RESUMEN
Proteins become S-glutathionylated as a result of the derivatization of their cysteine thiols with the thiolate anion derivative of glutathione; this process is frequently linked to diseases and protein misbehavior. Along with the other well-known oxidative modifications like S-nitrosylation, S-glutathionylation has quickly emerged as a major contributor to a number of diseases, with a focus on neurodegeneration. The immense clinical significance of S-glutathionylation in cell signaling and the genesis of diseases are progressively coming to light with advanced research, which is also creating new opportunities for prompt diagnostics that utilize this phenomenon. In-depth investigation in recent years has revealed other significant deglutathionylases in addition to glutaredoxin, necessitating the hunt for their specific substrates. The precise catalytic mechanisms of these enzymes must also be understood, along with how the intracellular environment affects their impact on protein conformation and function. These insights must then be extrapolated to the understanding of neurodegeneration and the introduction of novel and clever therapeutic approaches to clinics. Clarifying the importance of the functional overlap of glutaredoxin and other deglutathionylases and examining their complementary functions as defense systems in the face of stress are essential prerequisites for predicting and promoting cell survival under high oxidative/nitrosative stress.
Asunto(s)
Glutarredoxinas , Procesamiento Proteico-Postraduccional , Glutarredoxinas/metabolismo , Proteínas/metabolismo , Glutatión/metabolismo , Cisteína/metabolismo , Oxidación-Reducción , Estrés OxidativoRESUMEN
Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron-sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Hierro-Azufre , Trypanosoma brucei brucei , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Ligandos , Unión Proteica , Pliegue de Proteína , Trypanosoma brucei brucei/metabolismoRESUMEN
Plant class-II glutaredoxins (GRXs) are oxidoreductases carrying a CGFS active site signature and are able to bind iron-sulfur clusters in vitro. In order to explore the physiological functions of the 2 plastidial class-II isoforms, GRXS14 and GRXS16, we generated knockdown and overexpression Arabidopsis thaliana lines and characterized their phenotypes using physiological and biochemical approaches. Plants deficient in one GRX did not display any growth defect, whereas the growth of plants lacking both was slowed. Plants overexpressing GRXS14 exhibited reduced chlorophyll content in control, high-light, and high-salt conditions. However, when exposed to prolonged darkness, plants lacking GRXS14 showed accelerated chlorophyll loss compared to wild-type and overexpression lines. We observed that the GRXS14 abundance and the proportion of reduced form were modified in wild type upon darkness and high salt. The dark treatment also resulted in decreased abundance of proteins involved in the maturation of iron-sulfur proteins. We propose that the phenotype of GRXS14-modified lines results from its participation in the control of chlorophyll content in relation with light and osmotic conditions, possibly through a dual action in regulating the redox status of biosynthetic enzymes and contributing to the biogenesis of iron-sulfur clusters, which are essential cofactors in chlorophyll metabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Clorofila/metabolismo , Glutarredoxinas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Oscuridad , Ambiente , Regulación de la Expresión Génica de las Plantas , Proteínas Hierro-Azufre/metabolismo , Presión Osmótica , Oxidación-Reducción , Estrés Oxidativo , Extractos Vegetales/metabolismo , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plastidios/metabolismoRESUMEN
Glutaredoxins (Grxs) are ubiquitous thioltransferases and members of the thioredoxin (Trx) fold superfamily. They have multiple functions in cells including oxidative stress responses and cell signaling. A novel glutaredoxin from buckwheat (rbGrx) with higher catalytic activity was identified, cloned, and purified. The structures of glutathionylated rbGrx and an rbGrx mutant, in which cysteine 39 was mutated to alanine, were solved by x-ray diffraction at a resolution of 2.05Å and 2.29Å, respectively. In rbGrx, GSH (glutathione) is bound at the conserved GSH-binding site, and its structure shows that it has the potential to function as a scaffold protein for the assembly and delivery of GSH. The crystal structure shows that GSH does not bind to the C39A rbGrx mutant, and the C39A mutant had no catalytic activity, indicating that C39 is a key residue that is involved in both the binding of rbGrx to GSH and the regulation of its catalytic activity. The model showing the binding of GSH with rbGrx provides a basis for understanding its molecular function and its potential future applications in medicinal food science.
