Iron-frataxin involved in the protective effect of quercetin against alcohol-induced liver mitochondrial dysfunction.

Emerging evidence supports the beneficial effect of quercetin on liver mitochondrial disorders. However, the molecular mechanism by which quercetin protects mitochondria is limited, especially in alcoholic liver disease. In this study, C57BL/6N mice were fed with Lieber De Carli liquid diet (28% ethanol-derived calories) for 12 weeks plus a single binge ethanol and intervened with quercetin (100 mg/kg.bw). Moreover, HepG2CYP2E1+/+ were stimulated with ethanol (100 mM) and quercetin (50 µM) to investigate the effects of mitochondrial protein frataxin. The results indicated that quercetin alleviated alcohol-induced histopathological changes and mitochondrial functional disorders in mice livers. Consistent with increased PINK1, Parkin, Bnip3 and LC3II as well as decreased p62, TOM20 and VDAC1 expression, the inhibition of mitophagy by ethanol was blocked by quercetin. Additionally, quercetin improved the imbalance of iron metabolism-related proteins expression in alcohol-fed mice livers. Compared with ethanol-treated Lv-empty HepG2CYP2E1+/+ cells, frataxin deficiency further exacerbated the inhibition of mitochondrial function. Conversely, restoration of frataxin expression ameliorated the effect of ethanol. Furthermore, frataxin deficiency reduced the protective effects of quercetin on mitochondria disordered by ethanol. Attentively, ferric ammonium citrate (FAC) and deferiprone decreased or increased frataxin expression in HepG2CYP2E1+/+, respectively. Notably, we further found FAC reversed the increasing effect of quercetin on frataxin expression. Ultimately, silencing NCOA4 attenuated the inhibition of quercetin on LDH release and mitochondrial membrane potential increase, and similar results were observed by adding FAC. Collectively, these findings demonstrated quercetin increased frataxin expression through regulating iron level, thereby mitigating ethanol-induced mitochondrial dysfunction.

Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase.

Fanconi anemia (FA) is a unique DNA damage repair pathway. To date, 22 genes have been identified that are associated with the FA pathway. A defect in any of those genes causes genomic instability, and the patients bearing the mutation become susceptible to cancer. In our earlier work, we identified that Fanconi anemia protein G (FANCG) protects the mitochondria from oxidative stress. In this report, we have identified eight patients having a mutation (C.65G>C), which converts arginine at position 22 to proline (p.Arg22Pro) in the N terminus of FANCG. The mutant protein, hFANCGR22P, is able to repair the DNA and able to retain the monoubiquitination of FANCD2 in the FANCGR22P/FGR22P cell. However, it lost mitochondrial localization and failed to protect mitochondria from oxidative stress. Mitochondrial instability in the FANCGR22P cell causes the transcriptional downregulation of mitochondrial iron-sulfur cluster biogenesis protein frataxin (FXN) and the resulting iron deficiency of FA protein FANCJ, an iron-sulfur-containing helicase involved in DNA repair.

Iron regulatory protein deficiency compromises mitochondrial function in murine embryonic fibroblasts.

Iron is essential for growth and proliferation of mammalian cells. The maintenance of cellular iron homeostasis is regulated by iron regulatory proteins (IRPs) through binding to the cognate iron-responsive elements in target mRNAs and thereby regulating the expression of target genes. Irp1 or Irp2-null mutation is known to reduce the cellular iron level by decreasing transferrin receptor 1 and increasing ferritin. Here, we report that Irp1 or Irp2-null mutation also causes downregulation of frataxin and IscU, two of the core components in the iron-sulfur cluster biogenesis machinery. Interestingly, while the activities of some of iron-sulfur cluster-containing enzymes including mitochondrial aconitase and cytosolic xanthine oxidase were not affected by the mutations, the activities of respiratory chain complexes were drastically diminished resulting in mitochondrial dysfunction. Overexpression of human ISCU and frataxin in Irp1 or Irp2-null cells was able to rescue the defects in iron-sulfur cluster biogenesis and mitochondrial quality. Our results strongly suggest that iron regulatory proteins regulate the part of iron sulfur cluster biogenesis tailored specifically for mitochondrial electron transport chain complexes.

Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways.

Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.

Identification of CHEK1, SLC26A4, c-KIT, TPO and TG as new biomarkers for human follicular thyroid carcinoma.

