Elevated FGF21 secretion, PGC-1α and ketogenic enzyme expression are hallmarks of iron-sulfur cluster depletion in human skeletal muscle.

Iron-sulfur (Fe-S) clusters are ancient enzyme cofactors found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF21 (fibroblast growth factor 21). Enhanced muscle FGF21 expression was reflected by elevated circulating FGF21 levels in the patients, and robust FGF21 secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF21 levels.

Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes.

Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs(∗)13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.

Tissue specificity of a human mitochondrial disease: differentiation-enhanced mis-splicing of the Fe-S scaffold gene ISCU renders patient cells more sensitive to oxidative stress in ISCU myopathy.

BACKGROUND: ISCU myopathy is a disease caused by muscle-specific deficiency of the Fe-S cluster scaffold protein ISCU. RESULTS: MyoD expression enhanced ISCU mRNA mis-splicing, and oxidative stress exacerbated ISCU depletion in patient cells. CONCLUSION: ISCU protein deficiency in patients results from muscle-specific mis-splicing as well as oxidative stress. SIGNIFICANCE: Oxidative stress negatively influences the mammalian Fe-S cluster assembly machinery by destabilization of ISCU. Iron-sulfur (Fe-S) cluster cofactors are formed on the scaffold protein ISCU. ISCU myopathy is a disease caused by an intronic mutation that leads to abnormally spliced ISCU mRNA. We found that two predominant mis-spliced ISCU mRNAs produce a truncated and short-lived ISCU protein product in multiple patient cell types. Expression of the muscle-specific transcription factor MyoD further diminished normal splicing of ISCU mRNA in patient myoblasts, demonstrating that the process of muscle differentiation enhances the loss of normal ISCU mRNA splicing. ISCU protein was nearly undetectable in patient skeletal muscle, but was higher in patient myoblasts, fibroblasts, and lymphoblasts. We next treated patient cells with pro-oxidants to mimic the oxidative stress associated with muscle activity. Brief hydrogen peroxide treatment or incubation in an enriched oxygen atmosphere led to a marked further reduction of ISCU protein levels, which could be prevented by pretreatment with the antioxidant ascorbate. Thus, we conclude that skeletal muscle differentiation of patient cells causes a higher degree of abnormal ISCU splicing and that oxidative stress resulting from skeletal muscle work destabilizes the small amounts of normal ISCU protein generated in patient skeletal muscles.

Posttranslational stability of the heme biosynthetic enzyme ferrochelatase is dependent on iron availability and intact iron-sulfur cluster assembly machinery.

Mammalian ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, possesses an iron-sulfur [2Fe-2S] cluster that does not participate in catalysis. We investigated ferrochelatase expression in iron-deficient erythropoietic tissues of mice lacking iron regulatory protein 2, in iron-deficient murine erythroleukemia cells, and in human patients with ISCU myopathy. Ferrochelatase activity and protein levels were dramatically decreased in Irp2(-/-) spleens, whereas ferrochelatase mRNA levels were increased, demonstrating posttranscriptional regulation of ferrochelatase in vivo. Translation of ferrochelatase mRNA was unchanged in iron-depleted murine erythroleukemia cells, and the stability of mature ferrochelatase protein was also unaffected. However, the stability of newly formed ferrochelatase protein was dramatically decreased during iron deficiency. Ferrochelatase was also severely depleted in muscle biopsies and cultured myoblasts from patients with ISCU myopathy, a disease caused by deficiency of a scaffold protein required for Fe-S cluster assembly. Together, these data suggest that decreased Fe-S cluster availability because of cellular iron depletion or impaired Fe-S cluster assembly causes reduced maturation and stabilization of apo-ferrochelatase, providing a direct link between Fe-S biogenesis and completion of heme biosynthesis. We propose that decreased heme biosynthesis resulting from impaired Fe-S cluster assembly can contribute to the pathogenesis of diseases caused by defective Fe-S cluster biogenesis.

Iron control of erythroid development by a novel aconitase-associated regulatory pathway.

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in iron-deprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase Calpha/beta, or possibly protein kinase Cdelta, whose activities were regulated by iron, isocitrate, and erythropoietin.

Hyperactivation of mTOR and AKT in a cardiac hypertrophy animal model of Friedreich ataxia.

