Iron regulatory protein 1 is required for the propagation of inflammation in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are complex disorders. Iron accumulates in the inflamed tissue of IBD patients, yet neither a mechanism for the accumulation nor its implication on the course of inflammation is known. We hypothesized that the inflammation modifies iron homeostasis, affects tissue iron distribution, and that this in turn perpetuates the inflammation. This study analyzed human biopsies, animal models, and cellular systems to decipher the role of iron homeostasis in IBD. We found inflammation-mediated modifications of iron distribution, and iron-decoupled activation of the iron regulatory protein (IRP) 1. To understand the role of IRP1 in the course of this inflammation-associated iron pattern, a novel cellular coculture model was established, which replicated the iron-pattern observed in vivo, and supported involvement of nitric oxide in the activation of IRP1 and the typical iron pattern in inflammation. Importantly, deletion of IRP1 from an IBD mouse model completely abolished both, the misdistribution of iron and intestinal inflammation. These findings suggest that IRP1 plays a central role in the coordination of the inflammatory response in the intestinal mucosa and that it is a viable candidate for therapeutic intervention in IBD.

A novel treatment strategy to prevent Parkinson's disease: focus on iron regulatory protein 1 (IRP1).

We propose that neural damage in Parkinson's disease (PD) is due to dysregulation of iron utilization rather than to high iron levels per se. Iron deposits are associated with neuronal cell death in substantia nigra (SN) resulting in PD where high levels of iron in SNs are due to dysregulation of iron utilization. Cytosolic aconitase (ACO1) upon losing an iron-sulfur cluster becomes iron regulatory protein 1 (IRP1). Rotenone increases levels of IRP1 and induces PD in rats. An increase in iron leads to inactivation of IRP1. We propose a novel treatment strategy to prevent PD. Specifically in rats given rotenone by subcutaneous injections, iron, from iron carbonyl from which iron is slowly absorbed, given three times a day by gavage will keep iron levels constant in the gut whereby iron levels and iron utilization systematically can be tightly regulated. Rotenone adversely affects complex 1 iron-sulfur proteins. Iron supplementation will increase iron-sulfur cluster formation switching IRP1 to ACO1. With IRP1 levels kept constantly low, iron utilization will systematically be tightly regulated stopping dysregulation of complex 1 and the neural damage done by rotenone preventing PD.

PPARδ Activation Mitigates 6-OHDA-Induced Neuronal Damage by Regulating Intracellular Iron Levels.

Intracellular iron accumulation in dopaminergic neurons contributes to neuronal cell death in progressive neurodegenerative disorders such as Parkinson's disease. However, the mechanisms of iron homeostasis in this context remain incompletely understood. In the present study, we assessed the role of the nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) in cellular iron homeostasis. We identified that PPARδ inhibited 6-hydroxydopamine (6-OHDA)-triggered neurotoxicity in SH-SY5Y neuroblastoma cells. PPARδ activation with GW501516, a specific PPARδ agonist, mitigated 6-OHDA-induced neuronal damage. Further, PPARδ activation also suppressed iron accumulation, which contributes to 6-OHDA-induced neuronal damage. PPARδ activation attenuated 6-OHDA-induced neuronal damage in a similar manner to that of the iron chelator deferoxamine. We further elucidated that PPARδ modulated cellular iron homeostasis by regulating expression of divalent metal transporter 1, ferroportin 1, and ferritin, but not transferrin receptor 1, through iron regulatory protein 1 in 6-OHDA-treated cells. Interestingly, PPARδ activation suppressed 6-OHDA-triggered generation of reactive oxygen species and lipid peroxides. The effects of GW501516 were abrogated by shRNA knockdown of PPARδ, indicating that the effects of GW501516 were PPARδ-dependent. Taken together, these findings suggest that PPARδ attenuates 6-OHDA-induced neurotoxicity by preventing intracellular iron accumulation, thereby suppressing iron overload-associated generation of reactive oxygen species and lipid peroxides, key mediators of ferroptotic cell death.

Iron Regulatory Protein 1 Inhibits Ferritin Translation Responding to OsHV-1 Infection in Ark Clams, Scapharca Broughtonii.

