Alpha-synuclein affects certain iron transporters of BV2 microglia cell through its ferric reductase activity.

Alpha-synuclein (α-syn) is a major component of Lewy bodies, which is a biomarker of Parkinson's disease (PD). It accumulates in substantia nigra pars compacta (SNpc) to form insoluble aggregates and cause neurotoxicity, which is often accompanied by iron deposition. We compared the iron reductase activity between monomeric α-syn (M-α-syn) and oligomeric α-syn (O-α-syn) and investigated the effect of α-syn on iron metabolism of BV2 microglia cells as well. α-syn had ferric reductase activity, and O-α-syn had stronger enzyme activity than M-α-syn. M-α-syn upregulated iron uptake protein, divalent metal transporter1 (DMT1) expression, and iron influx but did not regulate iron release protein ferroportin1 (FPN1) expression and iron efflux. O-α-syn elevated the expression of both DMT1 and FPN1 and thus increased the iron influx and efflux in BV2 microglial cells, but the expressions of iron regulatory protein1 (IRP1) and hypoxia-inducible factor 2α (HIF-2α) had no significant change. Moreover, both M-α-syn and O-α-syn could increase the mRNA expressions of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in BV2 microglia cells. Both types of α-syn can activate microglia, which leads to increased expressions of proinflammatory factors. α-syn can affect DMT1 and FPN1 expressions in BV2 microglia cells, which might be through its ferric reductase activity.NEW & NOTEWORTHY The effects of monomeric α-syn (M-α-syn) and oligomeric α-syn (O-α-syn) on the iron metabolism of BV2 microglia cells were detected by exogenous α-syn treatment. This study provides a strong experimental basis for α-syn involvement in iron metabolism in microglia.

Diurnal control of iron responsive element containing mRNAs through iron regulatory proteins IRP1 and IRP2 is mediated by feeding rhythms.

BACKGROUND: Cellular iron homeostasis is regulated by iron regulatory proteins (IRP1 and IRP2) that sense iron levels (and other metabolic cues) and modulate mRNA translation or stability via interaction with iron regulatory elements (IREs). IRP2 is viewed as the primary regulator in the liver, yet our previous datasets showing diurnal rhythms for certain IRE-containing mRNAs suggest a nuanced temporal control mechanism. The purpose of this study is to gain insights into the daily regulatory dynamics across IRE-bearing mRNAs, specific IRP involvement, and underlying systemic and cellular rhythmicity cues in mouse liver. RESULTS: We uncover high-amplitude diurnal oscillations in the regulation of key IRE-containing transcripts in the liver, compatible with maximal IRP activity at the onset of the dark phase. Although IRP2 protein levels also exhibit some diurnal variations and peak at the light-dark transition, ribosome profiling in IRP2-deficient mice reveals that maximal repression of target mRNAs at this timepoint still occurs. We further find that diurnal regulation of IRE-containing mRNAs can continue in the absence of a functional circadian clock as long as feeding is rhythmic. CONCLUSIONS: Our findings suggest temporally controlled redundancy in IRP activities, with IRP2 mediating regulation of IRE-containing transcripts in the light phase and redundancy, conceivably with IRP1, at dark onset. Moreover, we highlight the significance of feeding-associated signals in driving rhythmicity. Our work highlights the dynamic nature and regulatory complexity in a metabolic pathway that had previously been considered well-understood.

Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.

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

MEMO1 binds iron and modulates iron homeostasis in cancer cells.

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

Iron regulatory proteins 1 and 2 have opposing roles in regulating inflammation in bacterial orchitis.

