RESUMO
Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with â¼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42-210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42-210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42-210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of â¼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42-210 to ISCU.
Assuntos
Liases de Carbono-Enxofre/química , Proteínas de Ligação ao Ferro/química , Proteínas Reguladoras de Ferro/química , Proteínas Ferro-Enxofre/química , Proteínas Mitocondriais/química , Simulação de Dinâmica Molecular , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , FrataxinaRESUMO
The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of â¼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.
Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enxofre/metabolismo , Cristalografia por Raios X , Proteínas de Ligação ao Ferro/química , Proteínas Ferro-Enxofre/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , FrataxinaRESUMO
Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.
Assuntos
Ferroquelatase/química , Ferroquelatase/metabolismo , Heme/biossíntese , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , FrataxinaRESUMO
Non-progressive cerebellar ataxias are a rare group of disorders that comprise approximately 10% of static infantile encephalopathies. We report the identification of mutations in PMPCA in 17 patients from four families affected with cerebellar ataxia, including the large Lebanese family previously described with autosomal recessive cerebellar ataxia and short stature of Norman type and localized to chromosome 9q34 (OMIM #213200). All patients present with non-progressive cerebellar ataxia, and the majority have intellectual disability of variable severity. PMPCA encodes α-MPP, the alpha subunit of mitochondrial processing peptidase, the primary enzyme responsible for the maturation of the vast majority of nuclear-encoded mitochondrial proteins, which is necessary for life at the cellular level. Analysis of lymphoblastoid cells and fibroblasts from patients homozygous for the PMPCA p.Ala377Thr mutation and carriers demonstrate that the mutation impacts both the level of the alpha subunit encoded by PMPCA and the function of mitochondrial processing peptidase. In particular, this mutation impacts the maturation process of frataxin, the protein which is depleted in Friedreich ataxia. This study represents the first time that defects in PMPCA and mitochondrial processing peptidase have been described in association with a disease phenotype in humans.
Assuntos
Metaloendopeptidases/genética , Proteínas Mitocondriais/metabolismo , Mutação/genética , Subunidades Proteicas/genética , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/metabolismo , Adulto , Criança , Humanos , Líbano , Linfócitos/metabolismo , Masculino , Metaloendopeptidases/metabolismo , Linhagem , Subunidades Proteicas/metabolismo , Adulto Jovem , Peptidase de Processamento MitocondrialRESUMO
Friedreich ataxia is an early-onset multisystemic disease linked to a variety of molecular defects in the nuclear gene FRDA. This gene normally encodes the iron-binding protein frataxin (FXN), which is critical for mitochondrial iron metabolism, global cellular iron homeostasis, and antioxidant protection. In most Friedreich ataxia patients, a large GAA-repeat expansion is present within the first intron of both FRDA alleles, that results in transcriptional silencing ultimately leading to insufficient levels of FXN protein in the mitochondrial matrix and probably other cellular compartments. The lack of FXN in turn impairs incorporation of iron into iron-sulfur cluster and heme cofactors, causing widespread enzymatic deficits and oxidative damage catalyzed by excess labile iron. In a minority of patients, a typical GAA expansion is present in only one FRDA allele, whereas a missense mutation is found in the other allele. Although it is known that the disease course for these patients can be as severe as for patients with two expanded FRDA alleles, the underlying pathophysiological mechanisms are not understood. Human cells normally contain two major mitochondrial isoforms of FXN (FXN(42-210) and FXN(81-210)) that have different biochemical properties and functional roles. Using cell-free systems and different cellular models, we show that two of the most clinically severe FXN point mutations, I154F and W155R, have unique direct and indirect effects on the stability, biogenesis, or catalytic activity of FXN(42-210) and FXN(81-210) under physiological conditions. Our data indicate that frataxin point mutations have complex biochemical effects that synergistically contribute to the pathophysiology of Friedreich ataxia.
Assuntos
Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Alelos , Substituição de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Sistema Livre de Células , Chlorocebus aethiops , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Humanos , Proteínas de Ligação ao Ferro/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação ao Ferro/química , Ferro/química , Multimerização Proteica , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Proteínas de Ligação ao Ferro/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Termodinâmica , FrataxinaRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive, multi-systemic degenerative disease that results from reduced synthesis of the mitochondrial protein frataxin. Frataxin has been intensely studied since its deficiency was linked to FRDA in 1996. The defining properties of frataxin - (i) the ability to bind iron, (ii) the ability to interact with, and donate iron to, other iron-binding proteins, and (iii) the ability to oligomerize, store iron and control iron redox chemistry - have been extensively characterized with different frataxin orthologs and their interacting protein partners. This very large body of biochemical and structural data [reviewed in (Bencze et al., 2006)] supports equally extensive biological evidence that frataxin is critical for mitochondrial iron metabolism and overall cellular iron homeostasis and antioxidant protection [reviewed in (Wilson, 2006)]. However, the precise biological role of frataxin remains a matter of debate. Here, we review seminal and recent data that strongly link frataxin to the synthesis of iron-sulfur cluster cofactors (ISC), as well as controversial data that nevertheless link frataxin to additional iron-related processes. Finally, we discuss how defects in ISC synthesis could be a major (although likely not unique) contributor to the pathophysiology of FRDA via (i) loss of ISC-dependent enzymes, (ii) mitochondrial and cellular iron dysregulation, and (iii) enhanced iron-mediated oxidative stress. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.
Assuntos
Ataxia de Friedreich/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Estresse Oxidativo , Animais , Ataxia de Friedreich/genética , Homeostase , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , FrataxinaRESUMO
Friedreich's Ataxia (FRDA) is a neuromuscular degenerative disorder caused by trinucleotide expansions in the first intron of the frataxin (FXN) gene, resulting in insufficient levels of functional FNX protein. Deficits in FXN involve mitochondrial disruptions including iron-sulfur cluster synthesis and impaired energetics. These studies were to identify unique protein-protein interactions with FXN to better understand its function and design therapeutics. Two complementary approaches were employed, BioID and Co-IP, to identify protein interactions with FXN at the direct binding, indirect binding, and non-proximal levels. Forty-one novel protein interactions were identified by BioID and IP techniques. The FXN protein landscape was further analyzed incorporating both interaction type and functional pathways using a maximum path of 6 proteins with a potential direct interaction between FXN and NFS1. Probing the intersection between FXN-protein landscape and biological pathways associated with FRDA, we identified 41 proteins of interest. Peroxiredoxin 3 (Prdx3) was chosen for further analysis because of its role in mitochondrial oxidative injury. Our data has demonstrated the strengths of employing complementary methods to identify a unique interactome for FXN. Our data provides new insights into FXN function and regulation, a potential direct interaction between FXN and NFS1, and pathway interactions between FXN and Prdx3.
RESUMO
BACKGROUND: Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS: Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 µL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS: The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%-13% and 9.8%-16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2-200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12-22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1-26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS: This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.
Assuntos
Ataxia de Friedreich/diagnóstico , Proteínas de Ligação ao Ferro/sangue , Adulto , Teste em Amostras de Sangue Seco , Feminino , Ataxia de Friedreich/sangue , Ensaios de Triagem em Larga Escala , Humanos , Imunoensaio/métodos , Recém-Nascido , Masculino , Valores de Referência , FrataxinaRESUMO
Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD(+)-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations.
Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Fibroblastos/enzimologia , Mutação de Sentido Incorreto , Estresse Oxidativo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Células Cultivadas , Di-Hidrolipoamida Desidrogenase/genética , Estabilidade Enzimática/genética , Feminino , Fibroblastos/patologia , Humanos , Masculino , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , NAD , Estrutura Terciária de Proteína , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Fe-S clusters (ISCs) are versatile cofactors utilized by many mitochondrial, cytoplasmic, and nuclear enzymes. Whereas mitochondria can independently initiate and complete ISC synthesis, other cellular compartments are believed to assemble ISCs from putative precursors exported from the mitochondria via an ATP binding cassette (ABC) transporter conserved from yeast (Atm1p) to humans (ABCB7). However, the regulatory interactions between mitochondrial and extramitochondrial ISC synthesis are largely unknown. In yeast, we found that mitochondrial ISC synthesis is regulated by the leucine biosynthetic pathway, which among other proteins involves an abundant cytoplasmic [4Fe-4S] enzyme, Leu1p. Enzymatic blocks in the pathway (i.e. leu1Δ or leu2Δ gene deletion mutations) induced post-transcriptional up-regulation of core components of mitochondrial ISC biosynthesis (i.e. the sulfur donor Nfs1p, the iron donor Yfh1p, and the ISC scaffold Isu1p). In leu2Δ cells, transcriptional mechanisms also led to dramatic up-regulation of Leu1p with concomitant down-regulation of mitochondrial aconitase (Aco1p), a [4Fe-4S] enzyme in the tricarboxylic acid cycle. Accordingly, the leu2Δ deletion mutation exacerbated Aco1p inactivation in cells with mutations in Yfh1p. These data indicate that defects in leucine biosynthesis promote the biogenesis of enzymatically active Leu1p at the expense of Aco1p activity. Surprisingly, this effect is independent of Atm1p; previous reports linking the loss of Leu1p activity to Atm1p depletion were confounded by the fact that LEU2 was used as a selectable marker to create Atm1p-depleted cells, whereas a leu2Δ allele was present in Atm1p-repleted controls. Thus, still largely unknown transcriptional and post-transcriptional mechanisms control ISC distribution between mitochondria and other cellular compartments.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Citoplasma/metabolismo , Proteína 1 Reguladora do Ferro/biossíntese , Leucina/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Respiração Celular , DNA Fúngico/metabolismo , Regulação para Baixo , Proteína 1 Reguladora do Ferro/metabolismo , Mitocôndrias/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.
Assuntos
Ataxia de Friedreich/metabolismo , Regulação da Expressão Gênica , Proteínas de Ligação ao Ferro/biossíntese , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Precursores de Proteínas/biossíntese , Adolescente , Adulto , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Linhagem Celular Transformada , Criança , Feminino , Ataxia de Friedreich/genética , Humanos , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Masculino , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , FrataxinaRESUMO
Chelatases catalyze the insertion of a specific metal ion into porphyrins, a key step in the synthesis of metalated tetrapyrroles that are essential for many cellular processes. Despite apparent common structural features among chelatases, no general reaction mechanism accounting for metal ion specificity has been established. We propose that chelatase-induced distortion of the porphyrin substrate not only enhances the reaction rate by decreasing the activation energy of the reaction but also modulates which divalent metal ion is incorporated into the porphyrin ring. We evaluate the recently recognized interaction between ferrochelatase and frataxin as a way to regulate iron delivery to ferrochelatase, and thus iron and heme metabolism. We postulate that the ferrochelatase-frataxin interaction controls the type of metal ion that is delivered to ferrochelatase.
Assuntos
Ferroquelatase/metabolismo , Animais , Catálise , Ferroquelatase/química , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Metais/química , Metais/metabolismo , Porfirinas/metabolismo , Ligação Proteica , FrataxinaRESUMO
Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.
RESUMO
The mitochondrion, conventionally thought to be an organelle specific to energy metabolism, is in fact multifunctional and implicated in many diseases, including cancer. To evaluate whether mitochondria-related genes are associated with increased risk for prostate cancer, we genotyped 24 single-nucleotide polymorphisms (SNP) within the mitochondrial genome and 376 tagSNPs localized to 78 nuclear-encoded mitochondrial genes. The tagSNPs were selected to achieve > or = 80% coverage based on linkage disequilibrium. We compared allele and haplotype frequencies in approximately 1,000 prostate cancer cases with approximately 500 population controls. An association with prostate cancer was not detected for any of the SNPs within the mitochondrial genome individually or for 10 mitochondrial common haplotypes when evaluated using a global score statistic. For the nuclear-encoded genes, none of the tagSNPs were significantly associated with prostate cancer after adjusting for multiple testing. Nonetheless, we evaluated unadjusted P values by comparing our results with those from the Cancer Genetic Markers of Susceptibility (CGEMS) phase I data set. Seven tagSNPs had unadjusted P < or = 0.05 in both our data and in CGEMS (two SNPs were identical and five were in strong linkage disequilibrium with CGEMS SNPs). These seven SNPs (rs17184211, rs4147684, rs4233367, rs2070902, rs3829037, rs7830235, and rs1203213) are located in genes MTRR, NDUFA9, NDUFS2, NDUFB9, and COX7A2, respectively. Five of the seven SNPs were further included in the CGEMS phase II study; however, none of the findings for these were replicated. Overall, these results suggest that polymorphisms in the mitochondrial genome and those in the nuclear-encoded mitochondrial genes evaluated are not substantial risk factors for prostate cancer.
