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

Clarke, Stephen; Vasanthakumar, Aparna; Anderson, Sheila A.; Pondarré, Corinne; Koh, Cheryl M.; Deck, Kathryn M.; Pitula, Joseph S.; Epstein, Charles J.; Fleming, Mark D.; Eisenstein, Richard S.

doi:10.1038/sj.emboj.7600954

Cited by 87 publications

(120 citation statements)

References 54 publications

(99 reference statements)

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“…4B). For IRP1 the decrease is readily explained by the common observation that apo-IRP1 lacking its Fe/S cluster is more prone to degradation (26,35). Because IRP2 protein levels are negatively regulated by the iron availability in the cytosol (34), our results suggest that cytosolic iron was diminished.…”

Section: Fdx2 Depletion Adversely Affects Cellular Iron Metabolism Insupporting

confidence: 46%

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

Sheftel

Stehling

Pierik

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

313

296

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Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.adrenodoxin | cytochrome P450 | iron | iron-sulfur cluster | IRP1

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Section: Fdx2 Depletion Adversely Affects Cellular Iron Metabolism Insupporting

confidence: 46%

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

Sheftel

Stehling

Pierik

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

313

296

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“…6A, second panel from top). We as well as others have observed that the apo form of IRP1 is less stable than the iron-replete form (18,33,34). Consistent with this, Irp1 protein levels also decline upon Iop1 knock-out (Fig.…”

Section: Resultssupporting

confidence: 72%

Mouse Knock-out of IOP1 Protein Reveals Its Essential Role in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis

Song

Lee

2011

Journal of Biological Chemistry

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Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.Iron-sulfur cluster proteins are an important class of proteins that contain iron-sulfur clusters in a variety of configurations, including [2Fe-2S] and [4Fe-4S] (1, 2). The distinctive redox and biophysical properties of these clusters make them suitable for a range of functions. Consequently, iron-sulfur proteins have diverse roles and participate in a broad range of biologic processes, including Krebs cycle reactions, oxidative phosphorylation, translation, iron regulatory pathways, and purine metabolism. Notably, these proteins exist in many cellular locales, including the cytosol, mitochondria, and nucleus.How iron-sulfur cluster proteins are assembled in different cellular compartments has been an area of active investigation (3, 4). Iron-sulfur cluster assembly for all compartments originates in the mitochondria, and the initial step is catalyzed by a cysteine desulfurase that removes elemental sulfur from cysteine. The incorporation of this sulfur into iron-sulfur clusters then occurs by mechanisms that depend, in part, on cell compartment-specific machineries. Such a pathway exists, for example, for cytosolic iron-sulfur proteins. Important insights into this pathway, the cytosolic iron-sulfur protein assembly (CIA) 2 pathway, have been obtained from studies in Saccharomyces cerevisiae (3, 5). These studies have revealed a series of proteins that, in a stepwise fashion, assemble and deliver ironsulfur clusters to target proteins. A current model is that the proteins Tah18 and Dre2 provide reducing equivalents for the assemb...

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“…1A). Thiol mutagenesis and modification analyses have revealed that IRP1 C437 is critical for not only [4Fe-4S] coordination but also IRP1 RNA binding and protein stability (7,23,38,51). Sequence alignment between IRP1 and IRP2 shows that seven of the nine cysteine residues in human IRP1 are conserved in human IRP2, including the three cysteines required for IRP1 [4Fe-4S] cluster binding (IRP2 C512, C578, and C581) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2

Zumbrennen¹,

Wallander²,

Romney

et al. 2009

Molecular and Cellular Biology

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Iron regulatory protein 2 (IRP2) is an RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as ferritin and transferrin receptor 1. IRP2 RNA-binding activity is primarily regulated by iron-mediated proteasomal degradation, but studies have suggested that IRP2 RNA binding is also regulated by thiol oxidation. We generated a model of IRP2 bound to RNA and found that two cysteines (C512 and C516) are predicted to lie in the RNA-binding cleft. Site-directed mutagenesis and thiol modification show that, while IRP2 C512 and C516 do not directly interact with RNA, both cysteines are located within the RNA-binding cleft and must be unmodified/reduced for IRP2-RNA interactions. Oxidative stress induced by cellular glucose deprivation reduces the RNA-binding activity of IRP2 but not IRP2-C512S or IRP2-C516S, consistent with the formation of a disulfide bond between IRP2 C512 and C516 during oxidative stress. Decreased IRP2 RNA binding is correlated with reduced transferrin receptor 1 mRNA abundance. These studies provide insight into the structural basis for IRP2-RNA interactions and reveal an iron-independent mechanism for regulating iron homeostasis through the redox regulation of IRP2 cysteines.Iron is an essential nutrient required for a variety of cellular processes, including DNA synthesis, respiration, and heme biosynthesis. However, ferrous iron readily reacts with hydroperoxides to produce hydroxyl radicals that can cause cellular damage. As both iron excess and deficiency are deleterious, cells have developed mechanisms to ensure that iron levels are sufficient for cellular need but at the same time limit iron toxicity.Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are the key iron sensors in mammalian cells (32,48). IRPs are cytosolic proteins that bind RNA stem-loops known as iron-responsive elements (IREs) located in the 5Ј or 3Ј untranslated regions of mRNAs encoding proteins involved in iron homeostasis. IRP binding to a 5Ј IRE present in ferritin (iron storage) or ferroportin (iron exporter) mRNAs represses protein translation. IRP binding to 3Ј IREs, such as those in transferrin receptor 1 (TfR-1) and divalent metal transporter 1 (DMT1) (iron importers), stabilizes the mRNA and increases protein expression. While both IRPs function as RNA-binding proteins when iron content is low, increased cellular iron regulates IRP1 and IRP2 by different mechanisms. Increased cellular iron results in the assembly of an [4Fe-4S] cluster in the RNA-binding cleft of IRP1, which allows IRP1 to function as a cytosolic aconitase. Unlike IRP1, IRP2 does not coordinate an [4Fe-4S] cluster or function as an aconitase. Instead, the RNA-binding activity of IRP2 is reduced by iron-dependent polyubiquitylation and proteasomal degradation. The role of IRP2 as the predominant RNA-binding protein in vivo has been established in murine knockout models in which Irp1 Ϫ/Ϫ mice display no overt phenotype, whereas Irp2 Ϫ/Ϫ mice develop microcytic anemia and locomotor deficits...

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

Cited by 87 publications

References 54 publications

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

Mouse Knock-out of IOP1 Protein Reveals Its Essential Role in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis

Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2

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