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1.
Blood Adv ; 2(20): 2732-2743, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30337301

RESUMO

Heme oxygenase 1 (HMOX1), the inducible enzyme that catabolizes the degradation of heme into biliverdin, iron, and carbon monoxide, plays an essential role in the clearance of senescent and damaged red blood cells, systemic iron homeostasis, erythropoiesis, vascular hemostasis, and oxidative and inflammatory stress responses. In humans, HMOX1 deficiency causes a rare and lethal disease, characterized by severe anemia, intravascular hemolysis, as well as vascular and tissue damage. Hmox1 knockout (KO) mice recapitulated the phenotypes of HMOX1-deficiency patients and could be rescued by bone marrow (BM) transplantation that engrafted donor's hematopoietic stem cells into the recipient animals after myeloablation. To find better therapy and elucidate the contribution of macrophages to the pathogenesis of HMOX1-deficiency disease, we infused wild-type (WT) macrophages into Hmox1 KO mice. Results showed that WT macrophages engrafted and proliferated in the livers of Hmox1 KO mice, which corrected the microcytic anemia, rescued the intravascular hemolysis, restored iron homeostasis, eliminated kidney iron overload and tissue damage, and provided long-term protection. These results showed that a single macrophage infusion delivered a long-term curative effect in Hmox1 KO mice, obviating the need for BM transplantation, and suggested that the HMOX1 disease stems mainly from the loss of viable reticuloendothelial macrophages. Our work provides new insights into the etiology of HMOX1 deficiency and demonstrates the potential of infusion of WT macrophages to prevent disease in patients with HMOX1 deficiency and potentially other macrophage-related diseases.


Assuntos
Anemia Hemolítica/complicações , Anemia/genética , Transtornos do Crescimento/complicações , Heme Oxigenase-1/deficiência , Hemólise/genética , Distúrbios do Metabolismo do Ferro/complicações , Fígado/fisiopatologia , Macrófagos/metabolismo , Animais , Humanos , Camundongos
2.
Blood ; 124(9): 1522-30, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-24963040

RESUMO

Loss-of-function mutation in the heme oxygenase 1 (Hmox1) gene causes a rare and lethal disease in children, characterized by severe anemia and intravascular hemolysis, with damage to endothelia and kidneys. Previously, we found that macrophages engaged in recycling of red cells were depleted from the tissues of Hmox1(-/-) mice, which resulted in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. Here, we report that subablative bone marrow transplantation (BMT) has a curative effect for disease in Hmox1(-/-) animals as a result of restoration of heme recycling by repopulation of the tissues with wild-type macrophages. Although engraftment was transient, BMT reversed anemia, normalized blood chemistries and iron metabolism parameters, and prevented renal damage. The largest proportion of donor-derived cells was observed in the livers of transplanted animals. These cells, identified as Kupffer cells with high levels of Hmox1 expression, persisted months after transient engraftment of the donor bone marrow and were responsible for the full restoration of heme-recycling ability in Hmox1(-/-) mice and reversing Hmox1-deficient phenotype. Our findings suggest that BMT or the development of specific cell therapies to repopulate patients' tissues with wild-type or reengineered macrophages represent promising approaches for HMOX1 deficiency treatment in humans.


Assuntos
Heme Oxigenase-1/deficiência , Células de Kupffer/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/deficiência , Anemia Hemolítica Congênita/metabolismo , Anemia Hemolítica Congênita/patologia , Anemia Hemolítica Congênita/terapia , Animais , Transplante de Medula Óssea , Modelos Animais de Doenças , Feminino , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Ferro/metabolismo , Rim/metabolismo , Rim/patologia , Células de Kupffer/transplante , Macrófagos/transplante , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo
3.
Cell Metab ; 17(2): 271-81, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23395173