The search for preoperative biomarkers for thyroid malignancies, in particular for follicular thyroid carcinoma (FTC) diagnostics, is of utmost clinical importance. We thus aimed at screening for potential biomarker candidates for FTC. To evaluate dynamic alterations in molecular patterns as a function of thyroid malignancy progression, a comparative analysis was conducted in clinically distinct subgroups of FTC and poorly differentiated thyroid carcinoma (PDTC) nodules. NanoString analysis of FFPE samples was performed in 22 follicular adenomas, 56 FTC and 25 PDTC nodules, including oncocytic and non-oncocytic subgroups. The expression levels of CHEK1, c-KIT, SLC26A4, TG and TPO were significantly altered in all types of thyroid carcinomas. Based on collective changes of these biomarkers which correlating among each other, a predictive score has been established, allowing for discrimination between benign and FTC samples with high sensitivity and specificity. Additional transcripts related to thyroid function, cell cycle, circadian clock, and apoptosis regulation were altered in the more aggressive oncocytic subgroups only, with expression levels correlating with disease progression. Distinct molecular patterns were observed for oncocytic and non-oncocytic FTCs and PDTCs. A predictive score correlation coefficient based on collective alterations of identified here biomarkers might help to improve the preoperative diagnosis of FTC nodules.

Efficient attenuation of Friedreich's ataxia (FRDA) cardiomyopathy by modulation of iron homeostasis-human induced pluripotent stem cell (hiPSC) as a drug screening platform for FRDA.

BACKGROUND: Friedreich's ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is caused by silencing of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. METHODS: Application of our previously established FRDA human induced pluripotent stem cell (hiPSC) derived cardiomyocytes model as a platform to assess the efficacy of treatment with either the antioxidant coenzyme Q10 analog, idebenone (IDE) or the iron chelator, deferiprone (DFP), which are both under clinical trial. RESULTS: DFP was able to more significantly suppress synthesis of reactive oxygen species (ROS) than IDE at the dosages of 25 µM and 10nM respectively which agreed with the reduced rate of intracellular accumulation of iron by DFP treatment from 25 to 50 µM. With regard to cardiac electrical-contraction (EC) coupling function, decay velocity of calcium handling kinetics in FRDA-hiPSC-cardiomyocytes was significantly improved by DFP treatment but not by IDE. Further mechanistic studies revealed that DFP also modulated iron induced mitochondrial stress as reflected by mitochondria network disorganization and decline level of respiratory chain protein, succinate dehydrogenase (CxII) and cytochrome c oxidase (COXIV). In addition, iron-response protein (IRP-1) regulatory loop was overridden by DFP as reflected by resumed level of ferritin (FTH) back to basal level and the attenuated transferrin receptor (TSFR) mRNA level suppression thereby reducing further iron uptake. CONCLUSIONS: DFP modulated iron homeostasis in FRDA-hiPSC-cardiomyocytes and effectively relieved stress-stimulation related to cardiomyopathy. The resuming of redox condition led to the significantly improved cardiac prime events, cardiac electrical-coupling during contraction.

Repression of the Low Affinity Iron Transporter Gene FET4: A NOVEL MECHANISM AGAINST CADMIUM TOXICITY ORCHESTRATED BY YAP1 VIA ROX1.

Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake.

Src inhibitors modulate frataxin protein levels.

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.

The role of frataxin in fission yeast iron metabolism: implications for Friedreich's ataxia.

BACKGROUND: The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron-sulfur cluster (Fe-S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe-S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function. METHODS: The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe-S cluster containing enzymes. RESULTS: Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe-S cluster containing enzyme activities. CONCLUSIONS: Despite the presence of oxidative stress and accumulated iron, activation of Fe-S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe-S cluster biogenesis in S. pombe. GENERAL SIGNIFICANCE: We provide evidence that suggests that dysregulated Fe-S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.

The mitochondrial protein frataxin is downregulated in hemodialysis patients.

BACKGROUND: The mitochondrial protein frataxin regulates iron metabolism for heme and iron sulfur cluster synthesis in the mitochondria and could be associated with the regulation of oxidative stress. To clarify the expression of frataxin and its association with uremia, we evaluated the mRNA and protein levels of frataxin in the polymorphonuclear leukocytes (PMNLs) of patients on hemodialysis (HD). METHODS: Uremic patients on HD (n = 18) and healthy control subjects (n = 18) were investigated. PMNLs were isolated by differential centrifugation. The mRNA levels of frataxin in isolated leukocytes were quantified by TaqMan real-time polymerase chain reaction. Frataxin protein expression in the cell lysate was evaluated using SDS-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The frataxin/glyceraldehyde-3-phosphate dehydrogenase mRNA ratio in PMNLs from uremic patients was significantly lower than that in control subjects. Frataxin protein expression in uremic patients was also significantly lower than that in controls. Multiple regression analysis showed that frataxin mRNA levels were independently associated with the serum levels of both the oxidative stress marker malondialdehyde and the proinflammatory cytokine tumor necrosis factor-α. CONCLUSION: The downregulation of frataxin seems to be linked with uremic status, which is usually associated with chronic inflammation and the acceleration of oxidative stress. Mitochondrial iron regulation may play a role in several comorbidities and in the poor prognosis in uremic patients. Further investigation is needed to elucidate whether reduced frataxin levels are linked to the pathological status of uremic patients and whether uremic substances affect frataxin expression.

Exposure to cigarette smoke induces overexpression of von Hippel-Lindau tumor suppressor in mouse skeletal muscle.

Cigarette smoke (CS) is a well-established risk factor in the development of chronic obstructive pulmonary disease (COPD). In contrast, the extent to which CS exposure contributes to the development of the systemic manifestations of COPD, such as skeletal muscle dysfunction and wasting, remains largely unknown. Decreased skeletal muscle capillarization has been previously reported in early stages of COPD and might play an important role in the development of COPD-associated skeletal muscle abnormalities. To investigate the effects of chronic CS exposure on skeletal muscle capillarization and exercise tolerance, a mouse model of CS exposure was used. The 129/SvJ mice were exposed to CS for 6 mo, and the expression of putative elements of the hypoxia-angiogenic signaling cascade as well as muscle capillarization were studied. Additionally, functional tests assessing exercise tolerance/endurance were performed in mice. Compared with controls, skeletal muscles from CS-exposed mice exhibited significantly enhanced expression of von Hippel-Lindau tumor suppressor (VHL), ubiquitin-conjugating enzyme E2D1 (UBE2D1), and prolyl hydroxylase-2 (PHD2). In contrast, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression was reduced. Furthermore, reduced muscle fiber cross-sectional area, decreased skeletal muscle capillarization, and reduced exercise tolerance were also observed in CS-exposed animals. Taken together, the current results provide evidence linking chronic CS exposure and induction of VHL expression in skeletal muscles leading toward impaired hypoxia-angiogenesis signal transduction, reduced muscle fiber cross-sectional area, and decreased exercise tolerance.

Cotransfected sodium iodide symporter and human tyroperoxidase genes following human telomerase reverse transcriptase promoter for targeted radioiodine therapy of malignant glioma cells.

INTRODUCTION: Radioiodine is a routine therapy for differentiated thyroid cancers. In principle, undifferentiated thyroid cancers as well as nonthyroid cancers can concentrate and, thus, be treated with radioiodine after transfection with the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter is an effective tumor-specific promoter of gene expression and, thus, may be useful in targeted gene therapy of cancer. METHODS: We used hTERT promoter-modulated expression of the hNIS and human thyroperoxidase (hTPO) genes in an experimental model of radioiodine-based treatment of malignant glioma. Cells were cotransfected by adenovirus in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the human cytomegalovirus (CMV) promoter (Ad-hTERT-hNIS and Ad-CMV-hTPO, respectively), and they were evaluated in cells thus transfecting transgene expression by western blots, (125)I uptake and influx, and clonogenecity after (131)I treatment. RESULTS: After cotransfection with two adenovirus, cells showed about 31-34 times higher (125)I uptake than the control cells transfected with Ad-CMV-EGFP (enhanced green fluorescent protein) and almost 1.3-1.4 times higher (125)I uptake than cells only transfected with Ad-hTERT-hNIS. Western blots revealed two bands of ∼70 and 110 kDa, respectively. The in vitro clonogenic assay indicated that, after exposure to 100-1000 µCi of (131)I-iodide for 12 hours, 91%-94% of cells cotransfected with the hNIS and hTPO genes, 88%-93% of cells transfected with the hNIS gene, and only 62%-68% of control (nontransfected) cells were killed. CONCLUSIONS: The experiments demonstrated that an effective therapy of (131)I was achieved in malignant glioma cell lines after induction of tumor-specific iodide uptake activity by the hTERT promoter-directed NIS expression in vitro. Cotransfection of the hNIS and hTPO genes can lead to longer retention of radioiodide, but did not increase cell killing over that achieved with transfection with the hNIS gene alone.

Silencing of frataxin gene expression triggers p53-dependent apoptosis in human neuron-like cells.

Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by mutations that produce a deficiency in frataxin. Despite the importance of neurodegeneration in FRDA, little is known about the consequences of frataxin deficiency in neuronal cells. Here we describe a neuronal cell model for FRDA based on the use of lentiviral vectors that carry minigenes encoding frataxin-specific shRNAs that silence the expression of this gene. These lentivectors can knockdown frataxin expression in human neuroblastoma SH-SY5Y cells, which results in large-scale cell death in differentiated neuron-like cells but not in undifferentiated neuroblastoma cells. Frataxin-deficient neuron-like cells appear to die through apoptosis that is accompanied by up-regulation of p53, PUMA and Bax and activation of caspase-3. No significant autophagy is observed in frataxin-deficient neuron-like cells and the pharmacological activation of autophagy does not significantly increase neuronal cell death in response to the frataxin deficiency. Cell death triggered by frataxin knockdown can be impaired by interference with p53, caspase inhibitors and gene transfer of FXN. These results suggest that frataxin gene silencing in human neuron-like cells may constitute a useful cell model to characterize the molecular changes triggered by frataxin deficiency in neurons, as well as to search for therapies that may protect against neurodegeneration.