Asunto(s)
Fagopyrum/metabolismo , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Proteínas de Plantas/metabolismo , Glutarredoxinas/genética , Glutatión/genética , Proteínas de Plantas/genética , Unión Proteica , Difracción de Rayos XRESUMEN
Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.
Asunto(s)
Antioxidantes/farmacología , Benzofuranos/farmacología , Medicamentos Herbarios Chinos/farmacología , Células Epiteliales/efectos de los fármacos , Glutarredoxinas/genética , Peróxido de Hidrógeno/antagonistas & inhibidores , Disulfuros/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Feto , Regulación de la Expresión Génica , Glutarredoxinas/antagonistas & inhibidores , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system.
Asunto(s)
Antioxidantes/administración & dosificación , Catequina/análogos & derivados , Glutatión Reductasa/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Selenio/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Animales , Antioxidantes/farmacología , Catequina/administración & dosificación , Catequina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Glutatión Reductasa/genética , Ratones , Factor 2 Relacionado con NF-E2/genética , Especies Reactivas de Oxígeno/metabolismo , Selenio/deficiencia , Transducción de Señal/efectos de los fármacos , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/metabolismoRESUMEN
Glutaredoxin 5 (GLRX5) is a 156 amino acid mitochondrial protein that plays an essential role in mitochondrial iron-sulfur cluster transfer. Mutations in this protein were reported to result in sideroblastic anemia and variant nonketotic hyperglycinemia in human. Recently, we have characterized a Chinese congenital sideroblastic anemia patient who has two compound heterozygous missense mutations (c. 301 A>C and c. 443 T>C) in his GLRX5 gene. Herein, we developed a GLRX5 knockout K562 cell line and studied the biochemical functions of the identified pathogenic mutations and other conserved amino acids with predicted essential functions. We observed that the K101Q mutation (due to c. 301 A>C mutation) may prevent the binding of [Fe-S] to GLRX5 protein, while L148S (due to c. 443 T>C mutation) may interfere with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase) and ferrochelatase. We also demonstrated that L148S is functionally complementary to the K51del mutant with respect to Fe/S-ferrochelatase, Fe/S-IRP1, Fe/S-succinate dehydrogenase, and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex. Furthermore, we demonstrated that the mutations of highly conserved amino acid residues in GLRX5 protein can have different effects on downstream Fe/S proteins. Collectively, our current work demonstrates that GLRX5 protein is multifunctional in [Fe-S] protein synthesis and maturation and defects of the different amino acids of the protein will lead to distinct effects on downstream Fe/S biosynthesis.
Asunto(s)
Glutarredoxinas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Línea Celular , Glutarredoxinas/genética , Humanos , Proteínas Hierro-Azufre/genética , Mutación/genética , Unión ProteicaRESUMEN
The aim of the current study was to evaluate the effect of chronic dehydroepiandrosterone (DHEA) administration on steroid hormones and antioxidant parameters in aged rats. To this end, three groups of Sprague-Dawley rats were compared: young (3 months of age) untreated; aged (19 months old) untreated; and aged rats treated with 20 mg/kg DHEA for 8 weeks. Major organs of aged rats in the untreated group demonstrated physiological atrophy, compared to those of young rats; this effect appeared to have been partially reversed by DHEA treatment. Testosterone and estradiol contents were significantly decreased and aldosterone significantly increased in aged untreated, compared to young untreated rats. Steroid hormone levels were obviously reversed, however, in aged rats treated with DHEA. Additionally, superoxide dismutase activity in serum, brain, heart, and liver was decreased, and maleic dialdehyde content in heart was markedly increased in untreated aged, compared to young, rats. Importantly, these changes in brain and heart of aged rats were reversed by DHEA treatment. Heme oxygenase mRNA levels were increased and inducible nitric oxide synthase mRNA levels decreased in aged, compared to young, rats; DHEA treatment appeared to reverse these changes. These results indicate that chronic DHEA administration may have effects on steroid hormone levels and antioxidant parameters in aged rats and result in postponement of the aging process.
