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1.
Chemistry ; 26(45): 10175-10184, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32097513

RESUMO

The thioredoxin system is highly conserved system found in all living cells and comprises NADPH, thioredoxin, and thioredoxin reductase. This system plays a critical role in preserving a reduced intracellular environment, and its involvement in regulating a wide range of cellular functions makes it especially vital to cellular homeostasis. Its critical role is not limited to healthy cells, it is also involved in cancer development, and is overexpressed in many cancers. This makes the thioredoxin system a promising target for cancer drug development. As such, over the last decade, many inhibitors have been developed that target the thioredoxin system, most of which are small molecules targeting the thioredoxin reductase C-terminal redox center. A few inhibitors of thioredoxin have also been developed. We believe that more efforts should be invested in developing protein/peptide-based inhibitors against both thioredoxin reductase and/or thioredoxin.


Assuntos
Antineoplásicos/farmacologia , NADP/química , Neoplasias/tratamento farmacológico , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/uso terapêutico , Antineoplásicos/química , Desenvolvimento de Medicamentos , Humanos , Oxirredução , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/metabolismo
2.
Inorg Chem ; 59(5): 3281-3289, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32073260

RESUMO

Metal complexes provide a versatile platform to develop novel anticancer pharmacophores, and they form stable compounds with N-heterocyclic carbene (NHC) ligands, some of which have been shown to inhibit the cancer-related selenoenzyme thioredoxin reductase (TrxR). To expand a library of isostructural NHC complexes, we report here the preparation of RhIII- and IrIII(Cp*)(NHC)Cl2 (Cp* = η5-pentamethylcyclopentadienyl) compounds and comparison of their properties to the RuII- and OsII(cym) analogues (cym = η6-p-cymene). Like the RuII- and OsII(cym) complexes, the RhIII- and IrIII(Cp*) derivatives exhibit cytotoxic activity with half maximal inhibitory concentration (IC50) values in the low micromolar range against a set of four human cancer cell lines. In studies on the uptake and localization of the compounds in cancer cells by X-ray fluorescence microscopy, the Ru and Os derivatives were shown to accumulate in the cytoplasmic region of treated cells. In an attempt to tie the localization of the compounds to the inhibition of the tentative target TrxR, it was surprisingly found that only the Rh complexes showed significant inhibitory activity at IC50 values of ∼1 µM, independent of the substituents on the NHC ligand. This indicates that, although TrxR may be a potential target for anticancer metal complexes, it is unlikely the main target or the sole target for the Ru, Os, and Ir compounds described here, and other targets should be considered. In contrast, Rh(Cp*)(NHC)Cl2 complexes may be a scaffold for the development of TrxR inhibitors.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Inibidores Enzimáticos/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Ligantes , Metais Pesados/química , Metais Pesados/farmacologia , Metano/análogos & derivados , Metano/química , Metano/farmacologia , Conformação Molecular , Relação Estrutura-Atividade , Tiorredoxina Dissulfeto Redutase/metabolismo
3.
J Enzyme Inhib Med Chem ; 35(1): 506-510, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31928252

RESUMO

The hypothesis that sulfocoumarin acting as inhibitors of human carbonic anhydrase (CA, EC 4.2.1.1) cancer-associated isoforms hCA IX and - hCA XII is being able to also inhibit thioredoxin reductase was verified and confirmed. The dual targeting of two cancer cell defence mechanisms, i.e. hypoxia and oxidative stress, may both contribute to the observed antiproliferative profile of these compounds against many cancer cell lines. This unprecedented dual anticancer mechanism may lead to a new approach for designing innovative therapeutic agents.


