Iron-sulfur Cluster-containing Proteins Research Articles

Abstract 424: FDX2-KO induces global down-regulation of iron-sulfur cluster-containing proteins and senescence-like growth arrest or death in ovarian cancer cells

Abstract Iron-Sulfur clusters (Fe-S) are synthetized in mainly mitochondria and binds to various proteins (Fe-S proteins). Fe-S proteins play important roles in various cellular functions including energy metabolism, nucleic acid synthesis or redox homeostasis. However, roles of Fe-S clusters in cancer cells are not fully understood. Here, we report that loss of Fe-S biosynthesis causes proliferation arrest with DNA-damage response or cell death depend on TP53 status in ovarian cancer (OVC) cells. CRISPR-screening of all metabolism-related genes identified that Fe-S biosynthesis is essential for OVC proliferation. We chose one of the Fe-S biosynthesis factors, FDX2, and developed a cell line that can maintain FDX2 expression doxycycline (Dox) -dependently. In these cells, FDX2-loss induced irreversible growth arrest. FDX2-KO cells also showed enlarged and flatten morphology, up-regulation of SASP factors, and DNA damage through p53/p21 pathway activation. These were all senescence like phenotypes. To know these mechanisms, we performed proteome analysis and found that FDX2-loss caused global but differential post-transcriptional down-regulation of Fe-S proteins, in turn perturbing mitochondrial respiration, iron-regulation and redox homeostasis. These results all associated with DNA damage. Furthermore, we found that FDX2-KO induced caspase dependent cell death in TP53-KO condition instead of growth arrests. These results suggest that Fe-S cluster biosynthesis is significant in OVC proliferation and fate of FDX2-KO cells depends on TP53 status (senescence or cell death). FDX2 or Fe-S biosynthesis may be a new therapeutic target of OVC. Citation Format: Shuko Miyahara, Mai Ohuchi, Miyuki Nomura, Kayoko Hayashi, Muneaki Shimada, Nobuo Yaegashi, Nobuhiro Tanuma. FDX2-KO induces global down-regulation of iron-sulfur cluster-containing proteins and senescence-like growth arrest or death in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 424.

Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing

Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here, we show that knockout (KO) of Dars2 significantly impairs the maintenance of hematopoietic stem and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3-labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster-containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathways in HSPCs. Our study reveals that Dars2 plays a crucial role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Determining the oxidation state of elements by X-ray crystallography.

Protein-mediated redox reactions play a critical role in many biological processes and often occur at centres that contain metal ions as cofactors. In order to understand the exact mechanisms behind these reactions it is important to not only characterize the three-dimensional structures of these proteins and their cofactors, but also to identify the oxidation states of the cofactors involved and to correlate this knowledge with structural information. The only suitable approach for this based on crystallographic measurements is spatially resolved anomalous dispersion (SpReAD) refinement, a method that has been used previously to determine the redox states of metals in iron-sulfur cluster-containing proteins. In this article, the feasibility of this approach for small, non-iron-sulfur redox centres is demonstrated by employing SpReAD analysis to characterize Sulfolobus tokodaii sulerythrin, a ruberythrin-like protein that contains a binuclear metal centre. Differences in oxidation states between the individual iron ions of the binuclear metal centre are revealed in sulerythrin crystals treated with H2O2. Furthermore, data collection at high X-ray doses leads to photoreduction of this metal centre, showing that careful control of the total absorbed dose is a prerequisite for successfully determining the oxidation state through SpReAD analysis.

Expression, purification and microscopic characterization of human ATP-binding cassette sub-family B member 7 protein

The ATP-binding cassette sub-family B member 7 (ABCB7) is a membrane transport protein located on the inner membrane of mitochondria, which could be involved in the transport of heme from the mitochondria to the cytosol. ABCB7 also plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins, and mutations can cause a series of mitochondrial defects. X-linked sideroblastic anemia and ataxia (XLSA-A) is a rare cause of early onset ataxia, which may be overlooked due to the usually mild asymptomatic anemia. The genetic defect has been identified as a mutation in the ABCB7 gene at Xq12-q13. Here, we report the expression, purification and the 2D projections derived from negatively stained electron micrographs of recombinant H. sapiens ABCB7 (hABCB7), paving the way from an atomic structure determination of ABCB7.

The cytotoxicity of gallium maltolate in glioblastoma cells is enhanced by metformin through combined action on mitochondrial complex 1.

New drugs are needed for glioblastoma, an aggressive brain tumor with a dismal prognosis. We recently reported that gallium maltolate (GaM) retards the growth of glioblastoma in a rat orthotopic brain tumor model by inhibiting mitochondrial function and iron-dependent ribonucleotide reductase (RR). However, GaM’s mechanism of action at the mitochondrial level is not known. Given the interaction between gallium and iron metabolism, we hypothesized that gallium might target iron-sulfur (Fe-S) cluster-containing mitochondrial proteins. Using Extracellular Flux Analyzer technology, we confirmed that after a 24-h incubation, GaM 50 μmol/L inhibited glioblastoma cell growth by <10% but inhibited cellular oxygen consumption rate by 44% and abrogated mitochondrial reserve capacity. GaM blocked mitochondrial complex I activity and produced a 2.9-fold increase in cellular ROS. NMR spectroscopy revealed that gallium binds to IscU, the bacterial scaffold protein for Fe-S cluster assembly and stabilizes its folded state. Gallium inhibited the rate of in vitro cluster assembly catalyzed by bacterial cysteine desulfurase in a reaction mixture containing IscU, Fe (II), DTT, and L-cysteine. Metformin, a complex I inhibitor, enhanced GaM’s inhibition of complex I, further increased cellular ROS levels, and synergistically enhanced GaM’s cytotoxicity in glioblastoma cells in 2-D and 3-D cultures. Metformin did not affect GaM action on cellular iron uptake or transferrin receptor1 expression nor did it enhance the cytotoxicity of the RR inhibitor Didox. Our results show that GaM inhibits complex I by disrupting iron-sulfur cluster assembly and that its cytotoxicity can be synergistically enhanced by metformin through combined action on complex I.

Exenatide induces frataxin expression and improves mitochondrial function in Friedreich ataxia.

Friedreich ataxia is an autosomal recessive neurodegenerative disease associated with a high diabetes prevalence. No treatment is available to prevent or delay disease progression. Friedreich ataxia is caused by intronic GAA trinucleotide repeat expansions in the frataxin-encoding FXN gene that reduce frataxin expression, impair iron-sulfur cluster biogenesis, cause oxidative stress, and result in mitochondrial dysfunction and apoptosis. Here we examined the metabolic, neuroprotective, and frataxin-inducing effects of glucagon-like peptide-1 (GLP-1) analogs in in vivo and in vitro models and in patients with Friedreich ataxia. The GLP-1 analog exenatide improved glucose homeostasis of frataxin-deficient mice through enhanced insulin content and secretion in pancreatic β cells. Exenatide induced frataxin and iron-sulfur cluster-containing proteins in β cells and brain and was protective to sensory neurons in dorsal root ganglia. GLP-1 analogs also induced frataxin expression, reduced oxidative stress, and improved mitochondrial function in Friedreich ataxia patients' induced pluripotent stem cell-derived β cells and sensory neurons. The frataxin-inducing effect of exenatide was confirmed in a pilot trial in Friedreich ataxia patients, showing modest frataxin induction in platelets over a 5-week treatment course. Taken together, GLP-1 analogs improve mitochondrial function in frataxin-deficient cells and induce frataxin expression. Our findings identify incretin receptors as a therapeutic target in Friedreich ataxia.

Metabolic Adaptation to Sulfur of Hyperthermophilic Palaeococcus pacificus DY20341T from Deep-Sea Hydrothermal Sediments.

