Protein IscU Research Articles

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

The Azotobacter vinelandii molybdenum nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires an existing [Fe-S] cluster to form a [Mo-Fe3-S4] group, which acts as a specific molybdenum donor during nitrogenase FeMo-co biosynthesis. Here, we show biochemical evidence supporting the role of NifU as the [Fe-S] cluster donor. Protein-protein interaction studies involving apo-NifQ and as-isolated NifU demonstrated their interaction, which was only effective when NifQ lacked its [Fe-S] cluster. Incubation of apo-NifQ with [Fe4-S4]-loaded NifU increased the iron content of the former, contingent on both proteins being able to interact with one another. As a result of this interaction, a [Fe4-S4] cluster was transferred from NifU to NifQ. In A. vinelandii, NifQ was preferentially metalated by NifU rather than by the [Fe-S] cluster scaffold protein IscU. These results indicate the necessity of co-expressing NifU and NifQ to efficiently provide molybdenum for FeMo-co biosynthesis when engineering nitrogenase in plants.

Downregulation of Iron-Sulfur Cluster Biogenesis May Contribute to Hyperglycemia-Mediated Diabetic Peripheral Neuropathy in Murine Models.

Diabetic peripheral neuropathy (DPN) is considered one of the most common chronic complications of diabetes. Impairment of mitochondrial function is regarded as one of the causes. Iron-sulfur clusters are essential cofactors for numerous iron-sulfur (Fe-S)-containing proteins/enzymes, including mitochondrial electron transport chain complex I, II, and III and aconitase. To determine the impact of hyperglycemia on peripheral nerves, we used Schwann-like RSC96 cells and classical db/db mice to detect the expression of Fe-S-related proteins, mitochondrially enzymatic activities, and iron metabolism. Subsequently, we treated high-glucose-induced RSC96 cells and db/db mice with pioglitazone (PGZ), respectively, to evaluate the effects on Fe-S cluster biogenesis, mitochondrial function, and animal behavior. We found that the core components of Fe-S biogenesis machinery, such as frataxin (Fxn) and scaffold protein IscU, significantly decreased in high-glucose-induced RSC96 cells and db/db mice, accompanied by compromised mitochondrial Fe-S-containing enzymatic activities, such as complex I and II and aconitase. Consequently, oxidative stress and inflammation increased. PGZ not only has antidiabetic effects but also increases the expression of Fxn and IscU to enhance mitochondrial function in RSC96 cells and db/db mice. Meanwhile, PGZ significantly alleviated sciatic nerve injury and improved peripheral neuronal behavior, accompanied by suppressed oxidative stress and inflammation in the sciatic nerve of the db/db mice. Iron-sulfur cluster deficiency may contribute to hyperglycemia-mediated DPN.

Iron–sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in Escherichiacoli

It was previously postulated that when intracellular free iron content is elevated in bacteria, the Ferric uptake regulator (Fur) binds its co-repressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content, and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur, and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.

Copper exerts cytotoxicity through inhibition of iron-sulfur cluster biogenesis on ISCA1/ISCA2/ISCU assembly proteins.

Copper is an essential mineral nutrient that provides the cofactors for some key enzymes. However, excess copper is paradoxically cytotoxic. Wilson's disease is an autosomal recessive hereditary disease characterized by pathological copper accumulation in many organs, with high mortality and disability. Nevertheless, many questions about the molecular mechanism in Wilson's disease remain unknown and there is an imperative need to address these questions to better exploit therapeutic strategy. In this study, we constructed the mouse model of Wilson's disease, ATP7A-/- immortalized lymphocyte cell line and ATP7B knockdown cells to explore whether copper could impair iron-sulfur cluster biogenesis in eukaryotic mitochondria. Through a series of cellular, molecular, and pharmacological analyses, we demonstrated that copper could suppress the assembly of Fe-S cluster, decrease the activity of the Fe-S enzyme and disorder the mitochondrial function both in vivo and in vitro. Mechanistically, we found that human ISCA1, ISCA2 and ISCU proteins have a strong copper-binding activity, which would hinder the process of iron-sulfur assembly. Of note, we proposed a novel mechanism of action to explain the toxicity of copper by providing evidence that iron-sulfur cluster biogenesis may be a primary target of copper toxicity both in cells and mouse models. In summary, the current work provides an in-depth study on the mechanism of copper intoxication and describes a framework for the further understanding of impaired Fe-S assembly in the pathological processes of Wilson's diseases, which helps to develop latent therapeutic strategies for the management of copper toxicity.