Assuntos
Antineoplásicos/farmacologia , Anidrase Carbônica IX/antagonistas & inibidores , Anidrases Carbônicas/metabolismo , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Anidrase Carbônica IX/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células MCF-7 , Estrutura Molecular , Relação Estrutura-Atividade , Tiorredoxina Dissulfeto Redutase/metabolismo , Células Tumorais Cultivadas
4.
Appl Environ Microbiol ; 86(6)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31924613

RESUMO

To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H2 oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor.IMPORTANCE This study has importance in that TrxR can use H2 as an electron donor with the aid of the frhAGB-encoded hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoded hydrogenase in a nonmethanogen; the hydrogenase can transfer electrons derived from oxidation of H2 to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F420-reducing hydrogenases of methanogens.


Assuntos
Proteínas Arqueais/metabolismo , Elétrons , Hidrogenase/metabolismo , Thermococcus/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Transporte de Elétrons , Oxirredução
5.
Psychopharmacology (Berl) ; 237(1): 127-136, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31473777

RESUMO

RATIONALE: Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are the most commonly used drugs for the treatment of depression. Studies have shown that chronic treatment with SSRIs and SNRIs produces a protective effect against oxidative stress. Thioredoxin (Trx) is an antioxidant protein that reverses protein cysteine oxidation and facilitates scavenging reactive oxygen species. OBJECTIVES: The current study is to determine whether the SSRI fluoxetine and the SNRI venlafaxine regulate Trx and protect neuronal cells against protein cysteine oxidation. METHODS: HT22 mouse hippocampal cells were incubated with fluoxetine or venlafaxine for 5 days. Protein levels of Trx, Trx reductase (TrxR), and Trx-interacting protein (Txnip) were measured by immunoblotting analysis. Trx and TrxR activities were analyzed by spectrophotometric method. Protein cysteine sulfenylation was measured by dimedone-conjugation assay, while nitrosylation was measured by biotin-switch assay. RESULTS: We found that treatment with fluoxetine or venlafaxine for 5 days increased Trx and TrxR protein levels but produced no effect on Txnip protein levels. These treatments also increased Trx and TrxR activities. Although treatment with fluoxetine or venlafaxine alone had no effect on sulfenylated and nitrosylated protein levels, both drugs inhibited H2O2-increased sulfenylated protein levels and nitric oxide donor nitrosoglutathione-increased nitrosylated protein levels. Stress increases risk of depression. We also found that treatment with fluoxetine or venlafaxine for 5 days inhibited stress hormone corticosterone-increased total sulfenylated and nitrosylated protein levels. CONCLUSIONS: Our findings suggest that chronic treatment with antidepressants may upregulate Trx, subsequently inhibiting protein sulfenylation and nitrosylation, which may contribute to the protective effect of antidepressants against oxidative stress. Our findings also indicate that thioredoxin is a potential therapeutic target for the treatment of depression.


Assuntos
Fluoxetina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxinas/metabolismo , Regulação para Cima/efeitos dos fármacos , Cloridrato de Venlafaxina/farmacologia , Animais , Antidepressivos/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Inibidores de Captação de Serotonina/farmacologia , Tiorredoxina Dissulfeto Redutase/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(1): 741-751, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871212

RESUMO

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.


Assuntos
Arabidopsis/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Germinação/fisiologia , Mitocôndrias/metabolismo , Sementes/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Oxirredução , Oxigênio/metabolismo , Plantas Geneticamente Modificadas , Proteômica/métodos , Sementes/citologia , Sementes/crescimento & desenvolvimento , Tiorredoxina h/genética , Tiorredoxina h/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-31801208

