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Grazia Isaya - One of the best experts on this subject based on the ideXlab platform.
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iron sulfur cluster synthesis iron homeostasis and oxidative stress in friedreich ataxia
Molecular and Cellular Neuroscience, 2013Co-Authors: Rachael A Vaubel, Grazia IsayaAbstract:Abstract 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’.
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lateral flow immunoassay for the Frataxin protein in friedreich s ataxia patients and carriers
Molecular Genetics and Metabolism, 2008Co-Authors: John H. Willis, Grazia Isaya, Oleksandr Gakh, Roderick A. Capaldi, Michael F. MarusichAbstract: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.
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Lateral-flow immunoassay for the Frataxin protein in Friedreich's ataxia patients and carriers.
Molecular genetics and metabolism, 2008Co-Authors: John H. Willis, Grazia Isaya, Oleksandr Gakh, Roderick A. Capaldi, Michael F. MarusichAbstract: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
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Partial conservation of functions between eukaryotic Frataxin and the Escherichia coli Frataxin homolog CyaY.
FEMS yeast research, 2007Co-Authors: Tibor Bedekovics, Gabriella B. Gajdos, Gyula Kispal, Grazia IsayaAbstract:Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic Frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic Frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe–2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of Frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast Frataxin protein (yfh1Δ). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Δ from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.
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mitochondrial iron detoxification is a primary function of Frataxin that limits oxidative damage and preserves cell longevity
Human Molecular Genetics, 2006Co-Authors: Oleksandr Gakh, Sungjo Park, Gang Liu, Lee Macomber, James A Imlay, Gloria C Ferreira, Grazia IsayaAbstract:Friedreich ataxia is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein Frataxin. Although Frataxin is ubiquitously expressed, Frataxin deficiency leads to a selective loss of dorsal root ganglia neurons, cardiomyocytes and pancreatic beta cells. How Frataxin normally promotes survival of these particular cells is the subject of intense debate. The predominant view is that Frataxin sustains mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron-sulfur cluster (ISC) assembly and repair. We have proposed that Frataxin not only promotes the biogenesis of iron-containing enzymes, but also detoxifies surplus iron thereby affording a critical anti-oxidant mechanism. These two functions have been difficult to tease apart, however, and the physiologic role of iron detoxification by Frataxin has not yet been demonstrated in vivo. Here, we describe mutations that specifically impair the ferroxidation or mineralization activity of yeast Frataxin, which are necessary for iron detoxification but do not affect the iron chaperone function of the protein. These mutations increase the sensitivity of yeast cells to oxidative stress, shortening chronological life span and precluding survival in the absence of the anti-oxidant enzyme superoxide dismutase. Thus, the role of Frataxin is not limited to promoting ISC assembly or heme synthesis. Iron detoxification is another function of Frataxin relevant to anti-oxidant defense and cell longevity that could play a critical role in the metabolically demanding environment of non-dividing neuronal, cardiac and pancreatic beta cells.
Diego F. Gomez-casati - One of the best experts on this subject based on the ideXlab platform.
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Plant Frataxin in Metal Metabolism.
Frontiers in plant science, 2018Co-Authors: Diego F. Gomez-casati, Maria V. Busi, Maria A. PaganiAbstract:Frataxin is a highly-conserved protein from prokaryotes to eukaryotes. Several functions related to iron metabolism have been postulated for this protein, including Fe-S cluster and heme synthesis, response to oxidative damage and oxidative phosphorylation. In plants, the presence of one or two isoforms of this protein with dual localization in mitochondria and chloroplasts has been reported. Frataxin deficiency affects iron metabolism in both organelles, leading to an impairment of mitochondrial respiration, and chlorophyll and photosynthetic electron transport deficiency in chloroplasts. In addition, plant Frataxins can react with Cu2+ ions and dimerize, which causes the reduction of free Cu ions. This could provide an additional defense mechanism against the oxidation of Fe-S groups by Cu ions. While there is a consensus on the involvement of Frataxin in iron homeostasis in most organisms, the interaction of plant Frataxins with Cu ions, the presence of different isoforms, and/or the localization in two plant organelles suggest that this protein might have additional functions in vegetal tissues.
