How Oxidation of a Unique Iron-Sulfur Cluster in FBXL5 Regulates IRP2 Levels and Promotes Regulation of Iron Metabolism Proteins.

In this issue of Molecular Cell, Wang et al. (2020) discover that the C-terminal substrate-binding domain of FBXL5 contains a redox-sensitive [2Fe-2S] cluster that, upon oxidation, promotes FBXL5 binding to IRP2 to effect its oxygen-dependent degradation, unveiling a novel and previously unrecognized mechanism involved in regulation of cellular iron homeostasis.

An iron-sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an Fe4S4 cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Outlining the Complex Pathway of Mammalian Fe-S Cluster Biogenesis.

Iron-sulfur (Fe-S) clusters (ISCs) are ubiquitous cofactors essential to numerous fundamental cellular processes. Assembly of ISCs and their insertion into apoproteins involves the function of complex cellular machineries that operate in parallel in the mitochondrial and cytosolic/nuclear compartments of mammalian cells. The spectrum of diseases caused by inherited defects in genes that encode the Fe-S assembly proteins has recently expanded to include multiple rare human diseases, which manifest distinctive combinations and severities of global and tissue-specific impairments. In this review, we provide an overview of our understanding of ISC biogenesis in mammalian cells, discuss recent work that has shed light on the molecular interactions that govern ISC assembly, and focus on human diseases caused by failures of the biogenesis pathway.

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1.

NFU1, a late-acting iron-sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease, multiple mitochondrial dysfunctions syndrome. In this work, using genetic and biochemical approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate the formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2. By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We observed that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochemical functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase, which supports the Fe-S-dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and the glycine cleavage complex.

Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAO1-mediated transfer to recipients.

Iron-sulfur (Fe-S) clusters are cofactors in hundreds of proteins involved in multiple cellular processes, including mitochondrial respiration, the maintenance of genome stability, ribosome biogenesis and translation. Fe-S cluster biogenesis is performed by multiple enzymes that are highly conserved throughout evolution, and mutations in numerous biogenesis factors are now recognized to cause a wide range of previously uncategorized rare human diseases. Recently, a complex formed of components of the cytoplasmic Fe-S cluster assembly (CIA) machinery, consisting of CIAO1, FAM96B and MMS19, was found to deliver Fe-S clusters to a subset of proteins involved in DNA metabolism, but it was unclear how this complex acquired its fully synthesized Fe-S clusters, because Fe-S clusters have been alleged to be assembled de novo solely in the mitochondrial matrix. Here, we investigated the potential role of the human cochaperone HSC20 in cytosolic Fe-S assembly and found that HSC20 assists Fe-S cluster delivery to cytosolic and nuclear Fe-S proteins. Cytosolic HSC20 (C-HSC20) mediated complex formation between components of the cytosolic Fe-S biogenesis pathway (ISC), including the primary scaffold, ISCU1, and the cysteine desulfurase, NFS1, and the CIA targeting complex, consisting of CIAO1, FAM96B and MMS19, to facilitate Fe-S cluster insertion into cytoplasmic and nuclear Fe-S recipients. Thus, C-HSC20 integrates initial Fe-S biosynthesis with the transfer activities of the CIA targeting system. Our studies demonstrate that a novel cytosolic pathway functions in parallel to the mitochondrial ISC to perform de novo Fe-S biogenesis, and to escort Fe-S clusters to cytoplasmic and nuclear proteins.

Absence of iron-responsive element-binding protein 2 causes a novel neurodegenerative syndrome.

Disruption of cellular iron homeostasis can contribute to neurodegeneration. In mammals, two iron-regulatory proteins (IRPs) shape the expression of the iron metabolism proteome. Targeted deletion of Ireb2 in a mouse model causes profoundly disordered iron metabolism, leading to functional iron deficiency, anemia, erythropoietic protoporphyria, and a neurodegenerative movement disorder. Using exome sequencing, we identified the first human with bi-allelic loss-of-function variants in the gene IREB2 leading to an absence of IRP2. This 16-year-old male had neurological and haematological features that emulate those of Ireb2 knockout mice, including neurodegeneration and a treatment-resistant choreoathetoid movement disorder. Cellular phenotyping at the RNA and protein level was performed using patient and control lymphoblastoid cell lines, and established experimental assays. Our studies revealed functional iron deficiency, altered post-transcriptional regulation of iron metabolism genes, and mitochondrial dysfunction, as observed in the mouse model. The patient's cellular abnormalities were reversed by lentiviral-mediated restoration of IRP2 expression. These results confirm that IRP2 is essential for regulation of iron metabolism in humans, and reveal a previously unrecognized subclass of neurodegenerative disease. Greater understanding of how the IRPs mediate cellular iron distribution may ultimately provide new insights into common and rare neurodegenerative processes, and could result in novel therapies.

Acute loss of iron-sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells.

Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a ∼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.