RESUMO

Methylmercury (MeHg) is a highly neurotoxic compound to which human populations are exposed via fish consumption. Once in cells, MeHg actively binds thiols and selenols, interfering with the activity of redox enzymes such as thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR) which integrate the thioredoxin system. In fact, it has been shown that inhibition of this system by MeHg is a critical step in the unfolding of cell death. Current clinical approaches to mitigate the toxicity of MeHg rely on the use of chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) which largely replaced British anti-Lewisite or 2,3-dimercapto-1-propanol (BAL) as the prime choice. However, therapeutic efficacy is limited and therefore new therapeutic options are necessary. In this work, we evaluated the efficacy of a macrocyclic chelator, 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN4S), in preventing MeHg toxicity, namely by looking at the effects over relevant molecular targets, i.e., the thioredoxin system, using both purified enzyme solutions and cell experiments with human neuroblastoma cells (SH-SY5Y). Results showed that [15]aneN4S had a similar efficacy to DMSA and BAL in reversing the inhibition of MeHg over purified TrxR and Trx by looking at both the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reduction assay and insulin reduction capability. In experiments with cells, none of the chelating agents could reverse the inhibition of TrxR by MeHg, which corroborates the high affinity of MeHg to the selenol in TrxR active site. [15]aneN4S and BAL, unlike DMSA, could prevent inhibition of Trx, which allows the maintenance of downstream functions, although BAL showed higher toxicity to cells. Overall these findings highlight the potential of using [15]aneN4S in the treatment of MeHg poisoning and encourage further studies, namely in vivo.


Assuntos
Compostos Aza/farmacologia , Quelantes/farmacologia , Compostos Macrocíclicos/farmacologia , Compostos de Metilmercúrio/toxicidade , Linhagem Celular Tumoral , Humanos , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
8.
Biomed Res Int ; 2019: 6502793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31828114

RESUMO

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.


Assuntos
Antineoplásicos/farmacologia , Mesilato de Imatinib/farmacologia , Células K562 , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Células K562/efeitos dos fármacos , Células K562/metabolismo , Fator 2 Relacionado a NF-E2/análise , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Tiorredoxina Dissulfeto Redutase/análise , Tiorredoxina Dissulfeto Redutase/metabolismo
9.
Plant Physiol ; 181(3): 976-992, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31527089

RESUMO

NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.


Assuntos
Frutas/enzimologia , Lycopersicon esculentum/enzimologia , Amido/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Metabolismo dos Carboidratos , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/fisiologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/fisiologia , Metabolômica , Oxirredução , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , Tiorredoxina Dissulfeto Redutase/genética
10.
J Biol Chem ; 294(38): 14105-14118, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31366732

RESUMO

Thioredoxin (Trx) is a conserved, cytosolic reductase in all known organisms. The enzyme receives two electrons from NADPH via thioredoxin reductase (TrxR) and passes them on to multiple cellular reductases via disulfide exchange. Despite the ubiquity of thioredoxins in all taxa, little is known about the functions of resurrected ancestral thioredoxins in the context of a modern mesophilic organism. Here, we report on functional in vitro and in vivo analyses of seven resurrected Precambrian thioredoxins, dating back 1-4 billion years, in the Escherichia coli cytoplasm. Using synthetic gene constructs for recombinant expression of the ancestral enzymes, along with thermodynamic and kinetic assays, we show that all ancestral thioredoxins, as today's thioredoxins, exhibit strongly reducing redox potentials, suggesting that thioredoxins served as catalysts of cellular reduction reactions from the beginning of evolution, even before the oxygen catastrophe. A detailed, quantitative characterization of their interactions with the electron donor TrxR from Escherichia coli and the electron acceptor methionine sulfoxide reductase, also from E. coli, strongly hinted that thioredoxins and thioredoxin reductases co-evolved and that the promiscuity of thioredoxins toward downstream electron acceptors was maintained during evolution. In summary, our findings suggest that thioredoxins evolved high specificity for their sole electron donor TrxR while maintaining promiscuity to their multiple electron acceptors.