Ferrodifferentiation regulates neurodevelopment via ROS generation.

Iron is important for life, and iron deficiency impairs development, but whether the iron level regulates neural differentiation remains elusive. In this study, with iron-regulatory proteins (IRPs) knockout embryonic stem cells (ESCs) that showed severe iron deficiency, we found that the Pax6- and Sox2-positive neuronal precursor cells and Tuj1 fibers in IRP1-/-IRP2-/- ESCs were significantly decreased after inducing neural differentiation. Consistently, in vivo study showed that the knockdown of IRP1 in IRP2-/- fetal mice remarkably affected the differentiation of neuronal precursors and the migration of neurons. These findings suggest that low intracellular iron status significantly inhibits neurodifferentiation. When supplementing IRP1-/-IRP2-/- ESCs with iron, these ESCs could differentiate normally. Further investigations revealed that the underlying mechanism was associated with an increase in reactive oxygen species (ROS) production caused by the substantially low level of iron and the down-regulation of iron-sulfur cluster protein ISCU, which, in turn, affected the proliferation and differentiation of stem cells. Thus, the appropriate amount of iron is crucial for maintaining normal neural differentiation that is termed ferrodifferentiation.

Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.

Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe-S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe-S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe-S cluster assembly process and establish that the initiation of Fe-S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

Molecular Details of the Frataxin-Scaffold Interaction during Mitochondrial Fe-S Cluster Assembly.

Iron–sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe–S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron–sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe–2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)2 complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe–S cluster assembly.

A novel role of the mitochondrial iron-sulfur cluster assembly protein ISCU-1/ISCU in longevity and stress response.

As an ancient cellular co-factor ubiquitously present in all domains of life, nearly all iron-sulfur ([Fe-S]) clusters are assembled in the mitochondrion. Although multiple mitochondrion-derived signalings are known to be key players in longevity regulation, whether the mitochondrial [Fe-S] cluster assembly machinery modulates lifespan is previously unknown. Here, we find that ISCU-1, the C. elegans ortholog of the evolutionarily conserved iron-sulfur cluster (ISC) assembly machinery central protein ISCU, regulates longevity and stress response. Specifically, ISCU-1 accelerates aging in the intestine. Moreover, we identify the Nrf2 transcription factor SKN-1 and a nuclear hormone receptor NHR-49 as the downstream factors of ISCU-1. Lastly, a mitochondrial outer membrane protein phosphatase PGAM-5 appears to link ISCU-1 to SKN-1 and NHR-49 in lifespan regulation. Together, we have identified a novel function of mitochondrial ISC assembly machinery in longevity modulation and stress response.

Mammalian mitochondrial iron-sulfur cluster biogenesis and transfer and related human diseases.

As a cofactor, iron-sulfur (Fe-S) cluster binds to proteins or enzymes that play important roles in various important biological processes, including DNA synthesis and repair, mitochondrial function, gene transcription and translation. In mammals, the core components involved in Fe-S cluster biosynthesis are considered to include the scaffold protein ISCU, cysteine desulfurase NFS1 and its accessory proteins ISD11 and ACP, and frataxin (FXN). Proteins involved in Fe-S cluster transfer have been found to include HSC20/HSPA9, as chaperone system, and Fe-S cluster carriers. The biosynthesis and transfer of Fe-S clusters to Fe-S recipients require fine-tune regulation. Recently, significant progress has been made in the structure and mechanism of mitochondrial Fe-S biosynthesis and transfer. Based on, especially, the development of DNA sequencing technology, bioinformatics, and gene editing technology, diseases caused by mutations of Fe-S cluster-related genes have been revealed in recent years, promoting the rapid development in the field of Fe-S and human health. This review focuses on the function of genes involved in Fe-S cluster biosynthesis and transfer and on the diseases caused by the mutations of the related genes. Finally, some questions we are facing are raised, new hypotheses presented, and the perspectives discussed.

Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron-sulfur cluster biosynthesis.

IscU is a central component of the ISC machinery and serves as a scaffold for de novo assembly of Fe-S clusters. The dedicated chaperone system composed of the Hsp70-chaperone HscA and the J-protein cochaperone HscB synergistically interacts with IscU and facilitates cluster transfer from IscU to recipient apo-proteins. Here, we report that the otherwise essential roles of HscA and HscB can be bypassed in vivo by a number of single amino acid substitutions in IscU. CD spectroscopic studies of the variant IscU proteins capable of this bypass activity revealed dynamic interconversion between two conformations: the denatured (D) and the structured (S) state in the absence and presence of Zn2+ , respectively, which was far more prominent than interconversion observed in wild-type IscU. Furthermore, we found that neither the S-shifted (more structured) variants of IscU nor the perpetually denatured variants could perform their in vivo role regardless of whether the chaperone system was present or not. The present study thus provides for the first time evidence that an in vivo D-state of IscU exists and implies that conformational interconversion between the S- and D-states of the scaffolding protein is a fundamental requirement for the assembly and transfer of the Fe-S cluster.

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.

Structure of the Human ACP-ISD11 Heterodimer.

In recent years, the mammalian mitochondrial protein complex for iron-sulfur cluster assembly has been the focus of important studies. This is partly because of its high degree of relevance in cell metabolism and because mutations of the involved proteins are the cause of several human diseases. Cysteine desulfurase NFS1 is the key enzyme of the complex. At present, it is well-known that the active form of NFS1 is stabilized by the small protein ISD11. In this work, the structure of the human mitochondrial ACP-ISD11 heterodimer was determined at 2.0 Å resolution. ACP-ISD11 forms a cooperative unit stabilized by several ionic interactions, hydrogen bonds, and apolar interactions. The 4'-phosphopantetheine-acyl chain, which is covalently bound to ACP, interacts with several residues of ISD11, modulating together with ACP the foldability of ISD11. Recombinant human ACP-ISD11 was able to interact with the NFS1 desulfurase, thus yielding an active enzyme, and the NFS1/ACP-ISD11 core complex was activated by frataxin and ISCU proteins. Internal motions of ACP-ISD11 were studied by molecular dynamics simulations, showing the persistence of the interactions between both protein chains. The conformation of the dimer is similar to that found in the context of the (NFS1/ACP-ISD11)2 supercomplex core, which contains the Escherichia coli ACP instead of the human variant. This fact suggests a sequential mechanism for supercomplex consolidation, in which the ACP-ISD11 complex may fold independently and, after that, the NFS1 dimer would be stabilized.

P.54Defects in iron-sulphur cluster assembly proteins ISCU and FDX2 cause characteristic mitochondrial myopathy

P.54Defects in iron-sulphur cluster assembly proteins ISCU and FDX2 cause characteristic mitochondrial myopathy

Understanding the Mechanism of [4Fe-4S] Cluster Assembly on Eukaryotic Mitochondrial and Cytosolic Aconitase.