RESUMO

Iron regulatory proteins (Irps) 1 and 2 posttranscriptionally control the expression of transcripts that contain iron-responsive element (IRE) sequences, including ferritin, ferroportin, transferrin receptor, and hypoxia-inducible factor 2α (HIF2α). We report here that mice with targeted deletion of Irp1 developed pulmonary hypertension and polycythemia that was exacerbated by a low-iron diet. Hematocrits increased to 65% in iron-starved mice, and many polycythemic mice died of abdominal hemorrhages. Irp1 deletion enhanced HIF2α protein expression in kidneys of Irp1(-/-) mice, which led to increased erythropoietin (EPO) expression, polycythemia, and concomitant tissue iron deficiency. Increased HIF2α expression in pulmonary endothelial cells induced high expression of endothelin-1, likely contributing to the pulmonary hypertension of Irp1(-/-) mice. Our results reveal why anemia is an early physiological consequence of iron deficiency, highlight the physiological significance of Irp1 in regulating erythropoiesis and iron distribution, and provide important insights into the molecular pathogenesis of pulmonary hypertension.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Deleção de Genes , Hipertensão Pulmonar/complicações , Proteína 1 Reguladora do Ferro/metabolismo , Policitemia/complicações , Biossíntese de Proteínas , Animais , Dieta , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotelina-1/genética , Endotelina-1/metabolismo , Eritropoetina/sangue , Hemorragia Gastrointestinal/sangue , Hemorragia Gastrointestinal/complicações , Hemorragia Gastrointestinal/patologia , Hematopoese Extramedular/efeitos dos fármacos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/patologia , Ferro/farmacologia , Proteína 1 Reguladora do Ferro/deficiência , Proteína 2 Reguladora do Ferro/metabolismo , Longevidade , Camundongos , Modelos Biológicos , Degeneração Neural/sangue , Degeneração Neural/complicações , Degeneração Neural/patologia , Especificidade de Órgãos/efeitos dos fármacos , Policitemia/sangue , Policitemia/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
4.
Biochim Biophys Acta ; 1823(2): 484-92, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22101253

RESUMO

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.


Assuntos
Ferredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Família Multigênica , Oxirredutases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Citosol/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Ferredoxinas/genética , Técnicas de Silenciamento de Genes , Heme/deficiência , Humanos , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Oxirredutases/genética , Interferência de RNA , Alinhamento de Sequência
5.
Blood ; 116(26): 6054-62, 2010 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-20844238

RESUMO

To better understand the tissue iron overload and anemia previously reported in a human patient and mice that lack heme oxygenase-1 (HO-1), we studied iron distribution and pathology in HO-1(Hmox1)(-/-) mice. We found that resident splenic and liver macrophages were mostly absent in HO-1(-/-) mice. Erythrophagocytosis caused the death of HO-1(-/-) macrophages in in vitro experiments, supporting the hypothesis that HO-1(-/-) macrophages died of exposure to heme released on erythrophagocytosis. Rupture of HO-1(-/-) macrophages in vivo and release of nonmetabolized heme probably caused tissue inflammation. In the spleen, initial splenic enlargement progressed to red pulp fibrosis, atrophy, and functional hyposplenism in older mice, recapitulating the asplenia of an HO-1-deficient patient. We postulate that the failure of tissue macrophages to remove senescent erythrocytes led to intravascular hemolysis and increased expression of the heme and hemoglobin scavenger proteins, hemopexin and haptoglobin. Lack of macrophages expressing the haptoglobin receptor, CD163, diminished the ability of haptoglobin to neutralize circulating hemoglobin, and iron overload occurred in kidney proximal tubules, which were able to catabolize heme with HO-2. Thus, in HO-1(-/-) mammals, the reduced function and viability of erythrophagocytosing macrophages are the main causes of tissue damage and iron redistribution.


Assuntos
Eritrócitos/patologia , Heme Oxigenase-1/fisiologia , Heme/metabolismo , Ferro/metabolismo , Fígado/patologia , Macrófagos/patologia , Baço/patologia , Anemia/metabolismo , Anemia/patologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Células Cultivadas , Eritrócitos/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fagocitose , Receptores de Superfície Celular/metabolismo , Baço/metabolismo , Distribuição Tecidual
6.
Hum Mol Genet ; 19(19): 3816-34, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20668094

RESUMO

The importance of mitochondrial iron-sulfur cluster (ISC) biogenesis for human health has been well established, but the roles of some components of this critical pathway still remain uncharacterized in mammals. Among them is human heat shock cognate protein 20 (hHSC20), the putative human homolog of the specialized DnaJ type co-chaperones, which are crucial for bacterial and fungal ISC assembly. Here, we show that the human HSC20 protein can complement for its counterpart in yeast, Jac1p, and interacts with its proposed human partners, hISCU and hHSPA9. hHSC20 is expressed in various human tissues and localizes mainly to the mitochondria in HeLa cells. However, small amounts were also detected extra-mitochondrially. RNA interference-mediated depletion of hHSC20 specifically reduced the activities of both mitochondrial and cytosolic ISC-containing enzymes. The recovery of inactivated ISC enzymes was markedly delayed after an oxidative insult of hHSC20-deficient cells. Conversely, overexpression of hHSC20 substantially protected cells from oxidative insults. These results imply that hHSC20 is an integral component of the human ISC biosynthetic machinery that is particularly important in the assembly of ISCs under conditions of oxidative stress. A cysteine-rich N-terminal domain, which clearly distinguishes hHSC20 from the specialized DnaJ type III proteins of fungi and most bacteria, was found to be important for the integrity and function of the human co-chaperone.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas Ferro-Enxofre/biossíntese , Chaperonas Moleculares/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Cisteína/metabolismo , Teste de Complementação Genética , Proteínas de Choque Térmico HSP40/química , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Chaperonas Moleculares/química , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo
7.
Cell Microbiol ; 12(10): 1463-79, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20497182