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Copper redox chemistry of plant Frataxins.
Journal of inorganic biochemistry, 2017Co-Authors: Manu Sánchez, Celeste Buchensky, Òscar Palacios, Mercè Capdevila, Sílvia Atrian, Maria A. Pagani, Diego F. Gomez-casati, Laura Sabio, José M. Domínguez-veraAbstract:The presence of a conserved cysteine residue in the C-terminal amino acid sequences of plant Frataxins differentiates these Frataxins from those of other kingdoms and may be key in Frataxin assembly and function. We report a full study on the ability of Arabidopsis (AtFH) and Zea mays (ZmFH-1 and ZmFH-2) Frataxins to assemble into disulfide-bridged dimers by copper-driven oxidation and to revert to monomers by chemical reduction. We monitored the redox assembly-disassembly process by electrospray ionization mass spectrometry, electrophoresis, UV-Vis spectroscopy, and fluorescence measurements. We conclude that plant Frataxins AtFH, ZmFH-1 and ZmFH-2 are oxidized by Cu2+ and exhibit redox cysteine monomer - cystine dimer interexchange. Interestingly, the tendency to interconvert is not the same for each protein. Through yeast phenotypic rescue experiments, we show that plant Frataxins are important for plant survival under conditions of excess copper, indicating that these proteins might be involved in copper metabolism.
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Identification of two Frataxin isoforms in Zea mays: Structural and functional studies.
Biochimie, 2017Co-Authors: Celeste Buchensky, Manuel Calderon De La Barca Sanchez, Martin Carrillo, Òscar Palacios, Mercè Capdevila, José M. Domínguez-vera, Maria V. Busi, Sílvia Atrian, Maria A. Pagani, Diego F. Gomez-casatiAbstract:Frataxin is a ubiquitous protein that plays a role in Fe-S cluster biosynthesis and iron and heme metabolism, although its molecular functions are not entirely clear. In non-photosynthetic eukaryotes, Frataxin is encoded by a single gene, and the protein localizes to mitochondria. Here we report the presence of two functional Frataxin isoforms in Zea mays, ZmFH-1 and ZmFH-2. We confirmed our previous findings regarding plant Frataxins: both proteins have dual localization in mitochondria and chloroplasts. Physiological, biochemical and biophysical studies show some differences in the expression pattern, protection against oxidants and in the aggregation state of both isoforms, suggesting that the two Frataxin homologs would play similar but not identical roles in plant cell metabolism. In addition, two specific features of plant Frataxins were evidenced: their ability to form dimers and their tendency to undergo conformational change under oxygen exposure.
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Expression and one-step purification of recombinant Arabidopsis thaliana Frataxin homolog (AtFH).
Protein expression and purification, 2006Co-Authors: Maria Victoria Maliandi, Maria V. Busi, Alejandro Araya, Marina Clemente, Eduardo Zabaleta, Diego F. Gomez-casatiAbstract:Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the Frataxin family has been described. To gain insight on the Frataxin role in plants, the Frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant Frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.
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Functional and molecular characterization of the Frataxin homolog from Arabidopsis thaliana.
FEBS letters, 2004Co-Authors: Maria V. Busi, Eduardo J Zabaleta, Alejandro Araya, Diego F. Gomez-casatiAbstract:Frataxin is a highly conserved protein from bacteria to mammals that has been proposed to participate in iron-sulfur cluster assembly and mitochondrial iron homeostasis. In higher organisms, the Frataxin gene is nuclear-encoded and the protein is required for maintenance of normal mitochondrial iron levels and respiration. We describe here AtFH, a plant gene with significant homology to other members of the Frataxin family. Plant Frataxin has five segments of beta regions and two alpha helices, which are characteristics of human Frataxin, as well as a potential N-terminal targeting peptide for the mitochondrial localization. Transcription analysis showed that AtFH is ubiquitously expressed with high levels in flowers. Complementation of a Saccharomyces cerevisiae mutant (Deltayfh) lacking the Frataxin gene proved that AtFH is a functional protein, because it restored normal rates of respiration, growth and sensitivity to H2O2 of the null mutant. Our results support the involvement of AtFH in mitochondrial respiration and survival during oxidative stress in plants. This is the first report of a functional Frataxin gene in plants.
