The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein (ISCU) Mutation Leads to Development of Mitochondrial Myopathy

Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044G→C), compound heterozygous patients with severe myopathy have been identified to carry the c.149G→A missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms.     

Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis,while complete loss of ISCU results in early embryonic death in mice

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe–S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe–S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe–S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.     

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

Background

Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis.

Methods and Findings

In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.

Conclusions

Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.     

MicroRNA-210 Controls Mitochondrial Metabolism during Hypoxia by Repressing the Iron-Sulfur Cluster Assembly Proteins ISCU1/2

Repression of mitochondrial respiration represents an evolutionarily ancient cellular adaptation to hypoxia and profoundly influences cell survival and function; however, the underlying molecular mechanisms are incompletely understood. Primarily utilizing pulmonary arterial endothelial cells as a representative hypoxic cell type, we identify the iron-sulfur cluster assembly proteins (ISCU1/2) as direct targets for repression by the hypoxia-induced microRNA-210 (miR-210). ISCU1/2 facilitate the assembly of iron-sulfur clusters, prosthetic groups that are critical for electron transport and mitochondrial oxidation-reduction reactions. Under in vivo conditions of upregulating miR-210 and repressing ISCU1/2, the integrity of iron-sulfur clusters is disrupted. In turn, by repressing ISCU1/2 during hypoxia, miR-210 decreases the activity of prototypical iron-sulfur proteins controlling mitochondrial metabolism, including Complex I and aconitase. Consequently, miR-210 represses mitochondrial respiration and associated downstream functions. These results identify important mechanistic connections among microRNA, iron-sulfur cluster biology, hypoxia, and mitochondrial function, with broad implications for cellular metabolism and adaptation to cellular stress.     

Human Mitochondrial Chaperone (mtHSP70) and Cysteine Desulfurase (NFS1) Bind Preferentially to the Disordered Conformation,Whereas Co-chaperone (HSC20) Binds to the Structured Conformation of the Iron-Sulfur Cluster Scaffold Protein (ISCU)

Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human cysteine desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU.     

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.     

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.     

Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression

Hereditary myopathy with lactic acidosis (HML) (OMIM #255125) presents in childhood with exercise intolerance and muscle pain on trivial exercise, lactic acidosis, dyspnoea, palpitations, and rhabdomyolysis which can be fatal. The disease is recessively inherited and caused by a deep intronic, single base transition in the iron-sulfur cluster scaffold, ISCU gene that causes retention of a pseudoexon and introduction of a premature termination codon. IscU protein deficiency causes secondary defects in several iron-sulfur dependant proteins, including enzymes involved in aerobic energy metabolism. We have shown in a previous study that the splice abnormality affects skeletal muscle more than other tissues, leading to the purely muscular phenotype.Antisense oligonucleotides (AOs) have been able to redirect mRNA splicing in a number of disease models, and show promise in clinical studies. We designed 2′O-methyl phosphorothioate AOs targeting either splice site of the detrimental HML pseudoexon. The acceptor site AO effectively redirected splicing towards the normal state in cultured muscle fibroblasts, whilst the donor site AO promoted pseudoexon inclusion in both patient and control cells. Our results show that AO therapy seems feasible in HML, but care must be taken to avoid adverse splicing effects.     

Characterization of iron-sulfur cluster assembly protein isca from Acidithiobacillus ferrooxidans

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes, but the mechanism of its function in the biogenesis of iron-sulfur cluster remains elusive. In this paper, we demonstrate that Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of 4·10²⁰ M^?1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form of IscA can be converted to iron-sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99, or Cys101 in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron-sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro. Taken together, these findings suggest that A. ferrooxidans IscA is not only an iron-sulfur protein, but also an iron binding protein that can act as an iron donor for biogenesis of iron-sulfur clusters.     

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

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.     

Methyl Sulfone Blocked Multiple Hypoxia- and Non-Hypoxia-Induced Metastatic Targets in Breast Cancer Cells and Melanoma Cells

Metastatic cancer causes 90% of cancer deaths. Unlike many primary tumors, metastatic tumors cannot be cured by surgery alone. Metastatic cancer requires chemotherapy. However, metastatic cells are not easily killed by chemotherapy. These problems with chemotherapy are caused in part by the metastatic cell niche: hypoxia. Here we show that the molecule, methyl sulfone, normalized metastatic metabolism of hypoxic breast cancer and melanoma cells by altering several metabolic functions of the cells. Under hypoxia, methyl sulfone decreased expression of the master regulator of hypoxia, HIF-1α, and reduced levels of the glycolytic enzymes, PKM2, LDHA, GLUT1, the pro-angiogenic protein, VEGF, and the iron-sulfur metabolism molecules, miR-210 and transferrin, all of which promote metastasis. Conversely, methyl sulfone increased levels of ISCU1/2 and ferroportin, proteins associated with iron-sulfur cluster biogenesis and iron homeostasis in normal cells. These data identify methyl sulfone as a multi-targeting molecule that blocks the survival/proliferative effect of hypoxia on metastatic cells and brings normality back to cellular metabolism.     

Interplay of IscA and IscU in biogenesis of iron-sulfur clusters

Increasing evidence suggests that sulfur in ubiquitous iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. In Escherichia coli, the major cysteine desulfurase activity for biogenesis of iron-sulfur clusters has been attributed to IscS. The gene that encodes IscS is a member of an operon iscSUA, which also encodes two highly conserved proteins: IscU and IscA. Previous studies suggested that both IscU and IscA may act as the iron-sulfur cluster assembly scaffold proteins. However, recent evidence indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU (Ding, H., Harrison, K., and Lu, J. (2005) J. Biol. Chem. 280, 30432-30437). To further elucidate the function of IscA in biogenesis of iron-sulfur clusters, we evaluate the iron-sulfur cluster binding activity of IscA and IscU under physiologically relevant conditions. When equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS, L-cysteine and dithiothreitol, iron-sulfur clusters are assembled in IscU, but not in IscA, suggesting that IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contrast, when equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS and dithiothreitol but without L-cysteine, nearly all iron is bound to IscA. The iron binding in IscA appears to prevent the formation of the biologically inaccessible ferric hydroxide under aerobic conditions. Subsequent addition of L-cysteine efficiently mobilizes the iron center in IscA and transfers the iron for the iron-sulfur cluster assembly in IscU. The results suggest an intriguing interplay between IscA and IscU in which IscA acts as an iron chaperon that recruits "free" iron and delivers the iron for biogenesis of iron-sulfur clusters in IscU under aerobic conditions.     

Expression, purification and molecular modelling of the Iro protein from Acidithiobacillus ferrooxidans Fe-1

The Iro protein was proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, it is a member of HiPIP family with the iron-sulfur cluster for electron transfer. The gene of Iro protein from A. ferrooxidans Fe-1 was cloned and then successfully expressed in Escherichia coli, finally purified by one-step affinity chromatography to homogeneity. The recombinant protein was observed to be dimer. The molecular mass of a monomer containing the [Fe4S4] cluster was 6847.35 Da by MALDI-TOF-MS. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Molecular modelling for the protein revealed that Cys20, Cys23, Cys32 and Cys45 were in ligation with the iron-sulfur cluster, and Tyr10 was important for the stability of the [Fe4S4] cluster. As we know, this is the first report of expression in E. coli of the Iro protein from A. ferrooxidans Fe-1.     

Repair of nitric oxide-modified ferredoxin [2Fe-2S] cluster by cysteine desulfurase (IscS)

Iron-sulfur proteins are among the sensitive targets of the nitric oxide cytotoxicity. When Escherichia coli cells are exposed to nitric oxide, iron-sulfur clusters are modified forming protein-bound dinitrosyl iron complexes. Such modified protein dinitrosyl iron complexes are stable in vitro but are efficiently repaired in aerobically growing E. coli cells even without any new protein synthesis. Here we show that cysteine desulfurase encoded by the gene iscS of E. coli can directly convert the ferredoxin dinitrosyl iron complex to the ferredoxin [2Fe-2S] cluster in the presence of L-cysteine in vitro. A reassembly of the [2Fe-2S] cluster in the ferredoxin dinitrosyl iron complex does not require any addition of iron or other protein components. Furthermore, a complete removal of the dinitrosyl iron complex from ferredoxin prevents reassembly of the [2Fe-2S] cluster in the protein. The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin [2Fe-2S] cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.     

IscA mediates iron delivery for assembly of iron-sulfur clusters in IscU under the limited accessible free iron conditions

Increasing evidence suggests that IscS, a cysteine desulfurase, provides sulfur for assembly of transient iron-sulfur clusters in IscU. IscU appears to act as a scaffold and eventually transfers the assembled clusters to target proteins. However, the iron donor for the iron-sulfur cluster assembly largely remains elusive. Here we find that Escherichia coli IscU fails to assemble iron-sulfur clusters when the accessible "free" iron in solution is limited by an iron chelator sodium citrate. Remarkably, IscA, an iron-sulfur cluster assembly protein with an iron association constant of 3.0 x 10(19) m(-1), is able to overcome the iron limitation due to sodium citrate and deliver iron for the IscS-mediated iron-sulfur cluster assembly in IscU. Substitution of the invariant cysteine residues Cys-99 or Cys-101 in IscA with serine completely abolishes the iron binding activity of the protein. The IscA mutants that fail to bind iron are unable to mediate iron delivery for the iron-sulfur cluster assembly in IscU under the limited accessible "free" iron conditions. The results suggest that IscA is capable of recruiting intracellular iron and providing iron for the iron-sulfur cluster assembly in IscU in cells in which the accessible "free" iron content is probably restricted.     

Yeast mitochondrial protein, Nfs1p, coordinately regulates iron-sulfur cluster proteins, cellular iron uptake, and iron distribution.

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.