cys-Gly-Pro-Cys-) CGPC redox-active site. EsTrx2 shares 62.3% identity with the swimming crab (Portunus trituberculatus) Trx2. The predicted three-dimensional structure of EsTrx2 consists of a thioredoxin fold. The high similarity and phylogenetic analysis have indicated that EsTrx2 is a member of the mitochondrial Trx2 sub-family. The recombinant EsTrx2 (rEsTrx2) was constructed and expressed in Escherichia coli BL21 (DE3). The rEsTrx2 protein showed high redox activity and antioxidant capacity at temperature from 4 to 37 °C. All results indicated that EsTrx2 was involved in the oxidative stress response of E. superba.


Assuntos
Euphausiacea/citologia , Euphausiacea/genética , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Antioxidantes/metabolismo , Sequência de Bases , Clonagem Molecular , Modelos Moleculares , Conformação Proteica , Análise de Sequência , Tiorredoxinas/química
17.
Redox Biol ; 21: 101125, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716538

RESUMO

AIM: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. RESULTS: TGFß1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. INNOVATION & CONCLUSION: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.


Assuntos
Espectrometria de Massas , NADPH Oxidase 4/metabolismo , Oxirredução , Animais , Expressão Gênica , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , NADPH Oxidase 4/genética , NADPH Oxidase 5/genética , NADPH Oxidase 5/metabolismo , Peroxirredoxinas/metabolismo , Podócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo
18.
Life Sci ; 220: 50-57, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30708098

RESUMO

AIMS: Oxidative stress caused by exposure to drugs of abuse such as ethanol or 3, 4 methylenedioxymethamphetamine (MDMA) may derive from direct or indirect effects in many organs including the heart. The aim of the present work was to evaluate cardiac sympathetic activity and the expression and activation of two antioxidant proteins: heat shock protein27 (HSP27) and thioredoxin-1 (Trx-1) after voluntary binge ethanol consumption, alone and in combination with MDMA. MATERIAL AND METHODS: Adolescent mice received MDMA, ethanol or both. Drinking in the dark (DID) procedure was used as a model of binge. HSP27 expression and phosphorylation at serine 82 (pHSP27), Trx-1 expression, tyrosine hydroxylase (TH) and TH phosphorylated at serine 31 (pTH) were evaluated in adolescent mice 48 h and 7 days after treatments in the right ventricle. TH, HSP27 expression and phosphorylation and Trx-1 expression were measured by quantitative blot immunolabeling using specific antibodies. KEY FINDINGS: The expression of HSP27, pHSP27, Trx-1, total TH and pTH in the right ventricle was increased after binge ethanol or MDMA alone. In addition, the combination of binge ethanol + MDMA enhanced TH expression and phosphorylation versus their individual administration. SIGNIFICANCE: These results indicate that this combination could produce higher activation of sympathetic pathways, which could trigger an increased cell stress. On the other hand, increased HSP27, pHSP27 and Trx-1 expression in the right ventricle by ethanol + MDMA could be a protective mechanism to reduce the adverse effects of oxidative stress caused by both drugs of abuse.


Assuntos
Proteínas de Choque Térmico HSP27/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Tiorredoxinas/efeitos dos fármacos , Animais , Bebedeira/metabolismo , Biomarcadores/metabolismo , Temperatura Corporal/efeitos dos fármacos , Cardiotoxicidade/metabolismo , Etanol/efeitos adversos , Etanol/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Camundongos , N-Metil-3,4-Metilenodioxianfetamina/efeitos adversos , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , Estresse Oxidativo , Fosforilação , Tiorredoxinas/metabolismo
19.
Plant Sci ; 279: 27-33, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30709489

RESUMO

Activation of plant immune responses is associated with rapid production of vast amounts of reactive oxygen and nitrogen species (ROS/RNS) that dramatically alter cellular redox homeostasis. Even though excessive ROS/RNS accumulation can cause widespread cellular damage and thus constitute a major risk, plant cells have evolved to utilise these molecules as important signalling cues. Particularly their ability to modify redox-sensitive cysteine residues has emerged as a key mechanism to control the activity, conformation, protein-protein interaction and localisation of a growing number of immune signalling proteins. Regulated reversal of cysteine oxidation is dependent on activities of the conserved superfamily of Thioredoxin (TRX) enzymes that function as cysteine reductases. The plant immune system recruits specific TRX enzymes that have the potential to functionally regulate numerous immune signalling proteins. Although our knowledge of different TRX immune targets is now expanding, little remains known about how these enzymes select their substrates, what range of oxidized residues they target, and if they function selectively in different redox-mediated immune signalling pathways. In this review we discuss these questions by examining evidence showing TRX enzymes exhibit novel activities that play important roles in diverse aspects of plant immune signalling.


Assuntos
Oxirredução , Imunidade Vegetal , Transdução de Sinais , Tiorredoxinas/metabolismo , Tiorredoxinas/fisiologia
20.
Biochimie ; 160: 55-60, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30796965

RESUMO

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophic factors that are vital to the survival and proliferation of neuron. Thioredoxin-1 (Trx-1) is a redox regulating protein and plays various roles in regulating transcript factors and inhibiting apoptosis. It has been reported that Trx-1 is required for nerve growth factor-mediated signal transduction and neurite outgrowth, and is involved in synaptic protein expression induced by BDNF. However, the molecular mechanism on BDNF inducing Trx-1 expression has not been fully verified. The present study investigated the expression of Trx-1 after treatment with BDNF in SH-SY5Y cells. We first demonstrated that cell viability and the expression of Trx-1 were increased by BDNF in SH-SY5Y cells, which were inhibited by the tyrosine kinase B (TrkB) inhibitor, K252a, and the phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002. Moreover, BDNF increased the activity of cAMP response element-binding protein (CREB) through TrkB/PI3-K/Akt pathway. Whereas the expression of Trx-1 induced by BDNF was suppressed by CREB siRNA. Thus, our data suggest that BDNF induces the expression of Trx-1 through the TrkB/Akt/CREB pathway.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tiorredoxinas/genética , Apoptose , Fator Neurotrófico Derivado do Encéfalo/genética , Sobrevivência Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Tiorredoxinas/metabolismo , Células Tumorais Cultivadas
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