Assuntos
Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , História Antiga , Cinética , NADP/metabolismo , Oxidantes/metabolismo , Oxirredução , Relação Estrutura-Atividade
11.
Int J Nanomedicine ; 14: 4991-5015, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371943

RESUMO

Purpose: This study evaluates the cytotoxicity of AuNPs coated with polyallylamine (AuNPs-PAA) and conjugated or not to the epidermal growth factor receptor (EGFR)-targeting antibody Cetuximab (AuNPs-PAA-Ctxb) in normal human kidney (HK-2), liver (THLE-2) and microvascular endothelial (TIME) cells, and compares it with two cancer cell lines that are EGFR-overexpressing (A431) or EGFR-negative (MDA-MB-453). Results: Conjugation of Cetuximab to AuNPs-PAA increased the AuNPs-PAA-Ctxb interactions with cells, but reduced their cytotoxicity. TIME cells exhibited the strongest reduction in viability after exposure to AuNPs-PAA(±Ctxb), followed by THLE-2, MDA-MB-453, HK-2 and A431 cells. This cell type-dependent sensitivity was strongly correlated to the inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR), and to the depolarization of the mitochondrial membrane potential. Both are suggested to initiate apoptosis, which was indeed detected in a concentration- and time-dependent manner. The role of oxidative stress in AuNPs-PAA(±Ctxb)-induced cytotoxicity was demonstrated by co-incubation of the cells with N-acetyl L-cysteine (NAC), which significantly decreased apoptosis and mitochondrial membrane depolarization. Conclusion: This study helps to identify the cells and tissues that could be sensitive to AuNPs and deepens the understanding of the risks associated with the use of AuNPs in vivo.


Assuntos
Antioxidantes/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/farmacologia , Endocitose/efeitos dos fármacos , Glutationa Redutase/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tamanho da Partícula , Poliaminas/química , Substâncias Protetoras/farmacologia , Eletricidade Estática , Tiorredoxina Dissulfeto Redutase/metabolismo
12.
Toxicol In Vitro ; 61: 104590, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31279089

RESUMO

The thioredoxin (Trx) system controls cellular redox in vascular smooth muscle cells. The present study investigated the roles of Trx1 and Trx reductase1 (TrxR1) proteins in regulation of cell growth, death, reactive oxygen species (ROS) and glutathione (GSH) levels in hydrogen peroxide (H2O2)-treated human pulmonary artery smooth muscle (HPASM) cells. H2O2 induced growth inhibition and cell death in HPASM cells over 24 h. Overexpression of Trx1 and TrxR1 using adenoviruses significantly weakened cell growth inhibition and cell death caused by H2O2. Increases in ROS levels including mitochondrial superoxide anion (O2•-) were observed as early as 5-30 min after H2O2 addition. Administration of adTrxR1 attenuated H2O2-induced increases in ROS levels at 30-180 min. adTrx1 and adTrxR1 significantly reduced the increases in O2•- level in H2O2-treated HPASM cells at 24 h. Furthermore, HPASM cells transfected with Trx1 or TrxR1 siRNA showed increases in ROS levels with or without H2O2 at 5 min. While H2O2 transiently decreased GSH level at 5 min, Trx1 and TrxR1 siRNA intensified the decrease in GSH level. In conclusion, upregulation of Trx1 and TrxR1 significantly attenuated cell growth inhibition and death in H2O2-treated HPASM cells. As a whole, Trx-related adenoviruses diminished H2O2-induced ROS level in HPASM cells whereas Trx-related siRNAs increased ROS levels and decreased GSH level in these cells.


Assuntos
Peróxido de Hidrogênio/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Glutationa/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , RNA Interferente Pequeno/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Transfecção , Regulação para Cima
13.
J Biol Chem ; 294(36): 13336-13343, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31320475

RESUMO

Dynamic control of thioredoxin (Trx) oxidoreductase activity is essential for balancing the need of cells to rapidly respond to oxidative/nitrosative stress and to temporally regulate thiol-based redox signaling. We have previously shown that cytokine stimulation of the respiratory epithelium induces a precipitous decline in cell S-nitrosothiol, which depends upon enhanced Trx activity and proteasome-mediated degradation of Txnip (thioredoxin-interacting protein). We now show that tumor necrosis factor-α-induced Txnip degradation in A549 respiratory epithelial cells is regulated by the extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase pathway and that ERK inhibition augments both intracellular reactive oxygen species and S-nitrosothiol. ERK-dependent Txnip ubiquitination and proteasome degradation depended upon phosphorylation of a PXTP motif threonine (Thr349) located within the C-terminal α-arrestin domain and proximal to a previously characterized E3 ubiquitin ligase-binding site. Collectively, these findings demonstrate the ERK mitogen-activated protein kinase pathway to be integrally involved in regulating Trx oxidoreductase activity and that the regulation of Txnip lifetime via ERK-dependent phosphorylation is an important mediator of this effect.


