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1.
mBio ; 12(4): e0209421, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34399606

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.


Assuntos
Proteases 3C de Coronavírus/metabolismo , Cisteína/química , Glutationa/química , Multimerização Proteica , SARS-CoV-2/metabolismo , Animais , COVID-19/patologia , Quirópteros/virologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Dimerização , Glutarredoxinas/metabolismo , Humanos , SARS-CoV-2/enzimologia
2.
Free Radic Biol Med ; 174: 73-83, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34332079

RESUMO

AIMS: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. RESULTS: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. CONCLUSIONS: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.


Assuntos
Proteínas Nucleares , Sirtuína 1 , Animais , Apoptose , Células Endoteliais/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio , Camundongos , Oxirredução , Sirtuína 1/genética , Sirtuína 1/metabolismo
3.
Atherosclerosis ; 328: 23-32, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34077868

RESUMO

BACKGROUND AND AIMS: The thiol transferase glutaredoxin 1 controls redox signaling and cellular functions by regulating the S-glutathionylation status of critical protein thiols. Here we tested the hypothesis that by derepressing the expression of glutaredoxin 1, inhibition of histone deacetylase 2 prevents nutrient stress-induced protein S-glutathionylation and monocyte dysfunction and protects against atherosclerosis. METHODS: Using both a pharmacological inhibitor and shRNA-mediated knockdown of histone deacetylase 2, we determine the role of this deacetylase on glutaredoxin 1 expression and nutrient stress-induced inactivation of mitogen-activated protein kinase phosphatase 1 activity and monocyte and macrophage dysfunction. To assess whether histone deacetylase 2 inhibition in myeloid cells protects against atherosclerosis, we fed eight-week-old female and male HDAC2-/-MyeloidLDLR-/- mice and age and sex-matched LysMcretg/wtLDLR-/- control mice a high-calorie diet for 12 weeks and assessed monocyte function and atherosclerotic lesion size. RESULTS: Myeloid histone deacetylase 2 deficiency in high-calorie diet-fed LDLR-/- mice reduced atherosclerosis in males by 39% without affecting plasma lipid and lipoprotein profiles or blood glucose levels but had no effect on atherogenesis in female mice. Macrophage content in plaques of male mice was reduced by 31%. Histone deacetylase 2-deficient blood monocytes from male mice showed increased acetylation on histone 3, and increased Grx1 expression, and was associated with increased MKP-1 activity and reduced recruitment of monocyte-derived macrophages, whereas in females, myeloid HDAC2 deficiency had no effect on Grx1 expression, did not prevent nutrient stress-induced loss of MKP-1 activity in monocytes and was not atheroprotective. CONCLUSIONS: Specific histone deacetylase 2 inhibitors may represent a potential novel therapeutic strategy for the prevention and treatment of atherosclerosis, but any benefits may be sexually dimorphic.


Assuntos
Aterosclerose , Monócitos , Animais , Aterosclerose/prevenção & controle , Dieta , Feminino , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Histona Desacetilase 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Oxirredução , Receptores de LDL/genética , Receptores de LDL/metabolismo , Compostos de Sulfidrila
4.
Free Radic Biol Med ; 172: 340-349, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34146665

RESUMO

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.


Assuntos
Glutarredoxinas , Glutationa , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa , Glutationa Redutase , Oxirredução
5.
Methods Mol Biol ; 2276: 113-127, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060036

