RESUMO
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.
Assuntos
Vesículas Extracelulares , Antígeno AC133 , Transporte Biológico , Autofagia , FerritinasRESUMO
Iron homeostasis is strictly regulated at both the systemic and cellular levels by complex mechanisms because of its indispensability and toxicity. Among the various iron-regulatory proteins, ferritin is the earliest discovered regulator of iron metabolism and is a molecule that safely retains excess intracellular iron in the cores of its shells. Two types of ferritin, cytosolic ferritin and mitochondrial ferritin (FTMT), have been identified in a range of organisms from plants to humans. FTMT was identified approximately 60 years after the discovery of cytosolic ferritin. Cytosolic ferritin expression is regulated in an iron-responsive manner. Recently, the molecular mechanisms of iron-dependent degradation of cytosolic ferritin or its secretion into serum have been clarified. FTMT, which shares a high degree of sequence homology with cytosolic ferritin, has distinct functions and is regulated in different ways from cytosolic ferritin. Although knowledge of the physiological role of FTMT is still incomplete, recent studies have shed light on the function and regulation of FTMT. The accumulating biological evidence of both ferritins has made it possible to deepen our knowledge about iron metabolism and its significance in diseases. In this review, we discuss the biological properties of both ferritins, focusing on their newly uncovered behaviors.
Assuntos
Ferritinas , Ferro , Humanos , Ferritinas/genética , Ferritinas/metabolismo , Ferro/metabolismoRESUMO
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.
Assuntos
Apoferritinas/metabolismo , Vesículas Extracelulares/metabolismo , Ferritinas/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Oxirredutases/metabolismo , Tetraspanina 30/metabolismo , Apoferritinas/genética , Linhagem Celular , Vesículas Extracelulares/genética , Ferritinas/genética , Inativação Gênica , Humanos , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Oxirredutases/genética , Transporte Proteico , RNA Mensageiro/genética , Tetraspanina 30/genética , Regulação para CimaRESUMO
Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. C. pneumoniae uses a cell contact-dependent type-III secretion (T3S) system to translocate pathogen effector proteins that manipulate host cellular functions. While several C. pneumoniae T3S effectors have been proposed, few have been experimentally confirmed in Chlamydia In this study, we expressed 382 C. pneumoniae genes in C. trachomatis as fusion proteins to an epitope tag derived from glycogen synthase kinase 3ß (GSK3ß) which is the target of phosphorylation by mammalian kinases. Based on the detection of the tagged C. pneumoniae protein with anti-phospho GSK3ß antibodies, we identified 49 novel C. pneumoniae-specific proteins that are translocated by C. trachomatis into the host cytoplasm and thus likely play a role as modifiers of host cellular functions. In this manner, we identified and characterized a new C. pneumoniae effector CPj0678 that recruits the host cell protein PACSIN2 to the plasma membrane. These findings indicate that C. trachomatis provides a powerful screening system to detect candidate effector proteins encoded by other pathogenic and endosymbiotic Chlamydia species.Importance Chlamydia injects numerous effector proteins into host cells to manipulate cellular functions important for bacterial survival. Based on findings in C. trachomatis, it has been proposed that between 5-10% of the C. pneumoniae genome, a related respiratory pathogen, encodes secreted effectors. However only a few C. pneumoniae effectors have been identified and experimentally validated. With the development of methods for the stable genetic transformation of C. trachomatis, it is now possible to use C. trachomatis shuttle plasmids to express and explore the function of proteins from other Chlamydia in a more relevant bacterial system. In this study, we experimentally determined that 49 C. pneumoniae-specific proteins are translocated into the host cytoplasm by Chlamydia secretion systems, and identify a novel effector required to recruit the host factor PACSIN2 to the plasma membrane during infection.
