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1.
Mol Cell ; 67(6): 962-973.e5, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28918898

RESUMO

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.


Assuntos
Retículo Endoplasmático/enzimologia , Proteínas Fúngicas/metabolismo , Glutationa/metabolismo , Glicoproteínas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Canais de Translocação SEC/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/enzimologia , Difusão Facilitada , Proteínas Fúngicas/genética , Dissulfeto de Glutationa/metabolismo , Glicoproteínas/genética , Proteínas de Choque Térmico HSP70/genética , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Tempo , Resposta a Proteínas não Dobradas
2.
J Biol Chem ; 299(12): 105419, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37923140

RESUMO

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.


Assuntos
Antígeno 2 Relacionado a Fos , Fatores de Transcrição GATA , Glutarredoxinas , Homeostase , Proteínas Ferro-Enxofre , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Humanos , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
3.
J Biol Inorg Chem ; 24(6): 809-815, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31493153

RESUMO

The Saccharomyces cerevisiae transcriptional activator Aft1 and its paralog Aft2 respond to iron deficiency by upregulating expression of proteins required for iron uptake at the plasma membrane, vacuolar iron transport, and mitochondrial iron metabolism, with the net result of mobilizing iron from extracellular sources and intracellular stores. Conversely, when iron levels are sufficient, Aft1 and Aft2 interact with the cytosolic glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, which promote Aft1/2 dissociation from DNA and subsequent export from the nucleus. Previous studies unveiled the molecular mechanism for iron-dependent inhibition of Aft1/2 activity, demonstrating that the [2Fe-2S]-bridged Grx3-Bol2 heterodimer transfers a cluster to Aft2, driving Aft2 dimerization and dissociation from DNA. Here, we provide further insight into the regulation mechanism by investigating the roles of conserved cysteines in Aft2 in iron-sulfur cluster binding and interaction with [2Fe-2S]-Grx3-Bol2. Using size exclusion chromatography and circular dichroism spectroscopy, these studies reveal that both cysteines in the conserved Aft2 Cys-Asp-Cys motif are essential for Aft2 dimerization via [2Fe-2S] cluster binding, while only one cysteine is required for interaction with the [2Fe-2S]-Grx3-Bol2 complex. Taken together, these results provide novel insight into the molecular details of iron-sulfur cluster transfer from Grx3-Bol2 to Aft2 which likely occurs through a ligand exchange mechanism. Loss of either cysteine in the Aft2 iron-sulfur binding site may disrupt this ligand-exchange process leading to the isolation of a trapped Aft2-Grx3-Bol2 intermediate, while the replacement of both cysteines abrogates both the iron-sulfur cluster exchange and the protein-protein interactions between Aft2 and Grx3-Bol2.


Assuntos
Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Oxirredutases/química , Oxirredutases/genética , Plasmídeos/genética , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/química
4.
J Biol Chem ; 292(38): 15990-15991, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28939752

RESUMO

The RNA-binding iron regulatory proteins IRP1 and IRP2 are inactivated by either Fe-S cluster insertion or protein degradation mediated by the E3 ligase component FBXL5. However, the mechanisms for coordination between Fe-S cluster assembly, FBXL5, and IRP1/IRP2 activity are poorly defined. A new study reveals that FBXL5 plays a critical role in limiting IRP1 and IRP2 overaccumulation when cytosolic Fe-S cluster assembly is impaired in order to maintain optimal iron levels for cell viability.


Assuntos
Ferro/metabolismo , Sobrevivência Celular , Citosol/metabolismo , Proteínas F-Box/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Enxofre/metabolismo
5.
Proc Natl Acad Sci U S A ; 111(11): 4043-8, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24591629

RESUMO

The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn(2+)-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe(2+)) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity. Furthermore, we demonstrate that the [2Fe-2S]-bridged heterodimer formed between glutaredoxin-3 and the BolA-like protein Fe repressor of activation-2 transfers a [2Fe-2S] cluster to Aft2 that facilitates Aft2 dimerization. Previous in vivo findings strongly support the [2Fe-2S] cluster-induced dimerization model; however, given the available evidence, Fe(2+)-induced Aft2 dimerization cannot be completely ruled out as an alternative Aft2 inhibition mechanism. Taken together, these data provide insight into the molecular mechanism for iron-dependent transcriptional regulation of Aft2 and highlight the key role of Fe-S clusters as cellular iron signals.


Assuntos
DNA/química , Modelos Moleculares , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Transativadores/química , Cromatografia em Gel , Clonagem Molecular , Cristalização , DNA/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Ferro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espalhamento a Baixo Ângulo , Transativadores/metabolismo , Ultracentrifugação
6.
Biochemistry ; 55(33): 4720-30, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27490699

RESUMO

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.4 and 37 °C results in the formation of putative S-tetrahydrothiophenium adducts at their catalytic Cys residues, followed by ß-elimination to yield dehydroalanine. Both proteins contain a second Cys, in their catalytic C-X-X-C motif, which reacts with the dehydroalanine, the initial Cys adduct with busulfan, or the S-tetrahydrothiophenium, to form novel intramolecular cross-links. The reactivity of the dehydroalanine (DHA) formed is further demonstrated by adduction with glutathione to yield a lanthionine and by a novel reaction with the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which yields a phosphine adduct via Michael addition to the DHA. Formation of a second quaternary organophosphonium salt via nucleophilic substitution with TCEP on the initial busulfan-protein adduct or on the THT(+)-Redoxin species is also observed. These results reveal a rich potential for reactions of busulfan with proteins in vitro, and likely in vivo. It is striking that several of the chemically altered protein products retain none of the atoms of busulfan, in contrast to typical drug-protein adducts or traditional protein modification reagents. In particular, the ability of a clinically used drug to convert Cys to dehydrolanine in intact proteins, and its subsequent reaction with biological thiols, is unprecedented.


Assuntos
Alanina/análogos & derivados , Bussulfano/química , Cisteína/química , Agonistas Mieloablativos/química , Sulfetos/química , Alanina/química , Humanos , Espectrometria de Massas em Tandem
7.
Biochemistry ; 55(49): 6869-6879, 2016 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-27951647

RESUMO

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Histidina/química , Proteínas Ferro-Enxofre/química , Fatores de Transcrição/química , Calorimetria , Dicroísmo Circular , Peso Molecular , Análise Espectral/métodos
8.
J Biol Chem ; 290(46): 27829-40, 2015 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-26396185

RESUMO

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.


Assuntos
Citosol/metabolismo , Glutationa/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Glutamato-Cisteína Ligase/metabolismo , Glutationa/deficiência , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética
9.
Biochem J ; 446(1): 59-67, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22651090

RESUMO

The intramolecular disulfide bond in hSOD1 [human SOD1 (Cu,Zn superoxide dismutase 1)] plays a key role in maintaining the protein's stability and quaternary structure. In mutant forms of SOD1 that cause familial ALS (amyotrophic lateral sclerosis), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalysed by CCS1 (copper chaperone for SOD1). Previous studies in yeast demonstrate that the yeast GSH/Grx (glutaredoxin) redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. In the present study, we probe further the interaction between hSOD1, GSH and Grxs to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that hGrx1 (human Grx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT (wild-type) hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared with WT hSOD1. Furthermore, the presence of metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT compared with ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Glutarredoxinas/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/genética , Dissulfetos/química , Glutarredoxinas/química , Glutarredoxinas/genética , Humanos , Cinética , Mutação , Oxirredução , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Termodinâmica
10.
Biochemistry ; 51(22): 4377-89, 2012 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-22583368

RESUMO

Monothiol glutaredoxins (Grxs) with a signature CGFS active site and BolA-like proteins have recently emerged as novel players in iron homeostasis. Elegant genetic and biochemical studies examining the functional and physical interactions of CGFS Grxs in the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe have unveiled their essential roles in intracellular iron signaling, iron trafficking, and the maturation of Fe-S cluster proteins. Biophysical and biochemical analyses of the [2Fe-2S] bridging interaction between CGFS Grxs and a BolA-like protein in S. cerevisiae provided the first molecular-level understanding of the iron regulation mechanism in this model eukaryote and established the ubiquitous CGFS Grxs and BolA-like proteins as novel Fe-S cluster-binding regulatory partners. Parallel studies focused on Escherichia coli and human homologues for CGFS Grxs and BolA-like proteins have supported the studies in yeast and provided additional clues about their involvement in cellular iron metabolism. Herein, we review recent progress in uncovering the cellular and molecular mechanisms by which CGFS Grxs and BolA-like proteins help regulate iron metabolism in both eukaryotic and prokaryotic organisms.


Assuntos
Evolução Molecular , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Animais , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glutarredoxinas/química , Humanos , Proteínas Ferro-Enxofre/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
11.
Biochemistry ; 51(8): 1687-96, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22309771

RESUMO

Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein, we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV-visible absorption and circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3-BolA2 complexes indicate that the Fe-S coordination environments in these complexes are virtually identical to those of the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2-Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx-BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans.


Assuntos
Proteínas de Transporte/química , Proteínas Ferro-Enxofre/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Dicroísmo Circular , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína
12.
J Biol Chem ; 286(1): 867-76, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-20978135

RESUMO

The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. To further characterize this unusual Fe-S-binding complex, site-directed mutagenesis was used to identify specific residues in Fra2 that influence Fe-S cluster binding and regulation of Aft1 activity in vivo. Here, we present spectroscopic evidence that His-103 in Fra2 is an Fe-S cluster ligand in the Fra2-Grx3 complex. Replacement of this residue does not abolish Fe-S cluster binding, but it does lead to a change in cluster coordination and destabilization of the [2Fe-2S] cluster. In vivo genetic studies further confirm that Fra2 His-103 is critical for control of Aft1 activity in response to the cellular iron status. Using CD spectroscopy, we find that ∼1 mol eq of apo-Fra2 binds tightly to the [2Fe-2S] Grx3 homodimer to form the [2Fe-2S] Fra2-Grx3 heterodimer, suggesting a mechanism for formation of the [2Fe-2S] Fra2-Grx3 heterodimer in vivo. Taken together, these results demonstrate that the histidine coordination and stability of the [2Fe-2S] cluster in the Fra2-Grx3 complex are essential for iron regulation in yeast.


Assuntos
Histidina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ferro/metabolismo , Oxirredutases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Enxofre/metabolismo , Sequência de Aminoácidos , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligantes , Camundongos , Dados de Sequência Molecular , Mutagênese , Mutação , Oxirredutases/química , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Análise Espectral , Fatores de Transcrição/metabolismo
13.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118847, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32910989

RESUMO

The synthesis and trafficking of iron-sulfur (Fe-S) clusters in both prokaryotes and eukaryotes requires coordination within an expanding network of proteins that function in the cytosol, nucleus, mitochondria, and chloroplasts in order to assemble and deliver these ancient and essential cofactors to a wide variety of Fe-S-dependent enzymes and proteins. This review focuses on the evolving roles of two ubiquitous classes of proteins that operate in this network: CGFS glutaredoxins and BolA proteins. Monothiol or CGFS glutaredoxins possess a Cys-Gly-Phe-Ser active site that coordinates an Fe-S cluster in a homodimeric complex. CGFS glutaredoxins also form [2Fe-2S]-bridged heterocomplexes with BolA proteins, which possess an invariant His and an additional His or Cys residue that serve as cluster ligands. Here we focus on recent discoveries in bacteria, fungi, humans, and plants that highlight the shared and distinct roles of CGFS glutaredoxins and BolA proteins in Fe-S cluster biogenesis, Fe-S cluster storage and trafficking, and Fe-S cluster signaling to transcriptional factors that control iron metabolism--.


Assuntos
Citosol/química , Glutarredoxinas/genética , Proteínas Ferro-Enxofre/genética , Transporte Proteico/genética , Glutarredoxinas/química , Humanos , Proteínas Ferro-Enxofre/química , Ligantes , Modelos Moleculares , Células Procarióticas/química , Transdução de Sinais/genética , Enxofre/metabolismo
14.
Curr Opin Chem Biol ; 55: 189-201, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32234663

RESUMO

Iron homeostasis in fungi involves balancing iron uptake and storage with iron utilization to achieve adequate, nontoxic levels of this essential nutrient. Extensive work in the nonpathogenic yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe has uncovered unique iron regulation networks for each organism that control iron metabolism via distinct molecular mechanisms. However, common themes have emerged from these studies. The activities of all fungal iron-sensing transcription factors characterized to date are regulated via iron-sulfur cluster signaling. Furthermore, glutaredoxins often play a key role in relaying the intracellular iron status to these DNA-binding proteins. Recent work with fungal pathogens, including Candida and Aspergillus species and Cryptococcus neoformans, has revealed novel iron regulation mechanisms, yet similar roles for iron-sulfur clusters and glutaredoxins in iron signaling have been confirmed. This review will focus on these recent discoveries regarding iron regulation pathways in both pathogenic and nonpathogenic fungi.


Assuntos
Proteínas de Bactérias/metabolismo , Fungos/metabolismo , Glutarredoxinas/metabolismo , Ferro/química , Ferro/metabolismo , Enxofre/química , Transporte Biológico , Técnicas Biossensoriais , Proteínas de Ligação a DNA/metabolismo , Homeostase , Domínios Proteicos , Transdução de Sinais , Fatores de Transcrição/metabolismo
15.
Biochemistry ; 48(40): 9569-81, 2009 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-19715344

RESUMO

The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S](2+) cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV-visible absorption and CD, resonance Raman, EPR, ENDOR, Mossbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.


Assuntos
Cisteína/metabolismo , Glutarredoxinas/química , Histidina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Ferro-Enxofre/química , Complexos Multiproteicos/química , Oxirredutases/química , Proteínas de Saccharomyces cerevisiae/química , Cisteína/genética , Dimerização , Estabilidade Enzimática/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glutarredoxinas/biossíntese , Glutarredoxinas/genética , Histidina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/genética , Ligantes , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/genética , Mutagênese Sítio-Dirigida , Oxirredutases/biossíntese , Oxirredutases/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
16.
Methods Enzymol ; 599: 327-353, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746245

RESUMO

Monothiol glutaredoxins (Grxs) with a conserved Cys-Gly-Phe-Ser (CGFS) active site are iron-sulfur (Fe-S) cluster-binding proteins that interact with a variety of partner proteins and perform crucial roles in iron metabolism including Fe-S cluster transfer, Fe-S cluster repair, and iron signaling. Various analytical and spectroscopic methods are currently being used to monitor and characterize glutaredoxin Fe-S cluster-dependent interactions at the molecular level. The electronic, magnetic, and vibrational properties of the protein-bound Fe-S cluster provide a convenient handle to probe the structure, function, and coordination chemistry of Grx complexes. However, some limitations arise from sample preparation requirements, complexity of individual techniques, or the necessity for combining multiple methods in order to achieve a complete investigation. In this chapter, we focus on the use of UV-visible circular dichroism spectroscopy as a fast and simple initial approach for investigating glutaredoxin Fe-S cluster-dependent interactions.


Assuntos
Dicroísmo Circular/métodos , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glutarredoxinas/química , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ferro-Enxofre/química , Cinética , Oxirredutases/química , Oxirredutases/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
17.
Metallomics ; 9(8): 1096-1105, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28725905

RESUMO

The fission yeast Schizosaccharomyces pombe expresses the CCAAT-binding factor Php4 in response to iron deprivation. Php4 forms a transcription complex with Php2, Php3, and Php5 to repress the expression of iron proteins as a means to economize iron usage. Previous in vivo results demonstrate that the function and location of Php4 are regulated in an iron-dependent manner by the cytosolic CGFS type glutaredoxin Grx4. In this study, we aimed to biochemically define these protein-protein and protein-metal interactions. Grx4 was found to bind a [2Fe-2S] cluster with spectroscopic features similar to other CGFS glutaredoxins. Grx4 and Php4 also copurify as a complex with a [2Fe-2S] cluster that is spectroscopically distinct from the cluster on Grx4 alone. In vitro titration experiments suggest that these Fe-S complexes may not be interconvertible in the absence of additional factors. Furthermore, conserved cysteines in Grx4 (Cys172) and Php4 (Cys221 and Cys227) are necessary for Fe-S cluster binding and stable complex formation. Together, these results show that Grx4 controls Php4 function through binding of a bridging [2Fe-2S] cluster.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Cisteína/metabolismo , Regulação Fúngica da Expressão Gênica , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fator de Ligação a CCAAT/genética , Glutarredoxinas/genética , Proteínas Ferro-Enxofre/genética , Modelos Moleculares , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais
18.
Biochem J ; 388(Pt 1): 93-101, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15641941

