Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

Neurite growth could be impaired by ETFDH mutation but restored by mitochondrial cofactors.

INTRODUCTION: c.250G>A (p.Ala84Thr) in ETFDH is the most common mutation that causes later-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD) in the southern Chinese population. No functional study has targeted this mutation. METHODS: Using cells expressing ETFDH-wild-type (WT) or ETFDH-mutant (p.Ala84Thr), reactive oxygen species (ROS) production and neurite length were analyzed, followed by pathomechanism exploration and drug screening. RESULTS: Increased ROS production and marked neurite shortening were observed in the cells expressing the ETFDH-mutant, compared with WT. Further studies demonstrated that suberic acid, an accumulated intermediate metabolite in MADD, could significantly impair neurite outgrowth of NSC34 cells, but neurite shortening could be restored by supplementation with carnitine, riboflavin, or Coenzyme Q10. CONCLUSIONS: Neurite shortening caused by the c.250G>A mutation in ETFDH suggests that neural defects could be underdiagnosed in human patients with MADD. This impairment might be treatable with mitochondrial cofactor supplementation. Muscle Nerve 56: 479-485, 2017.

Molecular view of an electron transfer process essential for iron-sulfur protein biogenesis.

Biogenesis of iron-sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron-sulfur protein assembly machinery, two human key proteins--NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin--form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron-sulfur cluster proteins. The Ndor1-anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.

Understanding the genetic and molecular pathogenesis of Friedreich's ataxia through animal and cellular models.

In 1996, a link was identified between Friedreich's ataxia (FRDA), the most common inherited ataxia in men, and alterations in the gene encoding frataxin (FXN). Initial studies revealed that the disease is caused by a unique, most frequently biallelic, expansion of the GAA sequence in intron 1 of FXN. Since the identification of this link, there has been tremendous progress in understanding frataxin function and the mechanism of FRDA pathology, as well as in developing diagnostics and therapeutic approaches for the disease. These advances were the subject of the 4th International Friedreich's Ataxia Conference held on 5th-7th May in the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France. More than 200 scientists gathered from all over the world to present the results of research spanning all areas of investigation into FRDA (including clinical aspects, FRDA pathogenesis, genetics and epigenetics of the disease, development of new models of FRDA, and drug discovery). This review provides an update on the understanding of frataxin function, developments of animal and cellular models of the disease, and recent advances in trying to uncover potential molecules for therapy.

Stage- and tissue-specific expression of rice OsIsu1 gene encoding a scaffold protein for mitochondrial iron-sulfur-cluster biogenesis.

Isu is a scaffold protein involved in mitochondrial iron-sulfur-cluster biogenesis, which affects redox and iron homeostasis in human and yeast cells. A BLASTP search identified two putative Isu genes in rice, and we designated one of them as OsIsu1. When expressed in onion epidermal cells, OsIsu1::GFP was localized to the mitochondria. Northern analysis showed that OsIsu1 was down-regulated in iron-deficient rice root. OsIsu1 promoter-GUS was introduced into Arabidopsis thaliana and histochemical GUS-staining showed that OsIsu1 expression was regulated in a stage- and tissue-specific manner. OsIsu1 was expressed ectopically in Arabidopsis under the control of the CaMV35S promoter, which increased weight of plants.

Small RNA ArrF regulates the expression of sodB and feSII genes in Azotobacter vinelandii.

Azotobacter vinelandii contains a prrF-like sequence in a noncoding region of the chromosome. Like the Pseudomonas aeruginosa PrrF small RNA-encoding genes, the expression of the sequence, herein named arrF (Azotobacter regulatory RNA involving Fe), was increased 100-fold in wild-type cells in response to iron depletion. The level of ArrF was also increased to the same degree in the iron-replete fur mutant, but down back to a wild-type level when this fur mutant was complemented with the wild-type fur gene. These results, with the location of arrF gene in a noncoding region, suggest that this gene encodes an iron-responsive small RNA whose expression is negatively regulated by the Fur-Fe(2+) complex. Disruption of this arrF gene upregulated the expression of iron-containing superoxide dismutase and FeSII protein, whereas fur mutation or iron depletion decreased the level of their transcript. A short region in the 5'-untranslated region of each transcript was predicted to be quite complementary to the core sequence of ArrF, assuming that ArrF represses the expression of the genes under Fur control by an antisense RNA mechanism. However, unlike the P. aeruginosa PrrF that has extensive targets in the tricarboxylic acid cycle and glyoxylate cycle, ArrF had little effect on those genes. The findings that there is a poor overlap between ArrF and PrrF targets and that the FeSII gene, which is present only in the chromosome of nitrogen-fixing bacterial species, is controlled by ArrF suggest that ArrF might have unique targets, some of which are involved in N(2) fixation.

Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system.

The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.

Iron-sulfur cluster biosynthesis: characterization of Schizosaccharomyces pombe Isa1.

Eukaryotic Isa1 is one of several mitochondrial proteins that have been implicated in Fe-S cluster assembly paths in vivo. We report the first biochemical characterization of an eukaryotic member of this family and discuss this in the context of results from in vivo studies and studies of bacterial homologues. Schizosaccharomyces pombe Isa1 is a multimeric protein carrying [2Fe-2S](2+) clusters that have been characterized by Mössbauer and optical spectroscopic studies. Complex formation with a redox-active ferredoxin has been identified through crosslinking experiments and the coordination chemistry and stability of the native clusters has been investigated through site-directed mutagenesis and spectroscopic analysis. Electronic supplementary material to this paper, containing Mössbauer and UV-visible spectra for mutant Isa1 proteins, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0330-2.

Identification of human and mouse HIRA-interacting protein-5 (HIRIP5), two mammalian representatives in a family of phylogenetically conserved proteins with a role in the biogenesis of Fe/S proteins.

The human HIRA protein is encoded from a region of chromosome 22q that is critical for the DiGeorge syndrome and the velocardiofacial syndrome. We have previously reported that it directly interacts with core histones, with a novel histone-binding protein, HIRIP3, and during mouse embryogenesis, with the developmentally regulated homeodomain protein Pax3, suggesting a promoter-targeted function at the chromatin level. We here report on HIRA-interacting protein 5 (HIRIP5), a small acidic protein that interacted with HIRA in a double-hybrid screen performed in yeast and in in vitro protein interaction experiments. HIRIP5 has highly conserved homologs in both prokaryotes and eukaryotes, including the NFU1 gene product which has been implicated in iron metabolism in mitochondria of the yeast Saccharomyces cerevisiae. By radioactive in situ hybridization, the HIRIP5 gene was mapped to the 2p13-p15 chromosomal region, separate from any region previously associated with DiGeorge syndrome.

The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase.

Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent. In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.

Changes in gene expression with iron loading and chelation in cardiac myocytes and non-myocytic fibroblasts.

Iron overload is associated with long-term cardiac iron accumulation and tissue changes such as fibrosis. To determine short-term iron-dependent changes in expression of genes associated with iron homeostasis and fibrosis we measured mRNA on Northern blots prepared from cultured rat neonatal cardiomyocytes and non-myocytes (fibroblasts) as a function of iron loading and chelation. Transferrin receptor mRNA was reduced in myocytes exposed to various concentrations of iron for 3 days and this decline was associated with a 63% decline in iron-response element (IRE) binding of iron regulatory protein-1, indicating that myocytes utilize IRE-dependent mechanisms to modulate gene expression. In myocytes iron caused a dose-dependent decline in mRNAs coding for transforming growth factor- beta(1)(TGF- beta(1)), biglycan, and collagen type I while plasminogen activator inhibitor-1 mRNA was unaffected by iron loading and decorin mRNA doubled. Total TGF- beta bioactivity was also decreased by iron loading. Thus, the effects of iron loading on genes related to cardiac fibrosis are gene-specific. Addition of deferoxamine for 1 day did not have any significant effect on any of these genes. Parallel changes in gene expression were exhibited by non-myocytes (fibroblasts), where chelation also decreased TGF- beta(1)mRNA and activity, and mRNA for collagen type I and biglycan, and collagen synthesis. In addition to these changes in transcripts associated with matrix formation the mRNA of the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase was unaffected by iron loading but doubled in both cell types upon treatment with deferoxamine. These findings suggest that in both cardiac myocytes and non-myocyte fibroblasts gene expression is coupled to intracellular iron pools by gene-specific and IRE-dependent and idependent mechanisms. This linkage may influence matrix deposition, a significant component of cardiac injury.

A single nuclear transcript encoding mitochondrial RPS14 and SDHB of rice is processed by alternative splicing: common use of the same mitochondrial targeting signal for different proteins.

The rice mitochondrial genome has a sequence homologous to the gene for ribosomal protein S14 (rps14), but the coding sequence is interrupted by internal stop codons. A functional rps14 gene was isolated from the rice nuclear genome, suggesting a gene-transfer event from the mitochondrion to the nucleus. The nuclear rps14 gene encodes a long N-terminal extension showing significant similarity to a part of mitochondrial succinate dehydrogenase subunit B (SDHB) protein from human and a malarial parasite (Plasmodium falciparum). Isolation of a functional rice sdhB cDNA and subsequent sequence comparison to the nuclear rps14 indicate that the 5' portions of the two cDNAs are identical. The sdhB genomic sequence shows that the SDHB-coding region is divided into two exons. Surprisingly, the RPS14-coding region is located between the two exons. DNA gel blot analysis indicates that both sdhB and rps14 are present at a single locus in the rice nucleus. These findings strongly suggest that the two gene transcripts result from a single mRNA precursor by alternative splicing. Protein blot analysis shows that the size of the mature RPS14 is 16.5 kDa, suggesting removal of the N-terminal 22.6-kDa peptide region. Considering that the rice mitochondrial genome lacks the sdhB gene but contains the rps14-related sequence, transfer of the sdhB gene seems to have occurred before the transfer of the rps14 gene. The migration of the mitochondrial rps14 sequence into the already existing sdhB gene could bestow the capacity for nuclear expression and mitochondrial targeting.

The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium.

Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH-supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe-4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX-Fe(II) complexes as ultimate by-products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein-1 (IRP-1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP-1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol-dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP-1 or to reincorporate iron within new Fe-S motifs. This damage is mediated by DOX-Fe(II) complexes and reflects oxidative modifications of -SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP-1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP-1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.

Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase.

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) in the inner mitochondrial membrane accepts electrons from electron-transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64-kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF-QO and used these as probes to clone a full-length human ETF-QO cDNA. The deduced human ETF-QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin-like iron-sulfur clusters. A cleavable 33-amino-acid sequence is also predicted at the amino terminus of the 67-kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67-kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF-QO is synthesized as a 67-kDa precursor which is targeted to mitochondria and processed in a single step to a 64-kDa mature form located in the mitochondrial membrane. The detergent-solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron-sulfur cluster are properly inserted into the heterologously expressed protein.