Asunto(s)
Envejecimiento/sangre , Deshidroepiandrosterona/farmacología , Aldosterona/sangre , Animales , Antioxidantes/metabolismo , Peso Corporal/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Estradiol/sangre , Expresión Génica/efectos de los fármacos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Masculino , Malondialdehído/sangre , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especificidad de Órganos , Oxidación-Reducción , Ratas Sprague-Dawley , Superóxido Dismutasa/sangre , Testosterona/sangreRESUMEN
Docosahexaenoic acid (DHA, 22:6n-3) is a major constituent of nerve cell membrane phospholipids. Besides a role in membrane architecture, DHA is a pleiotropic molecule involved in multiple facets of neuronal biology and also in neuroprotection. We show here that supplementation with DHA (but not arachidonic acid) to mouse hippocampal HT22 cells modulates the expression of genes encoding for antioxidant proteins associated with thioredoxin/peroxiredoxin and glutathione/glutaredoxin systems. Thus, within the thioredoxin system, DHA increased Txn1-2, Trxrd1-2, Prdx3, and Srxn1 gene expression. Paralleling these changes, DHA increased thioredoxin reductase activity, the main enzyme involved in thioredoxin regeneration. For the glutathione system, the most important change triggered by DHA was the upregulation of Gpx4 gene, encoding for the nuclear, cytosolic and mitochondrial isoforms of phospholipid-hydroperoxide glutathione peroxidase (PH-GPx/GPx4, the main enzyme protecting cell membranes against lipid peroxidation), which was followed by a significant increase in total glutathione peroxidase and GPx4 activities. Noticeably, DHA also upregulated a new Gpx4 splicing variant that retained part of the first intronic region. Finally, we demonstrate that DHA treatment, under the same time course, protects HT22 cells from the oxitoxic exposure to amyloid beta (Aß25-35 ) peptide. Altogether, our data pinpoint to a role of DHA as Indirect Antioxidant that modulates neuronal defences in neuroprotection. DHA improves the antioxidant capacity of cultured hippocampal HT22 cells. We propose that DHA supplementation induces the remodelling of membrane phospholipids, and also triggers a transcriptional program to increase the expression of members of the glutathione and thioredoxin systems. We postulate that this transcriptional effect is mediated by a signal arising from non-enzymatic oxidation of DHA.
Asunto(s)
Ácido Araquidónico/farmacología , Ácidos Docosahexaenoicos/farmacología , Expresión Génica/efectos de los fármacos , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Tiorredoxinas/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Antioxidantes/farmacología , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Hipocampo/citología , Ratones , Fragmentos de Péptidos/farmacología , Peroxirredoxinas/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factores de TiempoRESUMEN
Although conventional radiotherapy can directly damage DNA and other organic molecules within cells, most of the damage and the cytotoxicity of such ionising radiation, comes from the production of ions and free radicals produced via interactions with water. This 'indirect effect', a form of oxidative stress, can be modulated by a variety of systems within cells that are in place to, in normal situations, maintain homeostasis and redox balance. If cancer cells express high levels of antioxidant redox proteins, they may be more resistant to radiation and so targeting such systems may be a profitable strategy to increase therapeutic efficacy of conventional radiotherapy. An overview, with exemplars, of the main systems regulating redox homeostasis is supplied and discussed in relation to their use as prognostic and predictive biomarkers, and how targeting such proteins and systems may increase radiosensitivity and, potentially, improve the radiotherapeutic response.
