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Background: Ischemic stroke (IS) is known for its high morbidity, disability and mortality rates, and studies designed to explore its pathophysiological mechanisms and identify novel therapeutic strategies are urgently needed. We aimed to probe the effects of the deubiquitinase OTUD3-IRP2-p53/PTGS2 pathway on cerebral ischemia‒reperfusion (I/R) injury and hippocampal neuron ferroptosis. Methods: A cerebral I/R mouse model was established. Furthermore, lentiviral vectors that overexpressed OTUD3 and knocked down IRP2 were constructed, and a series of assays were performed to probe the OTUD3/IRP2/p53/PTGS2 mechanism. An oxygen‒glucose deprivation and reoxygenation (OGD/R) model of mouse hippocampal neurons was constructed. Then, OTUD3 and IRP2 were knocked down and overexpressed, and p53 was overexpressed to explore the mechanism of the OTUD3/IRP2/p53/PTGS2 pathway. Results: OTUD3 and IRP2 were expressed at low levels in cerebral I/R models. OTUD3 promoted IRP2 expression to protect damaged hippocampal neurons. Moreover, IRP2 affected ferroptosis in hippocampal neurons. In addition, IRP2 inhibited p53. After IRP2 and p53 were overexpressed, IRP2 regulated the p53/PTGS2 pathway and affected ferroptosis in hippocampal neurons. In vivo, after overexpressing OTUD3 and knocking down IRP2, we found that overexpression of OTUD3 promoted IRP2 expression to reduce ferroptosis in hippocampal neurons and improve cerebral I/R injury via the inhibition of the p53/PTGS2 pathway. Conclusions: The deubiquitinase OTUD3 stabilized IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to subsequently ameliorate cerebral I/R injury. [ABSTRACT FROM AUTHOR]

Abstract Background Ischemic stroke (IS) is known for its high morbidity, disability and mortality rates, and studies designed to explore its pathophysiological mechanisms and identify novel therapeutic strategies are urgently needed. We aimed to probe the effects of the deubiquitinase OTUD3-IRP2-p53/PTGS2 pathway on cerebral ischemia‒reperfusion (I/R) injury and hippocampal neuron ferroptosis. Methods A cerebral I/R mouse model was established. Furthermore, lentiviral vectors that overexpressed OTUD3 and knocked down IRP2 were constructed, and a series of assays were performed to probe the OTUD3/IRP2/p53/PTGS2 mechanism. An oxygen‒glucose deprivation and reoxygenation (OGD/R) model of mouse hippocampal neurons was constructed. Then, OTUD3 and IRP2 were knocked down and overexpressed, and p53 was overexpressed to explore the mechanism of the OTUD3/IRP2/p53/PTGS2 pathway. Results OTUD3 and IRP2 were expressed at low levels in cerebral I/R models. OTUD3 promoted IRP2 expression to protect damaged hippocampal neurons. Moreover, IRP2 affected ferroptosis in hippocampal neurons. In addition, IRP2 inhibited p53. After IRP2 and p53 were overexpressed, IRP2 regulated the p53/PTGS2 pathway and affected ferroptosis in hippocampal neurons. In vivo, after overexpressing OTUD3 and knocking down IRP2, we found that overexpression of OTUD3 promoted IRP2 expression to reduce ferroptosis in hippocampal neurons and improve cerebral I/R injury via the inhibition of the p53/PTGS2 pathway. Conclusions The deubiquitinase OTUD3 stabilized IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to subsequently ameliorate cerebral I/R injury.

Colorectal cancer and Crohn's disease patients develop pyogenic liver abscesses due to failures of immune cells to fight off bacterial infections. Here, we show that mice lacking iron regulatory protein 2 (Irp2), globally (Irp2-/-) or myeloid cell lineage (Lysozyme 2 promoter-driven, LysM)-specifically (Irp2ΔLysM), are highly susceptible to liver abscesses when the intestinal tissue was injured with dextran sodium sulfate treatment. Further studies demonstrated that Irp2 is required for lysosomal acidification and biogenesis, both of which are crucial for bacterial clearance. In Irp2-deficient liver tissue or macrophages, the nuclear location of transcription factor EB (Tfeb) was remarkably reduced, leading to the downregulation of Tfeb target genes that encode critical components for lysosomal biogenesis. Tfeb mislocalization was reversed by hypoxia-inducible factor 2 inhibitor PT2385 and, independently, through inhibition of lactic acid production. These experimental findings were confirmed clinically in patients with Crohn's disease and through bioinformatic searches in databases from Crohn's disease or ulcerative colitis biopsies showing loss of IRP2 and transcription factor EB (TFEB)-dependent lysosomal gene expression. Overall, our study highlights a mechanism whereby Irp2 supports nuclear translocation of Tfeb and lysosomal function, preserving macrophage antimicrobial activity and protecting the liver against invading bacteria during intestinal inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Iron is an essential element to all living organisms and plays a vital role in many cellular processes, such as DNA synthesis and energy production. The Mdm2 oncogene is an E3 ligase and known to promote tumor growth. However, the role of Mdm2 in iron homeostasis is not certain. Here, we showed that Mdm2 expression was increased by iron depletion but decreased by iron repletion. We also showed that Iron Regulatory Protein 2 (IRP2) mediated iron-regulated Mdm2 expression. Specifically, Mdm2 expression was increased by ectopic IRP2 but decreased by knockdown or knockout of IRP2 in human cancer cells as well as in mouse embryonic fibroblasts. In addition, we showed that IRP2-regulated Mdm2 expression was independent of tumor suppressor p53. Mechanistically, we found that IRP2 stabilized Mdm2 transcript via binding to an iron response element (IRE) in the 3'UTR of Mdm2 mRNA. Finally, we showed that Mdm2 is required for IRP2-mediated cell proliferation and Mdm2 expression is highly associated with IRP2 in both the normal and cancerous liver tissues. Together, we uncover a novel regulation of Mdm2 by IRP2 via mRNA stability and that the IRP2-Mdm2 axis may play a critical role in cell growth.

Abstract Background Although β-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment. Methods High-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated β-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of β-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify β-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in β-catenin-activated cells. Online databases were analyzed for correlation between β-catenin activity and IRP2-TfR1 axis in human cancers. Results Iron chelators were identified as selective inhibitors against β-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed β-catenin-activated cell proliferation and tumor formation in mice. Mechanically, β-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired β-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced β-catenin-associated cell viability and tumor formation. Conclusions β-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of β-catenin-potentiated cancer.

Abstract Background Hepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored. Methods We generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis. Results KLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency. Conclusion KLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has evolved mechanisms to evade the host's nutritional immunity and thus promote bacterial growth by using the iron in the host. However, the detailed mechanisms of S. Typhimurium induce dysregulation of iron homeostasis and whether Lactobacillus johnsonii L531 can alleviate the iron metabolism disorder caused by S. Typhimurium has not been fully elucidated. Here, we show that S. Typhimurium activated the expression of iron regulatory protein 2 (IRP2), transferrin receptor 1, and divalent metal transporter protein 1 and suppressed the expression of iron exporter ferroportin, which resulted in iron overload and oxidative stress, inhibiting the key antioxidant proteins NF-E2-related factor 2, Heme Oxygenase-1, and Superoxide Dismutase in vitro and in vivo. L. johnsonii L531 pretreatment effectively reversed these phenomena. IRP2 knockdown inhibited iron overload and oxidative damage induced by S. Typhimurium in IPEC-J2 cells, while IRP2 overexpression promoted iron overload and oxidative damage caused by S. Typhimurium. Interestingly, the protective effect of L. johnsonii L531 on iron homeostasis and antioxidant function was blocked following IRP2 overexpression in Hela cells, demonstrating that L. johnsonii L531 attenuates disruption of iron homeostasis and consequent oxidative damage caused by S. Typhimurium via the IRP2 pathway, which contributes to the prevention of S. Typhimurium diarrhea in mice. [ABSTRACT FROM AUTHOR]

Background: Iron regulatory proteins (IRPs) maintain cellular iron homeostasis. Due to aberrant tissue-iron distribution, Irp2-deficient mice suffer microcytic anemia and neurodegeneration, while iron overload occurs in the liver and intestine. We previously found that Irp2 deficiency-induced Hif2 plays an important role in neurodegeneration. Methods: To test the role of Hif2 in Irp2 deficiency-induced anemia, we used Irp2 global knockout mice. Following Hif2 inhibition, routine blood tests, iron availability in bone marrow, histological assays, and biochemical analysis were performed to assess anemia improvement and tissue iron distribution. Results: We found that Hif2 inhibition improved anemia. The increased iron bioavailability for erythropoiesis was mainly derived from hepatic iron release, and secondly from enhanced intestinal absorption. We further demonstrate that nuclear receptor coactivator 4 (Ncoa4) was upregulated for iron release via the process of ferritinophagy. The released iron was utilized not only for intracellular Fe-S biogenesis but also for erythropoiesis after being exported from the liver to circulation. The hepatic iron export reduced hepcidin expression to further support iron absorption through the hepcidin-ferroportin axis to alleviate intestinal iron overload. Conclusion: Irp2 not only regulates cellular iron homeostasis but also tissue iron distribution by managing the involvement of Hif2-Ncoa4. [ABSTRACT FROM AUTHOR]

Because of its peculiar redox properties, iron is an essential element in living organisms, being involved in crucial biochemical processes such as oxygen transport, energy production, DNA metabolism, and many others. However, its propensity to accept or donate electrons makes it potentially highly toxic when present in excess and inadequately buffered, as it can generate reactive oxygen species. For this reason, several mechanisms evolved to prevent both iron overload and iron deficiency. At the cellular level, iron regulatory proteins, sensors of intracellular iron levels, and post-transcriptional modifications regulate the expression and translation of genes encoding proteins that modulate the uptake, storage, utilization, and export of iron. At the systemic level, the liver controls body iron levels by producing hepcidin, a peptide hormone that reduces the amount of iron entering the bloodstream by blocking the function of ferroportin, the sole iron exporter in mammals. The regulation of hepcidin occurs through the integration of multiple signals, primarily iron, inflammation and infection, and erythropoiesis. These signals modulate hepcidin levels by accessory proteins such as the hemochromatosis proteins hemojuvelin, HFE, and transferrin receptor 2, the serine protease TMPRSS6, the proinflammatory cytokine IL6, and the erythroid regulator Erythroferrone. The deregulation of the hepcidin/ferroportin axis is the central pathogenic mechanism of diseases characterized by iron overload, such as hemochromatosis and iron-loading anemias, or by iron deficiency, such as IRIDA and anemia of inflammation. Understanding the basic mechanisms involved in the regulation of hepcidin will help in identifying new therapeutic targets to treat these disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

Background: Although β-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment. Methods: High-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated β-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of β-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify β-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in β-catenin-activated cells. Online databases were analyzed for correlation between β-catenin activity and IRP2-TfR1 axis in human cancers. Results: Iron chelators were identified as selective inhibitors against β-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed β-catenin-activated cell proliferation and tumor formation in mice. Mechanically, β-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired β-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced β-catenin-associated cell viability and tumor formation. Conclusions: β-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of β-catenin-potentiated cancer. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored. Methods: We generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis. Results: KLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency. Conclusion: KLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target. [ABSTRACT FROM AUTHOR]

IntroductionNeurons require iron to support their metabolism, growth, and differentiation, but are also susceptible to iron-induced oxidative stress and cytotoxicity. Ferritin, a cytosolic iron storage unit, mediates cellular adaptation to fluctuations in iron delivery. NCOA4 has been characterized as a selective autophagic cargo receptor facilitating the mobilization of intracellular iron from ferritin. This process named ferritinophagy results in the degradation of ferritin and the consequent release of iron into the cytosol.MethodsHere we demonstrate that NCOA4 is important for the adaptation of the HT22 mouse hippocampal neuronal cell line to cellular iron restriction. Additionally, we determined the pathophysiological implications of impaired ferritinophagy via functional analysis of the omics profile of HT22 cells deficient in NCOA4.ResultsNCOA4 silencing impaired ferritin turnover and was cytotoxic when cells were restricted of iron. Quantitative proteomics identified IRP2 accumulation among the most prominent protein responses produced by NCOA4 depletion in HT22 cells, which is indicative of functional iron deficiency. Additionally, proteins of apoptotic signaling pathway were enriched by those responsive to NCOA4 deficiency. Transcriptome profiles of NCOA4 depletion revealed neuronal cell death, differentiation of neurons, and development of neurons as potential diseases and bio functions affected by impaired ferritinophagy, particularly, when iron was restricted.DiscussionThese findings identify an integral role of NCOA4-mediated ferritinophagy in the maintenance of iron homeostasis by HT22 cells, and its potential implications in controlling genetic pathways of neurodevelopment and neurodegenerative diseases.

Objective To investigate the effects of bovine lactoferrin (bLF)in inhibiting iron metabolism and lipid peroxidation in rats with intracerebral hemorrhage, and to explore its mechanism of action. Methods Ninety adult healthy male SD rats were randomly divided into sham-operated group, model group and bLF intervention group, with 30 rats in each. The two-step autologous blood right caudate nucleus injection was used to establish the intracerebral hemorrhage models. After successful modeling, the rats in the bLF group were given bLF 85 mg/ (kg-d)by gavage, and the rats in the sham-operated group and the model group were given the same amount of normal saline by gavage for 7 d. The neurological deficits were evaluated by the modified Neurological Severity Score (mNSS)at 1, 3 and 7 d after modeling, respec⁃ tively. After executing rats to extract brain tissues, the histopathological changes were observed by HE staining; iron and reactive oxygen species (ROS)content were detected using a bioreagent kit. The expression levels of ferritin, transferrin receptor (TFR), divalent iron metal transporter (DMT1), iron regulatory protein 2 (IRP2)and the antioxidant glutathione peroxidase 4 (GPX4)in the IRE/IRP regulatory system were detected by Western blotting. The correlation between the iron metabolism-related proteins ferritin, TFR, DMT1, IRP2 and the antioxidant enzyme GPX4 in the bLF intervention group was analyzed by Pearson correlation analysis. Results Compared with the sham-operated group, brain histopathological damage was significantly more severe in the model group; mNSS increased (all P<0. 05)and decreased after reaching the highest score on day 3; iron and ROS content levels increased (all P<0. 05), but there was no statistically signifi⁃ cant difference between the third day and the seventh day; IRE/IRP regulatory system-related proteins (ferritin, TFR, DMT1, IRP2)expression increased (P<0. 05), and it increased to the highest level on day 3 and then decreased; the ex⁃ pression of antioxidant GPX4 decreased (P<0. 05), and it reached the lowest level on day 3 and then increased. Com⁃ pared with the model group, the bLF group showed and itreduced brain histopathological damage and decreased mNSS score (both P<0. 05), decreased expression of iron and ROS content, IRE/IRP regulatory system-related proteins (ferritin, TFR, DMT1, IRP2) (all P<0. 05), and increased expression of antioxidant GPX4 (P<0. 05). Correlation analysis showed that the antioxidant GPX4 was negatively correlated with the expression of ferritin, TFR, DMT1, and IRP2 in the bLF group (all P<0. 05). Conclusions Bovine lactoferrin may down-regulate the expression of ferritin, TFR, DMT1, and IRP2 in the IRE/IRP regulatory system and reduce iron and ROS content, and meanwhile, it up-regulates the expres⁃ sion of the antioxidant GPX4, thereby reducing lipid peroxidation and inhibiting ferroptosis after intracerebral hemorrhage and exerting neuroprotective effects. [ABSTRACT FROM AUTHOR]

Ferredoxin reductase (FDXR), a target of p53, modulates p53-dependent apoptosis and is necessary for steroidogenesis and biogenesis of iron-sulfur clusters. To determine the biological function of FDXR, we generated a Fdxr-deficient mouse model and found that loss of Fdxr led to embryonic lethality potentially due to iron overload in developing embryos. Interestingly, mice heterozygous in Fdxr had a short life span and were prone to spontaneous tumors and liver abnormalities, including steatosis, hepatitis, and hepatocellular carcinoma. We also found that FDXR was necessary for mitochondrial iron homeostasis and proper expression of several master regulators of iron metabolism, including iron regulatory protein 2 (IRP2). Surprisingly, we found that p53 mRNA translation was suppressed by FDXR deficiency via IRP2. Moreover, we found that the signal from FDXR to iron homeostasis and the p53 pathway was transduced by ferredoxin 2, a substrate of FDXR. Finally, we found that p53 played a role in iron homeostasis and was required for FDXR-mediated iron metabolism. Together, we conclude that FDXR and p53 are mutually regulated and that the FDXR-p53 loop is critical for tumor suppression via iron homeostasis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has evolved mechanisms to evade the host’s nutritional immunity and thus promote bacterial growth by using the iron in the host. However, the detailed mechanisms of S. Typhimurium induce dysregulation of iron homeostasis and whether Lactobacillus johnsonii L531 can alleviate the iron metabolism disorder caused by S. Typhimurium has not been fully elucidated. Here, we show that S. Typhimurium activated the expression of iron regulatory protein 2 (IRP2), transferrin receptor 1, and divalent metal transporter protein 1 and suppressed the expression of iron exporter ferroportin, which resulted in iron overload and oxidative stress, inhibiting the key antioxidant proteins NF-E2-related factor 2, Heme Oxygenase-1, and Superoxide Dismutase in vitro and in vivo. L. johnsonii L531 pretreatment effectively reversed these phenomena. IRP2 knockdown inhibited iron overload and oxidative damage induced by S. Typhimurium in IPEC-J2 cells, while IRP2 overexpression promoted iron overload and oxidative damage caused by S. Typhimurium. Interestingly, the protective effect of L. johnsonii L531 on iron homeostasis and antioxidant function was blocked following IRP2 overexpression in Hela cells, demonstrating that L. johnsonii L531 attenuates disruption of iron homeostasis and consequent oxidative damage caused by S. Typhimurium via the IRP2 pathway, which contributes to the prevention of S. Typhimurium diarrhea in mice.

Background: Iron regulatory proteins (IRPs) maintain cellular iron homeostasis. Due to aberrant tissue-iron distribution, Irp2-deficient mice suffer microcytic anemia and neurodegeneration, while iron overload occurs in the liver and intestine. We previously found that Irp2 deficiency-induced Hif2 plays an important role in neurodegeneration. Methods: To test the role of Hif2 in Irp2 deficiency-induced anemia, we used Irp2 global knockout mice. Following Hif2 inhibition, routine blood tests, iron availability in bone marrow, histological assays, and biochemical analysis were performed to assess anemia improvement and tissue iron distribution. Results: We found that Hif2 inhibition improved anemia. The increased iron bioavailability for erythropoiesis was mainly derived from hepatic iron release, and secondly from enhanced intestinal absorption. We further demonstrate that nuclear receptor coactivator 4 (Ncoa4) was upregulated for iron release via the process of ferritinophagy. The released iron was utilized not only for intracellular Fe-S biogenesis but also for erythropoiesis after being exported from the liver to circulation. The hepatic iron export reduced hepcidin expression to further support iron absorption through the hepcidin-ferroportin axis to alleviate intestinal iron overload. Conclusion: Irp2 not only regulates cellular iron homeostasis but also tissue iron distribution by managing the involvement of Hif2-Ncoa4.

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.

Because of its peculiar redox properties, iron is an essential element in living organisms, being involved in crucial biochemical processes such as oxygen transport, energy production, DNA metabolism, and many others. However, its propensity to accept or donate electrons makes it potentially highly toxic when present in excess and inadequately buffered, as it can generate reactive oxygen species. For this reason, several mechanisms evolved to prevent both iron overload and iron deficiency. At the cellular level, iron regulatory proteins, sensors of intracellular iron levels, and post-transcriptional modifications regulate the expression and translation of genes encoding proteins that modulate the uptake, storage, utilization, and export of iron. At the systemic level, the liver controls body iron levels by producing hepcidin, a peptide hormone that reduces the amount of iron entering the bloodstream by blocking the function of ferroportin, the sole iron exporter in mammals. The regulation of hepcidin occurs through the integration of multiple signals, primarily iron, inflammation and infection, and erythropoiesis. These signals modulate hepcidin levels by accessory proteins such as the hemochromatosis proteins hemojuvelin, HFE, and transferrin receptor 2, the serine protease TMPRSS6, the proinflammatory cytokine IL6, and the erythroid regulator Erythroferrone. The deregulation of the hepcidin/ferroportin axis is the central pathogenic mechanism of diseases characterized by iron overload, such as hemochromatosis and iron-loading anemias, or by iron deficiency, such as IRIDA and anemia of inflammation. Understanding the basic mechanisms involved in the regulation of hepcidin will help in identifying new therapeutic targets to treat these disorders.

Arsenic contamination is extremely threatening to the global public health. It was reported that sodium arsenite exposure induces serious kidney injury. However, the underlying mechanism is unclear. Ferroptosis is a newly characterized form of iron-dependent programmed cell death, which is implicated in the pathogenesis of various human diseases, including kidney injury. The lethal accumulation of iron-catalyzed lipid peroxidation is the fundamental biochemical characteristic of ferroptosis. Herein we report that sodium arsenite exposure initiates ferroptosis in mammalian HEK293, MEF and HT1080 cells, and induces ferroptosis-associated acute kidney injury in mice. RNA-binding protein G3BP1, the switch component of stress granules, is indispensable for sodium arsenite-induced ferroptosis in a stress granule-independent manner. Mechanistically, G3BP1 stabilizes IRP2, the master regulator of cellular iron homeostasis, through binding to and suppressing the translation of FBXL5 mRNA, which encodes the E3 ligase component to mediate IRP2 ubiquitination and proteasomal degradation. Sodium arsenite intoxication expedites this G3BP1- FBXL5 -IRP2 axis and elevates cellular labile free iron, which is responsible for sodium arsenite exposure-induced lipid peroxidation and ferroptotic cell death. In summary, this study highlights a regulatory module comprising G3BP1- FBXL5 -IRP2 axis in determining sodium arsenite-induced ferroptosis and ferroptosis-associated acute kidney injury in mice. [Display omitted] • NaAsO 2 exposure triggers ferroptosis in mammalian cells and induces ferroptosis-associated acute kidney injury in mice. • NaAsO 2 -induced ferroptosis is dependent on RNA-binding protein G3BP1, while dynamics of stress granule is not involved. • G3BP1- FBXL5 -IRP2 axis is responsible for the sodium arsenite-induced ferroptosis. [ABSTRACT FROM AUTHOR]

Currently, the existing treatments have not cured the liver fibrosis thoroughly. Ferroptosis is a newly discovered way of cell death, which is closely related to many diseases. Previous studies have shown that ferroptosis plays an important role in the occurrence and development of liver fibrosis, but the further mechanism remains to be discovered. LX-2 cells were used as the research object, fibrosis activation index was detected by Western blot, PCR and Immunofluorescence, ferroptosis was detected by kits, the binding and interaction between IRP2 (iron regulatory protein 2) and STUB1 (STIP1 homology and U-box containing protein 1) were detected by Immunoprecipitation and ubiquitin test, and IRP2 knockdown mice were constructed by interfering plasmid to verify the results of in vitro experiment. Our research showed that ART (artemether) had a good anti-fibrosis effect in vivo and in vitro, and ferroptosis played an important role in this process. Further studies have found that ART could lead to the accumulation of IRP 2 a in hepatic stellate cell by inhibiting the ubiquitination of it, thus inducing the increase of iron in HSC (hepatic stellate cell), which could product a large number of ROS (reactive oxide species), resulting the occurrence of ferroptosis in cells. Our findings provided an experimental basis for ART to become a drug for the treatment of liver fibrosis. Our results show that IRP2-Iron-ROS axis is necessary for ART to induce ferroptosis in HSC and play an anti-fibrotic effect. Image 1 • ART exerts its anti-fibrosis effect through ferroptosis pathway. • IRP2 is essential for ART to exert its anti-fibrotic effect. • STUB1 is a new ubiquitin ligase of IRP2 in hepatic stellate cells. • ROS accumulation caused by iron overload is the main cause of ferroptosis in hepatic stellate cells induced by ART. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second neurodegenerative disease that lacks effective treatments. Buddlejasaponin IVb (BJP-IVb) is the main bioactive component of herbs in genus Clinopodium which display antioxidative, anti-inflammatory and neuroprotective activities. However, the role of BJP-IVb in PD still remains unknown. This study aimed to evaluate the effect of BJP-IVb on dopaminergic neurodegeneration in PD and clarified the underlying mechanisms from the aspect of iron overload-mediated ferroptosis. One-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models were established in this study. Behavioral tests, cell cytotoxicity assay, tyrosine hydroxylase (TH) and Nissl staining were performed to evaluate the antiparkinsonian effect of BJP-IVb. Cellular ultrastructure, iron content and lipid peroxidation were detected to evaluate iron overload-mediated dopaminergic neuron ferroptosis. Iron regulatory protein 2 (IRP2) and iron transport-related proteins were detected by immunofluorescence and Western blot to evaluated iron transport. Finally, plasmid vector-mediated IRP2 overexpression were performed to further clarify the molecular mechanism. BJP-IVb alleviated MPP+-induced neurotoxicity in vitro and improved MPTP-induced dopaminergic neuron loss and motor dysfunctions of PD mice, confirming an effect of BJP-IVb against dopaminergic neurodegeneration of PD. Further results revealed that BJP-IVb protected against PD by suppressing iron overload-mediated dopaminergic neuron ferroptosis, as evidenced by the attenuated lipid peroxidation, decreased iron content and changes in cellular ultrastructure. Finally, the decreased iron regulatory protein (IRP2) was confirmed to be responsible for BJP-IVb-mediated ferroptosis suppression by modulating iron transport-related proteins and alleviating iron overload. BJP-IVb suppressed iron overload-mediated dopaminergic neuron ferroptosis and improved motor dysfunctions in PD, which was achieved by inhibiting IRP2-mediated iron overload. This study provided a potential drug candidate for the treatment of PD. [Display omitted] • BJP-IVb alleviates MPP+-induced neurotoxicity in vitro. • BJP-IVb improves MPTP-induced motor dysfunctions and dopaminergic neuron loss in vivo. • BJP-IVb suppresses dopaminergic neurons ferroptosis by mitigating iron overload in PD models. • BJP-IVb suppresses iron overload by regulating iron transport in PD models. • BJP-IVb suppresses iron overload by suppressing IRP2-mediated iron transport in PD models. [ABSTRACT FROM AUTHOR]

Iron regulatory protein 2 (IRP2) plays a key role in the cellular iron homeostasis and could be regulated by a variety of factors, such as oxidative stress, hypoxia and iron, etc. IRP2 depletion results in neurodegenerative movement disorder with the loss of neurons and accumulations of iron. Since oxidative stress extensively exists in several neurodegenerative diseases where iron accumulation also exists, it is important to clarify the mechanisms underlying the effects of oxidative stress on IRP2 expression and its consequence. 200 and 300 μM H2O2 could result in the reduced cell viability in SH-SY5Y cells. The intracellular levels of reactive oxygen species (ROS) were increased by 52.2 and 87.3% with 200 and 300 μM H2O2 treatments, respectively. The decreased levels of mitochondrial transmembrane potential (ΔΨm) were only observed in 300 μM H2O2-treated group. The protein levels of IRP2, but not for its mRNA levels, were observed decreased in both groups, which resulted in the lower TfR1 expression and decreased iron uptake in these cells. Pretreatment with MG132, the decreased IRP2 levels caused by H2O2 treatment could be antagonized. The protein levels of F box and leucine-rich repeat protein 5 (FBXL5), the only E3 ligase of IRP2, were observed decreased accordingly. When knockdown the intracellular FBXL5 levels by si-FBXL5, the protein levels of IRP2 were found increased with H2O2 treatment. Our results suggest that FBXL5 is involved in the degradation of IRP2 under oxidative stress in dopaminergic-like neuroblastoma cells, which implies that its role in the neuronal regulation of IRP2 in neurodegenerative diseases.

Abstract Because of essentiality and toxicity of iron in our body, iron metabolism is tightly regulated in cells. In mammalian cells, iron regulatory protein 1 and 2 (IRP1 and IRP2) are the central regulators of cellular iron metabolism. IRPs regulate iron metabolism by interacting with the RNA stem-loop structures, iron-responsive elements (IREs), found on the transcripts encoding proteins involved in iron metabolism only in iron depleted condition. It is also well-known that the ubiquitin system plays central roles in cellular iron regulation because both IRPs having the IRE binding activity are recognized and ubiquitinated by the SCFFBXL5 ubiquitin ligase in condition of iron-replete. FBXL5, which is a substrate recognition subunit of SCFFBXL5, senses iron availability via its hemerythrin-like domain. In this small article, current understanding of the roles of SCFFBXL5-mediated degradation of IRPs played in cellular iron metabolism is discussed. Graphical abstract fx1 Highlights • The IRE/IRP system is one of the central regulators of cellular iron metabolism. • IRPs bind to IREs on mRNAs encoding iron metabolism in iron-depleted condition. • The abundance of IRPs having IRE-biding activity is regulated by the SCFFBXL5 ligase. • The abundance of FBXL5 is regulated by availability of iron ion in cells. [ABSTRACT FROM AUTHOR]

Iron is essential for energy metabolism, and states of iron deficiency or excess are detrimental for organisms and cells. Therefore, iron and carbohydrate metabolism are tightly regulated. Serum iron and glucose levels are subjected to hormonal regulation by hepcidin and insulin, respectively. Hepcidin is a liver-derived peptide hormone that inactivates the iron exporter ferroportin in target cells, thereby limiting iron efflux to the bloodstream. Insulin is a protein hormone secreted from pancreatic β-cells that stimulates glucose uptake and metabolism via insulin receptor signaling. There is increasing evidence that systemic, but also cellular iron and glucose metabolic pathways are interconnected. This review article presents relevant data derived primarily from mouse models and biochemical studies. In addition, it discusses iron and glucose metabolism in the context of human disease.

Abstract: Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn‐FLAG in LD infected J774 macrophage confirms that Fpn down‐regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by 35S‐methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5′UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F‐box and leucine‐rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD‐infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2‐FBXL5 axis to inhibit host Fpn expression. [ABSTRACT FROM AUTHOR]

Although iridium diphosphide (IrP 2) has emerged as a promising electrocatalyst, the harsh synthetic conditions requiring high temperature (≥ 850 ℃) limits its wide application. This paper demonstrates a facile synthetic approach of single-phase IrP 2 nanoparticles under a mild condition. Instead of commonly used iridium chloride precursors (e.g., IrCl x), Ir(OH) 3 formed via acid/base precipitation reaction was employed as the iridium source. The following phosphorization step at 650 °C under argon atmosphere successfully produced IrP 2 nanoparticles. Moreover, the electroactivity of as-prepared IrP 2 nanoparticles for electrochemical oxidation of hydrogen sulfide (H 2 S) was investigated for the first time. IrP 2 nanoparticles showed an excellent amperometric response to H 2 S with ca. 2.5-fold greater sensitivity than iridium phosphide (Ir 2 P), presenting the feasibility of IrP 2 as an efficient electrode material for electrochemical H 2 S sensing. [Display omitted] • Single phase P-rich IrP 2 nanoparticles (IrP 2 NPs) were synthesized. • A facile synthetic methodology of IrP 2 at moderate temperature (650 ℃). • IrP 2 NPs shows an excellent amperometric response for H 2 S oxidation. [ABSTRACT FROM AUTHOR]

A few tightly regulated transport proteins mediate iron absorption across the intestinal epithelium. At the basolateral border of intestinal cells there is one identified transporter, ferroportin, for the transfer of intracellular iron to the vascular system. Here, we investigate the effects of ascorbate (vitamin C) on the regulation of ferroportin in human intestinal Caco-2 cells using ELISA and Western Blot analyses. The results indicate that ferroportin protein levels peak at 100 μM of added ascorbate with an increase of 274% (p = 0.02). At 150 μM of ascorbate, the increase was only 28% (p = 0.04), and at 200 μM there was no significant change from the baseline control. In addition, the ascorbate-induced, (at 150 μM) up-regulated ferroportin levels were associated with increased 55Fe transport across the basolateral border (19%, p = 0.03). Ascorbate-induced up-regulation of cellular ferroportin levels (no added iron) was associated with increased levels of the iron regulatory protein IRP2 (230%, p = 0.0009), and the hypoxia-inducible factor HIF2α (69%, p = 0.03). Thus, iron transport across the basal border via ferroportin is influenced by the intracellular status of ascorbate and IRP2 and HIF2α are involved. We discuss possible reasons for the ascorbate-effects and the dependence of cellular growth conditions for iron transport-related protein expression.

Ovarian endometrial cysts cause some kinds of ovarian cancer, and iron is considered as one factor of carcinogenesis. In contrast, hypoxia is associated with progression, angiogenesis, metastasis, and resistance to therapy in cancer. We investigated hypoxia-induced perturbation of iron homeostasis in terms of labile iron, iron deposition, and iron regulatory protein (IRP) in ovarian endometrial cysts. Iron deposition, expression of IRPs, and a protein marker of hypoxia in human ovarian endometrial cysts were analyzed histologically. The concentration of free iron and the pO 2 level of the cyst fluid of human ovarian cysts (n = 9) were measured. The expression of IRP2 under hypoxia was investigated in vitro by using Ishikawa cells as a model of endometrial cells. Iron deposition and the expression of IRP2 and Carbonic anhydrase 9 (CA9) were strong in endometrial stromal cells in the human ovarian endometrial cysts. The average concentration of free iron in the cyst fluid was 8.1 ± 2.9 mg/L, and the pO 2 was 22.4 ± 5.2 mmHg. A cell-based study using Ishikawa cells revealed that IRP2 expression was decreased by an overload of Fe(II) under normoxia but remained unchanged under hypoxia even in the presence of excess Fe(II). An increase in the expression of IRP2 caused upregulation of intracellular iron as a result of the response to iron deficiency, whereas the protein was degraded under iron-rich conditions. We found that iron-rich regions existed in ovarian endometrial cysts concomitantly with the high level of IRP2 expression, which should generally be decomposed upon an overload of iron. We revealed that an insufficient level of oxygen in the cysts is the main factor for the unusual stabilization of IRP2 against iron-mediated degradation, which provides aberrant uptake of iron in ovarian endometrial stromal cells and can potentially lead to carcinogenesis. [ABSTRACT FROM AUTHOR]

Iron is an essential nutrient that is potentially toxic due to its redox reactivity. Insufficient iron supply to erythroid cells, the major iron consumers in the body, leads to various forms of anemia. On the other hand, iron overload (hemochromatosis) is associated with tissue damage and diseases of liver, pancreas, and heart. Physiological iron balance is tightly controlled at the cellular and systemic level by iron regulatory proteins (IRP1, IRP2) and the iron regulatory hormone hepcidin, respectively. Underlying mechanisms often intersect to achieve optimal iron utilization, to control immune responses, and to prevent iron toxicity. This review focuses on systemic iron homeostasis in the context of erythropoiesis, a highly iron-demanding process. We discuss the function and regulation of hepcidin by various stimuli, and highlight hepcidin-dependent and -independent mechanisms that link iron utilization with maturation of erythroid progenitor cells. © 2017 IUBMB Life, 69(6):399-413, 2017 [ABSTRACT FROM AUTHOR]

Eighty-five isolates of Klebsiella pneumoniae and Enterobacter spp., originating from hospital- and community-acquired infections and from oropharyngeal and faecal microbiota from patients in Recife-PE, Brazil, were analyzed regarding the presence of irp2 gene. This is a Yersinia typical gene involved in the synthesis of siderophore yersiniabactin. DNA sequencing confirmed the identity of irp2 gene in five K. pneumoniae , five Enterobacter aerogenes and one Enterobacter amnigenus isolates. To our knowledge in the current literature, this is the first report of the irp2 gene in E. amnigenus , a species considered an unusual human pathogen, and in K. pneumoniae and E. aerogenes isolates from the normal microbiota and from community infections, respectively. Additionally, the analyses of nucleotide and amino acid sequences suggest the irp2 genes derived from isolates used in this study are more closely related to that of Yersinia pestis P.CE882 than to that of Yersinia enterocolitica 8081. These data demonstrated that K. pneumoniae and Enterobacter spp. from normal microbiota and from community- and hospital-acquired infections possess virulence factors important for the establishment of extra-intestinal infections. [ABSTRACT FROM AUTHOR]

The purpose of this study was to characterize the virulence potential and determine the molecular epidemiology of extended-spectrum β-lactamases (ESBLs) in CTX-M-producing Escherichia coli isolated from piglets with diarrhea in China. A total of 62 E. coli isolates were obtained among which 49 and 13 were collected from diarrheic and healthy piglets, respectively. Cefotaxime resistant strains were screened for the presence of ESBL, adhesin and exotoxin genes as well as for their biofilm-forming ability. Characterization of bla CTX-M plasmids was determined by conjugation along with the determination of genetic relatedness and plasmid replicon type. CTX-M producers were found in 36 isolates with 6 different subtypes: bla CTX-M-14,27,65 from CTX-M-9G ( n = 27) and bla CTX-M-55, 15,79 from CTX-M-1G ( n = 22). This also included 13 isolates that carried two different CTX-M genes. Thirty of 36 CTX-M producers and 12 of 13 multiple CTX-M alleles were confirmed from diarrheic piglets. The presence of the iron regulatory gene irp2 as well as EAST1 was found in 83.3% (25/30) of CTX-M-producing isolates from diarrheic piglets and these were significantly better biofilm formers. PFGE profiles of CTX-M-positive isolates indicated the spread of multidrug resistance was primarily horizontal and spread via transferable plasmids. Most bla CTX-M-9G genes (10/17) were located on the IncFIB type plasmid with sizes of 40–145 kb, while the bla CTX-M-1G (11/16) genes were located on the ∼100 kb IncN-type plasmid. Together, our findings demonstrate that CTX-M ESBL-producing E . coli from diarrheic piglets were associated with serious multidrug resistance, increased biofilm-forming ability and the irp2 gene of HPI. Our findings highlight the need to urgent control the spread of resistant strains through food chain. [ABSTRACT FROM AUTHOR]

Iron accumulates in the CNS of patients with multiple sclerosis, but our understanding of the mechanism accounting for this accumulation is unclear. Mouse models of cerebral experimental autoimmune encephalomyelitis (EAE) in C57BL/6 and SJL mice were used together with a histochemical stain for iron and immunohistochemical stains for transferrin receptor, synaptophysin, iron regulatory protein 1 (IRP1) and/or IRP2 to investigate the role of disease activity on CNS iron metabolism. The expression of transferrin receptor, but not IRP1 or IRP2, increased in the medial habenula, which is adjacent to the third ventricle, in response to both types of cerebral EAE. In the habenula, the elevated expression of transferrin receptor in C57BL/6 mice with cerebral EAE was generally restricted to the medial habenula while the expression in SJL mice with cerebral EAE was more diffusely expressed. Iron levels were increased in all regions of the habenula in C57BL/6 mice with cerebral EAE, and in the medial and medial lateral but not the lateral habenula in SJL mice with cerebral EAE. Synaptophysin, which has been observed previously in endocytic vesicles together with the transferrin receptor, was concentrated at the medial habenula, but its levels did not increase with disease in C57BL/6 mice with cerebral EAE. Our results support the model that the medial habenula responds to disease activity by upregulating transferrin receptor to facilitate the movement of iron into the brain from the third ventricle, raising the possibility that a similar mechanism accounts for iron accumulation in deep gray matter structures in patients with multiple sclerosis. [ABSTRACT FROM AUTHOR]

The article presents a study of the association between the core gene irp2, fyuA deletion of the high pathogenicity island and avian pathogenic Escherichia coli (APEC). The study used the Red homologous recombination method to construct the irp2 gene deletion and irp2, fyuA genes deletion strains and real-time polymerase chain reaction (PCR) to evaluate their effects on the messenger RNA (mRNA) levels. It shows the importance of irp2, fyuA genes delection effect to the pathogenicity in APEC.

The irp2 gene codes for a 190 kDa protein (HMWP2) synthesidez when highly pathogenic Yersinia are grown under conditions of iron starvation. In this work, the presence of irp2 in strains of Y. pestis isolated from different hosts during several plague outbreaks in the foci of Northeast Brazil wasstudied. For this purpose, 53 strains were spotted onto nylon filters and their DNA was hybridized with the A13 probe which is a 1 kb fragment of the irp2 coding sequence. All strains except two hybridized with the probe. However, when the initial stock culture of these two strains were analyzed, they both proved to bepositive with the A13 probe, indicating that the locus was lost after subculturein vitro but was always present in vivo. To examine the degree of conservation of the chromosomal fragment carrying irp2 among Brazilian strains, the hybridization profiles of 15 strains from different outbreaks, different hosts and different foci were compared. The hybridization profiles of these strains were all identical when their DNA was digested with either EcoRI, EcorRV or AvaII, indicatingthat the restriction sites surrounding the irp2 locus are very well conserved among Northeast Brazilian strains of Y. pestis. Altogether, these results suggest that the irp2 chromosomal region should be of prime importance for the bacteria during their multiplication in the host.

Abstract: This review is focused on the mammalian SLC11 and SLC40 families and their roles in iron homeostasis. The SLC11 family is composed of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the lysosomal compartment of macrophages and in the tertiary granules of neutrophils, playing a key role in innate resistance against infection by intracellular microbes. SLC11A2 is a key player in iron metabolism and is ubiquitously expressed, most notably in the proximal duodenum, immature erythroid cells, brain, placenta and kidney. Intestinal iron absorption is mediated by SLC11A2 at the apical membrane of enterocytes, followed by basolateral exit via SLC40A1. To meet the daily requirement for iron, approximately 80% of the iron comes from the breakdown of hemoglobin following macrophage phagocytosis of senescent erythrocytes (iron recycling). Both SLC11A1 and SLC11A2 play an important role in macrophage iron recycling. SLC11A2 also transports iron into the cytosol across the membrane of endocytotic vesicles of the transferrin receptor-cycle. SLC40A1 is the sole member of the SLC40 family and is involved in the only cellular iron efflux mechanism described. SLC40A1 is highly expressed in several tissues and cells that play a critical role in body iron homeostasis. The signaling pathways that regulate SLC11A2 and SLC40A1 expression at transcriptional, post-transcriptional and post-translational levels are discussed. The roles of SLC11A2 and/or SLC40A1 in iron-associated disorders such as hemochromatosis, neurodegenerative diseases, and breast cancer are also summarized. [Copyright &y& Elsevier]

Fifty-eight typical EAEC isolates from children with diarrhoea were examined for HEp-2 cell adherence assay, presence of dispersin ( aap), yersiniabactin ( irp2), plasmid encoded toxins ( pet), Shigella enterotoxin1 ( set1A) and cryptic open reading frame ( shf) putative virulence genes by polymerase chain reaction as well as for biofilm production. All the isolates showed aggregative adherence pattern on HEp-2 cells. All but five isolates (91.3 %) carried aap gene. While irp2, pet, set1A and shf genes were detected in 68.9, 5.1, 39.6, and 60.3 % isolates, respectively. Thirty-three (64.7 %) isolates out of 51 tested were found to produce biofilm which was found to be significantly associated only with set1A virulence gene ( P = 0.025). Highest amount of biofilm was produced by a strain that possessed all the genes studied. Out of 14 isolates in which the most frequent gene combination ( aap, irp2 and shf) was observed, only six produced biofilm. It is concluded that there is significant heterogeneity in putative virulence genes of EAEC isolates from diarrhoeic children and biofilm formation is associated with multiple genes. [ABSTRACT FROM AUTHOR]

Abstract: Constituting an integral part of a heme''s porphyrin ring, iron is essential for supplying cells and tissues with oxygen. Given tight links between oxygen delivery and iron availability, it is not surprising that iron deprivation and oxygen deprivation (hypoxia) have very similar consequences at the molecular level. Under hypoxia, the expression of major iron homeostasis genes including transferrin, transferrin receptor, ceruloplasmin, and heme oxygenase-1 is activated by hypoxia-inducible factors to provide increased iron availability for erythropoiesis in an attempt to enhance oxygen uptake and delivery to hypoxic cells. Iron-response proteins (IRP1 and IRP2) and “cap-n-collar” bZIP transcriptional factors (NE-F2 p45; Nrf1, 2, and 3; Bach1 and 2) also control gene and protein expression of the key iron homeostasis proteins. In this article, we give an overview of the mechanisms by which iron pathways are regulated by hypoxia at multiple levels. In addition, potential clinical benefits of manipulating iron pathways in the hypoxia-related conditions anemia and ischemia are discussed. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Background/Aims: Infection with hepatitis C virus (HCV) is associated with alterations in body iron homeostasis by poorly defined mechanisms. To seek for molecular links, we employed an established cell culture model for viral replication, and assessed how the expression of an HCV subgenomic replicon affects iron metabolism in host Huh7 hepatoma cells. Methods: The expression of iron metabolism genes and parameters defining the cellular iron status were analyzed and compared between parent and replicon Huh7 cells. Results: By using the IronChip microarray platform, we observed replicon-induced changes in expression profiles of iron metabolism genes. Notably, ceruloplasmin mRNA and protein expression were decreased in replicon cells. In addition, transferrin receptor 1 (TfR1) was also downregulated, while ferroportin levels were elevated, resulting in reduced iron uptake and increased iron release capacity of replicon cells. These responses were associated with an iron-deficient phenotype, manifested in decreased levels of the “labile iron pool” and concomitant induction of IRE-binding activity and IRP2 expression. Furthermore, hemin-treated replicon cells exhibited a defect in retaining iron. The clearance of the replicon by prolonged treatment with interferon-α only partially reversed the iron-deficient phenotype but almost completely restored the capacity of cured cells to retain iron. Conclusions: We propose that Huh7 cells undergo genetic reprogramming to permit subgenomic viral replication that results in reduction of intracellular iron levels. This response may provide a mechanism to bypass iron-mediated inactivation of the viral RNA polymerase NS5B. [Copyright &y& Elsevier]

The catalytic role of iron in the Haber–Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals. [ABSTRACT FROM AUTHOR]