FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

HIF-1 regulates iron homeostasis in Caenorhabditis elegans by activation and inhibition of genes involved in iron uptake and storage.

Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1-dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage.

Mammalian iron metabolism and its control by iron regulatory proteins.

Cellular iron homeostasis is maintained by iron regulatory proteins 1 and 2 (IRP1 and IRP2). IRPs bind to iron-responsive elements (IREs) located in the untranslated regions of mRNAs encoding protein involved in iron uptake, storage, utilization and export. Over the past decade, significant progress has been made in understanding how IRPs are regulated by iron-dependent and iron-independent mechanisms and the pathological consequences of IRP2 deficiency in mice. The identification of novel IREs involved in diverse cellular pathways has revealed that the IRP-IRE network extends to processes other than iron homeostasis. A mechanistic understanding of IRP regulation will likely yield important insights into the basis of disorders of iron metabolism. This article is part of a Special Issue entitled: Cell Biology of Metals.

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.

An Erythropoietin-Independent Mechanism of Erythrocytic Precursor Proliferation Underlies Hypoxia Tolerance in Sea Nomads.

The Bajau Sea Nomads were recently demonstrated to have evolved larger spleens as an adaptation to millennia of a marine foraging lifestyle. The large-spleen phenotype appears to derive from increases in thyroid hormone (TH) production as a result of reduced expression of phosphodiesterase 10A (PDE10A), though the exact mechanism remains unknown. Through pharmacological inhibition of PDE10A using the selective inhibitor MP-10 in mice, we were able to mimic the Bajau adaptation and show that treated mice had significantly larger spleens than control animals. This difference appears connected to an excess of early stage erythrocytes and an apparent increase in red blood cell (RBC) precursor proliferation in response to increased TH. However, we determined that the stimulation of RBC production in the mouse model via TH is Erythropoietin (EPO)-independent, unlike in the altitude (chronic hypoxemia) response. We confirmed this using human GWAS data; although the Bajau PDE10A variants are significantly associated with increased TH levels and RBC count, they are not associated with EPO levels, nor are other strongly thyroid-associated SNPs. We therefore suggest that an EPO-independent mechanism of stimulating RBC precursor proliferation via TH upregulation underlies the increase in spleen size observed in Sea Nomad populations.

Irp2 regulates insulin production through iron-mediated Cdkal1-catalyzed tRNA modification.

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in ß cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2-/- ß cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.

HOIL-1 is not required for iron-mediated IRP2 degradation in HEK293 cells.

Iron regulatory protein 2 (IRP2) binds to iron-responsive elements (IREs) to regulate the translation and stability of mRNAs encoding several proteins involved in mammalian iron homeostasis. Increases in cellular iron stimulate the polyubiquitylation and proteasomal degradation of IRP2. One study has suggested that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) binds to a unique 73-amino acid (aa) domain in IRP2 in an iron-dependent manner to regulate IRP2 polyubiquitylation and degradation. Other studies have questioned the role of the 73-aa domain in iron-dependent IRP2 degradation. We investigated the potential role of HOIL-1 in the iron-mediated degradation of IRP2 in human embryonic kidney 293 (HEK293) cells. We found that transiently expressed HOIL-1 and IRP2 interact via the 73-aa domain, but this interaction is not iron-dependent, nor does it enhance the rate of IRP2 degradation by iron. In addition, stable expression of HOIL-1 does not alter the iron-dependent degradation or RNA-binding activity of endogenous IRP2. Reduction of endogenous HOIL-1 by siRNA has no affect on the iron-mediated degradation of endogenous IRP2. These data demonstrate that HOIL-1 is not required for iron-dependent degradation of IRP2 in HEK293 cells, and suggest that a HOIL-1 independent mechanism is used for IRP2 degradation in most cell types.

NHR-14 loss of function couples intestinal iron uptake with innate immunity in C. elegans through PQM-1 signaling.

Iron is essential for survival of most organisms. All organisms have thus developed mechanisms to sense, acquire and sequester iron. In C. elegans, iron uptake and sequestration are regulated by HIF-1. We previously showed that hif-1 mutants are developmentally delayed when grown under iron limitation. Here we identify nhr-14, encoding a nuclear receptor, in a screen conducted for mutations that rescue the developmental delay of hif-1 mutants under iron limitation. nhr-14 loss upregulates the intestinal metal transporter SMF-3 to increase iron uptake in hif-1 mutants. nhr-14 mutants display increased expression of innate immune genes and DAF-16/FoxO-Class II genes, and enhanced resistance to Pseudomonas aeruginosa. These responses are dependent on the transcription factor PQM-1, which localizes to intestinal cell nuclei in nhr-14 mutants. Our data reveal how C. elegans utilizes nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as a component of the innate immune response.

Molecular control of vertebrate iron homeostasis by iron regulatory proteins.

Both deficiencies and excesses of iron represent major public health problems throughout the world. Understanding the cellular and organismal processes controlling iron homeostasis is critical for identifying iron-related diseases and in advancing the clinical treatments for such disorders of iron metabolism. Iron regulatory proteins (IRPs) 1 and 2 are key regulators of vertebrate iron metabolism. These RNA binding proteins post-transcriptionally control the stability or translation of mRNAs encoding proteins involved in iron homeostasis thereby controlling the uptake, utilization, storage or export of iron. Recent evidence provides insight into how IRPs selectively control the translation or stability of target mRNAs, how IRP RNA binding activity is controlled by iron-dependent and iron-independent effectors, and the pathological consequences of dysregulation of the IRP system.

Alteration of iron regulatory proteins (IRP1 and IRP2) and ferritin in the brains of scrapie-infected mice.

Considerable evidence suggests that oxidative stress may be involved in the pathogenesis of Transmissible Spongiform Encephalopathies (TSEs). To investigate the involvement of iron metabolism in TSEs, we examined the expression levels of iron regulatory proteins (IRPs), ferritins, and binding activities of IRPs to iron-responsive element (IRE) in scrapie-infected mice. We found that the IRPs-IRE-binding activities and ferritins were increased in the astrocytes of hippocampus and cerebral cortex in the brains of scrapie-infected mice. These results suggest that alteration of iron metabolism contributes to development of neurodegeneration and that some protective mechanisms against iron-induced oxidative damage may occur during the pathogenesis of TSEs.

Mechanisms of iron metabolism in Caenorhabditis elegans.

Iron is involved in many biological processes essential for sustaining life. In excess, iron is toxic due to its ability to catalyze the formation of free radicals that damage macromolecules. Organisms have developed specialized mechanisms to tightly regulate iron uptake, storage and efflux. Over the past decades, vertebrate model organisms have led to the identification of key genes and pathways that regulate systemic and cellular iron metabolism. This review provides an overview of iron metabolism in the roundworm Caenorhabditis elegans and highlights recent studies on the role of hypoxia and insulin signaling in the regulation of iron metabolism. Given that iron, hypoxia and insulin signaling pathways are evolutionarily conserved, C. elegans provides a genetic model organism that promises to provide new insights into mechanisms regulating mammalian iron metabolism.

Abnormal brain iron metabolism in Irp2 deficient mice is associated with mild neurological and behavioral impairments.

Iron Regulatory Protein 2 (Irp2, Ireb2) is a central regulator of cellular iron homeostasis in vertebrates. Two global knockout mouse models have been generated to explore the role of Irp2 in regulating iron metabolism. While both mouse models show that loss of Irp2 results in microcytic anemia and altered body iron distribution, discrepant results have drawn into question the role of Irp2 in regulating brain iron metabolism. One model shows that aged Irp2 deficient mice develop adult-onset progressive neurodegeneration that is associated with axonal degeneration and loss of Purkinje cells in the central nervous system. These mice show iron deposition in white matter tracts and oligodendrocyte soma throughout the brain. A contrasting model of global Irp2 deficiency shows no overt or pathological signs of neurodegeneration or brain iron accumulation, and display only mild motor coordination and balance deficits when challenged by specific tests. Explanations for conflicting findings in the severity of the clinical phenotype, brain iron accumulation and neuronal degeneration remain unclear. Here, we describe an additional mouse model of global Irp2 deficiency. Our aged Irp2-/- mice show marked iron deposition in white matter and in oligodendrocytes while iron content is significantly reduced in neurons. Ferritin and transferrin receptor 1 (TfR1, Tfrc), expression are increased and decreased, respectively, in the brain from Irp2-/- mice. These mice show impairments in locomotion, exploration, motor coordination/balance and nociception when assessed by neurological and behavioral tests, but lack overt signs of neurodegenerative disease. Ultrastructural studies of specific brain regions show no evidence of neurodegeneration. Our data suggest that Irp2 deficiency dysregulates brain iron metabolism causing cellular dysfunction that ultimately leads to mild neurological, behavioral and nociceptive impairments.

Cysteine oxidation regulates the RNA-binding activity of iron regulatory protein 2.

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.

Control of iron homeostasis by an iron-regulated ubiquitin ligase.

Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.

An iron enhancer element in the FTN-1 gene directs iron-dependent expression in Caenorhabditis elegans intestine.

Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5 '-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATA-binding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.

Iron-independent phosphorylation of iron regulatory protein 2 regulates ferritin during the cell cycle.

Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

Effects of iron regulatory protein regulation on iron homeostasis during hypoxia.