Asunto(s)
Neoplasias/metabolismo , Neoplasias/radioterapia , Tolerancia a Radiación/fisiología , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/metabolismo , Suplementos Dietéticos , Radicales Libres/metabolismo , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Oxidación-Reducción , Peroxirredoxinas/metabolismo , Pronóstico , Fármacos Sensibilizantes a Radiaciones/farmacología , Transducción de Señal , Superóxido Dismutasa/metabolismo , Tiorredoxinas/metabolismoRESUMEN
Glutaredoxins have been characterised as enzymes regulating the redox status of protein thiols via cofactors GSSG/GSH. However, such a function has not been demonstrated with physiologically relevant protein substrates in in vitro experiments. Their active sites frequently feature a Cys-xx-Cys motif that is predicted not to bind metal ions. Such motifs are also present in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone. In this work, we present the first demonstration that: (i) human glutaredoxin 1 (hGrx1) efficiently catalyses interchange of the dithiol and disulfide forms of the Cys(12)-xx-Cys(15) fragment in Atox1 but does not act upon the isolated single residue Cys(41); (ii) the direction of catalysis is regulated by the GSSG/2GSH ratio and the availability of Cu(I); (iii) the active site Cys(23)-xx-Cys(26) in hGrx1 can bind Cu(I) tightly with femtomolar affinity (K(D) = 10(-15.5) M) and possesses a reduction potential of E(o)' = -118 mV at pH 7.0. In contrast, the Cys(12)-xx-Cys(15) motif in Atox1 has a higher affinity for Cu(I) (K(D) = 10(-17.4) M) and a more negative potential (E(o)' = -188 mV). These differences may be attributed primarily to the very low pKa of Cys23 in hGrx1 and allow rationalisation of conclusion (ii) above: hGrx1 may catalyse the oxidation of Atox1(dithiol) by GSSG, but not the complementary reduction of the oxidised Atox1(disulfide) by GSH unless Cu(aq)(+) is present at a concentration that allows binding of Cu(I) to reduced Atox1 but not to hGrx1. In fact, in the latter case, the catalytic preferences are reversed. Both Cys residues in the active site of hGrx1 are essential for the high affinity Cu(I) binding but the single Cys(23) residue only is required for the redox catalytic function. The molecular properties of both Atox1 and hGrx1 are consistent with a correlation between copper homeostasis and redox sulfur chemistry, as suggested by recent cell experiments. These proteins appear to have evolved the features necessary to fill multiple roles in redox regulation, Cu(I) buffering and Cu(I) transport.
Asunto(s)
Cobre/metabolismo , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Metalochaperonas/metabolismo , Azufre/metabolismo , Cobre/química , Proteínas Transportadoras de Cobre , Glutarredoxinas/química , Glutatión/química , Humanos , Metalochaperonas/química , Modelos Moleculares , Chaperonas Moleculares , Oxidación-Reducción , Azufre/química , TermodinámicaRESUMEN
AIMS: Dietary supplementation with ursolic acid (UA) prevents monocyte dysfunction in diabetic mice and protects mice against atherosclerosis and loss of renal function. The goal of this study was to determine the molecular mechanism by which UA prevents monocyte dysfunction induced by metabolic stress. METHODS AND RESULTS: Metabolic stress sensitizes or "primes" human THP-1 monocytes and murine peritoneal macrophages to the chemoattractant MCP-1, converting these cells into a hyper-chemotactic phenotype. UA protected THP-1 monocytes and peritoneal macrophages against metabolic priming and prevented their hyper-reactivity to MCP-1. UA blocked the metabolic stress-induced increase in global protein-S-glutathionylation, a measure of cellular thiol oxidative stress, and normalized actin-S-glutathionylation. UA also restored MAPK phosphatase-1 (MKP1) protein expression and phosphatase activity, decreased by metabolic priming, and normalized p38 MAPK activation. Neither metabolic stress nor UA supplementation altered mRNA or protein levels of glutaredoxin-1, the principal enzyme responsible for the reduction of mixed disulfides between glutathione and protein thiols in these cells. However, the induction of Nox4 by metabolic stress, required for metabolic priming, was inhibited by UA in both THP-1 monocytes and peritoneal macrophages. CONCLUSION: UA protects THP-1 monocytes against dysfunction by suppressing metabolic stress-induced Nox4 expression, thereby preventing the Nox4-dependent dysregulation of redox-sensitive processes, including actin turnover and MAPK-signaling, two key processes that control monocyte migration and adhesion. This study provides a novel mechanism for the anti-inflammatory and athero- and renoprotective properties of UA and suggests that dysfunctional blood monocytes may be primary targets of UA and related compounds.
