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 DobradasRESUMO
Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe-S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe-S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe-S cluster assembly process and establish that the initiation of Fe-S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.
Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Cisteína/química , Proteínas de Escherichia coli/química , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Compostos de Sulfonilureia , Enxofre/metabolismoRESUMO
Friedreich ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, we generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. We were able to bypass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, we observed a progressive increase of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in our fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, we observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, our model provides the opportunity to elucidate in vivo, the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance.
Assuntos
Acetilcisteína/farmacologia , Sistemas CRISPR-Cas/genética , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/patologia , Humanos , Íntrons/genética , Estresse Oxidativo/genética , RNA-Seq , Expansão das Repetições de Trinucleotídeos/genética , FrataxinaRESUMO
Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect FriedreichÌs ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.
Assuntos
Cinetina/farmacologia , Purinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção , Relação Dose-Resposta a Droga , Humanos , Cinetina/síntese química , Cinetina/química , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Purinas/síntese química , Purinas/química , Relação Estrutura-AtividadeRESUMO
In Saccharomyces cerevisiae, Yap1 regulates an H2O2-inducible transcriptional response that controls cellular H2O2 homeostasis. H2O2 activates Yap1 by oxidation through the intermediary of the thiol peroxidase Orp1. Upon reacting with H2O2, Orp1 catalytic cysteine oxidizes to a sulfenic acid, which then engages into either an intermolecular disulfide with Yap1, leading to Yap1 activation, or an intramolecular disulfide that commits the enzyme into its peroxidatic cycle. How the first of these two competing reactions, which is kinetically unfavorable, occurs was previously unknown. We show that the Yap1-binding protein Ybp1 brings together Orp1 and Yap1 into a ternary complex that selectively activates condensation of the Orp1 sulfenylated cysteine with one of the six Yap1 cysteines while inhibiting Orp1 intramolecular disulfide formation. We propose that Ybp1 operates as a scaffold protein and as a sulfenic acid chaperone to provide specificity in the transfer of oxidizing equivalents by a reactive sulfenic acid species.
Assuntos
Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Ácidos Sulfênicos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Reactive oxygen species (ROS) have been shown to be toxic but also function as signalling molecules. This biological paradox underlies mechanisms that are important for the integrity and fitness of living organisms and their ageing. The pathways that regulate ROS homeostasis are crucial for mitigating the toxicity of ROS and provide strong evidence about specificity in ROS signalling. By taking advantage of the chemistry of ROS, highly specific mechanisms have evolved that form the basis of oxidant scavenging and ROS signalling systems.
Assuntos
Homeostase/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , OxirreduçãoRESUMO
Glutathione contributes to thiol-redox control and to extra-mitochondrial iron-sulphur cluster (ISC) maturation. To determine the physiological importance of these functions and sort out those that account for the GSH requirement for viability, we performed a comprehensive analysis of yeast cells depleted of or containing toxic levels of GSH. Both conditions triggered an intense iron starvation-like response and impaired the activity of extra-mitochondrial ISC enzymes but did not impact thiol-redox maintenance, except for high glutathione levels that altered oxidative protein folding in the endoplasmic reticulum. While iron partially rescued the ISC maturation and growth defects of GSH-depleted cells, genetic experiments indicated that unlike thioredoxin, glutathione could not support by itself the thiol-redox duties of the cell. We propose that glutathione is essential by its requirement in ISC assembly, but only serves as a thioredoxin backup in cytosolic thiol-redox maintenance. Glutathione-high physiological levels are thus meant to insulate its cytosolic function in iron metabolism from variations of its concentration during redox stresses, a model challenging the traditional view of it as prime actor in thiol-redox control.
Assuntos
Glutationa/metabolismo , Ferro/metabolismo , Saccharomyces cerevisiae/metabolismo , Compostos de Sulfidrila/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Dobramento de Proteína , Processamento de Proteína Pós-TraducionalRESUMO
During rotavirus infection, replication and packaging of the viral genome occur in viral factories, termed viroplasms. The viral nonstructural protein NSP5 is a major building block of viroplasms; it recruits the viral polymerase VP1, the core protein VP2, and the ATPase NSP2 inside the viroplasm to form the viral replication complex. Here we report that NSP5 is a unique viral metalloprotein that coordinates a [2Fe-2S] iron-sulfur cluster as demonstrated by the metal and labile sulfide contents, UV-visible light absorption, and electron paramagnetic resonance. Point mutations in NSP5 allowed us to identify C171 and C174, arranged in a CXC motif, as essential residues for cluster coordination. When coexpressed with NSP2, an NSP5 mutant devoid of the iron-sulfur cluster still forms viroplasm-like structures. The cluster is therefore neither involved in the interaction with NSP2 nor in the formation of viroplasm-like structures and thus presumably in viroplasm formation. Finally, we show using microscale thermophoresis that the iron-sulfur cluster modulates the affinity of NSP5 for single-stranded RNA. Because the cluster is near the binding sites of both the polymerase VP1 and the ATPase NSP2, we anticipate that this cluster is crucial for NSP5 functions, in either packaging or replication of the viral genome.
