RESUMO
Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum-Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body-exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.
Assuntos
Ferritinas/metabolismo , Vesículas Secretórias/metabolismo , Motivos de Aminoácidos , Animais , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Exossomos/ultraestrutura , Ferritinas/sangue , Ferritinas/química , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7RESUMO
[This corrects the article on p. 194 in vol. 5, PMID: 25202274.].
RESUMO
Epithelial barriers are found in many tissues such as the intestine, kidney and brain where they separate the external environment from the body or a specific compartment from its periphery. Due to the tight junctions that connect epithelial barrier-cells (EBCs), the transport of compounds takes place nearly exclusively across the apical or basolateral membrane, the cell-body and the opposite membrane of the polarized EBC, and is regulated on numerous levels including barrier-specific adapted trafficking-machineries. Iron is an essential element but toxic at excess. Therefore, all iron-requiring organisms tightly regulate iron concentrations on systemic and cellular levels. In contrast to most cell types that control just their own iron homeostasis, EBCs also regulate homeostasis of the compartment they enclose or the body as a whole. Iron is transported across EBCs by specialized transporters such as the transferrin receptor and ferroportin. Recently, the iron storage protein ferritin was also attributed a role in the regulation of systemic iron homeostasis and we gathered evidence from the literature and original data that ferritin is polarized in EBC, suggesting also a role for ferritin in iron trafficking across EBCs.
RESUMO
The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.
Assuntos
Células Germinativas/metabolismo , Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Ferritinas/metabolismo , Imunofluorescência , Células Germinativas/efeitos dos fármacos , Proteína da Hemocromatose , Hepcidinas , Antígenos de Histocompatibilidade Classe I/genética , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Sobrecarga de Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Mutação/fisiologia , Receptores da Transferrina/metabolismo , Túbulos Seminíferos/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismoRESUMO
Heme-oxygenase 1 is an endoplasmic reticulum-anchored enzyme that breaks down heme into iron, carbon monoxide and biliverdin. Heme is a hydrophobic co-factor in many proteins, including hemoglobin. Free heme is highly cytotoxic and, therefore, both heme synthesis and breakdown are tightly regulated. During turnover of heme proteins, heme is released in the phago-lysosomal compartment or the cytosol. The subcellular location of the heme-oxygenase 1 active site has not been clarified. Using constructs of heme-oxygenase 1 with fluorescent proteins, and the endogenous heme-oxygenase 1 in two variations of protease protection assays, we determined that heme-oxygenase 1 is membrane-bound and faces the cytosol in non-activated macrophages in vivo. These findings imply that in quiescent macrophages, heme breakdown products are generated in the cytosol. This facilitates iron recycling to ferroportin for iron export and to ferritin for iron storage.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Heme Oxigenase-1/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Heme/metabolismo , Camundongos , Transporte ProteicoRESUMO
BACKGROUND: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. DESIGN AND METHODS: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. RESULTS: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. CONCLUSIONS: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.
Assuntos
Envelhecimento Eritrocítico/fisiologia , Eritrócitos/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Animais , Biomarcadores/análise , Biotinilação , Transfusão de Eritrócitos , Eritrócitos/citologia , Eritropoese/fisiologia , Feminino , Citometria de Fluxo , Humanos , Ferro/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ferritin is known as a well-conserved iron detoxification and storage protein that is found in the cytosol of many prokaryotic and eukaryotic organisms. In insects and worms, ferritin has evolved into a classically secreted protein that transports iron systemically. Mammalian ferritins are found intracellularly in the cytosol, as well as in the nucleus, the endo-lysosomal compartment and the mitochondria. Extracellular ferritin is found in fluids such as serum and synovial and cerebrospinal fluids. We recently characterized the biophysical properties, secretion mechanism and cellular origin of mouse serum ferritin, which is actively secreted by a non-classical pathway involving lysosomal processing. Here, we review the data to support a hypothesis that intracellular and extracellular ferritin may play a role in intra- and intercellular redistribution of iron.
Assuntos
Ferritinas/metabolismo , Ferro/metabolismo , Animais , Transporte Biológico/fisiologia , Lisossomos/metabolismo , CamundongosRESUMO
The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway.
Assuntos
Ferritinas/sangue , Ferro/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Via Secretória , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Glicosilação , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Subunidades Proteicas , Homologia de Sequência de AminoácidosRESUMO
Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.
Assuntos
Técnicas de Tipagem Bacteriana , Repetições Minissatélites/genética , Filogenia , Vibrio cholerae/classificação , Cólera , Eletroforese em Gel de Campo Pulsado , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Vibrio cholerae/genética , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Vibrio cholerae O139/classificação , Vibrio cholerae O139/genética , Vibrio cholerae não O1/classificação , Vibrio cholerae não O1/genéticaRESUMO
Identification and typing of spoilage and pathogenic microorganisms have become major objectives over the past decade in microbiology. In food, strain typing is necessary to ensure food safety and for linking cases of foodborne infections to suspected items. Recent advances in molecular biology have resulted in the development of numerous DNA-based methods for discrimination among bacterial strains. Here, we present the use of Simple Sequence Repeats (SSR, or Microsatellites) for bacterial typing. SSRs are a class of short DNA sequence motifs that are tandemly repeated at a specific locus. Computer-based screen of the complete genomic DNA sequences of various prokaryotes showed the existence of tens of thousands well distributed SSR tracts. Mono Nucleotides Repeats (MNRs) are the majority of SSR tracts in bacteria, therefore selected MNR loci were analyzed for variation among strains belonging to three bacterial species: Escherichia coli, Listeria monocytogenes and Vibrio cholerae. High levels of polymorphism in the number of repeats was observed. The finding that most of the MNR tracts are variable in bacterial genomes, but stable at the strain level, allows the use of MNRs for bacterial strains identification. The variation in MNR tracts enables the separation between virulent and non-virulent strain groups and further discriminates among bacterial isolates, in the three tested bacterial species. The uncovered MNR polymorphism is important as a genome-wide source of variation, both in practical applications (e.g. rapid strain identification) and in evolutionary studies. This multi-locus MNR strategy could be applied for high throughput bacterial typing by assigning an "identity number" for each strain based on MNR variations. The developed typing technology should include the fingerprint database for large bacterial strain collections and a high throughput scanner. This accurate and rapid tool can have a major role in decreasing the incidences of food-related outbreaks and will contribute to limit epidemics.