Acute bacterial orchitis (AO) is a prevalent cause of intrascrotal inflammation, often resulting in sub- or infertility. A frequent cause eliciting AO is uropathogenic Escherichia coli (UPEC), a gram negative pathovar, characterized by the expression of various iron acquisition systems to survive in a low-iron environment. On the host side, iron is tightly regulated by iron regulatory proteins 1 and 2 (IRP1 and -2) and these factors are reported to play a role in testicular and immune cell function; however, their precise role remains unclear. Here, we showed in a mouse model of UPEC-induced orchitis that the absence of IRP1 results in less testicular damage and a reduced immune response. Compared with infected wild-type (WT) mice, testes of UPEC-infected Irp1-/- mice showed impaired ERK signaling. Conversely, IRP2 deletion led to a stronger inflammatory response. Notably, differences in immune cell infiltrations were observed among the different genotypes. In contrast with WT and Irp2-/- mice, no increase in monocytes and neutrophils was detected in testes of Irp1-/- mice upon UPEC infection. Interestingly, in Irp1-/- UPEC-infected testes, we observed an increase in a subpopulation of macrophages (F4/80+CD206+) associated with antiinflammatory and wound-healing activities compared with WT. These findings suggest that IRP1 deletion may protect against UPEC-induced inflammation by modulating ERK signaling and dampening the immune response.

Cell density impacts the susceptibility to ferroptosis by modulating IRP1-mediated iron homeostasis.

Ferroptosis has been implicated in several neurological disorders and may be therapeutically targeted. However, the susceptibility to ferroptosis varies in different cells, and inconsistent results have been reported even using the same cell line. Understanding the effects of key variables of in vitro studies on ferroptosis susceptibility is of critical importance to facilitate drug discoveries targeting ferroptosis. Here, we showed that increased cell seeding density leads to enhanced resistance to ferroptosis by reducing intracellular iron levels. We further identified iron-responsive protein 1 (IRP1) as the key protein affected by cell density, which affects the expression of ferroportin or transferrin receptor and results in altered iron levels. Such observations were consistent across different cell lines, indicating that cell density should be tightly controlled in studies of ferroptosis. Since cell densities vary in different brain regions, these results may also shed light on selective regional vulnerability observed in neurological disorders.

Nuclear translocation of STAT5 initiates iron overload in huntington's disease by up-regulating IRP1 expression.

Mutant huntingtin (mHtt) proteins interact to form aggregates, disrupting cellular functions including transcriptional dysregulation and iron imbalance in patients with Huntington's disease (HD) and mouse disease models. Previous studies have indicated that mHtt may lead to abnormal iron homeostasis by upregulating the expression of iron response protein 1 (IRP1) in the striatum and cortex of N171-82Q HD transgenic mice, as well as in HEK293 cells expressing the N-terminal fragment of mHtt containing 160 CAG repeats. However, the mechanism underlying the upregulation of IRP1 remains unclear. We investigated the levels and phosphorylation status of signal transducer and activator of transcription 5 (STAT5) in the brains of N171-82Q HD transgenic mice using immunohistochemistry staining. We also assessed the nuclear localization of STAT5 protein through western blot and immunofluorescence, and measured the relative RNA expression levels of STAT5 and IRP1 using RT-PCR in both N171-82Q HD transgenic mice and HEK293 cells expressing the N-terminal fragment of huntingtin. Our findings demonstrate that the transcription factor STAT5 regulates the transcription of the IPR1 gene in HEK293 cells. Notably, both the brains of N171-82Q mice and 160Q HEK293 cells exhibited increased nuclear content of STAT5, despite unchanged total STAT5 expression. These results suggest that mHtt promotes the nuclear translocation of STAT5, leading to enhanced expression of IRP1. The nuclear translocation of STAT5 initiates abnormal iron homeostatic pathways, characterized by elevated IRP1 expression, increased levels of transferrin and transferrin receptor, and iron accumulation in the brains of HD mice. These findings provide valuable insights into potential therapeutic strategies targeting iron homeostasis in HD.