Elemental iron is an indispensable prosthetic group of DNA replication relative enzymes. The upregulation of ferritin translation by iron regulatory proteins (IRP1) in host cells is a nutritional immune strategy to sequester available iron to pathogens. The efficient replication of Ostreid herpesvirus 1 (OsHV-1), a lethal dsDNA virus among bivalves, depends on available iron. OsHV-1 infection was found to trigger iron limitation in ark clams; however, it is still an enigma how OsHV-1 successfully conducted rapid replication, escaping host iron limitations. In this study, we identified the IRP1 protein (designated as SbIRP-1) in the ark clam (Scapharca broughtonii) and found it could bind to the iron-responsive element (IRE) of ferritin (SbFn) mRNA based on electrophoretic mobility shift assay (EMSA). Knockdown of SbIRP-1 expression (0.24 ± 1.82-fold of that in NC group, p < 0.01) by RNA interference resulted in the accumulation of SbFn in hemocytes (1.79 ± 0.01-fold, p < 0.01) post-24 h of enhanced RNA interference injection. During OsHV-1 infection, SbFn mRNA was significantly upregulated in hemocytes from 24 h to 60 h, while its protein level was significantly reduced from 24 h to 48 h, with the lowest value at 36 h post-infection (0.11 ± 0.01-fold, p < 0.01). Further analysis by RNA immunoprecipitation assays showed that OsHV-1 could enhance the binding of SbIRP-1 with the SbFn IRE, which was significantly increased (2.17 ± 0.25-fold, p < 0.01) at 36 h post-infection. Consistently, SbIRP-1 protein expression was significantly increased in hemocytes from 12 h to 48 h post OsHV-1 infection (p < 0.01). In conclusion, the results suggest that OsHV-1 infection could suppress post-transcriptional translation of SbFn through the regulation of SbIRP-1, which likely contributes to OsHV-1 evasion of SbFn-mediating host iron limitation.

6-Hydroxydopamine Induces Abnormal Iron Sequestration in BV2 Microglia by Activating Iron Regulatory Protein 1 and Inhibiting Hepcidin Release.

Disrupted iron homeostasis in the substantia nigra pars compacta (SNpc) is an important pathological mechanism in Parkinson's disease (PD). It is unclear what role microglia play in iron metabolism and selective iron deposition in the SNpc of PD brain. In this study, we observed that 6-hydroxydopamine (6-OHDA) induced the expression of divalent metal transporter-1 (DMT1) and iron influx in BV2 microglia cells, which might be associated with the upregulation of iron regulatory protein 1 (IRP1) expression. Moreover, we found that 6-OHDA had no significant effect on the expression of ferroportin 1 (FPN1) and iron efflux in BV2 microglial cells, which might be the combined action of IRP1 upregulation and reduced hepcidin levels. Furthermore, 6-OHDA treatment activated BV2 microglia and enhanced the release of pro-inflammatory cytokines. Interestingly, iron overloading suppressed IRP1 expression, thus downregulating DMT1 and upregulating FPN1 levels in these microglial cells. On the contrary, iron deficiency activated IRP1, leading to increased expression of DMT1 and decreased expression of FPN1-which indicates that activated IRP1 induces iron overloading in 6-OHDA-treated microglia, but not iron overloading modulates the expression of IRP1. Taken together, our data suggest that 6-OHDA can regulate the expression of DMT1 and FPN1 by activating IRP1 and inhibiting hepcidin release, thus leading to abnormal iron sequestration in microglia. In addition, 6-OHDA can activate microglia, which leads to increased release of pro-inflammatory factors that can further induce genome damage in dopaminergic neurons.

Baicalein Attenuates Brain Iron Accumulation through Protecting Aconitase 1 from Oxidative Stress in Rotenone-Induced Parkinson's Disease in Rats.

Aconitase 1 (ACO1) links oxidative stress and iron accumulation in Parkinson's disease (PD). ACO1 loses its aconitase activity and turns into iron regulatory protein 1 (IRP1) upon oxidative stress. IRP1 plays an important role in the accumulation of intracellular iron. Baicalein is a flavonoid isolated from the roots of Scutellaria baicalensis. The present results show that baicalein could bind to ACO1 and protect its isoform from the oxidative stress induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Furthermore, baicalein promoted aconitase activity and inhibited IRP1 activation in rotenone-induced PD models. Additionally, baicalein decreased the hydroxyl radicals generated by iron. In conclusion, baicalein attenuated iron accumulation and iron-induced oxidative stress in the brain of PD rats by protecting ACO1.