Assuntos
Metaloproteínas/química , RNA Viral/química , Rotavirus/química , Proteínas não Estruturais Virais/química , Humanos , Ferro/química , Ferro/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Mutação Puntual , RNA Viral/genética , RNA Viral/metabolismo , Rotavirus/fisiologia , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo , Espectrofotometria Ultravioleta , Enxofre/química , Enxofre/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/fisiologia , Replicação Viral/fisiologiaRESUMO
Ironsulfur (FeS) clusters constitute ancient cofactors that accompany a versatile range of fundamental biological reactions across eukaryotes and prokaryotes. Several cellular pathways exist to coordinate iron acquisition and sulfur mobilization towards a scaffold protein during the tightly regulated synthesis of FeS clusters. The mechanism of mitochondrial eukaryotic [2Fe-2S] cluster synthesis is coordinated by the Iron-Sulfur Cluster (ISC) machinery and its aberrations herein have strong implications to the field of disease and medicine which is therefore of particular interest. Here, we describe our current knowledge on the step-by-step mechanism leading to the production of mitochondrial [2Fe2S] clusters while highlighting the recent developments in the field alongside the challenges that are yet to be overcome.
RESUMO
Tah18-Dre2 is a recently identified yeast protein complex, which is highly conserved in human and has been implicated in the regulation of oxidative stress induced cell death and in cytosolic Fe-S proteins synthesis. Tah18 is a diflavin oxido-reductase with binding sites for flavin mononucleotide, flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which is able to transfer electrons to Dre2 Fe-S clusters. In this work we characterized in details the interaction between Tah18 and Dre2, and analysed how it conditions yeast viability. We show that Dre2 C-terminus interacts in vivo and in vitro with the flavin mononucleotide- and flavin adenine dinucleotide-binding sites of Tah18. Neither the absence of the electron donor nicotinamide adenine dinucleotide phosphate-binding domain in purified Tah18 nor the absence of Fe-S in aerobically purified Dre2 prevents the binding in vitro. In vivo, when this interaction is affected in a dre2 mutant, yeast viability is reduced. Conversely, enhancing artificially the interaction between mutated Dre2 and Tah18 restores cellular viability despite still reduced cytosolic Fe-S cluster biosynthesis. We conclude that Tah18-Dre2 interaction in vivo is essential for yeast viability. Our study may provide new insight into the survival/death switch involving this complex in yeast and in human cells.
Assuntos
Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Viabilidade Microbiana , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Ferro-Enxofre/genética , Oxirredutases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Friedreich's ataxia (FRDA) is the most prevalent autosomic recessive ataxia and is associated with a severe cardiac hypertrophy and less frequently diabetes. It is caused by mutations in the gene encoding frataxin (FXN), a small mitochondrial protein. The primary consequence is a defective expression of FXN, with basal protein levels decreased by 70-98%, which foremost affects the cerebellum, dorsal root ganglia, heart and liver. FXN is a mitochondrial protein involved in iron metabolism but its exact function has remained elusive and highly debated since its discovery. At the cellular level, FRDA is characterized by a general deficit in the biosynthesis of iron-sulfur (Fe-S) clusters and heme, iron accumulation and deposition in mitochondria, and sensitivity to oxidative stress. Based on these phenotypes and the proposed ability of FXN to bind iron, a role as an iron storage protein providing iron for Fe-S cluster and heme biosynthesis was initially proposed. However, this model was challenged by several other studies and it is now widely accepted that FXN functions primarily in Fe-S cluster biosynthesis, with iron accumulation, heme deficiency and oxidative stress sensitivity appearing later on as secondary defects. Nonetheless, the biochemical function of FXN in Fe-S cluster biosynthesis is still debated. Several roles have been proposed for FXN: iron chaperone, gate-keeper of detrimental Fe-S cluster biosynthesis, sulfide production stimulator and sulfur transfer accelerator. A picture is now emerging which points toward a unique function of FXN as an accelerator of a key step of sulfur transfer between two components of the Fe-S cluster biosynthetic complex. These findings should foster the development of new strategies for the treatment of FRDA. We will review here the latest discoveries on the biochemical function of frataxin and the implication for a potential therapeutic treatment of FRDA.