Helene Puccio - One of the best experts on this subject based on the ideXlab platform.
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Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of Friedreich Ataxia
Molecular therapy : the journal of the American Society of Gene Therapy, 2018Co-Authors: Françoise Piguet, Charline De Montigny, Nadège Vaucamps, Laurence Reutenauer, Aurélie Eisenmann, Helene PuccioAbstract:Friedreich ataxia (FA) is a rare mitochondrial disease characterized by sensory and spinocerebellar ataxia, hypertrophic cardiomyopathy, and diabetes, for which there is no treatment. FA is caused by reduced levels of Frataxin (FXN), an essential mitochondrial protein involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Despite significant progress in recent years, to date, there are no good models to explore and test therapeutic approaches to stop or reverse the ganglionopathy and the sensory neuropathy associated to Frataxin deficiency. Here, we report a new conditional mouse model with complete Frataxin deletion in parvalbumin-positive cells that recapitulate the sensory ataxia and neuropathy associated to FA, albeit with a more rapid and severe course. Interestingly, although fully dysfunctional, proprioceptive neurons can survive for many weeks without Frataxin. Furthermore, we demonstrate that post-symptomatic delivery of Frataxin-expressing AAV allows for rapid and complete rescue of the sensory neuropathy associated with Frataxin deficiency, thus establishing the pre-clinical proof of concept for the potential of gene therapy in treating FA neuropathy.
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prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of friedreich s ataxia
Nature Medicine, 2014Co-Authors: Morgane Perdomini, Laurence Reutenauer, Nadia Messaddeq, Brahim Belbellaa, Laurent Monassier, Nathalie Cartier, Ronald G Crystal, Patrick Aubourg, Helene PuccioAbstract:In Friedreich's ataxia, caused by mutation of the gene encoding the mitochondrial protein Frataxin, the major cause of mortality is heart failure. Using mice lacking Frataxin in the heart, Helene Puccio and her colleagues demonstrate that Frataxin gene therapy can correct mitochondrial metabolism and reverse heart damage, raising the possibility of a gene therapy treatment for this disease.
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Frataxin: a protein in search for a function.
Journal of Neurochemistry, 2013Co-Authors: Annalisa Pastore, Helene PuccioAbstract:Reduced levels of the protein Frataxin cause the neurodegenerative disease Friedreich's ataxia. Pathology is associated with disruption of iron-sulfur cluster biosynthesis, mitochondrial iron overload, and oxidative stress. Frataxin is a highly conserved iron-binding protein present in most organisms. Despite the intense interest generated since the determination of its pathology, identification of the cellular function of Frataxin has so far remained elusive. In this review, we revisit the most significant milestones that have led us to our current understanding of Frataxin and its functions. The picture that emerges is that Frataxin is a crucial element of one of the most essential cellular machines specialized in iron-sulfur cluster biogenesis. Future developments, therefore, can be expected from further advancements in our comprehension of this machine.
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mammalian Frataxin an essential function for cellular viability through an interaction with a preformed iscu nfs1 isd11 iron sulfur assembly complex
PLOS ONE, 2011Co-Authors: Stéphane Schmucker, Laurence Reutenauer, Alain Martelli, Florent Colin, Adeline Page, Marie Wattenhoferdonze, Helene PuccioAbstract:Background Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, Frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis. Methodology/Principal Findings We present data providing new insights into the interactions of mammalian Frataxin with the Fe-S assembly complex by combining in vitro and in vivo approaches. Through immunoprecipitation experiments, we show that the main endogenous interactors of a recombinant mature human Frataxin are ISCU, NFS1 and ISD11, the components of the core Fe-S assembly complex. Furthemore, using a heterologous expression system, we demonstrate that mammalian Frataxin interacts with the preformed core complex, rather than with the individual components. The quaternary complex can be isolated in a stable form and has a molecular mass of ≈190 kDa. Finally, we demonstrate that the mature human FXN81–210 form of Frataxin is the essential functional form in vivo. Conclusions/Significance Our results suggest that the interaction of Frataxin with the core ISCU/NFS1/ISD11 complex most likely defines the essential function of Frataxin. Our results provide new elements important for further understanding the early steps of de novo Fe-S cluster biosynthesis.
