RESUMEN
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.
Asunto(s)
Ritmo Circadiano , Proteína 1 Reguladora de Hierro , Proteína 2 Reguladora de Hierro , Hierro , Hígado , ARN Mensajero , Animales , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/genética , Ritmo Circadiano/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Ratones , Hígado/metabolismo , Hierro/metabolismo , Regulación de la Expresión Génica , Elementos de Respuesta , Ratones Endogámicos C57BL , Masculino , Conducta AlimentariaRESUMEN
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.
Asunto(s)
Proteína 1 Reguladora de Hierro , Hierro , Humanos , Animales , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Ratones , Proteína 2 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/genética , Biotinilación , Elementos de Respuesta , Fosforilación , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Reguladoras del Hierro/genética , Unión Proteica , Línea Celular TumoralRESUMEN
Acute bacterial orchitis (AO) is a prevalent cause of intrascrotal inflammation, often resulting in sub- or infertility. A frequent cause eliciting AO is uropathogenic Escherichia coli (UPEC), a gram negative pathovar, characterized by the expression of various iron acquisition systems to survive in a low-iron environment. On the host side, iron is tightly regulated by iron regulatory proteins 1 and 2 (IRP1 and -2) and these factors are reported to play a role in testicular and immune cell function; however, their precise role remains unclear. Here, we showed in a mouse model of UPEC-induced orchitis that the absence of IRP1 results in less testicular damage and a reduced immune response. Compared with infected wild-type (WT) mice, testes of UPEC-infected Irp1-/- mice showed impaired ERK signaling. Conversely, IRP2 deletion led to a stronger inflammatory response. Notably, differences in immune cell infiltrations were observed among the different genotypes. In contrast with WT and Irp2-/- mice, no increase in monocytes and neutrophils was detected in testes of Irp1-/- mice upon UPEC infection. Interestingly, in Irp1-/- UPEC-infected testes, we observed an increase in a subpopulation of macrophages (F4/80+CD206+) associated with antiinflammatory and wound-healing activities compared with WT. These findings suggest that IRP1 deletion may protect against UPEC-induced inflammation by modulating ERK signaling and dampening the immune response.
Asunto(s)
Proteína 1 Reguladora de Hierro , Proteína 2 Reguladora de Hierro , Orquitis , Animales , Masculino , Ratones , Inflamación , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/metabolismo , Orquitis/microbiología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/metabolismoRESUMEN
Ferroptosis has been implicated in several neurological disorders and may be therapeutically targeted. However, the susceptibility to ferroptosis varies in different cells, and inconsistent results have been reported even using the same cell line. Understanding the effects of key variables of in vitro studies on ferroptosis susceptibility is of critical importance to facilitate drug discoveries targeting ferroptosis. Here, we showed that increased cell seeding density leads to enhanced resistance to ferroptosis by reducing intracellular iron levels. We further identified iron-responsive protein 1 (IRP1) as the key protein affected by cell density, which affects the expression of ferroportin or transferrin receptor and results in altered iron levels. Such observations were consistent across different cell lines, indicating that cell density should be tightly controlled in studies of ferroptosis. Since cell densities vary in different brain regions, these results may also shed light on selective regional vulnerability observed in neurological disorders.
Asunto(s)
Ferroptosis , Homeostasis , Proteína 1 Reguladora de Hierro , Hierro , Hierro/metabolismo , Ferroptosis/fisiología , Homeostasis/fisiología , Humanos , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Recuento de Células , Animales , Receptores de Transferrina/metabolismo , Receptores de Transferrina/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , RatonesRESUMEN
Mutant huntingtin (mHtt) proteins interact to form aggregates, disrupting cellular functions including transcriptional dysregulation and iron imbalance in patients with Huntington's disease (HD) and mouse disease models. Previous studies have indicated that mHtt may lead to abnormal iron homeostasis by upregulating the expression of iron response protein 1 (IRP1) in the striatum and cortex of N171-82Q HD transgenic mice, as well as in HEK293 cells expressing the N-terminal fragment of mHtt containing 160 CAG repeats. However, the mechanism underlying the upregulation of IRP1 remains unclear. We investigated the levels and phosphorylation status of signal transducer and activator of transcription 5 (STAT5) in the brains of N171-82Q HD transgenic mice using immunohistochemistry staining. We also assessed the nuclear localization of STAT5 protein through western blot and immunofluorescence, and measured the relative RNA expression levels of STAT5 and IRP1 using RT-PCR in both N171-82Q HD transgenic mice and HEK293 cells expressing the N-terminal fragment of huntingtin. Our findings demonstrate that the transcription factor STAT5 regulates the transcription of the IPR1 gene in HEK293 cells. Notably, both the brains of N171-82Q mice and 160Q HEK293 cells exhibited increased nuclear content of STAT5, despite unchanged total STAT5 expression. These results suggest that mHtt promotes the nuclear translocation of STAT5, leading to enhanced expression of IRP1. The nuclear translocation of STAT5 initiates abnormal iron homeostatic pathways, characterized by elevated IRP1 expression, increased levels of transferrin and transferrin receptor, and iron accumulation in the brains of HD mice. These findings provide valuable insights into potential therapeutic strategies targeting iron homeostasis in HD.
Asunto(s)
Enfermedad de Huntington , Sobrecarga de Hierro , Proteína 1 Reguladora de Hierro , Ratones Transgénicos , Factor de Transcripción STAT5 , Regulación hacia Arriba , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Animales , Humanos , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Células HEK293 , Ratones , Sobrecarga de Hierro/metabolismo , Factor de Transcripción STAT5/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Núcleo Celular/metabolismo , Encéfalo/metabolismoRESUMEN
Disrupted iron homeostasis in the substantia nigra pars compacta (SNpc) is an important pathological mechanism in Parkinson's disease (PD). It is unclear what role microglia play in iron metabolism and selective iron deposition in the SNpc of PD brain. In this study, we observed that 6-hydroxydopamine (6-OHDA) induced the expression of divalent metal transporter-1 (DMT1) and iron influx in BV2 microglia cells, which might be associated with the upregulation of iron regulatory protein 1 (IRP1) expression. Moreover, we found that 6-OHDA had no significant effect on the expression of ferroportin 1 (FPN1) and iron efflux in BV2 microglial cells, which might be the combined action of IRP1 upregulation and reduced hepcidin levels. Furthermore, 6-OHDA treatment activated BV2 microglia and enhanced the release of pro-inflammatory cytokines. Interestingly, iron overloading suppressed IRP1 expression, thus downregulating DMT1 and upregulating FPN1 levels in these microglial cells. On the contrary, iron deficiency activated IRP1, leading to increased expression of DMT1 and decreased expression of FPN1-which indicates that activated IRP1 induces iron overloading in 6-OHDA-treated microglia, but not iron overloading modulates the expression of IRP1. Taken together, our data suggest that 6-OHDA can regulate the expression of DMT1 and FPN1 by activating IRP1 and inhibiting hepcidin release, thus leading to abnormal iron sequestration in microglia. In addition, 6-OHDA can activate microglia, which leads to increased release of pro-inflammatory factors that can further induce genome damage in dopaminergic neurons.
Asunto(s)
Hepcidinas , Proteína 1 Reguladora de Hierro , Hepcidinas/genética , Hepcidinas/metabolismo , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Microglía/metabolismo , Oxidopamina/metabolismo , Oxidopamina/farmacologíaRESUMEN
α-Enolase 1 (ENO1) is a critical glycolytic enzyme whose aberrant expression drives the pathogenesis of various cancers. ENO1 has been indicated as having additional roles beyond its conventional metabolic activity, but the underlying mechanisms and biological consequences remain elusive. Here, we show that ENO1 suppresses iron regulatory protein 1 (IRP1) expression to regulate iron homeostasis and survival of hepatocellular carcinoma (HCC) cells. Mechanistically, we demonstrate that ENO1, as an RNA-binding protein, recruits CNOT6 to accelerate the messenger RNA decay of IRP1 in cancer cells, leading to inhibition of mitoferrin-1 (Mfrn1) expression and subsequent repression of mitochondrial iron-induced ferroptosis. Moreover, through in vitro and in vivo experiments and clinical sample analysis, we identified IRP1 and Mfrn1 as tumor suppressors by inducing ferroptosis in HCC cells. Taken together, this study establishes an important role for the ENO1-IRP1-Mfrn1 pathway in the pathogenesis of HCC and reveals a previously unknown connection between this pathway and ferroptosis, suggesting a potential innovative cancer therapy.
Asunto(s)
Carcinoma Hepatocelular , Ferroptosis , Proteína 1 Reguladora de Hierro/metabolismo , Neoplasias Hepáticas , Biomarcadores de Tumor , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Ferroptosis/genética , Humanos , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Neoplasias Hepáticas/genética , Fosfopiruvato Hidratasa/genética , ARN Mensajero/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Chronic kidney disease involves disturbances in iron metabolism including anemia caused by insufficient erythropoietin (EPO) production. However, underlying mechanisms responsible for the dysregulation of cellular iron metabolism are incompletely defined. Using the unilateral ureteral obstruction (UUO) model in Irp1+/+ and Irp1-/- mice, we asked if iron regulatory proteins (IRPs), the central regulators of cellular iron metabolism and suppressors of EPO production, contribute to the etiology of anemia in kidney failure. We identified a significant reduction in IRP protein level and RNA binding activity that associates with a loss of the iron uptake protein transferrin receptor 1 (TfR1), increased expression of the iron storage protein subunits H- and L-ferritin, and a low but overall variable level of stainable iron in the obstructed kidney. This reduction in IRP RNA binding activity and ferritin RNA levels suggests the concomitant rise in ferritin expression and iron content in kidney failure is IRP dependent. In contrast, the reduction in the Epo mRNA level in the obstructed kidney was not rescued by genetic ablation of IRP1, suggesting disruption of normal hypoxia-inducible factor (HIF)-2α regulation. Furthermore, reduced expression of some HIF-α target genes in UUO occurred in the face of increased expression of HIF-α proteins and prolyl hydroxylases 2 and 1, the latter of which is not known to be HIF-α mediated. Our results suggest that the IRP system drives changes in cellular iron metabolism that are associated with kidney failure in UUO but that the impact of IRPs on EPO production is overridden by disrupted hypoxia signaling.NEW & NOTEWORTHY This study demonstrates that iron metabolism and hypoxia signaling are dysregulated in unilateral obstructive nephropathy. Expression of iron regulatory proteins (IRPs), central regulators of cellular iron metabolism, and the iron uptake (transferrin receptor 1) and storage (ferritins) proteins they target is strongly altered. This suggests a role of IRPs in previously observed changes in iron metabolism in progressive renal disease. Hypoxia signaling is disrupted and appeared to dominate the action of IRP1 in controlling erythropoietin expression.
Asunto(s)
Anemia/etiología , Hierro/metabolismo , Riñón/metabolismo , Insuficiencia Renal/etiología , Obstrucción Ureteral/complicaciones , Anemia/metabolismo , Anemia/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Modelos Animales de Enfermedad , Eritropoyetina/genética , Eritropoyetina/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Fibrosis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Extracellular vesicles (EVs) transfer functional molecules between cells. CD63 is a widely recognized EV marker that contributes to EV secretion from cells. However, the regulation of its expression remains largely unknown. Ferritin is a cellular iron storage protein that can also be secreted by the exosome pathway, and serum ferritin levels classically reflect body iron stores. Iron metabolism-associated proteins such as ferritin are intricately regulated by cellular iron levels via the iron responsive element-iron regulatory protein (IRE-IRP) system. Herein, we present a novel mechanism demonstrating that the expression of the EV-associated protein CD63 is under the regulation of the IRE-IRP system. We discovered a canonical IRE in the 5' untranslated region of CD63 messenger RNA that is responsible for regulating its expression in response to increased iron. Cellular iron loading caused a marked increase in CD63 expression and the secretion of CD63+ EVs from cells, which were shown to contain ferritin-H and ferritin-L. Our results demonstrate that under iron loading, intracellular ferritin is transferred via nuclear receptor coactivator 4 (NCOA4) to CD63+ EVs that are then secreted. Such iron-regulated secretion of the major iron storage protein ferritin via CD63+ EVs, is significant for understanding the local cell-to-cell exchange of ferritin and iron.
Asunto(s)
Apoferritinas/metabolismo , Vesículas Extracelulares/metabolismo , Ferritinas/metabolismo , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/metabolismo , Oxidorreductasas/metabolismo , Tetraspanina 30/metabolismo , Apoferritinas/genética , Línea Celular , Vesículas Extracelulares/genética , Ferritinas/genética , Silenciador del Gen , Humanos , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/genética , Oxidorreductasas/genética , Transporte de Proteínas , ARN Mensajero/genética , Tetraspanina 30/genética , Regulación hacia ArribaRESUMEN
Evidence is reviewed for the role of glutathione in providing a ligand for the cytosolic iron pool. The possibility of histidine and carnosine forming ternary complexes with iron(II)glutathione is discussed and the physiological significance of these interactions considered. The role of carnosine in muscle, brain, and kidney physiology is far from established and evidence is presented that the iron(II)-binding capability of carnosine relates to this role.
Asunto(s)
Carnosina/metabolismo , Glutatión/metabolismo , Histidina/metabolismo , Quelantes del Hierro/metabolismo , Hierro/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/metabolismo , Riñón/citología , Riñón/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Músculos/citología , Músculos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The development of thermogenic adipocytes concurs with mitochondrial biogenesis, an iron-dependent pathway. Iron regulatory proteins (IRP) 1 and 2 are RNA-binding proteins that regulate intracellular iron homeostasis. IRPs bind to the iron-response element (IRE) of their target mRNAs, balancing iron uptake and deposition at the posttranscriptional levels. However, IRP/IRE-dependent iron regulation in adipocytes is largely unknown. We hypothesized that iron demands are higher in brown/beige adipocytes than white adipocytes to maintain the thermogenic mitochondrial capacity. To test this hypothesis, we investigated the IRP/IRE regulatory system in different depots of adipose tissue. Our results revealed that 1) IRP/IRE interaction was increased in proportional to the thermogenic function of the adipose depot, 2) adipose iron content was increased in adipose tissue browning upon ß3-adrenoceptor stimulation, while decreased in thermoneutral conditions, and 3) modulation of iron content was linked with mitochondrial biogenesis. Moreover, the iron requirement was higher in HIB1B brown adipocytes than 3T3-L1 white adipocytes during differentiation. The reduction of the labile iron pool (LIP) suppressed the differentiation of brown/beige adipocytes and mitochondrial biogenesis. Using the 59Fe-Tf, we also demonstrated that thermogenic stimuli triggered cell-autonomous iron uptake and mitochondrial compartmentalization as well as enhanced mitochondrial respiration. Collectively, our work demonstrated that IRP/IRE signaling and subsequent adaptation in iron metabolism are a critical determinant for the thermogenic function of adipocytes.
Asunto(s)
Aconitato Hidratasa/metabolismo , Adipocitos/metabolismo , Hierro/metabolismo , Termogénesis/fisiología , Células 3T3-L1 , Aclimatación , Adipocitos Beige/metabolismo , Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Animales , Regulación de la Temperatura Corporal/fisiología , Diferenciación Celular , Homeostasis , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Biogénesis de Organelos , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Hemoglobin is the essential oxygen-carrying molecule in humans and is regulated by cellular iron and oxygen sensing mechanisms. To search for novel variants associated with hemoglobin concentration, we performed genome-wide association studies of hemoglobin concentration using a combined set of 684,122 individuals from Iceland and the UK. Notably, we found seven novel variants, six rare coding and one common, at the ACO1 locus associating with either decreased or increased hemoglobin concentration. Of these variants, the missense Cys506Ser and the stop-gained Lys334Ter mutations are specific to eight and ten generation pedigrees, respectively, and have the two largest effects in the study (EffectCys506Ser = -1.61 SD, CI95 = [-1.98, -1.35]; EffectLys334Ter = 0.63 SD, CI95 = [0.36, 0.91]). We also find Cys506Ser to associate with increased risk of persistent anemia (OR = 17.1, P = 2 × 10-14). The strong bidirectional effects seen in this study implicate ACO1, a known iron sensing molecule, as a major homeostatic regulator of hemoglobin concentration.
Asunto(s)
Eritropoyesis/genética , Mutación con Ganancia de Función , Hemoglobinas/metabolismo , Proteína 1 Reguladora de Hierro/genética , Mutación con Pérdida de Función , Biomarcadores/sangre , Bases de Datos Genéticas , Estudio de Asociación del Genoma Completo , Humanos , Islandia , Proteína 1 Reguladora de Hierro/metabolismo , Reino UnidoRESUMEN
The common clinical symptoms of Friedreich's ataxia (FRDA) include ataxia, muscle weakness, type 2 diabetes and heart failure, which are caused by impaired mitochondrial function due to the loss of frataxin (FXN) expression. Endurance exercise is the most powerful intervention for promoting mitochondrial function; however, its impact on FRDA has not been studied. Here we found that mice with genetic knockout and knock-in of the Fxn gene (KIKO mice) developed exercise intolerance, glucose intolerance and moderate cardiac dysfunction at 6 months of age. These abnormalities were associated with impaired mitochondrial respiratory function concurrent with reduced iron regulatory protein 1 (Irp1) expression as well as increased oxidative stress, which were not due to loss of mitochondrial content and antioxidant enzyme expression. Importantly, long-term (4 months) voluntary running in KIKO mice starting at a young age (2 months) completely prevented the functional abnormalities along with restored Irp1 expression, improved mitochondrial function and reduced oxidative stress in skeletal muscle without restoring Fxn expression. We conclude that endurance exercise training prevents symptomatic onset of FRDA in mice associated with improved mitochondrial function and reduced oxidative stress. These preclinical findings may pave the way for clinical studies of the impact of endurance exercise in FRDA patients.
Asunto(s)
Ataxia de Friedreich/prevención & control , Condicionamiento Físico Animal/métodos , Carrera , Animales , Ataxia de Friedreich/genética , Ataxia de Friedreich/fisiopatología , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Proteínas de Unión a Hierro/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Estrés Oxidativo , FrataxinaRESUMEN
The classic view is that iron regulatory proteins operate at the post-transcriptional level. Iron Regulatory Protein 1 (IRP1) shifts between an apo-form that binds mRNAs and a holo-form that harbors a [4Fe4S] cluster. The latter form is not considered relevant to iron regulation, but rather thought to act as a non-essential cytosolic aconitase. Recent work in Drosophila, however, shows that holo-IRP1 can also translocate to the nucleus, where it appears to downregulate iron metabolism genes, preparing the cell for a decline in iron uptake. The shifting of IRP1 between states requires a functional mitoNEET pathway that includes a glycogen branching enzyme for the repair or disassembly of IRP1's oxidatively damaged [3Fe4S] cluster. The new findings add to the notion that glucose metabolism is modulated by iron metabolism. Furthermore, we propose that ferritin ferroxidase activity participates in the repair of the IRP1 [3Fe4S] cluster leading to the hypothesis that cytosolic ferritin directly contributes to cellular iron sensing.
Asunto(s)
Proteína 1 Reguladora de Hierro/genética , Proteínas Reguladoras del Hierro/genética , Proteínas Hierro-Azufre/genética , Hierro/metabolismo , Aconitato Hidratasa/genética , Núcleo Celular/genética , Ceruloplasmina/genética , Citosol/metabolismo , Ferritinas/genética , Regulación de la Expresión Génica/genética , Proteínas Hierro-Azufre/química , Oxidación-Reducción , ARN Mensajero/genéticaRESUMEN
Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.
Asunto(s)
Cromosomas Humanos Par 9/genética , Citratos/farmacología , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Proteína 1 Reguladora de Hierro/deficiencia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citratos/uso terapéutico , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidores Enzimáticos/uso terapéutico , Femenino , Xenoinjertos , Humanos , Proteína 1 Reguladora de Hierro/antagonistas & inhibidores , Proteína 1 Reguladora de Hierro/genética , Melanoma/genética , Ratones , Ratones Desnudos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Neoplasias Cutáneas/genéticaRESUMEN
Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/genética , Proteínas de Drosophila/genética , Regulación de la Expresión Génica/genética , Proteína 1 Reguladora de Hierro/genética , Hierro/metabolismo , Porfirias/genética , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Transporte Activo de Núcleo Celular , Animales , Regulación hacia Abajo , Drosophila , Proteínas de Drosophila/metabolismo , Ecdisteroides/biosíntesis , Glándulas Endocrinas/metabolismo , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Hemo/metabolismo , Proteína 1 Reguladora de Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Larva/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Porfirias/metabolismo , ARN Mensajero/metabolismoRESUMEN
Sideroflexin4 (SFXN4) is a member of a family of nuclear-encoded mitochondrial proteins. Rare germline mutations in SFXN4 lead to phenotypic characteristics of mitochondrial disease including impaired mitochondrial respiration and hematopoetic abnormalities. We sought to explore the function of this protein. We show that knockout of SFXN4 has profound effects on Fe-S cluster formation. This in turn diminishes mitochondrial respiratory chain complexes and mitochondrial respiration and causes a shift to glycolytic metabolism. SFXN4 knockdown reduces the stability and activity of cellular Fe-S proteins, affects iron metabolism by influencing the cytosolic aconitase-IRP1 switch, redistributes iron from the cytosol to mitochondria, and impacts heme synthesis by reducing levels of ferrochelatase and inhibiting translation of ALAS2. We conclude that SFXN4 is essential for normal functioning of mitochondria, is necessary for Fe-S cluster biogenesis and iron homeostasis, and plays a critical role in mitochondrial respiration and synthesis of heme.
Asunto(s)
Hemo/biosíntesis , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Consumo de Oxígeno , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Técnicas de Inactivación de Genes , Glucólisis , Células HEK293 , Hemo/genética , Células Hep G2 , Humanos , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Células K562 , Proteínas de la Membrana/genética , Mitocondrias/genéticaRESUMEN
The purpose of this study was to determine whether the enteric coating process affects growth performance, Fe bioavailability, and gene expression levels that maintain iron balance in the body. The test was divided into the control group, ferrous sulfate group, ferrous fumarate group, ferrous glycine chelate(1:1) (Fe-Gly(1:1)) group, ferrous glycine chelate(2:1) (Fe-Gly(2:1)) group, enteric-coated Fe-Gly(1:1) group, and enteric-coated Fe-Gly(2:1) group. The results showed that the growth performance of the rats in each iron supplement group was no significant difference among them. The results of serum biochemical indicators showed that the antioxidant capacity of the rats in the iron supplement group after enteric coating increased. The iron supplementation effect of Fe-Gly(1:1) and Fe-Gly(2:1) was better than that of ferrous sulfate, and the effect of Fe-Gly(1:1) after enteric coating was enhanced. The expression levels of IRP1 and IRP2 in the genes of enteric-coated Fe-Gly(1:1) and enteric-coated Fe-Gly(2:1) were significantly higher than those of ferrous sulfate. The expression levels of IRP1 and IRP2 in the protein of enteric-coated Fe-Gly(1:1) group were significantly higher than those in the Fe-Gly(1:1) group. The above results show that Fe-Gly can improve the bioavailability and antioxidant capacity of iron and reduce the iron output of feces after enteric coating.
Asunto(s)
Compuestos Ferrosos/farmacología , Hierro/metabolismo , Animales , Antioxidantes , Disponibilidad Biológica , Suplementos Dietéticos/análisis , Compuestos Ferrosos/metabolismo , Homeostasis/genética , Homeostasis/fisiología , Proteína 1 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/genética , Ratas , Ratas Sprague-Dawley , Comprimidos Recubiertos/análisisRESUMEN
In Schizosaccharomyces pombe, there are two aconitases, Aco1 and Aco2, involved in the Krebs cycle in mitochondria. Interestingly, Aco2 is localized to nucleus as well. Here, we investigated the nuclear role of Aco2 by deleting its nuclear localization signal. The aco2ΔNLS mutation suppressed the gene-silencing defects of RNAi mutants at the centromere, where heterochromatin formation depends on RNAi pathway. In Δago1, the aco2ΔNLS mutation restored heterochromatin through elevating Chp1 binding. Aco2 physically interacted with Chp1 via the N-terminal chromodomain that binds to methylated histone H3K9. In the sub-telomeric region, where heterochromatin forms independent of RNAi pathway, the single aco2ΔNLS mutation caused extra gene silencing via elevating Chp1 binding, without increasing histone methylation. The anti-silencing effect did not require the catalytic function of aconitase. Taken together, Aco2 functions as an epigenetic regulator of gene expression, through associating with chromodomain of Chp1 to maintain heterochromatin.
Asunto(s)
Aconitato Hidratasa/genética , Proteínas de Ciclo Celular/genética , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Heterocromatina/química , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Aconitato Hidratasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrómero , Ensamble y Desensamble de Cromatina , ADN de Hongos/genética , ADN de Hongos/metabolismo , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Señales de Localización Nuclear , Unión Proteica , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Eliminación de SecuenciaRESUMEN
Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (Wâ¯=â¯A or U and Hâ¯=â¯A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.