RESUMO
Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Ferritinas/genética , Ferro/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , MitofagiaRESUMO
BACKGROUND: Intracellular iron transport is mediated by iron chaperone proteins known as the poly(rC)-binding proteins (PCBPs), which were originally identified as RNA/DNA-binding molecules. SCOPE OF REVIEW: PCBPs assume a role as not only as cytosolic iron carriers, but also as regulators of iron transport and recycling. PCBP1 is involved in the iron storage pathway that involves ferritin, while PCBP2 is involved in processes that include: iron transfer from the iron importer, divalent metal ion transporter 1; iron export mediated by ferroportin-1; and heme degradation via heme oxygenase 1. MAJOR CONCLUSIONS: Both PCBP1 and PCBP2 possess iron-binding activity and form hetero/homo dimer complexes. These iron chaperones have a subset of non-redundant functions and regulate iron metabolism independently. GENERAL SIGNIFICANCE: This intracellular iron chaperone system mediated by PCBPs provide a transport "gateway" of ferrous iron that may potentially link with dynamic, inter-organelle interactions to safely traffic intracellular iron.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Transporte Biológico , Citosol/metabolismo , Humanos , Modelos Moleculares , Organelas/metabolismoRESUMO
Heme and iron are essential to almost all forms of life. The strict maintenance of heme and iron homeostasis is essential to prevent cellular toxicity and the existence of systemic and intracellular regulation is fundamental. Cytosolic heme can be catabolized and detoxified by heme oxygenases (HOs). Interestingly, free heme detoxification through HOs results in the production of free ferrous iron, which can be potentially hazardous for cells. Recently, the intracellular iron chaperone, poly (rC)-binding protein 2 (PCBP2), has been identified, which can be involved in accepting iron after heme catabolism as well as intracellular iron transport. In fact, HO1, NADPH-cytochrome P450 reductase, and PCBP2 form a functional unit that integrates the catabolism of heme with the binding and transport of iron by PCBP2. In this review, we provide an overview of our understanding of the iron chaperones and discuss the mechanism how iron chaperones bind iron released during the process of heme degradation.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Ferro/metabolismo , Metalochaperonas/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Poli C/metabolismoRESUMO
Many past and recent advances in the field of iron metabolism have relied upon the discovery of divalent metal transporter 1, DMT1 in 1997. DMT1 is the major iron transporter and contributes non-heme iron uptake in most types of cell. Each DMT1 isoform exhibits different expression patterns in cell-type specificity and distinct subcellular distribution, which enables cells to uptake both transferrin-bound and non-transferrin-bound irons efficiently. DMT1 expression is regulated by iron through the translational and degradation pathways to ensure iron homeostasis. It is considered that mammalian iron transporters including DMT1 cannot transport ferric iron but ferrous iron. Being reduced to ferrous state is likely to damage cells and tissues through the production of reactive oxygen species. Recently, iron chaperones have been identified, which can provide an answer to how ferrous iron is transported safely in cytosol. We summarize DMT1 expression depending on the types of cell or tissue and the function and mechanism of one of the iron chaperones, PCBP2.
Assuntos
Transporte Biológico/genética , Proteínas de Transporte de Cátions/genética , Ferro/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Transporte de Íons/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transferrina/genética , Transferrina/metabolismoRESUMO
Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)-NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1-CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.
Assuntos
Heme Oxigenase-1/metabolismo , Heme/metabolismo , Ferro/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Transporte Biológico , Linhagem Celular , Deleção de Genes , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/química , Heme Oxigenase-1/genética , Humanos , Metaloporfirinas/metabolismo , Mutação , NADPH-Ferri-Hemoproteína Redutase/antagonistas & inibidores , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Interferência de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Ferroportin 1 (FPN1) is an iron export protein found in mammals. FPN1 is important for the export of iron across the basolateral membrane of absorptive enterocytes and across the plasma membrane of macrophages. The expression of FPN1 is regulated by hepcidin, which binds to FPN1 and then induces its degradation. Previously, we demonstrated that divalent metal transporter 1 (DMT1) interacts with the intracellular iron chaperone protein poly(rC)-binding protein 2 (PCBP2). Subsequently, PCBP2 receives iron from DMT1 and then disengages from the transporter. In this study, we investigated the function of PCBP2 in iron export. Mammalian genomes encode four PCBPs (i.e. PCBP1-4). Here, for the first time, we demonstrated using both yeast and mammalian cells that PCBP2, but not PCBP1, PCBP3, or PCBP4, binds with FPN1. Importantly, iron-loaded, but not iron-depleted, PCBP2 interacts with FPN1. The PCBP2-binding domain of FPN1 was identified in its C-terminal cytoplasmic region. The silencing of PCBP2 expression suppressed FPN1-dependent iron export from cells. These results suggest that FPN1 exports iron received from the iron chaperone PCBP2. Therefore, it was found that PCBP2 modulates cellular iron export, which is an important physiological process.
Assuntos
Proteínas de Transporte de Cátions/biossíntese , Regulação da Expressão Gênica/fisiologia , Ferro/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Biológico Ativo/fisiologia , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Hepcidinas/genética , Hepcidinas/metabolismo , Humanos , Chaperonas Moleculares/genética , Domínios Proteicos , Proteínas de Ligação a RNA/genéticaRESUMO
Kawasaki disease (KD; MIM#61175) is a systemic vasculitis syndrome with unknown etiology which predominantly affects infants and children. Recent findings of susceptibility genes for KD suggest possible involvement of the Ca(2+)/NFAT pathway in the pathogenesis of KD. ORAI1 is a Ca(2+) release activated Ca(2+) (CRAC) channel mediating store-operated Ca(2+) entry (SOCE) on the plasma membrane. The gene for ORAI1 is located in chromosome 12q24 where a positive linkage signal was observed in our previous affected sib-pair study of KD. A common non-synonymous single nucleotide polymorphism located within exon 2 of ORAI1 (rs3741596) was significantly associated with KD (P = 0.028 in the discovery sample set (729 KD cases and 1,315 controls), P = 0.0056 in the replication sample set (1,813 KD cases vs. 1,097 controls) and P = 0.00041 in a meta-analysis by the Mantel-Haenszel method). Interestingly, frequency of the risk allele of rs3741596 is more than 20 times higher in Japanese compared to Europeans. We also found a rare 6 base-pair in-frame insertion variant associated with KD (rs141919534; 2,544 KD cases vs. 2,414 controls, P = 0.012). These data indicate that ORAI1 gene variations are associated with KD and may suggest the potential importance of the Ca(2+)/NFAT pathway in the pathogenesis of this disorder.
Assuntos
Povo Asiático/genética , Canais de Cálcio/genética , Síndrome de Linfonodos Mucocutâneos/genética , Mutagênese Insercional , Polimorfismo de Nucleotídeo Único , Adolescente , Cálcio/metabolismo , Cromossomos Humanos Par 12/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença/genética , Humanos , Japão , Masculino , Síndrome de Linfonodos Mucocutâneos/patologia , Proteína ORAI1 , Irmãos , População Branca/genética , Adulto JovemRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive desmoplastic stromal response. Fibroblast activation protein-α (FAP) is best known for its presence in stromal cancer-associated fibroblasts (CAFs). Our aim was to assess whether FAP expression was associated with the prognosis of patients with PDAC and to investigate how FAP expressing CAFs contribute to the progression of PDAC. METHODS: FAP expression was immunohistochemically assessed in 48 PDAC specimens. We also generated a fibroblastic cell line stably expressing FAP, and examined the effect of FAP-expressing fibroblasts on invasiveness and the cell cycle in MiaPaCa-2 cells (a pancreatic cancer cell line). RESULTS: Stromal FAP expression was detected in 98% (47/48) of the specimens of PDAC, with the intensity being weak in 16, moderate in 19, and strong in 12 specimens, but was not detected in the 3 control noncancerous pancreatic specimens. Patients with moderate or strong FAP expression had significantly lower cumulative survival rates than those with negative or weak FAP expression (mean survival time; 352 vs. 497 days, P = 0.006). Multivariate analysis identified moderate to strong expression of FAP as one of the factors associated with the prognosis in patients with PDAC. The intensity of stromal FAP expression was also positively correlated to the histological differentiation of PDAC (P < 0.05). FAP-expressing fibroblasts promoted the invasiveness of MiaPaCa-2 cells more intensively than fibroblasts not expressing FAP. Coculture with FAP-expressing fibroblasts significantly activated cell cycle shift in MiaPaCa-2 cells compared to coculture with fibroblasts not expressing FAP. Furthermore, coculture with FAP expressing fibroblasts inactivated retinoblastoma (Rb) protein, an inhibitor of cell cycle progression, in MiaPaCa-2 cells by promoting phosphorylation of Rb. CONCLUSIONS: The present in vitro results and the association of FAP expression with clinical outcomes provide us with a better understanding of the effect of FAP-expressing CAFs on the progression of PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Fibroblastos/metabolismo , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas/patologia , Serina Endopeptidases/metabolismo , Idoso , Carcinoma Ductal Pancreático/química , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Progressão da Doença , Endopeptidases , Feminino , Gelatinases/análise , Gelatinases/genética , Humanos , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Pâncreas/química , Neoplasias Pancreáticas/química , Fosforilação , Prognóstico , Proteína do Retinoblastoma/metabolismo , Serina Endopeptidases/análise , Serina Endopeptidases/genética , Taxa de SobrevidaRESUMO
Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. After C. pneumoniae invades host cells, it disturbs the vesicle transport system to escape host lysosomal or autophagosomal degradation. By using a yeast mis-sorting assay, we found 10 C. pneumoniae candidate genes involved in aberrant vesicular trafficking in host cells. One of the candidate genes, CPj0783, was recognized by antibodies from C. pneumoniae-infected patients. The expression of CPj0783 was detected at mid to late-cycle time points and increased during the inclusion maturation. Two-hybrid screening in yeast cells revealed that CPj0783 interacted with Huntingtin-interacting protein 14 (HIP14). The specific interaction between CPj0783 and HIP14 could be demonstrated by an in vivo co-immunoprecipitation assay and an in vitro GST pull-down assay. It was also demonstrated that HIP14 was localized in the Golgi apparatus and colocalized with CPj0783. HIP14 has a palmitoyl transferase activity that is involved in the palmitoylation-dependent vesicular trafficking of several acylated proteins. These findings suggest that CPj0783 might cause abnormal vesicle-mediated transport by interacting with HIP14. [Int Microbiol 18(4):225-233 (2015)].
Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Chlamydophila/metabolismo , Chlamydophila pneumoniae/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Aciltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Bactérias/genética , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Feminino , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ferristatin II, discovered as an iron transport inhibitor, promotes the internalization and degradation of transferrin receptor 1 (TfR1). DMT1, which mediates iron transport across cell membranes, is located at the plasma membrane of enterocytes and imports dietary iron into the cytosol. TfR1 is not directly engaged in the intestinal absorption of free iron, and iron uptake by DMT1 is attenuated by ferristatin II treatment. In this study, we found another function for ferristatin II in iron uptake. Ferristatin II did not cause degradation of DMT1 but did induce DMT1 internalization from the plasma membrane. Dynasore, a small molecule inhibitor of dynamin, did not inhibit this internalization by ferristatin II, which might occur via a clathrin-independent pathway.
Assuntos
Transporte Biológico/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Membrana Celular/metabolismo , Ferro/metabolismo , Sulfonas/farmacologia , Fatores de Transcrição/metabolismo , Antígenos CD/metabolismo , Linhagem Celular , Clatrina/metabolismo , Citosol/metabolismo , Dinaminas/antagonistas & inibidores , Enterócitos/citologia , Enterócitos/metabolismo , Humanos , Hidrazonas/farmacologia , Microscopia de Fluorescência , Receptores da Transferrina/metabolismoRESUMO
Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). We investigated whether HCV affects mitophagy in terms of mitochondrial quality control. The effect of HCV on mitophagy was examined using HCV-Japanese fulminant hepatitis-1-infected cells and the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone as a mitophagy inducer. In addition, liver cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice were examined for mitophagy. Translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without a reduction of pentaerythritol tetranitrate-induced kinase 1 activity in the presence of HCV infection both in vitro and in vivo. Coimmunoprecipitation assays revealed that Parkin associated with the HCV core protein. Furthermore, a Yeast Two-Hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment. Silencing Parkin suppressed HCV core protein expression, suggesting a functional role for the interaction between the HCV core protein and Parkin in HCV propagation. The suppressed Parkin translocation to the mitochondria inhibited mitochondrial ubiquitination, decreased the number of mitochondria sequestered in isolation membranes, and reduced autophagic degradation activity. Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting Parkin translocation to the mitochondria. This inhibition may amplify and sustain HCV-induced mitochondrial injury.
Assuntos
Antígenos da Hepatite C/metabolismo , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Transporte Proteico , UbiquitinaçãoRESUMO
DMT1 (divalent metal transporter 1) is the main iron importer found in animals, and ferrous iron is taken up by cells via DMT1. Once ferrous iron reaches the cytosol, it is subjected to subcellular distribution and delivered to various sites where iron is required for a variety of biochemical reactions in the cell. Until now, the mechanism connecting the transporter and cytosolic distribution had not been clarified. In the present study, we have identified PCBP2 [poly(rC)-binding protein 2] as a DMT1-binding protein. The N-terminal cytoplasmic region of DMT1 is the binding domain for PCBP2. An interaction between DMT1 and PCBP1, which is known to be a paralogue of PCBP2, could not be demonstrated in vivo or in vitro. Iron uptake and subsequent ferritin expression were suppressed by either DMT1 or PCBP2 knockdown. Iron-associated DMT1 could interact with PCBP2 in vitro, whereas iron-chelated DMT1 could not. These results indicate that ferrous iron imported by DMT1 is transferred directly to PCBP2. Moreover, we demonstrated that PCBP2 could bind to ferroportin, which exports ferrous iron out of the cell. These findings suggest that PCBP2 can transfer ferrous iron from DMT1 to the appropriate intracellular sites or ferroportin and could function as an iron chaperone.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Ferro/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Compostos Ferrosos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Quelantes de Ferro/metabolismo , Transferrina/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The feline leukemia virus subgroup C receptor 1 (FLVCR1) is a heme exporter that maintains the intracellular heme concentration. FLVCR1 was previously assumed to be involved in Diamond-Blackfan anemia, and it was recently reported that mutations in the FLVCR1 gene are found in patients with posterior column ataxia and retinitis pigmentosa (PCARP). Four mutations in FLVCR1 (Asn121Asp, Cys192Arg, Ala241Thr, and Gly493Arg) are located within putative transmembrane domains; however, the effects of FLVCR1 mutations on PCARP are unclear. In this study, we analyzed the function of FLVCR1 mutants by using a fluorescent heme analog as a transporter substrate, and found that all 4 FLVCR1 mutants lost their heme export activity. To investigate the mechanism responsible for this loss of activity, we determined the subcellular localization of FLVCR1 mutants. FLVCR1 mutants did not localize to the plasma membrane and were observed in intracellular structures, including lysosomes. We hypothesize that the loss of function of FLVCR1 mutants is caused by their mislocation. We examined the half-life of FLVCR1 in cells, which was >16h for wild-type FLVCR1 compared with 2-4h for the mutants. Based on these results, we propose that FLVCR1 mutants failed to fold properly in the ER, were rapidly degraded in the lysosomes, and therefore, could not export heme out of cells. Thus, accumulation of heme in FLVCR1-mutant cells could cause cellular toxicity.
Assuntos
Substituição de Aminoácidos , Heme/metabolismo , Proteínas de Membrana Transportadoras , Mutação , Dobramento de Proteína , Receptores Virais , Retinose Pigmentar , Transtornos de Sensação , Degenerações Espinocerebelares , Animais , Transporte Biológico Ativo , Linhagem Celular , Cães , Heme/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Terciária de Proteína , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Transtornos de Sensação/genética , Transtornos de Sensação/metabolismo , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/metabolismoRESUMO
We performed a genome-wide association study (GWAS) of Kawasaki disease in Japanese subjects using data from 428 individuals with Kawasaki disease (cases) and 3,379 controls genotyped at 473,803 SNPs. We validated the association results in two independent replication panels totaling 754 cases and 947 controls. We observed significant associations in the FAM167A-BLK region at 8p22-23 (rs2254546, P = 8.2 × 10(-21)), in the human leukocyte antigen (HLA) region at 6p21.3 (rs2857151, P = 4.6 × 10(-11)) and in the CD40 region at 20q13 (rs4813003, P = 4.8 × 10(-8)). We also replicated the association of a functional SNP of FCGR2A (rs1801274, P = 1.6 × 10(-6)) identified in a recently reported GWAS of Kawasaki disease. Our findings provide new insights into the pathogenesis and pathophysiology of Kawasaki disease.
Assuntos
Povo Asiático/genética , Loci Gênicos , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Síndrome de Linfonodos Mucocutâneos/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , Receptores de IgG/genéticaRESUMO
Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C. pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C. pneumoniae, we screened 455 genes with unknown function in the genome of C. pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C. pneumoniae proteins were subjected to Western blot analysis using serum samples from C. pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C. pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C. pneumoniae infections and for the development of vaccines in future.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Western Blotting , Criança , Pré-Escolar , Infecções por Chlamydophila/sangue , Chlamydophila pneumoniae/química , Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/isolamento & purificação , Clonagem Molecular , Epitopos , Feminino , Genes Bacterianos , Genoma Bacteriano , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND AND OBJECTIVE: Since 2000, the prevalence of macrolide-resistant (MR) Mycoplasma pneumoniae has increased among paediatric patients in Japan. To determine the efficacy of macrolides against MR M. pneumoniae pneumonia, microbiological and clinical efficacies were compared during the antibiotic treatment. METHODS: Samples from a total of 30 children with M. pneumoniae pneumonia, as confirmed by PCR and serology, were analyzed. Primers for domain V of 23S rRNA were used, and DNA sequences of the PCR products were compared with the sequence of an M. pneumoniae reference strain. RESULTS: Isolates from 21 patients demonstrated point mutations, and these patients were defined as MR. The remaining nine patients, whose isolates showed no point mutations, were categorized as control (macrolide-sensitive) patients. The number of M. pneumoniae in nasopharyngeal samples from the control group decreased rapidly 48 h after initiation of macrolide treatment and showed a close relationship with clinical outcome. In contrast, the number of M. pneumoniae 48 h after initiation of macrolide treatment were significantly higher in samples from MR patients than in samples from macrolide-sensitive patients. In 15 of 21 MR patients, fever persisted for more than 48 h after the initiation of macrolide treatment. When treatment was changed to minocycline, fever disappeared within 48 h in all these MR patients. There were no differences between MR patients who demonstrated a reduction in fever and those in whom fever persisted after 48 h of macrolide treatment. CONCLUSIONS: The microbiological and clinical efficacies of macrolides for treating patients with MR M. pneumoniae pneumonia were low. These results show that macrolides are clearly less effective in patients with MR M. pneumoniae pneumonia.