Posiphen Reduces the Levels of Huntingtin Protein through Translation Suppression.

Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Posiphen's safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington's disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5'-untranslated regions (5'-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.

Iron regulatory protein 1 promotes ferroptosis by sustaining cellular iron homeostasis in melanoma.

Melanoma, the most aggressive skin cancer, is mainly treated with BRAF inhibitors or immunotheareapy. However, most patients who initially responded to BRAF inhibitors or immunotheareapy become resistant following relapse. Ferroptosis is a form of regulated cell death characterized by its dependence on iron ions and the accumulation of lipid reactive oxygen species (ROS). Recent studies have demonstrated that ferroptosis is a good method for tumor treatment, and iron homeostasis is closely associated with ferroptosis. Iron regulatory protein (IRP)1 and 2 play important roles in maintaining iron homeostasis, but their functions in ferroptosis have not been investigated. The present study reported that the expression of IRP1 and IRP2 was increased by the ferroptosis inducers erastin and RSL3 in melanoma cells. Depletion of IRP1 significantly suppressed erastin- and RSL3-induced ferroptosis. IRP2 had a weak effect but could enhance the promoting function of IRP1 on ferroptosis. Further, erastin and RSL3 promoted the transition of aconitase 1 to IRP1, which regulated downstream iron metabolism proteins, including transferrin receptor (TFRC), ferroportin (FPN) and ferritin heavy chain 1 (FTH1). Moreover, overexpression of TFRC and knockdown of FPN and FTH1 significantly promoted erastin- and RSL3-induced ferroptosis in IRP1 knockdown melanoma cells. Collectively, the present findings indicate that IRP1 plays an essential role in erastin- and RSL3-induced ferroptosis by regulating iron homeostasis.

Inflaming the Brain with Iron.

Iron accumulation and neuroinflammation are pathological conditions found in several neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Iron and inflammation are intertwined in a bidirectional relationship, where iron modifies the inflammatory phenotype of microglia and infiltrating macrophages, and in turn, these cells secrete diffusible mediators that reshape neuronal iron homeostasis and regulate iron entry into the brain. Secreted inflammatory mediators include cytokines and reactive oxygen/nitrogen species (ROS/RNS), notably hepcidin and nitric oxide (·NO). Hepcidin is a small cationic peptide with a central role in regulating systemic iron homeostasis. Also present in the cerebrospinal fluid (CSF), hepcidin can reduce iron export from neurons and decreases iron entry through the blood-brain barrier (BBB) by binding to the iron exporter ferroportin 1 (Fpn1). Likewise, ·NO selectively converts cytosolic aconitase (c-aconitase) into the iron regulatory protein 1 (IRP1), which regulates cellular iron homeostasis through its binding to iron response elements (IRE) located in the mRNAs of iron-related proteins. Nitric oxide-activated IRP1 can impair cellular iron homeostasis during neuroinflammation, triggering iron accumulation, especially in the mitochondria, leading to neuronal death. In this review, we will summarize findings that connect neuroinflammation and iron accumulation, which support their causal association in the neurodegenerative processes observed in AD and PD.

Hepatocyte growth factor protects PC12 cells against OGD/R-induced injury by reducing iron.

In the light of hepatocyte growth factor (HGF) the inhibiting role on the expression of hepcidin, we hypothesized that HGF might be able to reduce cell and tissue iron by increasing ferroportin 1 (Fpn1) content and Fpn1-mediated iron release from cells and tissues. The hypothesized ability of HGF to reduce iron might be one of the mechanisms associated with its neuroprotective action under the conditions of ischemia/reperfusion (I/R). Here, we investigated the effects of HGF on the expression of hepcidin as well as transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), Fpn1, ferritin and iron regulatory proteins (IRPs) in oxygen-glucose deprivation and reoxygenation (OGD/R)-treated PC12 cells by real-time PCR and Western blot analysis. We demonstrated that HGF could completely reverse the OGD/R-induced reduction in Fpn1 and IRP1 expression and increase in ferritin light chain protein and hepcidin mRNA levels in PC12 cells. It was concluded that HGF protects PC12 cells against OGD/R-induced injury mainly by reducing cell iron contents via the up-regulation of Fpn1 and increased Fpn1-mediated iron export from cells. Our findings suggested that HGF may also be able to ameliorate OGD/R or I/R-induced overloading of brain iron by promoting Fpn1 expression.