RESUMO

Disruptions in mitochondrial redox activity are implicated in maladies ranging from those in which cells degenerate to those in which cell division is unregulated. This is not surprising given the pivotal role of mitochondria as ATP producers, reactive oxygen species (ROS) generators, and gatekeepers of apoptosis. While increased ROS are implicated in such a wide variety of disorders, pinpointing the cause of their hyperproduction is challenging. Elevated levels of ROS can result from increases in their production and/or decreases in their turnover. Disruptions in and/or hyperactivity of NADH-ubiquinone oxidoreductase or ubiquinone-cytochrome c oxidoreductase can cause excessive ROS generation. Alternatively, if respiration is functioning in a homeostatic manner, decreases in levels or activity of antioxidants like glutathione, CuZn- and Mn-superoxide dismutase, and catalase could result in excessive ROS. Because of the diversity of disorders in which oxidative damage occurs, the most effective therapeutic strategies may be those that address the putatively diverse causes of increased ROS. Strategies for determining antioxidant activity typically involve semiquantitative measurement of relative protein levels using immunochemistry and mass spectrometry. These methods can be applied to a variety of samples, but they do not lend themselves to detection of cell-specific analyses within tissue like brain.Because we are interested in elucidating the cause of oxidative stress in selectively vulnerable brain neurons, we have taken advantage of the easily manipulatable genetics and high fecundity of the fly. Using a cell type-targeting approach, we have driven redox sensitive green fluorescent proteins (roGFP2 ) into the mitochondria of tyrosine hydroxylase-producing (dopaminergic) neurons. In oxidizing conditions, the fluorophore's maximal excitation wavelength reversibly shifts. Therefore, the relative amount of mitochondrial protein oxidation can be determined by taking the ratio of fluorescence excited with two different lasers. In addition, these GFPs have been independently fused to human glutaredoxin-1 (mito-roGFP2-Grx1) and yeast oxidant receptor peroxidase (mito-roGFP2-Orp1), facilitating measurements of relative mitochondrial glutathione redox potential and H2O2 levels, respectively. In order to obtain a more comprehensive observation of redox states, we capture 3D images of roGFP2 excited by two different lasers. Mito- and cytoplasmic-roGFP2 -Grx1 and -Orp1 expression can be driven by hundreds of genetic drivers in Drosophila , facilitating fixed or living whole organism or tissue- and cell-specific redox measurements.


Assuntos
Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Animais , Catalase/metabolismo , Drosophila , Complexo I de Transporte de Elétrons/metabolismo , Glutarredoxinas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peróxido de Hidrogênio/análise , Imageamento Tridimensional/métodos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069397

RESUMO

Drought stress is a major constraint in global maize production, causing almost 30-90% of the yield loss depending upon growth stage and the degree and duration of the stress. Here, we report that ectopic expression of Arabidopsis glutaredoxin S17 (AtGRXS17) in field grown maize conferred tolerance to drought stress during the reproductive stage, which is the most drought sensitive stage for seed set and, consequently, grain yield. AtGRXS17-expressing maize lines displayed higher seed set in the field, resulting in 2-fold and 1.5-fold increase in yield in comparison to the non-transgenic plants when challenged with drought stress at the tasseling and silking/pollination stages, respectively. AtGRXS17-expressing lines showed higher relative water content, higher chlorophyll content, and less hydrogen peroxide accumulation than wild-type (WT) control plants under drought conditions. AtGRXS17-expressing lines also exhibited at least 2-fold more pollen germination than WT plants under drought stress. Compared to the transgenic maize, WT controls accumulated higher amount of proline, indicating that WT plants were more stressed over the same period. The results present a robust and simple strategy for meeting rising yield demands in maize under water limiting conditions.


Assuntos
Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Estresse Fisiológico/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secas , Expressão Ectópica do Gene/genética , Grão Comestível/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal/genética , Estresse Fisiológico/fisiologia , Termotolerância/genética , Zea mays/genética
7.
Pestic Biochem Physiol ; 176: 104881, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34119223

RESUMO

Glutaredoxins (Grxs) and thioredoxin peroxidases (Tpxs) are major antioxidant enzyme families involved in regulating cellular redox homeostasis and in defense of enhanced oxidative stress through scavenging reactive oxygen species (ROS). However, the functions of these enzymes have not been reported in the oriental fruit moth, Grapholita molesta (Busck), a worldwide pest of stone and pome fruits. Here, we identified four new antioxidant genes, GmGrx, GmGrx3, GmGrx5, and GmTpx which were induced by exposure with emamectin benzoate, a commonly used biopesticide for G. molesta control. Other environmental factors (low and high temperatures, Escherichia coli and Metarhizium anisopliae) also significantly induced the expression of these genes. After GmGrx or GmTpx silenced by RNA interference (RNAi), the percentage of larval survival to emamectin benzoate were significantly decreased, demonstrating that GmGrx and GmTpx are involved in protecting G. molesta from stresses induced by emamectin benzoate. Furthermore, silenced GmGrx, GmGrx3, GmGrx5, or GmTpx significantly enhanced the enzymatic activities of superoxide dismutase (SOD) (except GmTpx) and peroxidase (POD), as well as the contents of hydrogen peroxide and metabolites ascorbate. Taken together, our results suggest that GmGrx, GmGrx3, GmGrx5, and GmTpx may play critical roles in antioxidant defense. Specially, GmGrx and GmTpx contribute to the defense of oxidative damage induced by exposure to emamectin benzoate through scavenging excessive ROS in G. molesta. Our findings provided a theoretical basis for understanding functions of insect glutaredoxin and peroxidase systems.