Iron-sulfur (Fe-S) clusters are common prosthetic groups that are found within a variety of proteins responsible for functions that include electron transfer, regulation of gene expression, and substrate binding and activation. Acquisition of a [4Fe-4S] cluster is essential for the functionality of many such roles, and dysfunctions in Fe-S cluster synthesis and trafficking often result in human disease, such as multiple mitochondrial dysfunctions syndrome. While the topic of [2Fe-2S] cluster biosynthesis and trafficking has been relatively well studied, the understanding of such processes involving [4Fe-4S] centers is less developed. Herein, we focus on the mechanism of the assembly of [4Fe-4S] clusters on two members of the aconitase family, differing also in organelle placement (mitochondrion and cytosol) and biochemical function. Two mechanistic models are evaluated by a combination of kinetic and spectroscopic models, namely, a consecutive model (I), in which two [2Fe-2S] clusters are sequentially delivered to the target, and a prereaction equilibrium model (II), in which a [4Fe-4S] cluster transiently forms on a donor protein before transfer to the target. The paper also addresses the issue of cluster nuclearity for functionally active forms of ISCU, NFU, and ISCA trafficking proteins, each of which has been postulated to exist in both [2Fe-2S] and [4Fe-4S] bound states. By the application of kinetic assays and electron paramagnetic resonance spectroscopy to examine delivery pathways from a variety of potential [2Fe-2S] donor proteins to eukaryotic forms of both aconitase and iron regulatory protein, we conclude that a consecutive model following the delivery of [2Fe-2S] clusters from NFU1 is the most likely mechanism for these target proteins.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich’s ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich’s ataxia therapies.

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein–protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.

Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS

AbstractIn macrophages, cellular iron metabolism status is tightly integrated with macrophage phenotype and associated with mitochondrial function. However, how molecular events regulate mitochondrial activity to integrate regulation of iron metabolism and macrophage phenotype remains unclear. Here, we explored the important role of the actin-regulatory protein glia maturation factor-γ (GMFG) in the regulation of cellular iron metabolism and macrophage phenotype. We found that GMFG was downregulated in murine macrophages by exposure to iron and hydrogen peroxide. GMFG knockdown altered the expression of iron metabolism proteins and increased iron levels in murine macrophages and concomitantly promoted their polarization toward an anti-inflammatory M2 phenotype. GMFG-knockdown macrophages exhibited moderately increased levels of mitochondrial reactive oxygen species (mtROS), which were accompanied by decreased expression of some mitochondrial respiration chain components, including the iron-sulfur cluster assembly scaffold protein ISCU as well as the antioxidant enzymes SOD1 and SOD2. Importantly, treatment of GMFG-knockdown macrophages with the antioxidant N-acetylcysteine reversed the altered expression of iron metabolism proteins and significantly inhibited the enhanced gene expression of M2 macrophage markers, suggesting that mtROS is mechanistically linked to cellular iron metabolism and macrophage phenotype. Finally, GMFG interacted with the mitochondrial membrane ATPase ATAD3A, suggesting that GMFG knockdown–induced mtROS production might be attributed to alteration of mitochondrial function in macrophages. Our findings suggest that GMFG is an important regulator in cellular iron metabolism and macrophage phenotype and could be a novel therapeutic target for modulating macrophage function in immune and metabolic disorders.

AMPK Regulates the Expression of the Fe-S Cluster Assembly Enzyme (ISCU) and ALAS2, Modulating Cellular Iron Metabolism and Increasing Hemoglobin Synthesis