Assuntos
Genes Mitocondriais/genética , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Idoso , Alelos , Estudos de Casos e Controles , Marcadores Genéticos , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Friedreich's Ataxia (FA) is an inherited neurodegenerative disease caused by reduction in levels of the mitochondrial protein frataxin. Currently there are no simple, reliable methods to accurately measure the concentrations of frataxin protein. We designed a lateral-flow immunoassay that quantifies frataxin protein levels in a variety of sample materials. Using recombinant frataxin we evaluated the accuracy and reproducibility of the assay. The assay measured recombinant human frataxin concentrations between 40 and 4000 pg/test or approximately 0.1-10 nM of sample. The intra and inter-assay error was <10% throughout the working range. To evaluate clinical utility of the assay we used genetically defined lymphoblastoid cells derived from FA patients, FA carriers and controls. Mean frataxin concentrations in FA patients and carriers were significantly different from controls and from one another (p=0.0001, p=0.003, p=0.005, respectively) with levels, on average, 29% (patients) and 64% (carriers) of the control group. As predicted, we observed an inverse relationship between GAA repeat number and frataxin protein concentrations within the FA patient cohort. The lateral flow immunoassay provides a simple, accurate and reproducible method to quantify frataxin protein in whole cell and tissue extracts, including primary samples obtained by non-invasive means, such as cheek swabs and whole blood. The assay is a novel tool for FA research that may facilitate improved diagnostic and prognostic evaluation of FA patients and could also be used to evaluate efficacy of therapies designed to cure FA by increasing frataxin protein levels.
Assuntos
Ataxia de Friedreich/diagnóstico , Heterozigoto , Imunoensaio/métodos , Proteínas de Ligação ao Ferro/metabolismo , Adolescente , Adulto , Idade de Início , Células Cultivadas , Criança , Estudos de Coortes , Feminino , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/análise , Proteínas de Ligação ao Ferro/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , FrataxinaRESUMO
Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.
Assuntos
Proteínas de Ligação ao Ferro/química , Ferro/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Transporte Biológico , Cristalografia por Raios X , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Microscopia Eletrônica , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , FrataxinaRESUMO
Although Fe-S clusters may assemble spontaneously from elemental iron and sulfur in protein-free systems, the potential toxicity of free Fe2+, Fe3+, and S2- ions in aerobic environments underscores the requirement for specialized proteins to oversee the safe assembly of Fe-S clusters in living cells. Prokaryotes first developed multiprotein systems for Fe-S cluster assembly, from which mitochondria later derived their own system and became the main Fe-S cluster suppliers for eukaryotic cells. Early studies in yeast and human mitochondria indicated that Fe-S cluster assembly in eukaryotes is centered around highly conserved Fe-S proteins (human ISCU) that serve as scaffolds upon which new Fe-S clusters are assembled from (i) elemental sulfur, provided by a pyridoxal phosphate-dependent cysteine desulfurase (human NFS1) and its stabilizing-binding partner (human ISD11), and (ii) elemental iron, provided by an iron-binding protein of the frataxin family (human FXN). Further studies revealed that all of these proteins could form stable complexes that could reach molecular masses of megadaltons. However, the protein-protein interaction surfaces, catalytic mechanisms, and overall architecture of these macromolecular machines remained undefined for quite some time. The delay was due to difficulties inherent in reconstituting these very large multiprotein complexes in vitro or isolating them from cells in sufficient quantities to enable biochemical and structural studies. Here, we describe approaches we developed to reconstitute the human Fe-S cluster assembly machinery in Escherichia coli and to define its remarkable architecture.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , Ferro/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Enxofre/química , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Escherichia coli/metabolismo , Humanos , Ferro/toxicidade , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Reguladoras de Ferro/química , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Mitocondriais/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Enxofre/toxicidade , FrataxinaRESUMO
Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed.