Mitochondrial Iron Research Articles

Altered sterol metabolism in budding yeast affects mitochondrial iron–sulfur (Fe-S) cluster synthesis

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial iron metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29 Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increases mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism.

CISD2 inhibition overcomes resistance to sulfasalazine-induced ferroptotic cell death in head and neck cancer

Sulfasalazine has been repurposed to induce ferroptotic cancer cell death via inhibition of xc−-cystine/glutamate antiporter (xCT). However, cancer cells are capable of developing mechanisms to evade cell death. Therefore, we sought to determine the molecular mechanisms underlying resistance to sulfasalazine-induced ferroptosis in head and neck cancer (HNC). The effects of sulfasalazine and pioglitazone were tested in various HNC cell lines. The effects of these drugs and inhibition and overexpression of CISD2 gene were determined by evaluating viability, cell death, lipid ROS production, mitochondrial iron, and mouse tumor xenograft models. SAS induced ferroptotic cell death in HNC at different levels. CISD2 expression showed an association between its expression and ferroptosis resistance. CISD2 overexpression conferred resistance to ferroptosis by sulfasalazine. Silencing CISD2 gene rendered resistant HNC cells susceptible to sulfasalazine-induced ferroptosis, with increased levels of lipid ROS and mitochondrial ferrous iron. Pioglitazone induced over-accumulation of mitochondrial iron and ROS and sensitized resistant HNC cells to sulfasalazine treatment in vitro and in a mouse tumor-xenograft model. CISD2 inhibition overcomes HNC resistance to ferroptotic cell death induced by sulfasalazine via increased accumulation of mitochondrial ferrous iron and lipid ROS.

Mitoferrin-1 is Involved in the Progression of Alzheimer’s Disease Through Targeting Mitochondrial Iron Metabolism in a Caenorhabditis elegans Model of Alzheimer’s Disease

In mammals, mitoferrin-1 and mitoferrin-2, two homologous proteins of the mitochondrial solute carrier family are required for iron delivery into mitochondria. However, there is only one kind, called W02B12 (mitoferrin-1 or mfn-1), in Caenorhabditis elegans and its regulatory mechanism is unknown. In this study, we used C. elegans strains CL2006 and GMC101 as models to investigate what role mitoferrin-1 played in Alzheimer’s disease (AD). We found that knockdown of mitoferrin-1 by feeding-RNAi treatment extended lifespans of both strains of C. elegans. In addition, it reduced the paralysis rate in the GMC101 strain. These results suggest that mitoferrin-1 may be involved in the progression of Alzheimer’s disease. Knockdown of mitoferrin-1 was seen to disturb mitochondrial morphology in the CB5600 strain. We tested whether knockdown of mitoferrin-1 could influence mitochondrial metabolism. Analysis of mitochondrial iron metabolism and mitochondrial ROS showed that knockdown of mitoferrin-1 could reduce mitochondrial iron content and reduce the level of mitochondrial ROS in the CL2006 and GMC101 strains. These results confirm that knockdown of mitoferrin-1 can slow the progress of disease in Alzheimer model of C. elegans and suggest that mitoferrin-1 plays a major role in mediating mitochondrial iron metabolism in this process.

Establishment of a cell model of X-linked sideroblastic anemia using genome editing

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.

ABCB7 is required for B lymphocyte development

Abstract ABCB7 is a mitochondrial iron transporter critical for cellular iron homeostasis, as its loss results in mitochondrial iron accumulation. ABCB7 has been demonstrated to be critical for embryogenesis and hematopoiesis, though its role in specific hematopoietic lineages is unexplored. We found that ABCB7 is required for lymphopoiesis, as CD2-iCre ABCB7 cKO mice have a severe block in both T and B lymphocyte development. The block in B cell development occurs at the Fr. C to Fr. C’ transition, where we found reduced expression of intracellular μ and pre-BCR, as well as reduced proliferation. We observed reduced expression of E2A, Ikaros, and Pax5, transcription factors essential for B cell lineage development. Previous literature has shown that deletion of ABCB7 results in mitochondrial iron accumulation. Because this is toxic to cells, we hypothesized that B cells increase heme synthesis in order to detoxify accumulating iron. In support of this, we found that HO-1, a surrogate marker for intracellular heme concentration, was elevated in B cells lacking ABCB7. Interestingly, heme levels regulate Bach2, a transcriptional repressor important for B cell development as it suppresses myeloid program in lymphocytes. Bach2 expression was found to be decreased in ABCB7-deficient B cells. We are currently examining the role of Bach2 and the mechanism responsible for the block in B cell development upon loss of ABCB7.

P-55 - Mitochondrial iron chelation as a novel anti-cancer strategy

Iron is an indispensable micronutrient required for function of key cellular enzymes required for DNA replication and repair, and for mitochondrial respiration and metabolism. Within cells, iron is primarily utilized in mitochondria, where it is essential for the synthesis of Fe-S clusters and heme. The role of iron in carcinogenesis is widely accepted, and tumor cells require higher amounts of iron for their survival and proliferation. Indeed, iron chelation has been proposed as an alternative strategy to target cancer cells. Therefore, we synthesized a mitochondrially targeted derivative of the iron chelator deferoxamine (mDFO) and evaluated its ability to eliminate cancer cells selectively. Our results show that mDFO is at least 100-fold more efficient than DFO in killing breast cancer cells. Moreover, mDFO was able to reduce mitochondrial respiration and aconitase activity, increase mitochondrial and cellular ROS production, and trigger the activation of mitophagy. Taken together, our data strongly support the role of mDFO as a novel and efficient compound in the selective elimination of cancer cells.

Involvement of cytosolic and mitochondrial iron in iron overload cardiomyopathy: an update.

Iron overload cardiomyopathy (IOC) is a major cause of death in patients with diseases associated with chronic anemia such as thalassemia or sickle cell disease after chronic blood transfusions. Associated with iron overload conditions, there is excess free iron that enters cardiomyocytes through both L- and T-type calcium channels thereby resulting in increased reactive oxygen species being generated via Haber-Weiss and Fenton reactions. It is thought that an increase in reactive oxygen species contributes to high morbidity and mortality rates. Recent studies have, however, suggested that it is iron overload in mitochondria that contributes to cellular oxidative stress, mitochondrial damage, cardiac arrhythmias, as well as the development of cardiomyopathy. Iron chelators, antioxidants, and/or calcium channel blockers have been demonstrated to prevent and ameliorate cardiac dysfunction in animal models as well as in patients suffering from cardiac iron overload. Hence, either a mono-therapy or combination therapies with any of the aforementioned agents may serve as a novel treatment in iron-overload patients in the near future. In the present article, we review the mechanisms of cytosolic and/or mitochondrial iron load in the heart which may contribute synergistically or independently to the development of iron-associated cardiomyopathy. We also review available as well as potential future novel treatments.

Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV, LC/MS/MS and ESR

To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron–sulphur (Fe−S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF2α and 5-iPF2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe–S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe–S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe–S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe–S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.

Brain mitochondrial iron accumulates in Huntington's disease, mediates mitochondrial dysfunction, and can be removed pharmacologically.

Mitochondrial bioenergetic dysfunction is involved in neurodegeneration in Huntington's disease (HD). Iron is critical for normal mitochondrial bioenergetics but can also contribute to pathogenic oxidation. The accumulation of iron in the brain occurs in mouse models and in human HD. Yet the role of mitochondria-related iron dysregulation as a contributor to bioenergetic pathophysiology in HD is unclear. We demonstrate here that human HD and mouse model HD (12-week R6/2 and 12-month YAC128) brains accumulated mitochondrial iron and showed increased expression of iron uptake protein mitoferrin 2 and decreased iron-sulfur cluster synthesis protein frataxin. Mitochondria-enriched fractions from mouse HD brains had deficits in membrane potential and oxygen uptake and increased lipid peroxidation. In addition, the membrane-permeable iron-selective chelator deferiprone (1 μM) rescued these effects ex-vivo, whereas hydrophilic iron and copper chelators did not. A 10-day oral deferiprone treatment in 9-week R6/2 HD mice indicated that deferiprone removed mitochondrial iron, restored mitochondrial potentials, decreased lipid peroxidation, and improved motor endurance. Neonatal iron supplementation potentiates neurodegeneration in mouse models of HD by unknown mechanisms. We found that neonatal iron supplementation increased brain mitochondrial iron accumulation and potentiated markers of mitochondrial dysfunction in HD mice. Therefore, bi-directional manipulation of mitochondrial iron can potentiate and protect against markers of mouse HD. Our findings thus demonstrate the significance of iron as a mediator of mitochondrial dysfunction and injury in mouse models of human HD and suggest that targeting the iron-mitochondrial pathway may be protective.

