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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.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Doença de Crohn , Proteína 2 Reguladora do Ferro , Lisossomos , Macrófagos , Animais , Lisossomos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Camundongos , Humanos , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Fígado/imunologia , Fígado/patologiaRESUMO
Airborne infection risk analysis is usually performed for enclosed spaces where susceptible individuals are exposed to infectious airborne respiratory droplets by inhalation. It is usually based on exponential, dose-response models of which a widely used variant is the Wells-Riley (WR) model. We revisit this infection-risk estimate and extend it to the population level. We use an epidemiological model where the mode of pathogen transmission, airborne or contact, is explicitly considered. We illustrate the link between epidemiological models and the WR and the Gammaitoni and Nucci models. We argue that airborne infection quanta are, up to an overall density, airborne infectious respiratory droplets modified by a parameter that depends on biological properties of the pathogen, physical properties of the droplet and behavioural properties of the individual. We calculate the time-dependent risk of being infected for two scenarios. We show how the epidemic infection risk depends on the viral latent period and the event time, the time infection occurs. Infection risk follows the dynamics of the infected population. As the latent period decreases, infection risk increases. The longer a susceptible is present in the epidemic, the higher its risk of infection for equal exposure time to the pathogen is.
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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.
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Ritmo Circadiano , Proteína 1 Reguladora do Ferro , Proteína 2 Reguladora do Ferro , Ferro , Fígado , RNA Mensageiro , Animais , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Ritmo Circadiano/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Camundongos , Fígado/metabolismo , Ferro/metabolismo , Regulação da Expressão Gênica , Elementos de Resposta , Camundongos Endogâmicos C57BL , Masculino , Comportamento AlimentarRESUMO
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.
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Proteína 1 Reguladora do Ferro , Ferro , Humanos , Animais , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 1 Reguladora do Ferro/genética , Camundongos , Proteína 2 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Biotinilação , Elementos de Resposta , Fosforilação , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Ligação Proteica , Linhagem Celular TumoralRESUMO
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.
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Injúria Renal Aguda , Arsenitos , Proteínas F-Box , Ferroptose , Compostos de Sódio , Humanos , Camundongos , Animais , DNA Helicases , Células HEK293 , Proteína 2 Reguladora do Ferro/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA , Ferro/metabolismo , Mamíferos/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas F-Box/química , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMO
BACKGROUND: Colon adenocarcinoma (COAD) is a major cause of cancer mortality worldwide. The occurrence and development of colon cancer is regulated by complex mechanisms that require further exploration. Recently, long non-coding RNAs (lncRNAs) were found to be related to the mortality of colon cancer patients through their participation in competing endogenous RNA (ceRNA) networks. Therefore, screening the lncRNAs involved in colon cancer may contribute to clarifying the complex mechanisms. METHODS: In this study, we explored the potential lncRNAs associated with colon cancer by establishing a ceRNA network using bioinformatics, followed by biological verification. RESULTS: RP11-197K6.1 and RP11-400N13.3 were screened out owing to their involvement in the expression of CDK2NA, a gene that potentially prevents colon cancer cells from high oxygen levels. CONCLUSIONS: Our work explored the mechanisms of recurrence and metastasis in colon cancer and provided potential targets for drug development.
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Adenocarcinoma , Neoplasias do Colo , MicroRNAs , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Adenocarcinoma/genética , Redes Reguladoras de Genes , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Biomarcadores Tumorais/genética , MicroRNAs/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Ferritin is a spherical nanocage protein for iron storage, composed of 24 light- or heavy-polypeptide chain subunits. A single ferritin molecule can carry up to 4500 iron atoms in its core, which plays an important role in suppressing intracellular iron toxicity. Serum ferritin levels are used as a marker for the total amount of iron stored in the body. Most serum ferritin is iron-free (apo-ferritin) and it is unclear how ferritin is released from cells. Ferritin is secreted into serum via extracellular vesicles (EVs) or the secretory autophagy pathway but not via the classical endoplasmic reticulum (ER)-to-Golgi secretion pathway. We recently discovered that the level of tetraspanin CD63, a common EV marker, is post-transcriptionally regulated by the intracellular iron level and both CD63 and ferritin expression is induced by iron loading. Ferritin is incorporated into CD63(+)-EVs through the ferritin-specific autophagy adapter molecule, NCOA4, and then secreted from cells. EV production differs drastically depending on cell type and physiological conditions. Extracellular matrix detached cells express pentaspanin prominin 2 and prominin 2(+)-EVs secrete ferritin independently of NCOA4 trafficking. Ferritin is tightly bound to iron in EVs and functions as an iron-carrier protein in the extracellular environment. Cells can suppress ferroptosis by secreting holo-ferritin, which reduces intracellular iron concentration. However, this exposes the neighboring cells receiving the secreted holo-ferritin to a large excess of iron. This results in cellular toxicity through increased generation of reactive oxygen species (ROS). Here we review the machinery by which ferritin is incorporated into EVs and its role as an intercellular communication molecule.
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Vesículas Extracelulares , Antígeno AC133 , Transporte Biológico , Autofagia , FerritinasRESUMO
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.
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Sulfeto de Hidrogênio , Sobrecarga de Ferro , Animais , Camundongos , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Músculo Liso Vascular/metabolismo , Homeostase , Ferro/metabolismo , Cistationina gama-Liase/metabolismoRESUMO
OBJECTIVE: Increasing evidence indicates that prolonged exposure to sulforaphane (SFN) can improve malignancies. However, the role of iron in SFN-triggered death in gastric carcinoma cells and the underlying molecular mechanisms remain unclear. Thus, the current study explored the effects of SFN on iron overload-mediated ferroptosis and the PI3K/IRP2/DMT1 pathway in gastric carcinoma cells. METHODS: We utilized the MGC-803 cell line to assess whether SFN affected iron metabolism and whether this effect contributed to cell death. Pharmacological inhibition of iron metabolism also was performed to determine the molecular mechanism underlying SFN-triggered iron overload and the disturbance in iron metabolism. RESULTS: Our data revealed that SFN treatment altered iron homeostasis and led to iron overload in vitro. Interestingly, SFN-stimulated cell death resulted from ferroptosis, a recently identified iron-dependent form of regulated cell death. Furthermore, an iron chelator, deferiprone, ameliorated the SFN-triggered mitochondrial dysfunction and reduced the iron overload. In addition, we found that the SFN-triggered iron overload was regulated by the PI3K/IRP2/DMT1 signaling pathway. CONCLUSION: We discovered that disturbance in iron metabolism might be involved in the SFN-triggered cell death in gastric carcinoma cells. Blockade of the PI3K/IRP2/DMT1 axis could provide a feedback effect on SFN-induced ferroptosis to protect tumor cells from growth.
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Carcinoma , Ferroptose , Sobrecarga de Ferro , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Sobrecarga de Ferro/metabolismo , Ferro/metabolismoRESUMO
Ransomware is a type of malware that employs encryption to target user files, rendering them inaccessible without a decryption key. To combat ransomware, researchers have developed early detection models that seek to identify threats before encryption takes place, often by monitoring the initial calls to cryptographic APIs. However, because encryption is a standard computational activity involved in processes, such as packing, unpacking, and polymorphism, the presence of cryptographic APIs does not necessarily indicate an imminent ransomware attack. Hence, relying solely on cryptographic APIs is insufficient for accurately determining a ransomware pre-encryption boundary. To this end, this paper is devoted to addressing this issue by proposing a Temporal Data Correlation method that associates cryptographic APIs with the I/O Request Packets (IRPs) based on the timestamp for pre-encryption boundary delineation. The process extracts the various features from the pre-encryption dataset for use in early detection model training. Several machine and deep learning classifiers are used to evaluate the accuracy of the proposed solution. Preliminary results show that this newly proposed approach can achieve higher detection accuracy compared to those reported elsewhere.
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BACKGROUND: The dysfunctions in the metabolism of iron have an important role in many pathological conditions, ranging from disease with iron deposition to cancer. Studies on malignant diseases of the breast reported irregular expression in genes associated with iron metabolism. The variations are related to findings that have prognostic significance. This study evaluated the relationship of the expression levels of transferrin receptor 1 (TFRC), iron regulatory protein 1 (IRP1), hepcidin (HAMP), ferroportin 1 (FPN1), hemojuvelin (HFE2), matriptase 2 (TMPRSS6), and miR-122 genes in the normal and malignant tissues of breast cancer patients. METHODS & RESULTS: The normal and malignant tissues from 75 women with breast malignancies were used in this study. The patients did not receive any treatment previously. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used in figuring the levels of gene expression associated with iron metabolism. When the malignant and normal tissues gene expression levels were analyzed, expression of TFRC increased (1.586-fold); IRP1 (0.594 fold) and miR-122 (0.320 fold) expression decreased; HAMP, FPN1, HFE2, and TMPRSS6 expressions did not change. FPN1 and IRP1 had a positive association, and this association was statistically significant (r = 0.266; p = 0.022). IRP1 and miR-122 had a positive association, and this association had statistical significance (r = 0.231; p = 0.048). CONCLUSIONS: Our study portrayed the important association between genes involved in iron hemostasis and breast malignancy. The results could be used to establish new diagnostic techniques in the management of breast malignancies. The alterations in the metabolism of malignant breast cells with normal breast cells could be utilized to achieve advantages in treatment.
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Neoplasias da Mama , MicroRNAs , Humanos , Feminino , Neoplasias da Mama/genética , Ferro/metabolismo , Homeostase/genética , MicroRNAs/genéticaRESUMO
Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.
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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.
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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.
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Sobrecarga de Ferro , Lactobacillus johnsonii , Salmonella enterica , Humanos , Animais , Camundongos , Salmonella typhimurium , Proteína 2 Reguladora do Ferro/metabolismo , Lactobacillus johnsonii/metabolismo , Antioxidantes/farmacologia , Células HeLa , Sorogrupo , Estresse Oxidativo , Ferro/metabolismo , Diarreia , HomeostaseRESUMO
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.
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Biotinilação , Proteínas de Transporte , Cromatografia de Afinidade , DNA , RNA , Humanos , Proteínas de Transporte/química , DNA/química , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/química , Proteína 2 Reguladora do Ferro/química , Fator 2 Relacionado a NF-E2/química , RNA/química , Estreptavidina/metabolismo , Cromatografia de Afinidade/métodosRESUMO
Introduction: Neurons 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. Methods: Here 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. Results: NCOA4 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. Discussion: These 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.
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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.
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Hepcidinas , Deficiências de Ferro , Sobrecarga de Ferro , Ferro , Animais , Hemocromatose/metabolismo , Hepcidinas/metabolismo , Inflamação , Ferro/metabolismo , Deficiências de Ferro/metabolismoRESUMO
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.
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Carcinoma Hepatocelular , Proteína 2 Reguladora do Ferro , Ferro , Fatores de Transcrição Kruppel-Like , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Flufenazina , Regulação Neoplásica da Expressão Gênica , Homeostase , Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismoRESUMO
BACKGROUND: Although ß-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 ß-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 ß-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 ß-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in ß-catenin-activated cells. Online databases were analyzed for correlation between ß-catenin activity and IRP2-TfR1 axis in human cancers. RESULTS: Iron chelators were identified as selective inhibitors against ß-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed ß-catenin-activated cell proliferation and tumor formation in mice. Mechanically, ß-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired ß-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced ß-catenin-associated cell viability and tumor formation. CONCLUSIONS: ß-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of ß-catenin-potentiated cancer.
Assuntos
Proteína 2 Reguladora do Ferro , Neoplasias , Camundongos , Humanos , Animais , Proteína 2 Reguladora do Ferro/metabolismo , beta Catenina/metabolismo , Ferro/metabolismo , Neoplasias/tratamento farmacológico , Quelantes de Ferro/farmacologia , Mitocôndrias/metabolismoRESUMO
BACKGROUND: Previously we reported the clinical safety and pharmacological activity of buntanetap (known as Posiphen or ANVS401) in healthy volunteers and mild cognitive impaired (MCI) patients (21). The data supported continued clinical evaluation of buntanetap for treating Alzheimer's Disease (AD). Neurodegenerative diseases such as AD and Parkinson's disease (PD) share several pathological manifestations, including increased levels of multiple neurotoxic protein aggregates. Therefore, a treatment strategy that targets toxic species common to both disorders can potentially provide better clinical outcomes than attacking one neurotoxic protein alone. To test this hypothesis, we recently completed a clinical study in early AD and early PD participants and report the data here. OBJECTIVES: We evaluated safety, pharmacokinetics, biomarkers, and efficacy of buntanetap in treating early AD and PD patients. DESIGN: Double-blind, placebo-controlled, multi-center study. SETTING: 13 sites in the US participated in this clinical trial. The registration number is NCT04524351 at ClinicalTrials.gov. PARTICIPANTS: 14 early AD patients and 54 early PD patients. INTERVENTION: AD patients were given either 80mg buntanetap or placebo QD. PD patients were given 5mg, 10mg, 20mg, 40mg, 80mg buntanetap or placebo QD. MEASUREMENTS: Primary endpoint is safety and tolerability; secondary endpoint is pharmacokinetics of buntanetap in plasma; exploratory endpoints are 1) biomarkers in cerebrospinal fluid (CSF) in both AD and PD patients 2) psychometric tests specific for AD (ADAS-Cogs and WAIS coding test) or PD (MDS-UPDRS and WAIS coding test). RESULTS: Buntanetap was safe and well tolerated. Biomarker data indicated a trend in lowering levels of neurotoxic proteins and inflammatory factors and improving axonal integrity and synaptic function in both AD and PD cohorts. Psychometric tests showed statistically significant improvements in ADAS-Cog11 and WAIS coding in AD patients and MDS-UPDRS and WAIS coding in PD patients. CONCLUSIONS: Buntanetap is well tolerated and safe at doses up to 80mg QD in both AD and PD patients. Cmax and AUC increase with dose without evidence for a plateau up to 80mg QD. The drug shows promising evidence in exploratory biomarker and efficacy measures. Further evaluation of buntanetap in larger, longer-term clinical trials for the treatment of AD and PD are warranted.