Dimeric ferrochelatase bridges ABCB7 and ABCB10 homodimers in an architecturally defined molecular complex required for heme biosynthesis.

Loss-of-function mutations in the ATP-binding cassette (ABC) transporter of the inner mitochondrial membrane, ABCB7, cause X-linked sideroblastic anemia with ataxia, a phenotype that remains largely unexplained by the proposed role of ABCB7 in exporting a special sulfur species for use in cytosolic iron-sulfur (Fe-S) cluster biogenesis. Here, we generated inducible ABCB7-knockdown cell lines to examine the time-dependent consequences of loss of ABCB7. We found that knockdown of ABCB7 led to significant loss of mitochondrial Fe-S proteins, which preceded the development of milder defects in cytosolic Fe-S enzymes. In erythroid cells, loss of ABCB7 altered cellular iron distribution and caused mitochondrial iron overload due to activation of iron regulatory proteins 1 and 2 in the cytosol and to upregulation of the mitochondrial iron importer, mitoferrin-1. Despite the exceptionally large amount of iron imported into mitochondria, erythroid cells lacking ABCB7 showed a profound hemoglobinization defect and underwent apoptosis triggered by oxidative stress. In ABCB7-depleted cells, defective heme biosynthesis resulted from translational repression of ALAS2 by iron regulatory proteins and from decreased stability of the terminal enzyme ferrochelatase. By combining chemical crosslinking, tandem mass spectrometry and mutational analyses, we characterized a complex formed of ferrochelatase, ABCB7 and ABCB10, and mapped the interfaces of interactions of its components. A dimeric ferrochelatase physically bridged ABCB7 and ABCB10 homodimers by binding near the nucleotide-binding domains of each ABC transporter. Our studies not only underscore the importance of ABCB7 for mitochondrial Fe-S biogenesis and iron homeostasis, but also provide the biochemical characterization of a multiprotein complex required for heme biosynthesis.

Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways.

Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.

Iron-sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery.

Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i.e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein-protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Iron­sulfur clusters in viral proteins: Exploring their elusive nature, roles and new avenues for targeting infections.

Viruses have evolved complex mechanisms to exploit host factors for replication and assembly. In response, host cells have developed strategies to block viruses, engaging in a continuous co-evolutionary battle. This dynamic interaction often revolves around the competition for essential resources necessary for both host cell and virus replication. Notably, iron, required for the biosynthesis of several cofactors, including iron­sulfur (FeS) clusters, represents a critical element in the ongoing competition for resources between infectious agents and host. Although several recent studies have identified FeS cofactors at the core of virus replication machineries, our understanding of their specific roles and the cellular processes responsible for their incorporation into viral proteins remains limited. This review aims to consolidate our current knowledge of viral components that have been characterized as FeS proteins and elucidate how viruses harness these versatile cofactors to their benefit. Its objective is also to propose that viruses may depend on incorporation of FeS cofactors more extensively than is currently known. This has the potential to revolutionize our understanding of viral replication, thereby carrying significant implications for the development of strategies to target infections.

CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes.

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming.

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

Loss of Function of the Cytoplasmic Fe-S Assembly Protein CIAO1 Causes a Neuromuscular Disorder with Compromise of Nucleocytoplasmic Fe-S Enzymes.

Cytoplasmic and nuclear iron-sulfur enzymes that are essential for genome maintenance and replication depend on the cytoplasmic iron-sulfur assembly (CIA) machinery for cluster acquisition. Here we report that patients with biallelic loss of function in CIAO1 , a key CIA component, develop proximal and axial muscle weakness, fluctuating creatine kinase elevation and respiratory insufficiency. In addition, they present with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, macrocytic anemia and gastrointestinal symptoms. Mutational analysis and functional assays revealed reduced stability of the variants compared to wild-type CIAO1. Loss of CIAO1 impaired DNA helicases, polymerases and repair enzymes which rely on the CIA complex to acquire their Fe-S cofactors, with lentiviral restoration reversing all patient-derived cellular abnormalities. Our study identifies CIAO1 as a novel human disease gene and provides insights into the broader implications of the iron-sulfur assembly pathway in human health and disease.

TEMPOL inhibits SARS-CoV-2 replication and development of lung disease in the Syrian hamster model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide outbreak, known as coronavirus disease 2019 (COVID-19). Alongside vaccines, antiviral therapeutics is an important part of the healthcare response to COVID-19. We previously reported that TEMPOL, a small molecule stable nitroxide, inactivated the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 by causing the oxidative degradation of its iron-sulfur cofactors. Here, we demonstrate that TEMPOL is effective in vivo in inhibiting viral replication in the Syrian hamster model. The inhibitory effect of TEMPOL on SARS-CoV-2 replication was observed in animals when the drug was administered 2 h before infection in a high-risk exposure model. These data support the potential application of TEMPOL as a highly efficacious antiviral against SARS-CoV-2 infection in humans.