Asunto(s)
Macrófagos Peritoneales/metabolismo , Monocitos/metabolismo , NADPH Oxidasas/metabolismo , Triterpenos/farmacología , Actinas/metabolismo , Animales , Quimiocina CCL2/metabolismo , Suplementos Dietéticos , Regulación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , NADPH Oxidasa 4 , Estrés Fisiológico/efectos de los fármacos , Ácido UrsólicoRESUMEN
In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 residues C31, which belongs to the redox active site (CX(2)C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glutarredoxinas/metabolismo , Mercurio/metabolismo , Oxidorreductasas/metabolismo , Synechocystis/efectos de los fármacos , Synechocystis/enzimología , Glutarredoxinas/química , Glutarredoxinas/genética , Mercurio/toxicidad , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Dominios y Motivos de Interacción de Proteínas , Synechocystis/genética , Synechocystis/crecimiento & desarrollo , Técnicas del Sistema de Dos Híbridos , Uranio/metabolismo , Uranio/toxicidadRESUMEN
The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-L-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glutarredoxinas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Sulfurtransferasas/metabolismo , Catálisis , Dicroismo Circular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Radicales Libres , Glutarredoxinas/genética , Proteínas Hierro-Azufre/genética , Mutación , Mapeo de Interacción de Proteínas , ARN de Transferencia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Sulfurtransferasas/genética , Resonancia por Plasmón de SuperficieRESUMEN
SIGNIFICANCE: Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. RECENT ADVANCES: The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. CRITICAL ISSUES: We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. FUTURE DIRECTIONS: It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.
Asunto(s)
Disulfuros/química , Selenocisteína/química , Selenocisteína/metabolismo , Compuestos de Sulfhidrilo/química , Catálisis , Disulfuros/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutatión Peroxidasa/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Oxidación-Reducción , Peroxidasa/metabolismo , Selenio/química , Selenio/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Azufre/química , Azufre/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
The switch from mitosis to meiosis is one of the most pivotal events in eukaryotes undergoing sexual reproduction. However, the mechanisms orchestrating meiosis initiation remain elusive, particularly in plants. Flowering plants are heterosporous, with male and female spore genesis adopting different developmental courses. We show here that plant pollen mother cells contain a specific meiosis initiation machinery through characterization of a rice (Oryza sativa) gene, MICROSPORELESS1 (MIL1). The mil1 mutant does not produce microspores in anthers but has the normal female fertility. Detailed molecular and cytological investigations demonstrate that mil1 anthers are defective in the meiotic entry of sporogenous cell progenies and in the differentiation of surrounding somatic cell layers, resulting in locules filled with somatic cells instead of microspores. Furthermore, analysis of mil1 msp1 double mutants reveals that due to the absence of MIL1, the cells in their anther locule center do not activate meiotic cell cycle either, generating a similar anther phenotype to mil1. MIL1 encodes a plant-specific CC-type glutaredoxin, which could interact with TGA transcription factors. These results suggest meiotic entry in microsporocytes is directed by an anther-specific mechanism, which requires MIL1 activity, and redox regulation might play important roles in this process.
Asunto(s)
Flores/crecimiento & desarrollo , Glutarredoxinas/metabolismo , Meiosis , Oryza/genética , Proteínas de Plantas/metabolismo , Polen/citología , Clonación Molecular , Flores/citología , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Glutarredoxinas/genética , Datos de Secuencia Molecular , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Filogenia , Proteínas de Plantas/genética , Polen/crecimiento & desarrollo , Factores de Transcripción/metabolismoRESUMEN
The ß(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(â¢)) that is essential for nucleotide reduction. The ß(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (ß) and Rnr4 (ß'). Although only ß is capable of iron binding and Tyr(â¢) formation, cells lacking ß' are either dead or exhibit extremely low Tyr(â¢) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that ß' is required for iron loading and Tyr(â¢) formation in ß in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent ß and are hypersensitive to iron depletion and the Tyr(â¢)-reducing agent hydroxyurea. Transient induction of ß' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(â¢) levels in ß. Tyr(â¢) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant ß' and is inhibited by adding an iron chelator prior to, but not after, ß' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(â¢) levels and ßß' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(â¢) cofactor in RNR.