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The first cellular models based on Frataxin missense mutations that reproduce spontaneously the defects associated with Friedreich ataxia.
PLoS ONE, 2009Co-Authors: Nadège Calmels, Michel Koenig, Nadège Vaucamps, Laurence Reutenauer, Stéphane Schmucker, Alain Martelli, Marie Wattenhofer-donzé, Nadia Messaddeq, Cécile Bouton, Helene PuccioAbstract:BACKGROUND: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of Frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the Frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation. METHODOLOGY: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a Frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous Frataxin and expressing missense-mutated human Frataxin. We showed that complete absence of murine Frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) Frataxin. Interestingly, cells expressing the mutated Frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients. CONCLUSIONS: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating Frataxin deficiency.
Ivano Condo - One of the best experts on this subject based on the ideXlab platform.
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drug repositioning screening identifies etravirine as a potential therapeutic for friedreich s ataxia
Movement Disorders, 2019Co-Authors: Giulia Alfedi, Ivano Condo, Monica Benini, Nicola Toschi, Damiano Sergio Massaro, Riccardo Luffarelli, Giorgia Pedini, Liliana Mannucci, Giorgia AlaimoAbstract:BACKGROUND Friedreich's ataxia is an autosomal-recessive cerebellar ataxia caused by mutation of the Frataxin gene, resulting in decreased Frataxin expression, mitochondrial dysfunction, and oxidative stress. Currently, no treatment is available for Friedreich's ataxia patients. Given that levels of residual Frataxin critically affect disease severity, the main goal of a specific therapy for Friedreich's ataxia is to increase Frataxin levels. OBJECTIVES With the aim to accelerate the development of a new therapy for Friedreich's ataxia, we took a drug repositioning approach to identify market-available drugs able to increase Frataxin levels. METHODS Using a cell-based reporter assay to monitor variation in Frataxin amount, we performed a high-throughput screening of a library containing 853 U.S. Food and Drug Administration-approved drugs. RESULTS Among the potentially interesting candidates isolated from the screening, we focused our attention on etravirine, an antiviral drug currently in use as an anti-human immunodeficiency virus therapy. Here, we show that etravirine can promote a significant increase in Frataxin levels in cells derived from Friedreich's ataxia patients, by enhancing Frataxin messenger RNA translation. Importantly, Frataxin accumulation in treated patient cell lines is comparable to Frataxin levels in unaffected carrier cells, suggesting that etravirine could be therapeutically relevant. Indeed, etravirine treatment restores the activity of the iron-sulphur cluster containing enzyme aconitase and confers resistance to oxidative stress in cells derived from Friedreich's ataxia patients. CONCLUSIONS Considering its excellent safety profile along with its ability to increase Frataxin levels and correct some of the disease-related defects, etravirine represents a promising candidate as a therapeutic for Friedreich's ataxia. © 2019 International Parkinson and Movement Disorder Society.