RESUMO

Prolonged exposure to hyperoxia represents a serious danger to cells, yet little is known about the specific cellular factors that affect hyperoxia stress. By screening the yeast deletion library, we have identified genes that protect against high-O2 damage. Out of approx. 4800 mutants, 84 were identified as hyperoxia-sensitive, representing genes with diverse cellular functions, including transcription and translation, vacuole function, NADPH production, and superoxide detoxification. Superoxide plays a significant role, since the majority of hyperoxia-sensitive mutants displayed cross-sensitivity to superoxide-generating agents, and mutants with compromised SOD (superoxide dismutase) activity were particularly vulnerable to hyperoxia. By comparison, factors known to guard against H2O2 toxicity were poorly represented amongst hyperoxia-sensitive mutants. Although many cellular components are potential targets, our studies indicate that mitochondrial glutathione is particularly vulnerable to hyperoxia damage. During hyperoxia stress, mitochondrial glutathione is more susceptible to oxidation than cytosolic glutathione. Furthermore, two factors that help maintain mitochondrial GSH in the reduced form, namely the NADH kinase Pos5p and the mitochondrial glutathione reductase (Glr1p), are critical for hyperoxia resistance, whereas their cytosolic counterparts are not. Our findings are consistent with a model in which hyperoxia toxicity is manifested by superoxide-related damage and changes in the mitochondrial redox state.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glutationa/fisiologia , Mitocôndrias/fisiologia , Mutação , Oxirredução , Estresse Oxidativo , Paraquat/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo
19.
J Mol Biol ; 323(5): 883-97, 2002 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-12417201

RESUMO

Zinc, a metal ion that functions in a wide variety of catalytic and structural sites in metalloproteins, is shown here to adopt a novel coordination environment in the Escherichia coli transport protein ZntA. The ZntA protein is a P-type ATPase that pumps zinc out of the cytoplasm and into the periplasm. It is physiologically selective for Zn(II) and functions with metalloregulatory proteins in the cell to keep the zinc quota within strict limits. Yet, the N-terminal cytoplasmic domain contains a region that is highly homologous to the yeast Cu(I) metallochaperone Atx1. To investigate how the structure of this region may influence its function, this fragment, containing residues 46-118, has been cloned out of the gene and overexpressed. We report here the solution structure of this fragment as determined by NMR. Both the apo and Zn(II)-ZntA(46-118) structures have been determined. It contains a previously unknown protein coordination site for zinc that includes two cysteine residues, Cys59 and Cys62, and a carboxylate residue, Asp58. The solvent accessibility of this site is also remarkably high, a feature that increasingly appears to be a characteristic of domains of heavy metal ion transport proteins. The participation of Asp58 in this ZntA metal ion binding site may play an important role in modulating the relative affinities and metal exchange rates for Zn(II)/Pb(II)/Cd(II) as compared with other P-type ATPases, which are selective for Cu(I) or Ag(I).


Assuntos
Adenosina Trifosfatases/química , Proteínas de Escherichia coli/química , Zinco/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Zinco/metabolismo
20.
Met Ions Life Sci ; 12: 241-78, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23595675

RESUMO

This chapter is focused on the iron metallome in eukaryotes at the cellular and subcellular level, including properties, utilization in metalloproteins, trafficking, storage, and regulation of these processes. Studies in the model eukaryote Saccharomyces cerevisiae and mammalian cells will be highlighted. The discussion of iron properties will center on the speciation and localization of intracellular iron as well as the cellular and molecular mechanisms for coping with both low iron bioavailability and iron toxicity. The section on iron metalloproteins will emphasize heme, iron-sulfur cluster, and non-heme iron centers, particularly their cellular roles and mechanisms of assembly. The section on iron uptake, trafficking, and storage will compare methods used by yeast and mammalian cells to import iron, how this iron is brought into various organelles, and types of iron storage proteins. Regulation of these processes will be compared between yeast and mammalian cells at the transcriptional, post-transcriptional, and post-translational levels.


Assuntos
Proteínas Ferro-Enxofre , Ferro , Animais , Heme , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae , Enxofre/metabolismo
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