Assuntos
Proteínas de Transporte/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Células A549 , Humanos , Espectrometria de Massas , Células Tumorais Cultivadas
14.
Biomed Khim ; 65(3): 165-179, 2019 Apr.
Artigo em Russo | MEDLINE | ID: mdl-31258141

RESUMO

Monocytes and macrophages play a key role in the development of inflammation: under the action of lipopolysaccharides (LPS), absorbed from the intestine, monocytes and macrophages form reactive oxygen species (ROS) and cytokines, this leads to the development of oxidative stress, inflammation and/or apoptosis in all types of tissues. In the cells LPS induce an "internal" TLR4-mediated MAP-kinase inflammatory signaling pathway and cytokines through the superfamily of tumor necrosis factor receptor (TNFR) and the "death domain" (DD) initiate an "external" caspase apoptosis cascade or necrosis activation that causes necroptosis. Many of the proteins involved in intracellular signaling cascades (MYD88, ASK1, IKKa/b, NF-kB, AP-1) are redox-sensitive and their activity is regulated by antioxidants thioredoxin, glutaredoxin, nitroredoxin, and glutathione. Oxidation of these signaling proteins induced by ROS enhances the development of inflammation and apoptosis, and their reduction with antioxidants, on the contrary, stabilizes the signaling cascades speed, preventing the vicious circle of oxidative stress, inflammation and apoptosis that follows it. Antioxidant (AO) enzymes thioredoxin reductase (TRXR), glutaredoxin reductase (GLRXR), glutathione reductase (GR) are required for reduction of non-enzymatic antioxidants (thioredoxin, glutaredoxin, nitroredoxin, glutathione), and AO enzymes (SOD, catalase, GPX) are required for ROS deactivation. The key AO enzymes (TRXR and GPX) are selenium-dependent; therefore selenium deficiency leads to a decrease in the body's antioxidant defense, the development of oxidative stress, inflammation, and/or apoptosis in various cell types. Nrf2-Keap1 signaling pathway activated by selenium deficiency and/or oxidative stress is necessary to restore redox homeostasis in the cell. In addition, expression of some genes is changed with selenium deficiency. Consequently, growth and proliferation of cells, their movement, development, death, and survival, as well as the interaction between cells, the redox regulation of intracellular signaling cascades of inflammation and apoptosis, depend on the selenium status of the body. Prophylactic administration of selenium-containing preparations (natural and synthetic (organic and inorganic)) is able to normalize the activity of AO enzymes and the general status of the body. Organic selenium compounds have a high bioavailability and, depending on their concentration, can act both as selenium donors to prevent selenium deficiency and as antitumor drugs due to their toxicity and participation in the regulation of signaling pathways of apoptosis. Known selenorganic compounds diphenyldiselenide and ethaselen share similarity with the Russian organo selenium compound, diacetophenonylselenide (DAPS-25), which serves as a source of bioavailable selenium, exhibits a wide range of biological activity, including antioxidant activity, that governs cell redox balance, inflammation and apoptosis regulation.


Assuntos
Apoptose , Inflamação/metabolismo , Estresse Oxidativo , Compostos de Selênio/metabolismo , Antioxidantes/metabolismo , Glutationa Redutase/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Selênio , Transdução de Sinais , Tiorredoxina Dissulfeto Redutase/metabolismo
15.
Cell Mol Life Sci ; 76(20): 3969-3985, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31218451

RESUMO

Cardiovascular diseases represent one of the most important health problems of developed countries. One of the main actors involved in the onset and development of cardiovascular diseases is the increased production of reactive oxygen species that, through lipid peroxidation, protein oxidation and DNA damage, induce oxidative stress and cell death. Basic and clinical research are ongoing to better understand the endogenous antioxidant mechanisms that counteract oxidative stress, which may allow to identify a possible therapeutic targeting/application in the field of stress-dependent cardiovascular pathologies. In this context, increasing attention is paid to the glutathione/glutathione-peroxidase and to the thioredoxin/thioredoxin-reductase systems, among the most potent endogenous antioxidative systems. These key enzymes, belonging to the selenoprotein family, have a well-established function in the regulation of the oxidative cell balance. The aim of the present review was to highlight the role of selenoproteins in cardiovascular diseases, introducing the emerging cardioprotective role of endoplasmic reticulum-resident members and in particular one of them, namely selenoprotein T or SELENOT. Accumulating evidence indicates that the dysfunction of different selenoproteins is involved in the susceptibility to oxidative stress and its associated cardiovascular alterations, such as congestive heart failure, coronary diseases, impaired cardiac structure and function. Some of them are under investigation as useful pathological biomarkers. In addition, SELENOT exhibited intriguing cardioprotective effects by reducing the cardiac ischemic damage, in terms of infarct size and performance. In conclusion, selenoproteins could represent valuable targets to treat and diagnose cardiovascular diseases secondary to oxidative stress, opening a new avenue in the field of related therapeutic strategies.


Assuntos
Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peptídeos/uso terapêutico , Selenocisteína/metabolismo , Selenoproteínas/genética , Animais , Antioxidantes/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/terapia , Regulação da Expressão Gênica , Glutationa Peroxidase/metabolismo , Humanos , Terapia de Alvo Molecular/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Selenoproteínas/agonistas , Selenoproteínas/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo
16.
Plant Cell Environ ; 42(9): 2627-2644, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31222760

RESUMO

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH-dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2-Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild-type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC-promoted growth, changes in root architecture, shifts in expression of VC-responsive CK- and ABA-regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc-Δ2cp plants with reduced 2-Cys peroxiredoxin expression. OxiTRAQ-based quantitative and site-specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis-related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.


Assuntos
Alternaria , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/metabolismo , Compostos Orgânicos Voláteis/farmacologia , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Fotossíntese/efeitos dos fármacos , Proteoma
17.
Drug Res (Stuttg) ; 69(10): 528-536, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31252433

RESUMO

BACKGROUND: In cancer cells, the intracellular antioxidant capacity and the redox homeostasis are mainly maintained by the glutathione- and thioredoxin-dependent systems which are considered as promising targets for anticancer drugs. Pyridazinones constitute an interesting source of heterocyclic compounds for drug discovery. The present investigation focused on studying the in-vitro antitumor activity of newly synthesized Pyridazin-3(2h)-ones derivatives against P815 (Murin mastocytoma) cell line. METHODS: The in-vitro cytotoxic activities were investigated toward the P815 cell line using tetrazolium-based MTT assay. Lipid peroxidation and the specific activities of antioxidant enzymes were also determined. RESULTS: The newly compounds had a selective dose-dependent cytotoxic effect without affecting normal cells (PBMCs). Apoptosis was further confirmed through the characteristic apoptotic morphological changes and DNA fragmentation. Two compounds (6F: and 7H: ) were highly cytotoxic and were submitted to extend biological testing to determine the likely mechanisms of their cytotoxicity. Results showed that these molecules may induce cytotoxicity via disturbing the redox homeostasis. Importantly, the anticancer activity of 6F: and 7H: could be due to the intracellular reactive oxygen species hypergeneration through significant loss of glutathione reductase and thioredoxin reductase activities. This eventually leads to oxidative stress-mediated P815 cell apoptosis. Furthermore, the co-administration of 6F: or 7H: with Methotrexate exhibited a synergistic cytotoxic effect. CONCLUSIONS: considering their significant anticancer activity and chemosensitivity, 6F: and 7H: may improve the therapeutic efficacy of the current treatment for cancer.


Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Piridazinas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Leucócitos Mononucleares , Peroxidação de Lipídeos/efeitos dos fármacos , Mastocitoma/tratamento farmacológico , Mastocitoma/patologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo
18.
Eur J Pharmacol ; 855: 112-123, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059712

RESUMO

Daucosterol (DS) is a plant phytosterol which is shown to induce oxidative stress mediated apoptosis in various cancer cell lines. However, the molecular mechanism underlying its cellular action has not been documented against Non- Small Cell Lung Cancer (NSCLC). Therefore, we attempted to decipher the mechanisms responsible for DS-induced anti-proliferation on human NSCLC cells. The present study showed, DS strongly inhibits the growth of A549 cells after 72 h time point with an IC50 value of ∼20.9 µM. Further DS elicits increased reactive oxygen species level and promote intrinsic apoptotic cell death on A549 cells as evidenced by increased expression of caspase-3, caspase-9, Bax, PARP inactivation, cytochrome-c release, and diminished expression of bcl-2 protein. DS failed to display its apoptotic actions upon pretreatment with the reactive oxygen species inhibitor NAC (N-acetyl cysteine). Indeed, apoptotic signal which was enhanced through p53/p21 activation and knockdown of p53 expression also moderately affected the DS induced apoptosis. In addition, DS preferentially inhibited the cell growth of p53 wild-type NSCLC cell lines than the mutant p53 models. Further, we show that inhibition of Thioredoxin (TrxR) redox system is principally associated with DS induced oxidative stress mediated apoptotic cell death on A549 cells. Moreover, we also demonstrated that DS stably interacted with serine residues in TrxR active sites. The obtained results confirmed that the anti-proliferative mechanism and increased reactive oxygen species level of DS was associated with down-regulation of TrxR1 pathway which triggers the p53 mediated intrinsic apoptotic mode of cell death in NSCLC cells.


Assuntos
Apoptose/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sitosteroides/farmacologia , Tiorredoxina Redutase 1/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Regulação para Baixo/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Oxirredução/efeitos dos fármacos , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sitosteroides/metabolismo , Termodinâmica , Tiorredoxina Redutase 1/química
19.
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
20.
Biochem J ; 476(7): 1159-1172, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988137

RESUMO

Photosynthesis is a highly regulated process in photoautotrophic cells. The main goal of the regulation is to keep the basic photosynthetic reactions, i.e. capturing light energy, conversion into chemical energy and production of carbohydrates, in balance. The rationale behind the evolution of strong regulation mechanisms is to keep photosynthesis functional under all conditions encountered by sessile plants during their lifetimes. The regulatory mechanisms may, however, also impair photosynthetic efficiency by overriding the photosynthetic reactions in controlled environments like crop fields or bioreactors, where light energy could be used for production of sugars instead of dissipation as heat and down-regulation of carbon fixation. The plant chloroplast has a high number of regulatory proteins called thioredoxins (TRX), which control the function of chloroplasts from biogenesis and assembly of chloroplast machinery to light and carbon fixation reactions as well as photoprotective mechanisms. Here, we review the current knowledge of regulation of photosynthesis by chloroplast TRXs and assess the prospect of improving plant photosynthetic efficiency by modification of chloroplast thioredoxin systems.


Assuntos
Tiorredoxinas de Cloroplastos/metabolismo , Fotossíntese/fisiologia , Plantas/metabolismo , Tiorredoxinas de Cloroplastos/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Luz , Modelos Biológicos , Oxirredução , Estresse Oxidativo , Fotossíntese/genética , Plantas/genética , Plantas Geneticamente Modificadas , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo
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