Contrasting Iron Metabolism in Undifferentiated Versus Differentiated MO3.13 Oligodendrocytes via IL-1ß-Induced Iron Regulatory Protein 1.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of iron in the substantia nigra. While iron accumulation and inflammation are implicated in PD pathogenesis, their impact on oligodendrocytes, the brain's myelin-forming cells, remains elusive. This study investigated the influence of interleukin-1ß (IL-1ß), an elevated proinflammatory cytokine in PD, on iron-related proteins in MO3.13 oligodendrocytes. We found that IL-1ß treatment in undifferentiated MO3.13 oligodendrocytes increased iron regulatory protein 1 and transferrin receptor 1 (TfR1) expression while decreasing ferroportin 1 (FPN1) expression. Consequently, iron uptake was enhanced, and iron release was reduced, leading to intracellular iron accumulation. Conversely, IL-1ß treatment in differentiated MO3.13 oligodendrocytes exhibited the opposite effect, with decreased TfR1 expression, increased FPN1 expression, and reduced iron uptake. These findings suggest that IL-1ß-induced dysregulation of iron metabolism in oligodendrocytes may contribute to the pathological processes observed in PD. IL-1ß can increase the iron content in undifferentiated oligodendrocytes, thus facilitating the differentiation of undifferentiated MO3.13 oligodendrocytes. In differentiated oligodendrocytes, IL-1ß may facilitate iron release, providing a potential source of iron for neighboring dopaminergic neurons, thereby initiating a cascade of deleterious events. This study provides valuable insights into the intricate interplay between inflammation, abnormal iron accumulation, and oligodendrocyte dysfunction in PD. Targeting the IL-1ß-mediated alterations in iron metabolism may hold therapeutic potential for mitigating neurodegeneration and preserving dopaminergic function in PD.

H2S regulation of iron homeostasis by IRP1 improves vascular smooth muscle cell functions.

Either H2S or iron is essential for cellular processes. Abnormal metabolism of H2S and iron has increased risk for cardiovascular diseases. The aim of the present study is to examine the mutual interplay of iron and H2S signals in regulation of vascular smooth muscle cell (SMC) functions. Here we found that deficiency of cystathionine gamma-lyase (CSE, a major H2S-producing enzyme in vascular system) induced but NaHS (a H2S donor) administration attenuated iron accumulation in aortic tissues from angiotensin II-infused mice. In vitro, iron overload induced labile iron levels, promoted cell proliferation, disrupted F-actin filaments, and inhibited protein expressions of SMC-specific markers (αSMA and calponin) more significantly in SMCs from CSE knockout mice (KO-SMCs) than the cells from wild-type mice (WT-SMCs), which could be reversed by exogenously applied NaHS. In contrast, KO-SMCs were more vulnerable to iron starvation-induced cell death. Either iron overload or NaHS did not affect elastin level and gelatinolytic activity. We further found that H2S induced more aconitase activity of iron regulatory protein 1 (IRP1) but inhibited its RNA binding activity accompanied with increased protein levels of ferritin and ferriportin, which would contribute to the lower level of labile iron level inside the cells. In addition, iron was able to suppress CSE-derived H2S generation, while iron also non-enzymatically induced H2S release from cysteine. This study reveals the mutual interaction between iron and H2S signals in regulating SMC phenotypes and functions; CSE/H2S system would be a target for preventing iron metabolic disorder-related vascular diseases.

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 response elements (IREs)-mRNA of Alzheimer's amyloid precursor protein binding to iron regulatory protein (IRP1): a combined molecular docking and spectroscopic approach.

The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M-1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (-25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (kon) and the rate of dissociation (koff) as 341 µM-1 s-1, and 11 s-1, respectively. The addition of Fe2+ has decreased the rate of association (kon) by ~ three-fold, whereas the rate of dissociation (koff) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.

Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2.

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein-RNA and protein-DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5'-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.

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.

Iron regulatory protein (IRP)-mediated iron homeostasis is critical for neutrophil development and differentiation in the bone marrow.

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. However, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory protein 1 (IRP1) and IRP2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

Liquid-Liquid Phase Separation Promotes Protein Aggregation and Its Implications in Ferroptosis in Parkinson's Disease Dementia.

The pathological features of PDD are represented by dopaminergic neuronal death and intracellular α-synuclein (α-syn) aggregation. The interaction of iron accumulation with α-syn and tau was further explored as an essential pathological mechanism of PDD. However, the links and mechanisms between these factors remain unclear. Studies have shown that the occurrence and development of neurodegenerative diseases such as PDD are closely related to the separation of abnormal phases. Substances such as proteins can form droplets through liquid-liquid phase separation (LLPS) under normal physiological conditions and even undergo further liquid-solid phase transitions to form solid aggregates under disease or regulatory disorders, leading to pathological phenomena. By analyzing the existing literature, we propose that LLPS is the crucial mechanism causing abnormal accumulation of α-syn, tau, and other proteins in PDD, and its interaction with iron metabolism disorder is the key factor driving ferroptosis in PDD. Therefore, we believe that LLPS can serve as one of the means to explain the pathological mechanism of PDD. Determining the significance of LLPS in neurodegenerative diseases such as PDD will stimulate interest in research into treatments based on interference with abnormal LLPS.

Human IRP1 Translocates to the Nucleus in a Cell-Specific and Iron-Dependent Manner.

Iron regulatory protein 1 (IRP1) is a bifunctional protein with mutually exclusive RNA-binding or enzymatic activities that depend on the presence of a 4Fe-4S cluster. While IRP1 is a well-established cytosolic protein, work in a Drosophila model suggested that it may also exhibit nuclear localization. Herein, we addressed whether mammalian IRP1 can likewise translocate to the nucleus. We utilized primary cells and tissues from wild type and Irp1-/- mice, as well as human cell lines and tissue biopsy sections. IRP1 subcellular localization was analyzed by Western blotting, immunofluorescence and immunohistochemistry. We did not detect presence of nuclear IRP1 in wild type mouse embryonic fibroblasts (MEFs), primary hepatocytes or whole mouse liver. However, we observed IRP1-positive nuclei in human liver but not ovary sections. Biochemical fractionation studies revealed presence of IRP1 in the nucleus of human Huh7 and HepG2 hepatoma cells, but not HeLa cervical cancer cells. Importantly, nuclear IRP1 was only evident in iron-replete cells and disappeared following pharmacological iron chelation. These data provide the first experimental evidence for nuclear IRP1 expression in mammals, which appears to be species- and cell-specific. Furthermore, they suggest that the nuclear translocation of IRP1 is mediated by an iron-dependent mechanism.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation.

Iron­sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron­sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5'-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

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.

ENO1 suppresses cancer cell ferroptosis by degrading the mRNA of iron regulatory protein 1.

α-Enolase 1 (ENO1) is a critical glycolytic enzyme whose aberrant expression drives the pathogenesis of various cancers. ENO1 has been indicated as having additional roles beyond its conventional metabolic activity, but the underlying mechanisms and biological consequences remain elusive. Here, we show that ENO1 suppresses iron regulatory protein 1 (IRP1) expression to regulate iron homeostasis and survival of hepatocellular carcinoma (HCC) cells. Mechanistically, we demonstrate that ENO1, as an RNA-binding protein, recruits CNOT6 to accelerate the messenger RNA decay of IRP1 in cancer cells, leading to inhibition of mitoferrin-1 (Mfrn1) expression and subsequent repression of mitochondrial iron-induced ferroptosis. Moreover, through in vitro and in vivo experiments and clinical sample analysis, we identified IRP1 and Mfrn1 as tumor suppressors by inducing ferroptosis in HCC cells. Taken together, this study establishes an important role for the ENO1-IRP1-Mfrn1 pathway in the pathogenesis of HCC and reveals a previously unknown connection between this pathway and ferroptosis, suggesting a potential innovative cancer therapy.