Iron-sulfur (Fe-S) clusters are iron-containing prosthetic groups and enzymatic cofactors. They are strong oxidants when unbound yet essential in many processes like facilitating ATP production in mitochondria, promoting DNA, RNA and protein syntheses during cell proliferation and enhancing DNA repair in antioxidant defense. In particular, Fe-S clusters are indispensable in erythropoiesis, where the majority of physiological iron is utilized and where Fe-S clusters are required for the heme synthesis. Deficient Fe-S cluster synthesis predisposes individual to various diseases, such as cancer, metabolic and neurodegeneration diseases and blood disorders. However, it is unclear how Fe-S cluster synthesis is regulated and coordinates with environmental and developmental needs to prevent oxidative damage.The 5' AMP-activated protein kinase (AMPK) is a kinase activated by oxidative stress and energy starvation and critical for maintaining redox and energy homeostasis. In this study, we investigated the role of AMPK on Fe-S clusters synthesis and function and extended our findings in normal and thalassemic erythroid cells.Through bioinformatic analysis, we found that the Fe-S cluster assembly enzyme (ISCU), a scaffold protein indispensable for Fe-S cluster biogenesis, contains putative AMPK phosphorylation motifs at serine (S) residues 14 and 29 (human numbering). Using the human cell line 293T, we confirmed that AMPK phosphorylates ISCU, while point mutations in these residues prevented this activity. Moreover, AMPK-mediated phosphorylation promoted ISCU binding to 14-3-3s, a family of proteins that, once associate with phosphorylated residues, modulates the stability and function of targeted proteins. Indeed, increased association with 14-3-3s stabilized ISCU proteins, corroborating the observation that AMPK promotes the activity of ISCU proteins.We extended our studies using A549 cells that do not have AMPK activity since they harbor a mutant LKB1 kinase, which is responsible for activating AMPK. By overexpression of wild type (WT)-LKB1 and LKB1 kinase-dead mutant (KDM), we found that only WT-LKB1 restored AMPK activity, binding of ISCU to 14-3-3s and stability of ISCU. Moreover, under hydrogen peroxide incubation and glucose starvation, ISCU protein levels and Fe-S cluster synthesis were both increased only in the presence of LKB1-WT, but not in cells harboring KMD. LKB1-WT overexpressed cells also survived hydrogen peroxide incubation and glucose starvation better than those with KMD. Together, these data suggest that AMPK activation stabilizes ISCU protein and preserves Fe-S cluster synthesis to maintain a healthy redox and energy homeostasis.We then explored the effect of AMPK on Fe-S cluster synthesis in erythropoiesis by using the drug AICAR, an AMPK activator, in murine erythroleukemia (MEL) cells. We found that in MEL cells, AICAR treatment stabilized ISCU, increased Fe-S cluster levels and promoted the synthesis of the aminolevulinic acid synthase 2 (ALAS2) protein, which represents the rate-limiting enzyme in erythroid heme synthesis. Furthermore, this was associated with increased heme and globin chain synthesis, with a trend in increasing β-globin mRNA and proteins more than α-globin. We further confirmed these observations in Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP-2) and CD34+ cells derived from peripheral blood isolated from both healthy individuals and ß-thalassemic patients. In these cells, we found that AMPK upregulation by AICAR administration not only increased ALAS2 expression and erythroid heme levels, but also enhanced the synthesis of both a- and ß-globin chains, though with a preference for increasing β-globin levels. Analysis using specimens from thalassemic mice is in progress.In conclusion, our work demonstrates that under redox and energetic stress, activated AMPK phosphorylates and stabilizes ISCU protein, thereby enhancing Fe-S cluster synthesis and maintaining their function. Moreover, AMPK activation with AICAR treatment increases erythroid heme synthesis and hemoglobin expression. Given that AMPK is the major kinase that responds to oxidative and energetic cues, our work provides a mechanistic explanation for how erythropoiesis responds to energy starvation and redox stress as well as a potential novel therapeutic target to treat blood and metabolic disorders. DisclosuresRivella:Ionis Pharmaceuticals, Inc: Consultancy; MeiraGTx: Other: SAB; Protagonist: Consultancy; Disc Medicine: Consultancy.

MITOCHONDRIAL DISEASES (Posters): P.194Deficiency of the iron-sulphur cluster assembly protein ISCU causes impaired biogenesis or stability of respiratory chain complex I, II and IV in muscle

MITOCHONDRIAL DISEASES (Posters): P.194Deficiency of the iron-sulphur cluster assembly protein ISCU causes impaired biogenesis or stability of respiratory chain complex I, II and IV in muscle

Zinc(II) binding on human wild-type ISCU and Met140 variants modulates NFS1 desulfurase activity

Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity.