Global Identification of Genes Affecting Iron-Sulfur Cluster Biogenesis and Iron Homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-¹⁴C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-¹⁴C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.     

Biogenesis of iron-sulfur proteins in eukaryotes: components, mechanism and pathology

Iron-sulfur (Fe-S) clusters are ubiquitous co-factors of proteins that play an important role in metabolism, electron-transfer and regulation of gene expression. In eukaryotes mitochondria are the primary site of Fe-S cluster biogenesis. The organelles contain some ten proteins of the so-called iron-sulfur cluster (ISC) assembly machinery that is well-conserved in bacteria and eukaryotes. The ISC assembly machinery is responsible for biogenesis of Fe-S proteins within mitochondria. In addition, this machinery is involved in the maturation of extra-mitochondrial Fe-S proteins by cooperating with mitochondrial proteins with an exclusive function in this process. This review summarizes recent developments in our understanding of the biogenesis of cellular Fe-S proteins in eukaryotes. Particular emphasis is given to disorders in Fe-S protein biogenesis causing human disease.     

Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases –which include Friedreich’s ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome – affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease.     

线粒体铁代谢与人类疾病的研究进展

线粒体铁代谢的研究主要包括两个方面：铁在胞质和线粒体之间的转运和调控;铁硫簇和血红素在线粒体内的合成与转运。目前认为线粒体铁的转入主要是与mitoferrinl／2（MFRNl和MFRN2）和ABCBl0有关,运出可能与ABCB6和／或ABCB7有关,转运和调控的具体机制不是很清楚,推测与某种含有铁硫簇的信号分子有关。哺乳动物铁硫簇的合成可以发生在胞质和线粒体内,但以线粒体为主;真核生物中与铁硫簇合成相关的蛋白达二十多种,其中FXN、ISCS、ISDll和ISCU及其同系物被认为是核心组分。血红素的合成起始和终止发生在线粒体内,终止步骤为亚铁螯合酶将铁插入原卟啉IX,该酶活性又依赖于铁硫簇。因此,铁硫簇的合成与调控是线粒体铁代谢的核心,也是整个细胞铁运作的核心。本文主要围绕线粒体铁代谢特别是铁硫簇的合成异常引起的疾病做一简单的综述。     

Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics

Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from l-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.     

Lack of the ApbC or ApbE protein results in a defect in Fe-S cluster metabolism in Salmonella enterica serovar Typhimurium

The isc genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the isc operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the isc operon. Here we demonstrate that null mutations in two genes of unknown function, apbC and apbE, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the apbC and apbE mutants, distinguishing them from previously described isc mutants. The deficiencies caused by the apbC and isc mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.     

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.     

Enterococcus faecalis SufU scaffold protein enhances SufS desulfurase activity by acquiring sulfur from its cysteine-153

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.     

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.     

Friedreich's ataxia variants I154F and W155R diminish frataxin-based activation of the iron-sulfur cluster assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ～15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ～ I154F > W155F > W155A ～ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.     

AtNAP1 represents an atypical SufB protein in Arabidopsis plastids

The assembly of iron-sulfur (Fe-S) clusters involves several pathways and in prokaryotes the mobilization of the sulfur (SUF) system is paramount for Fe-S biogenesis and repair during oxidative stress. The prokaryotic SUF system consists of six proteins: SufC is an ABC/ATPase that forms a complex with SufB and SufD, SufA acts as a scaffold protein, and SufE and SufS are involved in sulfur mobilization from cysteine. Despite the importance of Fe-S proteins in higher plant plastids, little is known regarding plastidic Fe-S cluster assembly. We have recently shown that Arabidopsis harbors an evolutionary conserved plastidic SufC protein (AtNAP7) capable of hydrolyzing ATP and interacting with the SufD homolog AtNAP6. Based on this and the prokaryotic SUF system we speculated that a SufB-like protein may exist in plastids. Here we demonstrate that the Arabidopsis plastid-localized SufB homolog AtNAP1 can complement SufB deficiency in Escherichia coli during oxidative stress. Furthermore, we demonstrate that AtNAP1 can interact with AtNAP7 inside living chloroplasts suggesting the presence of a plastidic AtNAP1.AtNAP6.AtNAP7 complex and remarkable evolutionary conservation of the SUF system. However, in contrast to prokaryotic SufB proteins with no associated ATPase activity we show that AtNAP1 is an iron-stimulated ATPase and that AtNAP1 is capable of forming homodimers. Our results suggest that AtNAP1 represents an atypical plastidic SufB-like protein important for Fe-S cluster assembly and for regulating iron homeostasis in Arabidopsis.     

AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis

Iron-sulfur (Fe-S) clusters are vital prosthetic groups for Fe-S proteins involved in fundamental processes such as electron transfer, metabolism, sensing and signaling. In plants, sulfur (SUF) protein-mediated Fe-S cluster biogenesis involves iron acquisition and sulfur mobilization, processes suggested to be plastidic. Here we have shown that AtSufE in Arabidopsis rescues growth defects in SufE-deficient Escherichia coli. In contrast to other SUF proteins, AtSufE localizes to plastids and mitochondria interacting with the plastidic AtSufS and mitochondrial AtNifS1 cysteine desulfurases. AtSufE activates AtSufS and AtNifS1 cysteine desulfurization, and AtSufE activity restoration in either plastids or mitochondria is not sufficient to rescue embryo lethality in AtSufE loss-of-function mutants. AtSufE overexpression induces AtSufS and AtNifS1 expression, which in turn leads to elevated cysteine desulfurization activity, chlorosis and retarded development. Our data demonstrate that plastidic and mitochondrial Fe-S cluster biogenesis shares a common, essential component, and that AtSufE acts as an activator of plastidic and mitochondrial desulfurases in Arabidopsis.     

Iron-responsive degradation of iron-regulatory protein 1 does not require the Fe-S cluster

The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron-regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNA-binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori

The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.     

Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin,a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.