The hyperthermo-piezophilic archaeon Palaeococcus pacificus DY20341T, isolated from East Pacific hydrothermal sediments, can utilize elemental sulfur as a terminal acceptor to simulate growth. To gain insight into sulfur metabolism, we performed a genomic and transcriptional analysis of Pa. pacificus DY20341T with/without elemental sulfur as an electron acceptor. In the 2001 protein-coding sequences of the genome, transcriptomic analysis showed that 108 genes increased (by up to 75.1 fold) and 336 genes decreased (by up to 13.9 fold) in the presence of elemental sulfur. Palaeococcus pacificus cultured with elemental sulfur promoted the following: the induction of membrane-bound hydrogenase (MBX), NADH:polysulfide oxidoreductase (NPSOR), NAD(P)H sulfur oxidoreductase (Nsr), sulfide dehydrogenase (SuDH), connected to the sulfur-reducing process, the upregulation of iron and nickel/cobalt transfer, iron–sulfur cluster-carrying proteins (NBP35), and some iron–sulfur cluster-containing proteins (SipA, SAM, CobQ, etc.). The accumulation of metal ions might further impact on regulators, e.g., SurR and TrmB. For growth in proteinous media without elemental sulfur, cells promoted flagelin, peptide/amino acids transporters, and maltose/sugar transporters to upregulate protein and starch/sugar utilization processes and riboflavin and thiamin biosynthesis. This indicates how strain DY20341T can adapt to different living conditions with/without elemental sulfur in the hydrothermal fields.

Iron-Sulfur Cluster Repair Contributes to Yersinia pseudotuberculosis Survival within Deep Tissues.

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein YtfE contributes to the survival of Yersinia pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene hmp. ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE, the area of microcolonies was significantly smaller than that of the wild type (WT), consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and ΔytfE microcolonies. In the absence of NO-detoxifying activity by hmp, NO diffused across ΔytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE, indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.

A Vibrio cholerae BolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity.

BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

Aconitases: Non-redox Iron-Sulfur Proteins Sensitive to Reactive Species.

Mammalian aconitases (mitochondrial and cytosolic isoenzymes) are unique iron-sulfur cluster-containing proteins in which the metallic center participates in the catalysis of a non-redox reaction. Within the cubane iron-sulfur cluster of aconitases only three of the four iron ions have cysteine thiolate ligands; the fourth iron ion (Feα) is solvent exposed within the active-site pocket and bound to oxygen atoms from either water or substrates to be dehydrated. The catalyzed reaction is the reversible isomerization of citrate to isocitrate with an intermediate metabolite, cis-aconitate. The cytosolic isoform of aconitase is a moonlighting enzyme; when intracellular iron is scarce, the complete disassembly of the iron-sulfur cluster occurs and apo-aconitase acquires the function of an iron responsive protein and regulates the translation of proteins involved in iron metabolism. In the late 1980s and during the 1990s, cumulative experimental evidence pointed out that aconitases are main targets of reactive oxygen and nitrogen species such as superoxide radical (O2•-), hydrogen peroxide (H2O2), nitric oxide (•NO), and peroxynitrite (ONOO-). These intermediates are capable of oxidizing the cluster, which leads to iron release and consequent loss of the catalytic activity of aconitase. As the reaction of the Fe-S cluster with O2•- is fast (∼107 M-1 s-1), quite specific, and reversible in vivo, quantification of active aconitase has been used to evaluate O2•- formation in cells. While •NO is modestly reactive with aconitase, its reaction with O2•- yields ONOO-, a strong oxidant that readily leads to the disruption of the Fe-S cluster. In the case of cytosolic aconitase, it has been seen that H2O2 and •NO promote activation of iron responsive protein activity in cells. Proteomic advances in the 2000s confirmed that aconitases are main targets of reactive species in cellular models and in vivo, and other post-translational oxidative modifications such as protein nitration and carbonylation have been detected. Herein, we (1) outline the particular structural features of aconitase that make these proteins specific targets of reactive species, (2) characterize the reactions of O2•-, H2O2, •NO, and ONOO- and related species with aconitases, (3) discuss how different oxidative post-translational modifications of aconitase impact the different functions of aconitases, and (4) argue how these proteins might function as redox sensors within different cellular compartments, regulating citrate concentration and efflux from mitochondria, iron availability in the cytosol, and cellular oxidant production.

Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1.

Iron-sulfur (Fe/S) cluster-containing proteins constitute one of the largest protein classes, with highly varied function. Consequently, the biosynthesis of Fe/S clusters is evolutionarily conserved and mutations in intermediate Fe/S cluster scaffold proteins can cause disease, including multiple mitochondrial dysfunctions syndrome (MMDS). Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. In vitro investigation into the structure-function relationship of the Gly189Arg derivative, along with two other variants, reveals that substitution at position 189 triggers structural changes that increase flexibility, decrease stability, and alter the monomer-dimer equilibrium toward monomer, thereby impairing the ability of the Gly189X derivatives to receive an Fe/S cluster from physiologically relevant sources.

The unique regulation of Fe-S cluster biogenesis in a Gram-positive bacterium

The ubiquitous iron-sulfur (Fe-S) cluster-containing proteins are involved in countless biological routes and play crucial roles for the functioning of both prokaryotic and eukaryotic cells. The transcription factor IscR, of the helix–turn–helix family, was first implicated in repressing expression of the ISC (Iron Sulfur Cluster biogenesis) pathway and was shown to contain a [2Fe–2S] cluster. Promoters controlled by IscR belong to two distinct sequence groups [1] and the [2Fe-2S] cluster of IscR was shown to be essential for regulation of type-1 promoters (isc, yadR, yhgI), while apo-IscR is responsible for the regulation of type-2 promoters [2]. The proposed model for IscR action consists on a mechanism for Fe-S biogenesis fine-tuned by the cellular Fe-S cluster status. Despite recent advances in understanding the regulation of isc and suf operons by environmental signals, the features of IscR target-site recognition and the structural changes that alter the DNA binding specificity of IscR upon ligation of the [2Fe-2S] cluster are unknown. To understand how IscR recognizes two different DNA motifs, we solved the three-dimensional structures of free apo-IscR and of its complex with a type-2 target sequence, the hya promoter from the hydrogenase-1 operon [3]. These experimental models revealed some of the molecular details of the unusual environmentally modulated recognition of two distinct promoter consensus sequences by IscR, using a single predicted helix–turn–helix DNA binding motif. To our knowledge, IscR is the only transcription factor that is active in both the apo and holo-forms and which displays cofactor-mediated modulation of its DNA binding specificity. This work was funded by Fundação para a Ciência e a Tecnologia (Portugal) through grant PTDC/BBB-BEP/2127/2012 (EU-FEDER funding through COMPETE FCOMP-01-0124-FEDER-028116) and PhD fellowship SFRH/BD/66461/2009 to JAS.

150 years of Friedreich Ataxia: from its discovery to therapy

150 years of Friedreich Ataxia: from its discovery to therapy

Mammalian Frataxin Controls Sulfur Production and Iron Entry during de Novo Fe4S4 Cluster Assembly

Iron-sulfur (Fe-S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe-S clusters are synthesized by the mitochondrial iron-sulfur cluster (ISC) machinery and the cytosolic iron-sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe-S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreich's ataxia. Here, by comparing the biochemical and spectroscopic properties of quaternary (ISCU/NFS1/ISD11/FXN) and ternary (ISCU/NFS1/ISD11) complexes, we show that FXN stabilizes the quaternary complex and controls iron entry to the complex through activation of cysteine desulfurization. Furthermore, we show for the first time that in the presence of iron and L-cysteine, an [Fe(4)S(4)] cluster is formed within the quaternary complex that can be transferred to mammalian aconitase (mACO2) to generate an active enzyme. In the absence of FXN, although the ternary complex can assemble an Fe-S cluster, the cluster is inefficiently transferred to ACO2. Taken together, these data help to unravel further the Fe-S cluster assembly process and the molecular basis of Friedreich's ataxia.

