Composed of iron (Fe) and inorganic sulfur (S), iron-sulfur clusters (ISCs) are ancient cofactors present across all domains of life and in some viruses. Over the past 3 decades, cytosolic and nuclear Fe-S proteins have emerged as integral components of DNA replication and repair machineries, telomere maintenance pathways, transcriptional processes, cell cycle regulation, and protein synthesis. More recently, ISCs were identified in viral proteins, including multiple components of the SARS-CoV-2 replication and transcription complex (RTC), which collectively host seven experimentally verified Fe-S cofactors. The coexistence of multiple ISC-dependent enzymes within both cellular and viral replication machineries raises fundamental questions about how these metal cofactors coordinate genome maintenance, replication, and host-virus interactions. Here, we provide an inventory of known mammalian nucleocytoplasmic Fe-S proteins, discuss mechanisms of ISC acquisition, explore the potential roles of ISCs within cellular and viral replication complexes, and highlight critical gaps in our understanding of ISC delivery, coordination, and function among Fe-S proteins in large multi-subunit assemblies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has emerged as a global pandemic pathogen with high mortality. While treatments have been developed to reduce morbidity and mortality of COVID-19, more antivirals with broad-spectrum activities are still needed. Here, we identified lonafarnib (LNF), a Food and Drug Administration-approved inhibitor of cellular farnesyltransferase (FTase), as an effective anti-SARS-CoV-2 agent. LNF inhibited SARS-CoV-2 infection and acted synergistically with known anti-SARS antivirals. LNF was equally active against diverse SARS-CoV-2 variants. Mechanistic studies suggested that LNF targeted multiple steps of the viral life cycle. Using other structurally diverse FTase inhibitors and a LNF-resistant FTase mutant, we demonstrated a key role of FTase in the SARS-CoV-2 life cycle. To demonstrate in vivo efficacy, we infected SARS-CoV-2-susceptible humanized mice expressing human angiotensin-converting enzyme 2 (ACE2) and treated them with LNF. LNF at a clinically relevant dose suppressed the viral titer in the respiratory tract and improved pulmonary pathology and clinical parameters. Our study demonstrated that LNF, an approved oral drug with excellent human safety data, is a promising antiviral against SARS-CoV-2 that warrants further clinical assessment for treatment of COVID-19 and potentially other viral infections.

ABSTRACT Objectives To investigate the consequences of a pathogenic missense variant (c.838C>T; p.L280F) and a 5′‐UTR regulatory variant (c.‐122G>T) in BCS1L on disease pathogenesis and to understand how regulatory variants influence disease severity and clinical presentation. Methods Deep phenotyping, research‐based whole genome sequencing, biochemical characterization of identified variants, and studies in patient‐derived fibroblast cultures were applied to uncover the underlying genetic cause and molecular defects in siblings with a genetically uncharacterized complex neurologic condition. Results Genome sequencing identified a paternally inherited missense variant (c.838C>T; p.L280F) and a maternally inherited 5′‐UTR variant (c.‐122G>T) in BCS1L in two affected siblings. Although the missense variant disrupts complex III assembly, the 5′‐UTR variant allows residual wild‐type BCS1L expression, likely mitigating disease severity. Biochemical studies in patient‐derived fibroblasts confirmed the pathogenicity of both variants and demonstrated a moderate in vitro response to a coenzyme Q10 analog. Interpretation This study expands the clinical spectrum of BCS1L ‐related disorders to include a comparatively milder phenotype with central and peripheral nervous system involvement. Our findings demonstrate that the 5′‐UTR variant modulates disease severity by enabling residual wild‐type BCS1L expression, partially mitigating the pathogenic effects of the missense variant. These insights underscore the importance of evaluating both protein coding and regulatory variants in mitochondrial disease diagnostics and pathogenesis.

Abstract Iron regulatory proteins (IRP1 and IRP2) play a pivotal role in maintaining cellular iron homeostasis by binding to iron-responsive elements (IREs) of target messenger RNAs and regulating the expression of these iron-related genes. Mice and humans who lack functional IRP1 develop erythrocytosis due to erythropoietin (EPO) overproduction, whereas those who lack IRP2 develop microcytic anemia, believed to result from iron deficiency of erythroblasts. Here, we discovered that IRP2 deficiency reduced the expression of hypoxia-inducible factor 2α (HIF2α) and its transcriptional target, EPO, thereby compromising the stress erythropoiesis response to generate red blood cells upon anemia. The distinct consequences of IRP2 and IRP1 on EPO result from the higher binding affinity of the HIF2α IRE for IRP1 than IRP2. This difference in binding affinity arises from a bulge uridine in the upper stem of HIF2α IRE that impairs the ability of IRP2 to bind the IRE. These results reveal that IRP1 and IRP2 play distinct roles in erythropoiesis and unveil an unsuspected IRE binding preference that contributes to the divergent phenotypes observed in IRP1- and IRP2-deficient mammals.