A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells

Mitochondrial chelatable iron contributes to several injury processes, including ischemia/reperfusion injury, oxidative stress and the hepatotoxicity of acetaminophen. To measure mitochondrial chelatable iron in living cells and tissues, we synthesized a new fluorescent Fe2+ indicator, mitoferrofluor (MFeF), which has a reactive group that forms covalent adducts with mitochondrial proteins to allow retention of MFeF after subsequent mitochondrial depolarization (Hepatology 60 [Suppl], 812A). MFeF selectively accumulates in the polarized mitochondria of cultured rat hepatocytes. Here we assessed whether MFeF specifically reports mitochondrial iron independent of calcium. After loading cultured rat hepatocytes with 1 uM MFeF and 500 nM rhodamine 123 (Rh123) or 1 μM calcein‐AM for 30 min, confocal microscopy revealed red MFeF fluorescence that co‐localized with green mitochondrial Rh123. Membrane‐permeant Fe3+/8‐hydroxyquinoline(8‐HQ) (10–50 μM) progressively quenched mitochondrial MFeF fluorescence to ~80% within 15 min, but mitochondrial Rhodamine 123 was unchanged. Fe2+ specific calcein fluorescence in the cytosol was also quenched, confirming that Fe3+/8‐HQ undergoes rapid intracellular reduction to Fe2+. MFeF fluorescence increased when the lipophilic transition metal chelator pyridoxal isonicotinoyl hydrazone (PIH) was added to competitively bind mitochondrial chelatable iron. Importantly, MFeF was retained in mitochondria after mitochondrial de‐energization with CCCP (10 μM), oligomycin (10 μg/ml) and myxothiazol (10 μM), confirming mitochondrial retention of the covalently adducted MFeF. In hepatocytes loaded with MFeF, red fluorescence did not decrease after adding 2 μM of Ca2+‐mobilizing thapsigargin, whereas parallel experiments showed that Ca2+‐indicating mitochondrial Rhod‐2 fluorescence did increase, showing that MFeF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In acetaminophen‐induced hepatotoxicity, progressive quenching of MFeF began at ~4 h after overdose acetaminophen (10 mM) in mouse hepatocytes, signifying increased mitochondrial chelatable Fe2+. Mitochondria then depolarized after ~10 h. Dipyridyl, a membrane‐permeable iron chelator, partially restored MFeF fluorescence. In conclusion, MFeF is a ΔΨ‐independent iron indicator that selectively detects mitochondrial chelatable Fe2+ but not Ca2+.Support or Funding InformationNIH grants: DK073336, AA021191 and T32 DK083262This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial DNA Damage And Impaired Iron Homeostasis In Muscle Aging

Iron dyshomeostasis has been indicated as a major risk factor for the development and progression of several disease conditions. Impairment of iron balance is also an aging feature especially in post‐mitotic tissues. In particular, while age‐related systemic iron deficiency (anemia) compromise oxygen availability, increased cellular and mitochondrial iron levels have been involved in DNA mutation load.We purified total proteins and DNA from muscle samples of young (Y, n=10) and sedentary older adults, classified as high‐ (HFE, n=14) and low‐functioning (LFE, n=6) based on the Short Physical Performance Battery (SPPB). The effect of aging on key proteins involved in iron homeostasis and qualitative alterations of mtDNA and how these relate to physical performance was evaluated.A decrease in muscle volume, as quantified through 3D‐NMR, was found in LFE (38%) and HFE (30%) participants compared to the younger counterpart and positively correlated with physical performance. A lower expression of the iron import protein transferrin receptor (TfR1) but not Zip14 was also found in both groups of elderly participants, regardless of the SPPB score. The analysis of the mutation load of mitochondrial DNA (mtDNA) (e.g., single‐strand, double‐strand breaks and abasic sites), instead, showed an increase in mtDNA damage in HFE and LFE participants, with no differences between the two groups. This study showed an association between altered mitochondrial homeostasis and levels of intracellular iron in aged human muscles. The accumulation of mtDNA mutations and the decline in iron balance may therefore represent critical steps to muscle aging and possible targets for interventions against muscle wasting.Support or Funding InformationThis study was supported by the National Institute on Aging (NIA) (R01 AG17994), the Claude D. Pepper Older Americans Independence Center (1 P30 AG028740), “Centro Studi Achille e Linda Lorenzon”, and Fondazione Roma (NCDs Call for Proposal 2013).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite's ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/ΔLmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans.

Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges

Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a ‘cysteine pocket’. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H2O2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34–0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions.

RNA expression profiling in sulfamethoxazole-treated patients with a range of invitro lymphocyte cytotoxicity phenotypes.

The lymphocyte toxicity assay (LTA) is a proposed surrogate marker of sulfonamide antibiotic hypersensitivity. In the LTA, peripheral blood mononuclear cells (PBMCs) undergo apoptosis more readily in hypersensitive versus tolerant patients when exposed to drug‐hydroxylamine metabolites in vitro. The purpose of this study was to identify key gene transcripts associated with increased cytotoxicity from sulfamethoxazole‐hydroxylamine in human PBMCs in the LTA. The LTA was performed on PBMCs of 10 patients hypersensitive to trimethoprim‐sulfamethoxazole (HS) and 10 drug‐tolerant controls (TOL), using two cytotoxicity assays: YO‐PRO (n = 20) and MTT (n = 12). mRNA expression profiles of PBMCs, enriched for CD8+ T cells, were compared between HS and TOL patients. Transcript expression was interrogated for correlation with % cytotoxicity from YO‐PRO and MTT assays. Correlated transcripts of interest were validated by qPCR. LTA results were not significantly different between HS and TOL patients, and no transcripts were found to be differentially expressed between the two groups. 96 transcripts were correlated with cytotoxicity by YO‐PRO (r = ±.63‐.75, FDR 0.188). Transcripts were selected for validation based on mechanistic plausibility and three were significantly over‐expressed by qPCR in high cytotoxicity patients: multi‐specific organic anion transporter C (ABCC5), mitoferrin‐1 (SLC25A37), and Porimin (TMEM123). These data identify novel transcripts that could contribute to sulfonamide‐hydroxylamine induced cytotoxicity. These include SLC25A37, encoding a mitochondrial iron transporter, ABCC5, encoding an arylamine drug transporter, and TMEM123, encoding a transmembrane protein that mediates cell death.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism.

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

Splicing factor mutations in myelodysplastic syndromes

Splicing factor mutations represent a novel class of driver mutations in myelodysplastic syndromes, where four genes, including SF3B1, SRSF2, U2AF1, and ZRSR2, are most frequently affected. SF3B1 and SRSF2 mutations show prominent specificity to the syndrome subtypes characterized by increased ring sideroblasts and chronic myelomonocytic leukemia, respectively. These mutations are suspected to be involved in the pathogenesis of the above mentioned syndromes most likely via abnormal RNA splicing. However, the precise mechanism and target genes have not been fully understood. Splicing alterations induced by these mutations are extensively studied using RNA sequencing. SF3B1 mutations caused misrecognition of 3' splice sites. Target genes included those involved in mitochondrial iron metabolism or heme biosynthesis, such as ABCB7 and PPOX, suggesting a role in the abnormal erythropoiesis associated with increased ring sideroblasts. By contrast, SRSF2 and U2AF1 mutations were mainly associated with alternative exon usage in hundreds of genes. The targets included genes of which mutations are definitive or putative drivers in myeloid malignancies. Protein structure modeling and experiments with cell lines and mouse models supported these findings. ZRSR2 mutations were associated with the retention of U12-type introns, and this is consistent with the known role of ZRSR2 as an essential component of the U12-type spliceosome. Further studies are warranted to determine the biological effects of splicing alterations.

Quantitative proteomics in Friedreich's ataxia B-lymphocytes: A valuable approach to decipher the biochemical events responsible for pathogenesis

Friedreich's ataxia (FRDA) represents the most frequent type of autosomal-recessively inherited ataxia and is caused by the deficiency of frataxin, a mitochondrial protein. It is known that frataxin-deficiency leads to alterations in cellular and mitochondrial iron metabolism and impacts in the cell physiology at several levels. Frataxin is thought to play a role in iron–sulfur cluster biogenesis and heme synthesis. Currently, cellular antioxidant defense is dysregulated when frataxin is deficient, which exacerbates oxidative damage in FRDA. Moreover, alterations in lipid metabolism have been observed in several models of the disease.To better understand the biochemical sequelae of frataxin reduction, global protein expression analysis was performed using quantitative proteomic experiments in Friedreich's ataxia patient-derived B-lymphocytes as compared to controls. We were able to confirm a subset of changes in these cells and importantly, we observed previously unreported signatures of protein expression. Among the novel protein signatures that we have identified, the decrease in CHCHD4 might partly explain some aspects of the molecular pathogenesis of FRDA.The identification of a core set of proteins changing in the FRDA pathogenesis is a useful tool in trying to decipher the function(s) of frataxin in order to clarify the mitochondrial metabolic disease process.

Mitochondrial ABC Transporters and Iron Metabolism

Mitochondrial are a key organelle in iron metabolism and many metabolic processes involved in iron homeostasis occur in the mitochondria. Eukaryotic cells have developed different transport mechanisms to deal with coordinating movement of iron and iron-related molecules across membranes. Some of those transport mechanisms involve ATP-binding cassette (ABC) transporters. There are four mitochondrial ABC transporters Abcb6, Abcb7, Abcb8 and Abcb10. Abcb6 is localized to the outer membrane of mitochondria where it is involved in porphyrin transport. Abcb7, Abcb8 and Abcb10 are localized to the inner mitochondrial membrane and the exact molecule transported by each is still unclear. Here, we provide a brief review of what is known about each transporter and its role in mitochondrial iron homeostasis. We describe the human diseases associated with known mutations in the genes encoding these proteins and discuss the possible importance of these transporters in immune cell function.

Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells

The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1-3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.