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.

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.

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.

Disruption of cellular iron homeostasis by IREB2 missense variants causes severe neurodevelopmental delay, dystonia and seizures.

Altered brain iron homeostasis can contribute to neurodegeneration by interfering with the delivery of the iron needed to support key cellular processes, including mitochondrial respiration, synthesis of myelin and essential neurotransmitters. Intracellular iron homeostasis in mammals is maintained by two homologous ubiquitously expressed iron-responsive element-binding proteins (IRP1 and IRP2). Using exome sequencing, two patients with severe neurodegenerative disease and bi-allelic mutations in the gene IREB2 were first identified and clinically characterized in 2019. Here, we report the case of a 7-year-old male patient with compound heterozygous missense variants in IREB2, whose neurological features resembled those of the two previously reported IRP2-deficient patients, including a profound global neurodevelopmental delay and dystonia. Biochemical characterization of a lymphoblast cell line derived from the patient revealed functional iron deficiency, altered post-transcriptional regulation of iron metabolism genes and mitochondrial dysfunction. The iron metabolism abnormalities of the patient cell line were reversed by lentiviral-mediated restoration of IREB2 expression. These results, in addition to confirming the essential role of IRP2 in the regulation of iron metabolism in humans, expand the scope of the known IRP2-related neurodegenerative disorders and underscore that IREB2 pathological variants may impact the iron-responsive element-binding activity of IRP2 with varying degrees of severity. The three severely affected patients identified so far all suffered from complete loss of function of IRP2, raising the possibility that individuals with significant but incomplete loss of IRP2 function may develop less severe forms of the disease, analogous to other human conditions that present with a wide range of phenotypic manifestations.

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.].

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.

The autophagy protein ATG9A enables lipid mobilization from lipid droplets.

The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.

Mechanisms of cellular iron sensing, regulation of erythropoiesis and mitochondrial iron utilization.

To maintain an adequate iron supply for hemoglobin synthesis and essential metabolic functions while counteracting iron toxicity, humans and other vertebrates have evolved effective mechanisms to conserve and finely regulate iron concentration, storage, and distribution to tissues. At the systemic level, the iron-regulatory hormone hepcidin is secreted by the liver in response to serum iron levels and inflammation. Hepcidin regulates the expression of the sole known mammalian iron exporter, ferroportin, to control dietary absorption, storage and tissue distribution of iron. At the cellular level, iron regulatory proteins 1 and 2 (IRP1 and IRP2) register cytosolic iron concentrations and post-transcriptionally regulate the expression of iron metabolism genes to optimize iron availability for essential cellular processes, including heme biosynthesis and iron-sulfur cluster biogenesis. Genetic malfunctions affecting the iron sensing mechanisms or the main pathways that utilize iron in the cell cause a broad range of human diseases, some of which are characterized by mitochondrial iron accumulation. This review will discuss the mechanisms of systemic and cellular iron sensing with a focus on the main iron utilization pathways in the cell, and on human conditions that arise from compromised function of the regulatory axes that control iron homeostasis.

Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

Mitochondrial DNA alterations underlie an irreversible shift to aerobic glycolysis in fumarate hydratase-deficient renal cancer.

Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)-encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.

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

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

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.

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.

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.

Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase.

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.

Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins.

The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP, and the main scaffold ISCU, have shed light on the molecular interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron-sulfur (Fe-S) cluster (ISC) biogenesis in mammalian cells that have arisen from the crystal structures of the core ISC assembly complex. We will also discuss how ISCs are delivered to recipient proteins and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe-S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.