Regulating ferroportin-1 and transferrin receptor-1 expression: A novel function of hydrogen sulfide.

Hydrogen sulfide (H2 S) has a significant effect on the regulation of interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) activities, while IL-6 directly regulates hepcidin expression via STAT3. We therefore hypothesized that H 2 S has a role in body iron homeostasis by regulating the expression of iron transport proteins via the IL-6/STAT3/Hepcidin pathway. Here, we investigated the effects of two H 2 S donors sodium hydrosulfide and GYY4137 on the expression of ferroportin-1 (Fpn1), transferrin receptor-1 (TfR1), hepcidin, IL-6 and pSTAT3 in the spleen of mice in vivo and peritoneal macrophage in vitro. We also examined the effects of H 2 S on serum iron, transferrin saturation, and ferritin light chain contents in the spleen, and on nitrite content, nuclear factor erythroid 2-related factor-2 (Nrf2) and iron regulatory protein 1 (IRP1) in the macrophages. We demonstrated that H 2 S regulates the expression of TfR1 and Fpn1 in the spleen in vivo and in peritoneal macrophages in vitro predominantly via the IL-6/pSTAT3/hepcidin pathway, under the conditions of inflammation induced by lipopolysaccharides. We also provide evidence that under uninflamed conditions, the regulation of Fpn1 and TfR1 expression by H 2 S, both in vivo and in vitro, are mediated by the nitric oxide (NO)/Nrf2 and iron regulatory protein/iron responsive element pathways, respectively, which are independent of IL-6/pSTAT3/hepcidin signals. These findings show that H 2 S is a key player in iron homeostasis under not only the inflamed conditions but also uninflamed conditions.

The roles of hypoxia-inducible Factor-1 and iron regulatory protein 1 in iron uptake induced by acute hypoxia.

BACKGROUND INFORMATION: Divalent metal transporter 1 (DMT1) and transferrin receptor (TfR1) are vital proteins for cellular iron uptake. These proteins have hypoxia-responsive elements (HREs) in their 5'-regulatory region, and they are regulated by hypoxia-inducible factor 1α (HIF-1α) transcriptionally under hypoxic condition. Besides, iron regulatory protein 1 (IRP1) regulates DMT1 and TfR1 by binding to iron-responsive elements (IREs) present in their mRNAs to control cellular iron homeostasis. RESULTS: Here, we explored the effect of acute hypoxia on iron uptake. Ferrous iron uptake was elevated by DMT1(+IRE) and TfR1 under acute hypoxia. The luciferase activity analysis revealed that the functional HREs of DMT1 and TfR1 were increased. However, their IREs-dependent luciferase activities were reduced simultaneously. The mRNA stability of TfR1 and DMT1(+IRE) was suppressed under acute hypoxia. The mRNA levels of TfR1 and DMT1(+IRE) were restrain by silencing IRP1. In sharp contrast, HIF-1α overexpression enhanced the mRNA levels of TfR1 and DMT1(+IRE), which reversed the inhibition of IRP1 on both. HIF-1α konckdown suppressed the hypoxia-induced increase expression of TfR1 and DMT1(+IRE), whereas both proteins had little change when further decreased the IRP1 expression under hypoxia. Hypoxia upregulated the protein expression of Ferrtin-L in a time-dependent manner, yet there was no different when IRP1 silencing or overexperssion under hypoxia. The lactate dehydrogenase (LDH) release induced by hypoxia was increased by TfR1 siRNA silence. CONCLUSIONS: We propose that HIF-1/HRE system might play a principal part in hypoxia induced iron uptake.

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation.

Iron-regulatory protein 1 (IRP1) belongs to a family of RNA-binding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic iron-sulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRP13C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of iron-response element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.

Effect of iron on cholesterol 7α-hydroxylase expression in alcohol-induced hepatic steatosis in mice.

Both iron and lipids are involved in the progression of alcoholic fatty liver disease (AFLD), but the interaction between iron and lipids in AFLD is unclear. Here, we tested the hypothesis that iron regulates the expression of genes involved in lipid metabolism through iron regulatory proteins (IRPs), which interact with the iron-responsive elements (IREs) in the untranslated regions (UTRs) of genes, resulting in lipid accumulation. Using "RNA structure software", we predicted the mRNA secondary structures of more than 100 genes involved in lipid metabolism to investigate whether the IRE structure exists in novel mRNAs. Cholesterol 7α-hydroxylase (Cyp7a1) has an IRE-like stem-loop, a noncanonical IRE structure, in its 3'-UTR. Cyp7a1 expression can be regulated by in vivo and in vitro iron treatment. In addition, the noncanonical IRE motif can efficiently bind both to IRP1 and IRP2. The results indicate that hepatic iron overloading in AFLD mice decreased Cyp7a1 expression and resulted in cholesterol accumulation, providing a new mechanism of iron-regulated gene transcription and translation through the interaction between iron and a noncanonical IRE structure in Cyp7a1 mRNA. This finding has significant implications in studying a proposed mechanism for the regulation of cholesterol homeostasis by an Fe/IRP/noncanonical IRE axis.

Aspirin down Regulates Hepcidin by Inhibiting NF-κB and IL6/JAK2/STAT3 Pathways in BV-2 Microglial Cells Treated with Lipopolysaccharide.

Aspirin down regulates transferrin receptor 1 (TfR1) and up regulates ferroportin 1 (Fpn1) and ferritin expression in BV-2 microglial cells treated without lipopolysaccharides (LPS), as well as down regulates hepcidin and interleukin 6 (IL-6) in cells treated with LPS. However, the relevant mechanisms are unknown. Here, we investigate the effects of aspirin on expression of hepcidin and iron regulatory protein 1 (IRP1), phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3) and P65 (nuclear factor-κB), and the production of nitric oxide (NO) in BV-2 microglial cells treated with and without LPS. We demonstrated that aspirin inhibited hepcidin mRNA as well as NO production in cells treated with LPS, but not in cells without LPS, suppresses IL-6, JAK2, STAT3, and P65 (nuclear factor-κB) phosphorylation and has no effect on IRP1 in cells treated with or without LPS. These findings provide evidence that aspirin down regulates hepcidin by inhibiting IL6/JAK2/STAT3 and P65 (nuclear factor-κB) pathways in the cells under inflammatory conditions, and imply that an aspirin-induced reduction in TfR1 and an increase in ferritin are not associated with IRP1 and NO.

Human glutaredoxin 3 can bind and effectively transfer [4Fe-4S] cluster to apo-iron regulatory protein 1.

Glutaredoxin 3 (GLRX3) is a member of monothiol glutaredoxins with a CGFS active site that has been demonstrated to function in cellular iron sensing and trafficking via its bound iron-sulfur cluster. Human GLRX3 has been shown to form a dimer that binds two bridging [2Fe-2S] clusters with glutathione (GSH) as a ligand, assembling a compound 2GLRX3-2[2Fe-2S]-4GSH. Each iron of the iron-sulfur clusters is bound to the thiols of the cysteines, one of which is from the active site of GLRX3, the other from the noncovalently bound GSH. Here, we show that the recombinant human GLRX3 isolated anaerobically from Escherichia coli can incorporate [4Fe-4S] cluster in the absence of GSH, revealed by spectral and enzymatic analysis. [4Fe-4S] cluster-containing GLRX3 is competent for converting iron regulatory protein 1 (apo-IRP1) into aconitase within 30 min, via intact iron-sulfur cluster transfer. These in vitro studies suggest that human GLRX3 is important for cytosolic Fe-S protein maturation.

Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor inhibit ferrous iron influx via divalent metal transporter 1 and iron regulatory protein 1 regulation in ventral mesencephalic neurons.

Iron accumulation is observed in the substantia nigra of patients with Parkinson's disease. However, it is unknown whether neurotrophic factors, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) participate in the modulation of neuronal iron metabolism. Here, we investigated the effects and underlying mechanisms of BDNF and GDNF on the iron influx process in primary cultured ventral mesencephalic neurons. 6-hydroxydopamine-induced enhanced ferrous iron influx via improper up-regulation of divalent metal transporter 1 with iron responsive element (DMT1+IRE) was consistently relieved by BDNF and GDNF. Both the mRNA and protein levels of DMT1+IRE were down-regulated by BDNF or GDNF treatment alone. We further demonstrated the involvement of iron regulatory protein 1 (IRP1) in BDNF- and GDNF-induced DMT1+IRE expression. Extracellular-regulated kinase 1/2 (ERK1/2) and Akt were activated and participated in these processes. Inhibition of ERK1/2 and Akt phosphorylation abolished the down-regulation of IRP1 and DMT1+IRE induced by BDNF and GDNF. Taken together, these results show that BDNF and GDNF ameliorate iron accumulation via the ERK/Akt pathway, followed by inhibition of IRP1 and DMT1+IRE expression, which may provide new targets for the neuroprotective effects of these neurotrophic factors.

Cadmium-induced aggregation of iron regulatory protein-1.

Iron regulatory protein-1 (IRP-1) is central to regulation of iron homeostasis, and has been shown to be sensitive to Cd(2+) in vitro. Although Cd(2+) induces disulfide-bond formation in many proteins, the critical cysteine residues for iron binding in IRP-1 were shown not to be involved in Cd-induced IRP-1 aggregation in vitro. Here we show that Cd(2+) causes polymerization and aggregation of IRP-1 in vitro and in vivo, and decreases in a dose-dependent manner both its RNA-binding and aconitase enzymatic activities, as well as its cytosolic expression. We have used two-dimensional electrophoresis to demonstrate thiol-dependent self-association of purified recombinant IRP-1 treated with Cd(2+), as well as self-association in Cd(2+)-exposed mesangial cells. Circular dichroism spectra confirm significant conformational changes in the purified protein upon Cd(2+) exposure. Following Cd(2+) treatment, there is increased translocation of inactive IRP-1 to the actin cytoskeletal fraction, and this translocation is diminished by both antioxidant (BHA) treatment and inhibition of CaMK-II. These changes differ from those elicited by manipulation of iron levels. Cadmium-induced translocation of proteins to cellular compartments, and particularly to the cytoskeleton, is becoming a recognized event in Cd(2+) toxicity. Polymer-dependent translocation of IRP-1 in Cd(2+)-exposed cells may underlie effects of Cd(2+) on iron homeostasis.

The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1.

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis.

Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling, and metabolism of reactive oxygen species.

Aconitase, an enzyme possessing an iron-sulfur cluster that is sensitive to oxidation, is involved in the regulation of cellular metabolism. There are two isoenzymes of aconitase (Aco)--mitochondrial (mAco) and cytosolic (cAco) ones. The primary role of mAdco is believed to be to control cellular ATP production via regulation of intermediate flux in the Krebs cycle. The cytosolic Aco in its reduced form operates as an enzyme, whereas in the oxidized form it is involved in the control of iron homeostasis as iron regulatory protein 1 (IRP1). Reactive oxygen species (ROS) play a central role in regulation of Aco functions. Catalytic Aco activity is regulated by reversible oxidation of [4Fe-4S]²âº cluster and cysteine residues, so redox-dependent posttranslational modifications (PTMs) have gained increasing consideration as regards possible regulatory effects. These include modifications of cysteine residues by oxidation, nitrosylation and thiolation, as well as Tyr nitration and oxidation of Lys residues to carbonyls. Redox-independent PTMs such as phosphorylation and transamination also have been described. In the presence of a sustained ROS flux, redox-dependent PTMs may lead to enzyme damage and cell stress by impaired energy and iron metabolism. Aconitase has been identified as a protein that undergoes oxidative modification and inactivation in aging and certain oxidative stress-related disorders. Here we describe possible mechanisms of involvement of the two aconitase isoforms, cAco and mAco, in the control of cell metabolism and iron homeostasis, balancing the regulatory, and damaging effects of ROS.