RESUMO
Nitric oxide (NO), synthesized in eukaryotes by the NO synthases, has multiple roles in signalling pathways and in protection against pathogens. Pathogenic microorganisms have apparently evolved defence mechanisms that counteract the effects of NO and related reactive nitrogen species. Regulatory proteins that sense NO mediate the primary response to NO and nitrosative stress. The only regulatory protein in enteric bacteria known to serve exclusively as an NO-responsive transcription factor is the enhancer binding protein NorR (refs 9, 10-11). In Escherichia coli, NorR activates the transcription of the norVW genes encoding a flavorubredoxin (FlRd) and an associated flavoprotein, respectively, which together have NADH-dependent NO reductase activity. The NO-responsive activity of NorR raises important questions concerning the mechanism of NO sensing. Here we show that the regulatory domain of NorR contains a mononuclear non-haem iron centre, which reversibly binds NO. Binding of NO stimulates the ATPase activity of NorR, enabling the activation of transcription by RNA polymerase. The mechanism of NorR reveals an unprecedented biological role for a non-haem mononitrosyl-iron complex in NO sensing.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Óxido Nítrico/metabolismo , Transativadores/química , Transativadores/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Heme , Modelos Genéticos , Óxido Nítrico/farmacologia , Regiões Promotoras Genéticas/genética , Transativadores/genética , Ativação TranscricionalRESUMO
Cysteine-bound persulfides (Cys-SSH) in proteins are sulfur carrier intermediates in the synthesis of essential cofactors such as iron-sulfur clusters, molybdenum cofactor, vitamin (thiamine), and thionucleosides (thiolated tRNA). Protein-bound persulfides are also used for signaling purposes as a carrier of the "H2S" signal. Several methods have been developed to detect and quantify cysteine-bound persulfides in protein and monitor their exchange. The main challenge in developing these techniques is to discriminate persulfidated cysteine from cysteine and other cysteine modifications. It is also critical to develop ratiometric methods to quantify the level of persulfidation in the protein of interest. We describe here a ratiometric method to label and quantify protein-bound persulfides relying on alkylation and gel-shift assays. This method is based on the derivation of cysteine and persulfides with "heavy" alkylating agents, followed by specific cleavage of the sulfur-sulfur bond of the alkylated persulfide by a reducing agent and separation of the alkylated species by electrophoresis. A persulfide is thus revealed by the appearance of a species lacking one alkylation unit under reducing conditions. We call this alkylation-reduction band-shift (ARBS) assay. Moreover, the quantification of the bands corresponding to the persulfidated and non-persulfidated species in the same lane provides a ratiometric quantification allowing determination of the level of persulfidation of individual cysteine. Other cysteine modifications such as disulfides, sulfenic, sulfinic, sulfonic acids, nitrosothiols, and sulfenamides preclude alkylation. Thus, they may appear as false positives, but they are ruled out by the analysis under nonreducing conditions since these species do not behave as persulfides under these conditions.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética , Alquilação , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina , Cisteína/metabolismo , Dissulfetos , Proteínas , Sulfetos , Enxofre , Fatores de TempoRESUMO
Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.
Assuntos
Ferredoxinas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sulfetos/metabolismo , Liases de Carbono-Enxofre/metabolismo , Ferredoxinas/isolamento & purificação , Ataxia de Friedreich/patologia , Ferro/metabolismo , Proteínas de Ligação ao Ferro/isolamento & purificação , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Zinco/metabolismo , FrataxinaRESUMO
The prokaryotic transcriptional regulator NorR is unusual in that it utilizes a mononuclear ferrous iron center rather than a heme moiety as a means of sensing nitric oxide (NO). Binding of NO to the nonheme iron center in the amino-terminal GAF domain of NorR results in formation of a mononitrosyl iron complex and relieves intramolecular repression within NorR, allowing this regulatory protein, a member of the sigma(54)-dependent family of enhancer-binding proteins, to activate expression of genes required for NO detoxification. This chapter describes detailed protocols for measuring transcriptional activation by Escherichia coli NorR in vivo and in vitro. It also details spectroscopic methods for analysis of the interaction of NO with the nonheme iron center and determination of the NO-binding affinity constant.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Óxido Nítrico/farmacologia , Transativadores/química , Transativadores/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Eletrodos Seletivos de Íons/normas , Ferro/química , Óxido Nítrico/metabolismo , Nitritos/química , Ligação ProteicaRESUMO
Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide compounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1-ISD11-ISCU complex.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Compostos de Sulfidrila/metabolismo , Liases de Carbono-Enxofre/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cisteína/química , Cisteína/metabolismo , Glutationa/química , Glutationa/metabolismo , Humanos , Proteínas de Ligação ao Ferro/química , Espectrometria de Massas , Software , Compostos de Sulfidrila/química , Sulfetos/química , Sulfetos/metabolismo , FrataxinaRESUMO
SIGNIFICANCE: In bacteria, transcriptional responses to reactive oxygen and nitrogen species (ROS and RNS, respectively) are typically coordinated by regulatory proteins that employ metal centers or reactive thiols to detect the presence of those species. This review is focused on the structure, function and mechanism of three regulatory proteins (Fur, PerR, and NorR) that contain non-heme iron and regulate the transcription of target genes in response to ROS and/or RNS. The targets for regulation include genes encoding detoxification activities, and genes encoding proteins involved in the repair of the damage caused by ROS and RNS. RECENT ADVANCES: Three-dimensional structures of several Fur proteins and of PerR are revealing important details of the metal binding sites of these proteins, showing a surprising degree of structural diversity in the Fur family. CRITICAL ISSUES: Discussion of the interaction of Fur with ROS and RNS will illustrate the difficulty that sometimes exists in distinguishing between true physiological responses and adventitious reactions of a regulatory protein with a reactive ligand. FUTURE DIRECTIONS: Consideration of these three sensor proteins illuminates some of the key questions that remain unanswered, for example, the nature of the biochemical determinants that dictate the sensitivity and specificity of the interaction of the sensor proteins with their cognate signals.
Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Thiol-based peroxidases consist of the peroxiredoxins (Prx) and the related glutathione peroxidase (GPx)-like enzymes. Their catalytic function is to reduce peroxides by using the reactivity of the cysteine residue, and their presumed primary physiologic role is to protect living organisms from peroxide toxicity. However, as peroxide-metabolizing enzymes, they also regulate hydrogen peroxide (H2O2) signaling. We review here enzymatic and biochemical attributes of thiol peroxidases that specify both distinctive peroxide-scavenging functions and the property of regulating H2O2 signaling. We then discuss possible thiol peroxidase physiologic functions, based on selected observations made in microorganisms and mammals.
Assuntos
Peróxido de Hidrogênio/metabolismo , Peroxidases/metabolismo , Transdução de Sinais , Compostos de Sulfidrila/metabolismo , Animais , Dimerização , Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Peróxido de Hidrogênio/química , Modelos Biológicos , Peroxidases/química , Peroxidases/genética , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismoRESUMO
Identification of unknown proteins subsequent to a mass spectrometry signal is still a serious obstacle in the discovery of relevant biomarkers of diagnostic interest. In this report the evaluation of a rational process under optimized conditions is described for three unknown proteins representing important targets in their field of investigation. The process, involving few dozens of chromatographic sorbents and two buffers, allowed identifying prothrombin fragment 1, a minor glycoprotein of human serum with inhibitory activity associated with pathogenesis of calcium oxalate stones. The same technology demonstrated its efficiency for the separation of a recombinantly expressed yeast transcription factor in Escherichia coli with subsequent formal identification. In addition, a DNA-binding protein from the gastric pathogen Helicobacter pylori has been separated by the same technology and formally identified. The reported data show that the method is reliable and easily applicable to a large variety of cases with a standardized approach. Identity coverage and relative abundance after purification and removal of critical protein impurities are reported. Examples of protein isolation/identification are described, namely PTF1, recombinant YAP-1 transcription factor from E. coli and DNA-binding protein HU from H. pylori. Isolated proteins were pure enough for the purpose of formal identification by either peptide mass fingerprinting or sequencing.
Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Proteômica/métodos , Sequência de Aminoácidos , Biomarcadores , Proteínas Sanguíneas/química , Cromatografia/métodos , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Helicobacter pylori , Humanos , Dados de Sequência Molecular , Proteínas/química , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The NorR regulatory protein senses nitric oxide (NO) to activate genes required for NO detoxification under anaerobic and microaerobic conditions in Escherichia coli. NorR belongs to the sigma(54)-dependent family of transcriptional activators and contains an N-terminal regulatory GAF (cGMP phosphodiesterase, adenylate cyclase, FhlA) domain that controls the ATPase activity of the central AAA+ domain to regulate productive interactions with sigma(54). Binding of NO to a non-heme iron center in the GAF domain results in the formation of a mononitrosyl-iron complex and releases intramolecular repression of the AAA+ domain to enable activation of transcription. In this study, we have further characterized NorR spectroscopically and substituted conserved residues in the GAF domain. This analysis, in combination with structural modeling of the GAF domain, has identified five candidate ligands to the non-heme iron and suggests a model in which the metal ion is coordinated in a pseudo-octahedral environment by three aspartate residues, an arginine, and a cysteine.