Asunto(s)
Glutarredoxinas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Oxidorreductasas/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Tirosina/análogos & derivados , Glutarredoxinas/genética , Hidroxiurea , Proteínas Hierro-Azufre/genética , Mutación , Oxidorreductasas/genética , Ribonucleósido Difosfato Reductasa/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esferoplastos/genética , Esferoplastos/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMEN
ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Flores/crecimiento & desarrollo , Glutarredoxinas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Gametogénesis en la Planta , Regulación de la Expresión Génica de las Plantas , Germinación , Glutarredoxinas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Polen/crecimiento & desarrolloRESUMEN
The Grx (glutaredoxin) proteins are oxidoreductases with a central function in maintaining the redox balance within the cell. In the present study, we have explored the reactions between selenium compounds and the glutaredoxin system. Selenite, GS-Se-SG (selenodiglutathione) and selenocystine were all shown to be substrates of human Grx1, implying a novel role for the glutaredoxins in selenium metabolism. During the past few years, selenium has further evolved as a potential therapeutic agent in cancer treatment, and a leading mechanism of cytotoxicity is the generation of ROS (reactive oxygen species). Both selenite and GS-Se-SG were reduced by Grx1 and Grx2 in a non-stoichiometric manner due to redox cycling with oxygen, which in turn generated ROS. The role of Grx in selenium toxicity was therefore explored. Cells were treated with the selenium compounds in combination with transient overexpression of, or small interfering RNA against, Grx1. The results demonstrated an increased viability of the cells during silencing of Grx1, indicating that Grx1 is contributing to selenium toxicity. This is in contrast with TrxR (thioredoxin reductase), which previously was shown to protect cells from selenium cytotoxicity, verifying a diverse role between Grx and TrxR in selenium-mediated cytotoxicity. Furthermore, selenium treatment led to a marked increase in protein glutathionylation and cysteinylation that potentially can influence the activity and function of several proteins within the cell.
Asunto(s)
Glutarredoxinas/metabolismo , Compuestos de Selenio/metabolismo , Selenio/metabolismo , Selenio/toxicidad , Transducción de Señal/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Citotoxinas/metabolismo , Citotoxinas/toxicidad , Humanos , Especificidad por Sustrato/fisiologíaRESUMEN
Selenium is an essential micronutrient for humans and animals, and its deficiency can predispose to the development of pathological conditions. This study evaluates the effect of selenium deficiency on the thioredoxin system, consisting of NADPH, selenoprotein thioredoxin reductase (TrxR), and thioredoxin (Trx); and the glutathione system, including NADPH, glutathione reductase, glutathione, and glutaredoxin coupled with selenoprotein glutathione peroxidase (GPx). We particularly investigate whether inactive truncated TrxR is present under selenium-starvation conditions due to reading of the UGA codon as stop. Feeding rats a selenium-deficient diet resulted in a large decrease in activity of TrxR and GPx in rat liver but not in the levels of Trx1 and Grx1. However, selenium deficiency induced mitochondrial Grx2 10-fold and markedly changed the expression of some flavoproteins that are involved in the cellular folate, glucose, and lipid metabolism. Liver TrxR mRNA was nearly unchanged, but no truncated enzyme was found. Instead, a low-activity form of TrxR with a cysteine substituted for the penultimate selenocysteine in the C-terminal active site was identified in selenium-deficient rat liver. These results show a novel mechanism for decoding the UGA stop codon, inserting cysteine to make a full-length enzyme that may be required for selenium assimilation.