Assuntos
Glutarredoxinas , Mariposas , Animais , Glutarredoxinas/metabolismo , Ivermectina/análogos & derivados , Mariposas/genética , Mariposas/metabolismo , Estresse Oxidativo , Peroxirredoxinas
8.
Redox Biol ; 43: 101975, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33932870

RESUMO

Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.


Assuntos
Glutarredoxinas , Glutationa , Catálise , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Humanos , Oxirredução , Filogenia
9.
Nat Plants ; 7(3): 310-316, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33686225

RESUMO

The nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation1,2. However, the critical enzymes required for the post-translational activation of NRT2.1 remain to be identified. Here, we show that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating Ser501 of NRT2.1, a residue that functions as a negative phospho-switch in Arabidopsis2. CEPH is predominantly expressed in epidermal and cortex cells in roots and is upregulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signalling from shoots3,4. The loss of CEPH leads to marked decreases in high-affinity nitrate uptake, tissue nitrate content and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in the N-starvation-dependent activation of NRT2.1 and provide molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitratos/metabolismo , Arabidopsis/enzimologia , Glutarredoxinas/metabolismo , Fosforilação , Serina/metabolismo
10.
J Biotechnol ; 329: 192-203, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33610657

RESUMO

Soil salinity is one of the critical issue worldwide that adversely affect soil fertility. Salt stress significantly limits crop yield and grain quality; therefore, there is an urgent need to develop a strategy to improve salt stress tolerance. In present study, we reported that rice glutaredoxin (OsGrx_C7) plays a positive response in salt induced stress. Gene expression analysis, silencing, and overexpression of OsGrx_C7 gene were used to discover the role of OsGrx_C7 in response to salt stress. Gene expression analysis suggested that OsGrx_C7 expression was induced under salt stress and ubiquitously expressed in rice including root and shoot. The silencing of osgrx_c7 gene leads to increased sensitivity to salt stress, indicating its importance in salt stress tolerance. A gain-of-function approach showed that OsGrx_C7 may act as an important determinant in salt stress, compared with WT, and revealed higher biomass accumulation, improved root and plant growth under salt stress. Under salt stress condition, OsGrx_C7 overexpressing rice plants showed lower level of lipid peroxidation and Na+/K+ ratio, while proline accumulation, soluble sugar content and GSH/GSSG ratio was higher compared to WT. Furthermore, expression analysis suggested that OsGrx_C7 acted as positive regulator of salt tolerance by reinforcing the expression of transporters (OsHKT2;1, OsHKT1;5 and OsSOS1) engaged in Na+ homeostasis in overexpressing plants. Overall our study revealed that OsGrx_C7 emerged as a key mediator in response to salt stress in rice and could be used for engineering tolerance against salt stress in rice and other crops.


Assuntos
Glutarredoxinas , Oryza , Regulação da Expressão Gênica de Plantas , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Oryza/enzimologia , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Salinidade , Estresse Salino , Tolerância ao Sal/genética , Estresse Fisiológico
11.
Int Immunopharmacol ; 94: 107428, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33581580

RESUMO

Glutaredoxin 2 (GRX2) plays a cytoprotective role under various pathological conditions. However, whether GRX2 plays a role during myocardial ischemia-reperfusion injury has not been fully elucidated. In this work, we aimed to explore the detailed role and mechanism of GRX2 in modulating hypoxia/reoxygenation (H/R)-induced cardiac injury in vitro. H/R treatment resulted in a significant increase in GRX2 expression in cardiomyocytes. GRX2 knockdown enhanced the sensitivity of cardiomyocytes to H/R-induced apoptosis, oxidative stress, and inflammation, while GRX2 up-regulation exerted a cardioprotective role in H/R-injured cardiomyocytes. Further investigations revealed that GRX2 up-regulation enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling associated with upregulation of the phosphorylation of Akt and glycogen synthase kinase-3ß (GSK-3ß). Akt inhibition markedly abolished GRX2-mediated activation of Nrf2, while GSK-3ß inhibition reversed GRX2-knockdown-mediated inhibition of Nrf2. In addition, Nrf2 inhibition markedly abrogated GRX2-mediated protective effects against H/R-induced apoptosis, oxidative stress and inflammation. Overall, this work indicates that GRX2 protects cardiomyocytes from H/R-induced apoptosis, oxidative stress, and inflammation by enhancing Nrf2 activation via modulation of the Akt/GSK-3ß axis. Our study highlights a potential relevance of GRX2 in myocardial ischemia-reperfusion injury; it may serve as an attractive target for cardioprotection.