Zinc Pyrithione Inhibits Yeast Growth through Copper Influx and Inactivation of Iron-Sulfur Proteins

Zinc pyrithione (ZPT) is an antimicrobial material with widespread use in antidandruff shampoos and antifouling paints. Despite decades of commercial use, there is little understanding of its antimicrobial mechanism of action. We used a combination of genome-wide approaches (yeast deletion mutants and microarrays) and traditional methods (gene constructs and atomic emission) to characterize the activity of ZPT against a model yeast, Saccharomyces cerevisiae. ZPT acts through an increase in cellular copper levels that leads to loss of activity of iron-sulfur cluster-containing proteins. ZPT was also found to mediate growth inhibition through an increase in copper in the scalp fungus Malassezia globosa. A model is presented in which pyrithione acts as a copper ionophore, enabling copper to enter cells and distribute across intracellular membranes. This is the first report of a metal-ligand complex that inhibits fungal growth by increasing the cellular level of a different metal.

Expression, purification and spectroscopic analysis of an HdrC: An iron–sulfur cluster-containing protein from Acidithiobacillus ferrooxidans

Iron–sulfur cluster-containing proteins are present in all living organisms and are considered to be very ancient due to their ubiquity on the three domains of life, their importance in anaerobic metabolic pathways and energy conservation mechanisms. To date, there is little information regarding these proteins in Acidithiobacillus ferrooxidans, a bacterium involved in bioleaching processes. In this study are described the cloning, expression, purification and spectroscopic characterization of a new Fe–S cluster-containing protein, a putative HdrC homologue, from A. ferrooxidans strain LR. The oligomeric state of the protein was assessed by both dynamic light scattering and size-exclusion chromatography, demonstrating that it is present as a monomer in solution. Far-UV CD measurements revealed that this protein is extremely stable at acidic pHs, in which it assumes a different structure that exhibits a predominance of α-helixes. In contrast, at basic pHs, from 9 to 12, the protein showed a spectral profile which is characteristic for proteins with high content of beta structure and random coil. Thermal unfolding studies demonstrated that this protein tolerates high temperatures at acidic conditions, with a melting temperature of 95°C. Furthermore, bioinformatics analysis suggested a heterodisulfide reductase function for the analyzed protein and for the other identified subunits, HdrA and HdrB, which could be related to hydrogen and/or formate metabolism in A. ferrooxidans.

A Mammalian Siderophore Synthesized by an Enzyme with a Bacterial Homolog Involved in Enterobactin Production

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia

BackgroundFriedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression.Methodology/Principal FindingsWe thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide.Conclusions/SignificanceThus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress.

Proposed mechanism of inactivatingEscherichia coliO157:H7 by ultra-high pressure in combination withtert-butylhydroquinone

Investigating mechanisms of lethality enhancement when Escherichia coli O157:H7, and selected E. coli mutants, were exposed to tert-butylhydroquinone (TBHQ) during ultra-high pressure (UHP) treatment. Escherichia coli O157:H7 EDL-933, and 14 E. coli K12 strains with mutations in selected genes, were treated with dimethyl sulfoxide solution of TBHQ (15-30 ppm), and processed with UHP (400 MPa, 23 +/- 2 degrees C for 5 min). Treatment of wild-type E. coli strains with UHP alone inactivated 2.4-3.7 log CFU ml(-1), whereas presence of TBHQ increased UHP lethality by 1.1-6.2 log CFU ml(-1); TBHQ without pressure was minimally lethal (0-0.6 log reduction). Response of E. coli K12 mutants to these treatments suggests that iron-sulfur cluster-containing proteins ([Fe-S]-proteins), particularly those related to the sulfur mobilization (SUF system), nitrate metabolism, and intracellular redox potential, are critical to the UHP-TBHQ synergy against E. coli. Mutations in genes maintaining redox homeostasis and anaerobic metabolism were associated with UHP-TBHQ resistance. The redox cycling activity of cellular [Fe-S]-proteins may oxidize TBHQ, potentially leading to the generation of bactericidal reactive oxygen species. A mechanism is proposed for the enhanced lethality of UHP by TBHQ against E. coli O157:H7. The results may benefit food processors using UHP-based preservation, and biologists interested in piezophilic micro-organisms.