Coronaviruses rely on a multifunctional replication-transcription complex to ensure genome fidelity and support viral propagation. Within this complex, the nsp14-nsp10 heterodimer possesses 3'-5' exoribonuclease (ExoN) activity, while nsp14 alone functions as an N7-methyltransferase and the nsp16/nsp10 complex completes viral RNA capping via its 2'-O-methyltransferase. Here, we report that nsp14 and nsp10 ligate [Fe4S4] clusters when purified anoxically, in sites previously modeled as zinc centers. Quantum mechanics/molecular mechanics simulations revealed distinct reduction potentials for these iron-sulfur (Fe-S) clusters, and redox titrations demonstrated that changes in oxidation state modulate RNA binding by nsp14 and the nsp10/nsp16 complex. Functionally, Fe-S clusters enhance the methyltransferase activities of nsp14 and nsp10/nsp16, while leaving the ExoN activity unaffected. These findings uncover a redox-regulated role for Fe-S clusters in SARS-CoV-2 RNA processing and suggest that the viral core enzymatic functions may be modulated by the redox state of their Fe-S cofactors.

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.

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1–2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.

BCS1L is an essential chaperone for facilitating the assembly of complex III of the mitochondria respiratory chain, including incorporating the Rieske Fe–S protein UQCRFS1. Established phenotypes associated with biallelic disease‐causing variants in BCS1L are the lethal GRACILE syndrome and Björnstad syndrome (hearing loss and pili torti). We report two siblings presenting with a complex neurological disorder in whom we identified a paternally inherited missense variant (c.838C>T; L280F) and a maternally inherited 5’ UTR variant (c.-122G>T) in BCS1L. The index case is a 13-year-old boy who presented at age 16 months with progressive motor difficulties. He gradually developed hand dystonia, dysarthric speech, muscle weakness, and scoliosis. The older sibling, a 16-year-old male, was diagnosed in early childhood with depression, anxiety, and ADHD. His motor difficulties emerged later in childhood. Both siblings have learning disabilities, lifelong difficulty gaining weight, and report significantly low stamina. Their physical examinations were notable for mild proximal and moderate-severe distal muscle weakness; muscle ultrasound showed fasciculations and diffusely increased streak-like echogenicity. Electrodiagnostic studies confirmed a predominantly motor neuropathy/neuronopathy. Biochemical characterization of fibroblasts from the patients compared to control showed decreased BCS1L levels, decreased respiratory complexes I and III, and reduced basal oxygen consumption rates and respiratory spare capacity. Characterization of the homozygous L280F variant in cells was equivalent to the homozygous S78G variant causing the classic GRACILE syndrome. Thus, the milder phenotype is likely the result of the compound heterozygous state with the 5’ UTR variant. This study expands the clinical spectrum of BSC1L-related disorders to now include a milder phenotype with CNS and PNS involvement and combines genetic and biochemical characterization of the disease pathomechanism.

Iron is an important cofactor for many proteins and is used to create Fe-S clusters and heme prosthetic groups that enzymes use to catalyze enzymatic reactions. Proteins involved in the import, export, and sequestration of iron are regulated by Iron Regulatory Proteins (IRPs). Recently, a patient with bi-allelic loss of function mutations in IREB2 leading to the absence of IRP2 protein was discovered. The patient failed to achieve developmental milestones and was diagnosed with dystonic cerebral palsy, epilepsy, microcytic hypochromic anemia, and frontal lobe atrophy. Several more IREB2 deficient patients subsequently identified manifested similar neurological problems. To better understand the manifestations of this novel neurological disease, we subjected an Irp2-null mouse model to extensive behavioral testing. Irp2-null mice had a significant motor deficit demonstrated by reduced performance on rotarod and hanging wire tests. Somatosensory function was also compromised in hot and cold plate assays. Their spatial search strategy was impaired in the Barnes maze and they exhibited a difficulty in flexibly adapting their response in the operant touchscreen reversal learning task. The latter is a cognitive behavior known to require an intact prefrontal cortex. These results suggest that loss of Irp2 in mice causes motor and behavioral deficits that faithfully reflect the IREB2 patient's neurodegenerative disorder.

Iron is an essential nutrient and necessary for biological functions from DNA replication and repair to transcriptional regulation, mitochondrial respiration, electron transfer, oxygen transport, photosynthesis, enzymatic catalysis, and nitrogen fixation. However, due to iron’s propensity to generate toxic radicals which can cause damage to DNA, proteins, and lipids, multiple processes regulate the uptake and distribution of iron in living systems. Understanding how intracellular iron metabolism is optimized and how iron is utilized to regulate other intracellular processes is important to our overall understanding of a multitude of biological processes. One of the tools that the cell utilizes to regulate a multitude of functions is the ligation of the iron–sulfur (Fe-S) cluster cofactor. Fe-S clusters comprised of iron and inorganic sulfur are ancient components of living matter on earth that are integral for physiological function in all domains of life. FeS clusters that function as biological sensors have been implicated in a diverse group of life from mammals to bacteria, fungi, plants, and archaea. Here, we will explore the ways in which cells and organisms utilize Fe-S clusters to sense changes in their intracellular environment and restore equilibrium.

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 iron‑sulfur (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.

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.

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 FDX2, encoding the essential iron-sulfur (Fe-S) cluster biogenesis component ferredoxin 2, has been reported to cause optic atrophy, myopathy, partially reversible leukoencephalopathy, and sensorineural axonal neuropathy. We describe the first patient with FDX2 loss of function presenting with blindness (no light perception) at age 17 months. Our evaluation revealed clinical and muscle imaging evidence of a myopathy. The patient was found to harbor novel FDX2 variants: c.271C>T, p.L91F; c.344A>G, p.H115R. In vitro studies in the patient's fibroblasts revealed defective interactions of the FDX2 variants with the Fe-S assembly components, resulting in dysfunction of the mitochondrial oxidative phosphorylation system. We obtained a Single Patient Expanded Access IND for Idebenone, a Co-enzyme Q10 analog with increased solubility, which the patient started at age 3 years. Titration of Idebenone was limited by neutropenia, subsequently recognized to be independent of Idebenone. Treatment of patient fibroblasts with mitoquinol mesylate (MitoQ), a Co-enzyme Q10 analog targeted to the mitochondrial matrix, increased: ATP-linked and maximal respiration, basal oxygen consumption rate, and spare respiratory capacity. A second Single Patient Expanded Access IND was obtained for MitoQ, which the patient started at age 5 years and has tolerated at a dose of 10mg twice daily. Anecdotally, the patient's stamina has increased, and her walking ability is stable-to-improved. While the patient's roving eye movements have decreased following exposure to Idebenone and MitoQ, formal ophthalmology examinations demonstrate continued no light perception, and optical coherence tomography (OCT) reveals retinal and ganglion cell layer thinning while photoreceptors remain intact centrally. L-carnitine, biotin, alpha-lipoic acid, riboflavin and vitamin E have been added to the regimen of MitoQ, and in vitro testing in the patient's fibroblasts of these supplements is ongoing. Loss of function of FDX2, encoding the essential iron-sulfur (Fe-S) cluster biogenesis component ferredoxin 2, has been reported to cause optic atrophy, myopathy, partially reversible leukoencephalopathy, and sensorineural axonal neuropathy. We describe the first patient with FDX2 loss of function presenting with blindness (no light perception) at age 17 months. Our evaluation revealed clinical and muscle imaging evidence of a myopathy. The patient was found to harbor novel FDX2 variants: c.271C>T, p.L91F; c.344A>G, p.H115R. In vitro studies in the patient's fibroblasts revealed defective interactions of the FDX2 variants with the Fe-S assembly components, resulting in dysfunction of the mitochondrial oxidative phosphorylation system. We obtained a Single Patient Expanded Access IND for Idebenone, a Co-enzyme Q10 analog with increased solubility, which the patient started at age 3 years. Titration of Idebenone was limited by neutropenia, subsequently recognized to be independent of Idebenone. Treatment of patient fibroblasts with mitoquinol mesylate (MitoQ), a Co-enzyme Q10 analog targeted to the mitochondrial matrix, increased: ATP-linked and maximal respiration, basal oxygen consumption rate, and spare respiratory capacity. A second Single Patient Expanded Access IND was obtained for MitoQ, which the patient started at age 5 years and has tolerated at a dose of 10mg twice daily. Anecdotally, the patient's stamina has increased, and her walking ability is stable-to-improved. While the patient's roving eye movements have decreased following exposure to Idebenone and MitoQ, formal ophthalmology examinations demonstrate continued no light perception, and optical coherence tomography (OCT) reveals retinal and ganglion cell layer thinning while photoreceptors remain intact centrally. L-carnitine, biotin, alpha-lipoic acid, riboflavin and vitamin E have been added to the regimen of MitoQ, and in vitro testing in the patient's fibroblasts of these supplements is ongoing.

Iron homeostasis disruption has increasingly been implicated in various neurological disorders. In this review, we present an overview of our current understanding of iron metabolism in the central nervous system. We examine the consequences of both iron accumulation and deficiency in various disease contexts including neurodegenerative, neurodevelopmental, and neuropsychological disorders. The history of animal models of iron metabolism misregulation is also discussed followed by a comparison of three patients with a newly discovered neurodegenerative disorder caused by mutations in iron regulatory protein 2.

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.

The authors declare no conflict of interest.