Cardiomyopathy is a primary cause of death in Friedreich ataxia (FRDA) patients with defective iron-sulfur cluster (ISC) biogenesis due to loss of functional frataxin and in rare patients with functional loss of other ISC biogenesis factors. The mechanistic target of rapamycin (mTOR) and AKT signaling cascades that coordinate eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors, are crucial regulators of cardiovascular growth and homeostasis. We observed increased phosphorylation of AKT and dysregulation of multiple downstream effectors of mTORC1, including S6K1, S6, ULK1 and 4EBP1, in a cardiac/skeletal muscle specific FRDA conditional knockout (cKO) mouse model and in human cell lines depleted of ISC biogenesis factors. Knockdown of several mitochondrial metabolic proteins that are downstream targets of ISC biogenesis, including lipoyl synthase and subunit B of succinate dehydrogenase, also resulted in activation of mTOR and AKT signaling, suggesting that mTOR and AKT hyperactivations are part of the metabolic stress response to ISC deficiencies. Administration of rapamycin, a specific inhibitor of mTOR signaling, enhanced the survival of the Fxn cKO mice, providing proof of concept for the potential of mTOR inhibition to ameliorate cardiac disease in patients with defective ISC biogenesis. However, AKT phosphorylation remained high in rapamycin-treated Fxn cKO hearts, suggesting that parallel mTOR and AKT inhibition might be necessary to further improve the lifespan and healthspan of ISC deficient individuals.

Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis.

Iron-sulfur (Fe-S) clusters are required for the functions of mitochondrial aconitase, mammalian iron regulatory protein 1, and many other proteins in multiple subcellular compartments. Recent studies in Saccharomyces cerevisiae indicated that Fe-S cluster biogenesis also has an important role in mitochondrial iron homeostasis. Here we report the functional analysis of the mitochondrial and cytosolic isoforms of the human Fe-S cluster scaffold protein, ISCU. Suppression of human ISCU by RNAi not only inactivated mitochondrial and cytosolic aconitases in a compartment-specific manner but also inappropriately activated the iron regulatory proteins and disrupted intracellular iron homeostasis. Furthermore, endogenous ISCU levels were suppressed by iron deprivation. These results provide evidence for a coordinated response to iron deficiency that includes activation of iron uptake, redistribution of intracellular iron, and decreased utilization of iron in Fe-S proteins.

Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis.

The LYR family consists of proteins of diverse functions that contain the conserved tripeptide 'LYR' near the N-terminus, and it includes Isd11, which was previously observed to have an important role in iron-sulfur (Fe-S) cluster biogenesis in Saccharomyces cerevisiae. Here, we have cloned and characterized human ISD11 and shown that human ISD11 forms a stable complex in vivo with the human cysteine desulfurase (ISCS), which generates the inorganic sulfur needed for Fe-S protein biogenesis. Similar to ISCS, we have found that ISD11 localizes to the mitochondrial compartment, as expected, but also to the nucleus of mammalian cells. Using RNA-interference techniques, we have shown that suppression of human ISD11 inactivated mitochondrial and cytosolic aconitases. In addition, ISD11 suppression activated iron-responsive element-binding activity of iron regulatory protein 1, increased protein levels of iron regulatory protein 2, and resulted in abnormal punctate ferric iron accumulations in cells. These results indicate that ISD11 is important in the biogenesis of Fe-S clusters in mammalian cells, and its loss disrupts normal mitochondrial and cytosolic iron homeostasis.

Iron-sulfur cluster biogenesis and human disease.

Iron-sulfur (Fe-S) clusters are essential for numerous biological processes, including mitochondrial respiratory chain activity and various other enzymatic and regulatory functions. Human Fe-S cluster assembly proteins are frequently encoded by single genes, and inherited defects in some of these genes cause disease. Recently, the spectrum of diseases attributable to abnormal Fe-S cluster biogenesis has extended beyond Friedreich ataxia to include a sideroblastic anemia with deficiency of glutaredoxin 5 and a myopathy associated with a deficiency of a Fe-S cluster assembly scaffold protein, ISCU. Mutations within other mammalian Fe-S cluster assembly genes could be causative for human diseases that manifest distinctive combinations of tissue-specific impairments. Thus, defects in the iron-sulfur cluster biogenesis pathway could underlie many human diseases.

Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance.

A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.

Tempol-mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice.