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e3 ligase rnf126 directly ubiquitinates Frataxin promoting its degradation identification of a potential therapeutic target for friedreich ataxia
Cell Reports, 2017Co-Authors: Monica Benini, Dario Serio, Silvia Fortuni, Ivano Condo, Florence Malisan, Giulia Alfedi, Nicola Toschi, Damiano Sergio Massaro, Gaetano ArcuriAbstract:Friedreich ataxia (FRDA) is a severe genetic neurodegenerative disease caused by reduced expression of the mitochondrial protein Frataxin. To date, there is no therapy to treat this condition. The amount of residual Frataxin critically affects the severity of the disease; thus, attempts to restore physiological Frataxin levels are considered therapeutically relevant. Frataxin levels are controlled by the ubiquitin-proteasome system; therefore, inhibition of the Frataxin E3 ligase may represent a strategy to achieve an increase in Frataxin levels. Here, we report the identification of the RING E3 ligase RNF126 as the enzyme that specifically mediates Frataxin ubiquitination and targets it for degradation. RNF126 interacts with Frataxin and promotes its ubiquitination in a catalytic activity-dependent manner, both in vivo and in vitro. Most importantly, RNF126 depletion results in Frataxin accumulation in cells derived from FRDA patients, highlighting the relevance of RNF126 as a new therapeutic target for Friedreich ataxia.
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Src inhibitors modulate Frataxin protein levels
Human molecular genetics, 2015Co-Authors: Fabio Cherubini, Dario Serio, Ilaria Guccini, Silvia Fortuni, Gaetano Arcuri, Ivano Condo, Alessandra Rufini, Shadman Moiz, Serena Camerini, Marco CrescenziAbstract:Defective expression of Frataxin is responsible for the inherited, progressive degenerative disease Friedreich's Ataxia (FRDA). There is currently no effective approved treatment for FRDA and patients die prematurely. Defective Frataxin expression causes critical metabolic changes, including redox imbalance and ATP deficiency. As these alterations are known to regulate the tyrosine kinase Src, we investigated whether Src might in turn affect Frataxin expression. We found that Frataxin can be phosphorylated by Src. Phosphorylation occurs primarily on Y118 and promotes Frataxin ubiquitination, a signal for degradation. Accordingly, Src inhibitors induce accumulation of Frataxin but are ineffective on a non-phosphorylatable Frataxin-Y118F mutant. Importantly, all the Src inhibitors tested, some of them already in the clinic, increase Frataxin expression and rescue the aconitase defect in Frataxin-deficient cells derived from FRDA patients. Thus, Src inhibitors emerge as a new class of drugs able to promote Frataxin accumulation, suggesting their possible use as therapeutics in FRDA.
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Frataxin participates to the hypoxia-induced response in tumors
Cell death & disease, 2011Co-Authors: Ilaria Guccini, Dario Serio, Ivano Condo, Alessandra Rufini, Barbara Tomassini, Annunziato Mangiola, Giulio Maira, Carmelo Anile, D Fina, Francesco PalloneAbstract:Defective expression of Frataxin is responsible for the degenerative disease Friedreich's ataxia. Frataxin is a protein required for cell survival since complete knockout is lethal. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. We therefore sought to investigate the pathways through which Frataxin enhances stress resistance in tumor cells. We found that Frataxin expression is upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, Frataxin upregulation in response to hypoxia is dependent on hypoxia-inducible factors expression and modulates the activation of the tumor-suppressor p53. Importantly, we show for the first time that Frataxin is in fact increased in human tumors in vivo. These results show that Frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could have a critical role in tumor cell survival and/or progression.
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Preventing the ubiquitin/proteasome-dependent degradation of Frataxin, the protein defective in Friedreich’s Ataxia
Human molecular genetics, 2011Co-Authors: Alessandra Rufini, Dario Serio, Silvia Fortuni, Gaetano Arcuri, Ivano Condo, Natascia Ventura, Florence Malisan, Ottaviano Incani, Roberto TestiAbstract:Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein Frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual Frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate Frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that Frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for Frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable Frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent Frataxin ubiquitination and degradation, and increase Frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the Frataxin degradation pathway.
Annalisa Pastore - One of the best experts on this subject based on the ideXlab platform.
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Chemical shift assignment of a thermophile Frataxin.