Assuntos
Hipóxia Celular , Glutarredoxinas/metabolismo , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Apoptose , Linhagem Celular , Citocinas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Inflamação/metabolismo , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
12.
Biochim Biophys Acta Bioenerg ; 1862(1): 148317, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980338

RESUMO

Among the thioredoxin superfamily of proteins, the observation that numerous glutaredoxins bind iron-sulphur (Fe/S) clusters is one of the more recent and major developments concerning their functional properties. Glutaredoxins are present in most organisms. All members of the class II subfamily (including most monothiol glutaredoxins), but also some members of the class I (mostly dithiol glutaredoxins) and class III (land plant-specific monothiol or dithiol glutaredoxins) are Fe/S proteins. In glutaredoxins characterised so far, the [2Fe2S] cluster is coordinated by two active-site cysteine residues and two molecules of non-covalently bound glutathione in homo-dimeric complexes bridged by the cluster. In contrast to dithiol glutaredoxins, monothiol glutaredoxins possess no or very little oxidoreductase activity, but have emerged as important players in cellular iron metabolism. In this review we summarise the recent developments of the most prominent Fe/S glutaredoxins in eukaryotes, the mitochondrial single domain monothiol glutaredoxin 5, the chloroplastic single domain monothiol glutaredoxin S14 and S16, the nuclear/cytosolic multi-domain monothiol glutaredoxin 3, and the mitochondrial/cytosolic dithiol glutaredoxin 2.


Assuntos
Glutarredoxinas , Proteínas Ferro-Enxofre , Doenças das Plantas , Proteínas de Plantas , Plantas/enzimologia , Glutarredoxinas/química , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Ferro/química , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Relação Estrutura-Atividade , Enxofre/química , Enxofre/metabolismo
13.
Plant Physiol Biochem ; 159: 135-147, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360237

RESUMO

Reaumuria trigyna, an endangered recretohalophyte, is a small archaic wild shrub endemic to arid and semiarid plateau regions of Inner Mongolia, China. Based on salt-related transcriptomic data, we isolated a GRX family gene, glutaredoxin like protein (RtGRL1), from R. trigyna that is associated with the removal of active oxygen and regulation of redox status. RtGRL1 encodes a plasma membrane and chloroplast-localized protein induced by salt, cold, drought stress, ABA, and H2O2. In Arabidopsis thaliana, ectopically expressed RtGRL1 positively regulated biomass accumulation, chlorophyll content, germination rate, and primary root length under salt and drought stress. Overexpression of RtGRL1 induced expression of genes related to antioxidant enzymes and proline biosynthesis, thus increasing glutathione biosynthesis, glutathione-dependent detoxification of reactive oxygen species (ROS), and proline content under stress. Changes in RtGRL1 expression consistently affected glutathione/oxidizedglutathione and ascorbate/dehydroascorbate ratios and H2O2 concentrations. Furthermore, RtGRL1 promoted several GSH biosynthesis gene transcripts, decreased leaf Na+ content, and maintained lower Na+/K+ ratios in transgenic A. thaliana compared to wild type plants. These results suggest a critical link between RtGRL1 and ROS modulation, and contribute to a better understanding of the mechanisms governing plant responses to drought and salt stress.


Assuntos
Glutationa , Peróxido de Hidrogênio , Proteínas de Plantas , Estresse Fisiológico , Tamaricaceae , Arabidopsis/genética , China , Secas , Regulação da Expressão Gênica de Plantas , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Tamaricaceae/genética , Tamaricaceae/metabolismo
14.
Int J Biochem Cell Biol ; 131: 105904, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33359085

RESUMO

Nitric Oxide is a very well known gaseous second messenger molecule and vasorelaxant agent involved in a variety of signaling in the body such as neurotransmission, ion channel modulation, and inflammation modulation. However, it's reversible covalent attachment to thiol groups of cysteine residues under nitrosative stress leading to aberrant protein S-nitrosylation (PSNO) has been reported in several pathological conditions in the body stemming from neurodegenerative diseases, cancer, cardiovascular system, and immune system disorders. In the cell, PSNOs are partly unstable and transit to a more stable disulfide state serving as an intermediate step towards disulfide formation thus eliciting the biological response. Scientists have identified several cellular thiol-dependent disulfide reductases that have the intrinsic capability to reverse the modification by reducing the stable disulfides formed in PSNOs and thereby rescue S-nitrosylation-induced altered proteins. The physiological roles of these major cellular ubiquitous S-denitrosylases and their probable implementations have not been fully explored. Gaining knowledge from current research and development this review provides a deeper insight into understanding the interplay and role of the major ubiquitous S-denitrosylases in maintaining cellular redox homeostasis. This review umbrellas the mechanism of Thioredoxin, TRP14, and Glutaredoxin systems and highlights their substrates specificities at different cellular conditions, physiological roles, and importance in diseased conditions that would allow researchers to investigate effective therapeutic interventions for nitrosative stress-related diseases and disorders.


Assuntos
Doenças Cardiovasculares/enzimologia , Glutarredoxinas/metabolismo , Doenças do Sistema Imunitário/enzimologia , Neoplasias/enzimologia , Doenças Neurodegenerativas/enzimologia , Tiorredoxinas/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Cisteína/metabolismo , Regulação da Expressão Gênica , Glutarredoxinas/genética , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/patologia , Neoplasias/genética , Neoplasias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Óxido Nítrico/metabolismo , Nitrosação , Estresse Nitrosativo/genética , Oxirredução , S-Nitrosotióis/metabolismo , Transdução de Sinais , Especificidade por Substrato , Tiorredoxinas/genética
15.
Int J Mol Sci ; 21(23)2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33287436

RESUMO

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]2+ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]2+ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]2+ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]2+ clusters, via two-electron reductive coupling of two [2Fe-2S]2+ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Glutarredoxinas/química , Glutarredoxinas/isolamento & purificação , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/isolamento & purificação , Mitocôndrias/química , Mitocôndrias/genética , Ligação Proteica , Análise Espectral
16.
Front Immunol ; 11: 580934, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33329553

RESUMO

Glutaredoxin is central to cellular redox chemistry and regulates redox homeostasis and malignant progression of many cancers. In glioma, the role of its coding gene (GLRX) remains unclear. We aimed to elucidate the role of glutaredoxin at the transcriptome level and its clinical prognostic value in glioma. In total, we evaluated 1,717 glioma samples with transcriptome data and corresponding clinical data as well as single-cell sequencing data from 6 glioma patients from publicly available databases. Gene set variation analysis and gene ontology analysis were performed to reveal the biological function of GLRX. The immune cell enrichment score was calculated by GSVA analysis. Single-cell sequencing data was visualized by t-distributed stochastic neighbor embedding analysis. The prognostic value of GLRX in glioma was verified by the Kaplan-Meier curve and multivariate COX analysis. GLRX was found to be highly enriched in gliomas of higher grades with wild-type IDH, without 1p/19q co-deletion, and with a methylated MGMT promoter. Moreover, GLRX could be a potential marker for the mesenchymal molecular subtype of gliomas. The expression of GLRX was closely related to the tumor immune process, immune checkpoints, and inflammatory factors with GLRX being specifically expressed in M0 macrophages. GLRX is also shown to be an independent prognostic factor in glioma. Altogether, our study outcomes show that GLRX is highly enriched in malignant gliomas and is closely related to the tumor immune microenvironment. Therefore, GLRX-targeted cell redox regulatory therapy may enhance the efficacy of glioma immunotherapy.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Glutarredoxinas/metabolismo , Macrófagos/imunologia , Adulto , Antígenos de Neoplasias/imunologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/imunologia , Glioma/mortalidade , Glutarredoxinas/genética , Humanos , Imunidade , Masculino , Pessoa de Meia-Idade , Oxirredução , Análise de Sobrevida , Células THP-1
17.
Biol Chem ; 401(12): 1407-1428, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33031050

RESUMO

The physiological roles of the intracellular iron and redox regulatory systems are intimately linked. Iron is an essential trace element for most organisms, yet elevated cellular iron levels are a potent generator and amplifier of reactive oxygen species and redox stress. Proteins binding iron or iron-sulfur (Fe/S) clusters, are particularly sensitive to oxidative damage and require protection from the cellular oxidative stress protection systems. In addition, key components of these systems, most prominently glutathione and monothiol glutaredoxins are involved in the biogenesis of cellular Fe/S proteins. In this review, we address the biochemical role of glutathione and glutaredoxins in cellular Fe/S protein assembly in eukaryotic cells. We also summarize the recent developments in the role of cytosolic glutaredoxins in iron metabolism, in particular the regulation of fungal iron homeostasis. Finally, we discuss recent insights into the interplay of the cellular thiol redox balance and oxygen with that of Fe/S protein biogenesis in eukaryotes.


Assuntos
Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Oxirredução
18.
Biochem Biophys Res Commun ; 533(4): 1385-1392, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33097183

RESUMO

Rice is a particularly widely consumed food crop globally, but its yield is seriously damaged by bacterial blight due to Xanthomonas oryzae pv. oryzae (Xoo) and bakanae disease due to Fusarium fujikuroi (F. fujikuroi). However, broad-spectrum resistance (BSR) to both Xoo and F. fujikuroi remains largely elusive. In this study, we showed that rice monothiol glutaredoxin GRXS15 localizes in mitochondria and the nucleus, and its transcription is induced by Xoo. Transgenic rice lines constitutively expressing OsGRXS15 showed enhanced disease resistance to Xoo and F. fujikuroi, while CRISPR/Cas9-based knockout mutants showed reduced resistance compared with the wild-type plants. The transcription of pathogenesis-related (PR) genes was significantly induced in OsGRXS15-expressing plants. The rice transcription factor OsWRKY65 was identified as a binding partner, and it directly interacted with OsGRXS15 in the nucleus. Moreover, we revealed that the interaction of OsGRXS15 and OsWRKY65 results in the upregulation of OsPR1. These results suggested that OsGRXS15 interacts with transcription factors, and it confers BSR through regulating the expression of genes related to pathogen response. This is the first report on the nuclear function associated with the monothiol glutaredoxin GRXS15.


Assuntos
Fusarium/patogenicidade , Glutarredoxinas/metabolismo , Oryza/genética , Oryza/microbiologia , Xanthomonas/patogenicidade , Núcleo Celular/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Glutarredoxinas/genética , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Mitocôndrias/metabolismo , Oryza/citologia , Oryza/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas
19.
Mol Plant Microbe Interact ; 33(12): 1381-1393, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32970520

RESUMO

Glutaredoxin (GRX) plays an essential role in the control of the cellular redox state and related pathways in many organisms. There is limited information on GRXs from the model nitrogen (N2)-fixing bacterium Azorhizobium caulinodans. In the present work, we identified and performed functional analyses of monothiol and dithiol GRXs in A. caulinodans in the free-living state and during symbiosis with Sesbania rostrata. Our data show that monothiol GRXs may be very important for bacterial growth under normal conditions and in response to oxidative stress due to imbalance of the redox state in grx mutants of A. caulinodans. Functional redundancies were also observed within monothiol and dithiol GRXs in terms of different physiological functions. The changes in catalase activity and iron content in grx mutants were assumed to favor the maintenance of bacterial resistance against oxidants, nodulation, and N2 fixation efficiency in this bacterium. Furthermore, the monothiol GRX12 and dithiol GRX34 play a collective role in symbiotic associations between A. caulinodans and Sesbania rostrata. Our study provided systematic evidence that further investigations are required to understand the importance of glutaredoxins in A. caulinodans and other rhizobia.


Assuntos
Azorhizobium caulinodans , Glutarredoxinas , Homeostase , Simbiose , Azorhizobium caulinodans/genética , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Homeostase/genética , Oxirredução
20.
Plant J ; 104(5): 1423-1436, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32896918

RESUMO

To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant-specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novel ROXY1 function in the root meristem. Root cells that lack chlorophyll reducing plant-specific background problems that can hamper colocalization 3D microscopy. Thus far, a super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-dSTORM) approach has mainly been applied in animal studies. We established 3D-dSTORM using the roxy1 mutant complemented with green fluorescence protein-ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D-dSTORM was coupled with the coordinate-based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H2 O2 treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual-color 3D-dSTORM/coordinate-based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50 nm, which will enable the detection of novel subcellular protein associations and functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glutarredoxinas/metabolismo , Microscopia/métodos , Imagem Molecular/métodos , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , RNA Polimerase II/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/análise , Núcleo Celular/genética , Núcleo Celular/metabolismo , Glutarredoxinas/análise , Proteínas de Fluorescência Verde/genética , Peróxido de Hidrogênio/farmacologia , Isoenzimas/metabolismo , Meristema/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , RNA Polimerase II/análise , Processos Estocásticos , Transcrição Genética
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