Normal and Friedreich ataxia cells express different isoforms of frataxin with complementary roles in iron-sulfur cluster assembly.

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.

Protective effects of transduced PEP-1-Frataxin protein on oxidative stress-induced neuronal cell death.

Reactive oxygen species (ROS) actively contribute to the development of a number of human diseases including ischemia. In response to oxidative stress, frataxin has a significant ability to improve cell survival though its biological function is unclear in relation to ischemia. To explore frataxin's role in protecting against ischemic cell death, we constructed PEP-1-Frataxin cell-permeable fusion protein. In a dose- and time-dependent manner PEP-1-Frataxin rapidly transduced into astrocyte cells and protected them against oxidative stress-induced cell death. Further, using an animal model, immunohistochemical analysis revealed that PEP-1-Frataxin prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. These results demonstrate that transduced PEP-1-Frataxin protects against cell death in vitro and in vivo, suggesting that transduction of PEP-1-Frataxin could be useful as a therapeutic agent for various human diseases related to oxidative stress.

Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress.

Friedreich's ataxia is generally associated with defects in [Fe-S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich's ataxia to study the physiological consequences of modulating the expression of the frataxin gene (YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2muYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe-S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2muYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2muYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2muYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe-S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different.

Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein.

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO(2) and H(2). The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation.

Iron-dependent regulation of frataxin expression: implications for treatment of Friedreich ataxia.

Friedreich ataxia (FA) is a progressive neurodegenerative disease caused by expansion of a trinucleotide repeat within the first intron of the gene that encodes frataxin. In our study, we investigated the regulation of frataxin expression by iron and demonstrated that frataxin mRNA levels decrease significantly in multiple human cell lines treated with the iron chelator, desferal (DFO). In addition, frataxin mRNA and protein levels decrease in fibroblast and lymphoblast cells derived from both normal controls and from patients with FA when treated with DFO. Lymphoblasts and fibroblasts of FA patients have evidence of cytosolic iron depletion, as indicated by increased levels of iron regulatory protein 2 (IRP2) and/or increased IRE-binding activity of IRP1. We postulate that this inferred cytosolic iron depletion occurs as frataxin-deficient cells overload their mitochondria with iron, a downstream regulatory effect that has been observed previously when mitochondrial iron-sulfur cluster assembly is disrupted. The mitochondrial iron overload and presumed cytosolic iron depletion potentially further compromise function in frataxin-deficient cells by decreasing frataxin expression. Thus, our results imply that therapeutic efforts should focus on an approach that combines iron removal from mitochondria with a treatment that increases cytosolic iron levels to maximize residual frataxin expression in FA patients.

Limited role for iron regulation in Coxiella burnetii pathogenesis.

In gram-negative bacteria, iron acquisition proteins are commonly regulated by Fur (ferric uptake regulator), which binds iron-regulated promoters (the Fur box). We hypothesized that Coxiella burnetii requires iron and employs an iron-regulatory system and used various approaches to define a Fur regulon. Cloned C. burnetii fur complemented an Escherichia coli fur deletion mutant. A ferrous iron transporter gene (CBU1766), a putative iron binding protein-encoding gene (CBU0970), and a cation efflux pump gene (CBU1362) were identified by genome annotation and using a Fur titration assay. Bioinformatically predicted Fur box-containing promoters were tested for transcriptional control by iron. Five genes demonstrated at least a twofold induction with minimal iron. Putatively regulated genes were evaluated in a two-plasmid regulator/promoter heterologous expression system. These data suggested a very limited Fur-regulated system in C. burnetii. In an in vitro tissue culture model, a significant increase in bacterial growth was observed with infected cells treated with deferoxamine in comparison to growth under iron-replete conditions. In an iron-overloaded animal model in vivo, the level of bacterial growth detected in the iron-injected animals was significantly decreased in comparison to growth in control animals. In a low-iron-diet animal model, a significant increase in splenomegaly was observed, but no significant change in bacterial growth was identified. The small number of predicted iron acquisition systems, few Fur-regulated genes, and enhanced replication under a decreased iron level predict a requirement of a low level of iron for survival, perhaps to avoid creation of additional reactive oxygen radicals.

Identification of an iron-responsive protein that is antigenic in patients with Chlamydia trachomatis genital infections.

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.