Iron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that affect the translation and stabilization of specific mRNAs by binding to stem-loop structures known as iron responsive elements (IREs). IREs are found in the 5'-untranslated region (UTR) of ferritin (Ft) and mitochondrial aconitase (m-Aco) mRNAs, and in the 3'-UTR of transferrin receptor (TfR) and divalent metal transporter-1 (DMT1) mRNAs. Our previous studies show that besides iron, IRPs are regulated by hypoxia. Here we describe the consequences of IRP regulation and show that iron homeostasis is regulated in 2 phases during hypoxia: an early phase where IRP1 RNA-binding activity decreases and iron uptake and Ft synthesis increase, and a late phase where IRP2 RNA-binding activity increases and iron uptake and Ft synthesis decrease. The increase in iron uptake is independent of DMT1 and TfR, suggesting an unknown transporter. Unlike Ft, m-Aco is not regulated during hypoxia. During the late phase of hypoxia, IRP2 RNA-binding activity increases, becoming the dominant regulator responsible for decreasing Ft synthesis. During reoxygenation (ReO2), Ft protein increases concomitant with a decrease in IRP2 RNA-binding activity. The data suggest that the differential regulation of IRPs during hypoxia may be important for cellular adaptation to low oxygen tension.

Oxygen and iron regulation of iron regulatory protein 2.

Iron regulatory protein 2 (IRP2) is a central regulator of cellular iron homeostasis due to its regulation of specific mRNAs encoding proteins of iron uptake and storage. Iron regulates IRP2 by mediating its rapid proteasomal degradation, where hypoxia and the hypoxia mimetics CoCl2 and desferrioxamine (DFO) stabilize it. Previous studies showed that iron-mediated degradation of IRP2 requires the presence of critical cysteines that reside within a 73-amino acid unique region. Here we show that a mutant IRP2 protein lacking this 73-amino acid region degraded at a rate similar to that of wild-type IRP2. In addition, DFO and hypoxia blocked the degradation of both the wild-type and mutant IRP2 proteins. Recently, members of the 2-oxoglutarate (2-OG)-dependent dioxygenase family have been shown to hydroxylate hypoxia-inducible factor-1 alpha (HIF-1 alpha), a modification required for its ubiquitination and proteasomal degradation. Since 2-OG-dependent dioxygenases require iron and oxygen, in addition to 2-OG, for substrate hydroxylation, we hypothesized that this activity may be involved in the regulation of IRP2 stability. To test this we used the 2-OG-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG) and showed that it blocked iron-mediated IRP2 degradation. In addition, hypoxia, DFO and DMOG blocked IRP2 ubiquitination. These data indicate that the region of IRP2 that is involved in IRP2 iron-mediated degradation lies outside of the 73-amino acid unique region and suggest a model whereby 2-OG-dependent dioxygenase activity may be involved in the oxygen and iron regulation of IRP2 protein stability.

Cytosolic aconitase and ferritin are regulated by iron in Caenorhabditis elegans.

Iron regulatory protein-1 (IRP-1) is a cytosolic RNA-binding protein that is a regulator of iron homeostasis in mammalian cells. IRP-1 binds to RNA structures, known as iron-responsive elements, located in the untranslated regions of specific mRNAs, and it regulates the translation or stability of these mRNAs. Iron regulates IRP-1 activity by converting it from an RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting aconitase activity. IRP-1 is widely found in prokaryotes and eukaryotes. Here, we report the biochemical characterization and regulation of an IRP-1 homolog in Caenorhabditis elegans (GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of the hypodermis and the intestine. Like mammalian IRP-1/aconitases, GEI-22/ACO-1 exhibits aconitase activity and is post-translationally regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian ferritin H and L mRNAs are translationally regulated by iron, the amounts of C. elegans ftn-1 and ftn-2 mRNAs are increased by iron and decreased by iron chelation. Excess iron did not significantly alter worm development but did shorten their life span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates.

Developmental changes in the expression of iron regulatory proteins and iron transport proteins in the perinatal rat brain.

The perinatal brain requires a tightly regulated iron transport system. Iron regulatory proteins (IRPs) 1 and 2 are cytosolic proteins that regulate the stability of mRNA for the two major cellular iron transporters, transferrin receptor (TfR) and divalent metal transporter-1 (DMT-1). We studied the localization of IRPs, their change in expression during perinatal development, and their relationship to TfR and DMT-1 in rat brain between postnatal days (PND) 5 and 15. Twelve-micron frozen coronal sections of fixed brain tissue were obtained from iron-sufficient Sprague-Dawley rat pups on PND 5, 10, and 15, and were visualized at 20 to 1,000x light microscopy for diaminobenzidine activity after incubation with specific primary IRP-1, IRP-2, DMT-1, and TfR antibodies and a universal biotinylated secondary and tertiary antibody system. IRP and transport protein expression increased in parallel over time. IRP1, IRP2, and DMT-1 were partially expressed in the choroid plexus epithelial cells at PND 5 and 10, and fully expressed at PND 15. The cerebral blood vessels and ependymal cells strongly expressed IRP1, IRP2, and DMT-1 as early as PND 5. Substantive TfR staining was not seen in the choroid plexus or ependyma until PND 15. Glial and neuronal expression of IRP1, IRP2, DMT-1, and TfR in cortex, hippocampal subareas and striatum increased over time, but showed variability in cell number and intensity of expression based on brain region, cell type, and age. These developmental changes in IRP and transporter expression suggest potentially different time periods of brain structure vulnerability to iron deficiency or iron overload.