In mammals, two homologous cytosolic regulatory proteins, iron regulatory protein 1 (also known as IRP1 and Aco1) and iron regulatory protein 2 (also known as IRP2 and Ireb2), sense cytosolic iron levels and posttranscriptionally regulate iron metabolism genes, including transferrin receptor 1 (TfR1) and ferritin H and L subunits, by binding to iron-responsive elements (IREs) within target transcripts. Mice that lack IRP2 develop microcytic anemia and neurodegeneration associated with functional cellular iron depletion caused by low TfR1 and high ferritin expression. IRP1 knockout (IRP1(-/-)) animals do not significantly misregulate iron metabolism, partly because IRP1 is an iron-sulfur protein that functions mainly as a cytosolic aconitase in mammalian tissues and IRP2 activity increases to compensate for loss of the IRE binding form of IRP1. The neurodegenerative disease of IRP2(-/-) animals progresses slowly as the animals age. In this study, we fed IRP2(-/-) mice a diet supplemented with a stable nitroxide, Tempol, and showed that the progression of neuromuscular impairment was markedly attenuated. In cell lines derived from IRP2(-/-) animals, and in the cerebellum, brainstem, and forebrain of animals maintained on the Tempol diet, IRP1 was converted from a cytosolic aconitase to an IRE binding protein that stabilized the TfR1 transcript and repressed ferritin synthesis. We suggest that Tempol protected IRP2(-/-) mice by disassembling the cytosolic iron-sulfur cluster of IRP1 and activating IRE binding activity, which stabilized the TfR1 transcript, repressed ferritin synthesis, and partially restored normal cellular iron homeostasis in the brain.

Heme biosynthesis depends on previously unrecognized acquisition of iron-sulfur cofactors in human amino-levulinic acid dehydratase.

Heme biosynthesis and iron-sulfur cluster (ISC) biogenesis are two major mammalian metabolic pathways that require iron. It has long been known that these two pathways interconnect, but the previously described interactions do not fully explain why heme biosynthesis depends on intact ISC biogenesis. Herein we identify a previously unrecognized connection between these two pathways through our discovery that human aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme biosynthesis, is an Fe-S protein. We find that several highly conserved cysteines and an Ala306-Phe307-Arg308 motif of human ALAD are important for [Fe4S4] cluster acquisition and coordination. The enzymatic activity of human ALAD is greatly reduced upon loss of its Fe-S cluster, which results in reduced heme biosynthesis in human cells. As ALAD provides an early Fe-S-dependent checkpoint in the heme biosynthetic pathway, our findings help explain why heme biosynthesis depends on intact ISC biogenesis.

TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation.

Given the essential roles of iron-sulfur (Fe-S) cofactors in mediating electron transfer in the mitochondrial respiratory chain and supporting heme biosynthesis, mitochondrial dysfunction is a common feature in a growing list of human Fe-S cluster biogenesis disorders, including Friedreich ataxia and GLRX5-related sideroblastic anemia. Here, our studies showed that restriction of Fe-S cluster biogenesis not only compromised mitochondrial oxidative metabolism but also resulted in decreased overall histone acetylation and increased H3K9me3 levels in the nucleus and increased acetylation of α-tubulin in the cytosol by decreasing the lipoylation of the pyruvate dehydrogenase complex, decreasing levels of succinate dehydrogenase and the histone acetyltransferase ELP3, and increasing levels of the tubulin acetyltransferase MEC17. Previous studies have shown that the metabolic shift in Toll-like receptor (TLR)-activated myeloid cells involves rapid activation of glycolysis and subsequent mitochondrial respiratory failure due to nitric oxide (NO)-mediated damage to Fe-S proteins. Our studies indicated that TLR activation also actively suppresses many components of the Fe-S cluster biogenesis machinery, which exacerbates NO-mediated damage to Fe-S proteins by interfering with cluster recovery. These results reveal new regulatory pathways and novel roles of the Fe-S cluster biogenesis machinery in modifying the epigenome and acetylome and provide new insights into the etiology of Fe-S cluster biogenesis disorders.

Biochemical and biophysical methods for studying mitochondrial iron metabolism.

Iron is a heavily utilized element in organisms and numerous mechanisms accordingly regulate the trafficking, metabolism, and storage of iron. Despite the high regulation of iron homeostasis, several diseases and mutations can lead to the misregulation and often accumulation of iron in the cytosol or mitochondria of tissues. To understand the genesis of iron overload, it is necessary to employ various techniques to quantify iron in organisms and mitochondria. This chapter discusses techniques for determining the total iron content of tissue samples, ranging from colorimetric determination of iron concentrations, atomic absorption spectroscopy, inductively coupled plasma-optical emission spectroscopy, and inductively coupled plasma-mass spectrometry. In addition, we discuss in situ techniques for analyzing iron including electron microscopic nonheme iron histochemistry, electron energy loss spectroscopy, synchrotron X-ray fluorescence imaging, and confocal Raman microscopy. Finally, we discuss biophysical methods for studying iron in isolated mitochondria, including ultraviolet-visible, electron paramagnetic resonance, X-ray absorbance, and Mössbauer spectroscopies. This chapter should aid researchers to select and interpret mitochondrial iron quantifications.

Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery.

Iron sulfur (Fe-S) clusters, preassembled on the ISCU scaffold, are transferred to target proteins or to intermediate scaffolds by a dedicated chaperone-cochaperone system. However, the molecular mechanisms that underlie substrate discrimination and guide delivery of nascent clusters to specific subsets of Fe-S recipients are poorly understood. Here, we identified interacting partners of the cochaperone HSC20 and discovered that LYR motifs are molecular signatures of specific recipient Fe-S proteins or accessory factors that assist Fe-S cluster delivery. In succinate dehydrogenase B, two LYR motifs engage the ISCU-HSC20-HSPA9 complex to aid incorporation of three Fe-S clusters within the final structure of complex II. Moreover, we show that members of the LYR motif family which assist assembly of complexes II or III, SDHAF1 and LYRM7, respectively, are HSC20 binding partners. Our studies unveil a network of interactions between HSC20 and LYR motif-containing proteins that are key to the assembly and function of complexes I, II, and III.

Deletion of iron regulatory protein 1 causes polycythemia and pulmonary hypertension in mice through translational derepression of HIF2α.

Iron regulatory proteins (Irps) 1 and 2 posttranscriptionally control the expression of transcripts that contain iron-responsive element (IRE) sequences, including ferritin, ferroportin, transferrin receptor, and hypoxia-inducible factor 2α (HIF2α). We report here that mice with targeted deletion of Irp1 developed pulmonary hypertension and polycythemia that was exacerbated by a low-iron diet. Hematocrits increased to 65% in iron-starved mice, and many polycythemic mice died of abdominal hemorrhages. Irp1 deletion enhanced HIF2α protein expression in kidneys of Irp1(-/-) mice, which led to increased erythropoietin (EPO) expression, polycythemia, and concomitant tissue iron deficiency. Increased HIF2α expression in pulmonary endothelial cells induced high expression of endothelin-1, likely contributing to the pulmonary hypertension of Irp1(-/-) mice. Our results reveal why anemia is an early physiological consequence of iron deficiency, highlight the physiological significance of Irp1 in regulating erythropoiesis and iron distribution, and provide important insights into the molecular pathogenesis of pulmonary hypertension.

The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels.

Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells.

Tangled up in red: intertwining of the heme and iron-sulfur cluster biogenesis pathways.

A large-scale computational and genetic analysis study by Nilsson et al. (2009) has identified five genes that coexpress with heme biosynthetic enzymes and are required for normal heme synthesis. Several are implicated in iron-sulfur cluster biogenesis, and malfunction of these genes may repress heme synthesis by activating the IRE/IRP posttranscriptional regulatory system.

Metabolic regulation of citrate and iron by aconitases: role of iron-sulfur cluster biogenesis.

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe-S cluster biogenesis apparatus. Assembly and disassembly of Fe-S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe-S cluster biogenesis regulate the Fe-S cluster switch and modulate intracellular citrate flux.

Roles of the mammalian cytosolic cysteine desulfurase, ISCS, and scaffold protein, ISCU, in iron-sulfur cluster assembly.

Iron-sulfur clusters are prosthetic groups composed of sulfur and iron that are found in respiratory chain complexes and numerous enzymes. Iron-sulfur clusters are synthesized in a multistep process that utilizes cysteine desulfurases, scaffold proteins, chaperones, and iron donors. Assembly of iron-sulfur clusters occurs in the mitochondrial matrix of mammalian cells, but cytosolic isoforms of three major mammalian iron-sulfur cluster (ISC) assembly components have been found, raising the possibility that de novo iron-sulfur cluster biogenesis also occurs in cytosol. The human cysteine desulfurase, ISCS, has two isoforms, one of which targets to the mitochondria, whereas the other less abundant form is cytosolic and nuclear. The open-reading frame of cytosolic mammalian ISCS begins at the second AUG of the transcript and lacks mitochondrial targeting information. Yeast complementation experiments have suggested that the human cytosolic ISCS isoform (c-ISCS) cannot be functional. To evaluate function of c-ISCS, we overexpressed the human cytosolic ISCS in yeast Pichia pastoris and showed that the cytosolic form of ISCS is an active cysteine desulfurase that covalently binds 35S acquired from desulfuration of radiolabeled cysteine. Human cytosolic ISCS dimerized as efficiently as bacterial ISCS and formed a complex in vitro with overexpressed cytosolic human ISCU. When incubated with iron regulatory protein 1, cysteine, and iron, the cytosolic forms of ISCS and ISCU facilitated efficient formation of a [4Fe-4S] cluster on IRP1. Thus, the cytosolic form of ISCS is a functional cysteine desulfurase that can collaborate with cytosolic ISCU to promote de novo iron-sulfur cluster formation.