Biomolecular NMR assignments, 2017Co-Authors: Masooma Rasheed, Robert Yan, Geoff Kelly, Annalisa PastoreAbstract:Frataxin is the protein responsible for the genetically-inherited neurodegenerative disease Friedreich’s ataxia caused by partial silencing of the protein and loss of function. Although the Frataxin function is not yet entirely clear, it has been associated to the machine that builds iron–sulfur clusters, essential prosthetic groups involved in several processes and is strongly conserved in organisms from bacteria to humans. Two of its important molecular partners are the protein NFS1 (or IscS in bacteria), that is the desulfurase which converts cysteine to alanine and produces sulfur, and ISU (or IscU), the scaffold protein which transiently accepts the cluster. While bacterial Frataxin has been extensively characterized, only few eukaryotic Frataxins have been described. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of Frataxin from Chaetomium thermophilum, a thermophile increasingly used by virtue of its stability.
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Frataxin: a protein in search for a function.
Journal of Neurochemistry, 2013Co-Authors: Annalisa Pastore, Helene PuccioAbstract:Reduced levels of the protein Frataxin cause the neurodegenerative disease Friedreich's ataxia. Pathology is associated with disruption of iron-sulfur cluster biosynthesis, mitochondrial iron overload, and oxidative stress. Frataxin is a highly conserved iron-binding protein present in most organisms. Despite the intense interest generated since the determination of its pathology, identification of the cellular function of Frataxin has so far remained elusive. In this review, we revisit the most significant milestones that have led us to our current understanding of Frataxin and its functions. The picture that emerges is that Frataxin is a crucial element of one of the most essential cellular machines specialized in iron-sulfur cluster biogenesis. Future developments, therefore, can be expected from further advancements in our comprehension of this machine.
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effector role reversal during evolution the case of Frataxin in fe s cluster biosynthesis
Biochemistry, 2012Co-Authors: Jennifer Bridwellrabb, Annalisa Pastore, Clara Iannuzzi, David P. BarondeauAbstract:Human Frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and Frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the Frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the Frataxin homologue. These data are consistent with a model in which the Frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of Frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.
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Bacterial Frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS
Nature structural & molecular biology, 2009Co-Authors: Salvatore Adinolfi, Clara Iannuzzi, Filippo Prischi, Chiara Pastore, Stefania Iametti, Stephen R. Martin, Franco Bonomi, Annalisa PastoreAbstract:Frataxin is an essential mitochondrial protein whose reduced expression causes Friedreich's ataxia (FRDA), a lethal neurodegenerative disease. It is believed that Frataxin is an iron chaperone that participates in iron metabolism. We have tested this hypothesis using the bacterial Frataxin ortholog, CyaY, and different biochemical and biophysical techniques. We observe that CyaY participates in iron-sulfur (Fe-S) cluster assembly as an iron-dependent inhibitor of cluster formation, through binding to the desulfurase IscS. The interaction with IscS involves the iron binding surface of CyaY, which is conserved throughout the Frataxin family. We propose that Frataxins are iron sensors that act as regulators of Fe-S cluster formation to fine-tune the quantity of Fe-S cluster formed to the concentration of the available acceptors. Our observations provide new perspectives for understanding FRDA and a mechanistic model that rationalizes the available knowledge on Frataxin.
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the pathogenesis of friedreich ataxia and the structure and function of Frataxin
Journal of Neurology, 2009Co-Authors: Massimo Pandolfo, Annalisa PastoreAbstract:Understanding the role of Frataxin in mitochondria is key to an understanding of the pathogenesis of Friedreich ataxia. Frataxins are small essential proteins whose deficiency causes a range of metabolic disturbances, which include oxidative stress, deficit of iron-sulphur clusters, and defects in heme synthesis, sulfur amino acid and energy metabolism, stress response, and mitochondrial function. Structural studies carried out on different orthologues have shown that the Frataxin fold consists of a flexible N-terminal region present only in eukaryotes and in a highly conserved C-terminal globular domain. Frataxins bind iron directly but with very unusual properties: iron coordination is achieved solely by glutamates and aspartates exposed on the protein surface. It has been suggested that Frataxin function is that of a ferritin-like protein, an iron chaperone of the ironsulphur cluster machinery and heme metabolism and/or a controller of cellular oxidative stress. To understand FRDA pathogenesis and to design novel therapeutic strategies, we must first precisely identify the cellular role of Frataxin.