RESUMO

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações Hospedeiro-Patógeno , Poliovirus/fisiologia , Replicação Viral , Brefeldina A/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Células HeLa , Humanos , Estrutura Terciária de Proteína
8.
J Clin Invest ; 120(5): 1749-61, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20364084

RESUMO

Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element-binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate delta, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.


Assuntos
Anemia Sideroblástica/metabolismo , Citosol/metabolismo , Eritroblastos/metabolismo , Glutarredoxinas/deficiência , Glutarredoxinas/fisiologia , Heme/metabolismo , Ferro/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Sequência de Aminoácidos , Células HeLa , Humanos , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Fenótipo , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos
9.
Hum Mol Genet ; 17(15): 2265-73, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424449

RESUMO

Friedreich ataxia (FA) is a progressive neurodegenerative disease caused by expansion of a trinucleotide repeat within the first intron of the gene that encodes frataxin. In our study, we investigated the regulation of frataxin expression by iron and demonstrated that frataxin mRNA levels decrease significantly in multiple human cell lines treated with the iron chelator, desferal (DFO). In addition, frataxin mRNA and protein levels decrease in fibroblast and lymphoblast cells derived from both normal controls and from patients with FA when treated with DFO. Lymphoblasts and fibroblasts of FA patients have evidence of cytosolic iron depletion, as indicated by increased levels of iron regulatory protein 2 (IRP2) and/or increased IRE-binding activity of IRP1. We postulate that this inferred cytosolic iron depletion occurs as frataxin-deficient cells overload their mitochondria with iron, a downstream regulatory effect that has been observed previously when mitochondrial iron-sulfur cluster assembly is disrupted. The mitochondrial iron overload and presumed cytosolic iron depletion potentially further compromise function in frataxin-deficient cells by decreasing frataxin expression. Thus, our results imply that therapeutic efforts should focus on an approach that combines iron removal from mitochondria with a treatment that increases cytosolic iron levels to maximize residual frataxin expression in FA patients.


Assuntos
Ataxia de Friedreich/metabolismo , Sobrecarga de Ferro/metabolismo , Proteínas de Ligação ao Ferro/biossíntese , Ferro/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular , Citosol/metabolismo , Desferroxamina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ataxia de Friedreich/genética , Regulação da Expressão Gênica , Humanos , Quelantes de Ferro/farmacologia , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Mitocôndrias/efeitos dos fármacos , RNA Mensageiro/metabolismo , Frataxina
10.
J Virol ; 81(2): 558-67, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17079330

RESUMO

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.


Assuntos
Cisteína Endopeptidases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Poliovirus/patogenicidade , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Proteases Virais 3C , Animais , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HeLa , Humanos , Poliovirus/fisiologia , RNA Viral/metabolismo , Transfecção , Células Vero
11.
Parasitol Res ; 96(5): 312-20, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15918067

RESUMO

The eukaryotic flagellum is one of the most complex macromolecular structures found in cells, containing more than 250 proteins. One unique structure in the flagella of trypanomastids is the paraflagellar rod (PFR). The PFR constitutes a lattice of cytoskeletal filaments that lies alongside the axoneme in the flagella. This unique and complex structure is critical for cell motility, though little is known about its molecular assembly or its role in the lifecycle of trypanosomatids. These proteins are of particular importance in Trypanosoma cruzi, as purified or recombinant PFR proteins have been demonstrated to be immunogenic, protecting mice from a lethal challenge with the parasite. We have searched the T. cruzi databases and discovered two novel genes containing PFR domains. Both these genes are transcribed in vivo and are significantly larger than the previously described PFR genes identified in T. cruzi (>2 Kb). Real-time PCR was used to examine the relative expression levels of six PFR genes, including the two we describe here, in all three stages of T. cruzi's lifecycle. Database searches have further provided EST and genomic sequence support for the presence of these genes in two other pathogenic trypanosomatids, Trypanosoma brucei and Leishmania spp. One of these genes, designated PFR5 contains a carboxy terminal SH3 domain not previously seen in PFR family genes. We propose that this proline-binding SH3 domain may play an important role in the assembly of the PFR.


Assuntos
Genes de Protozoários , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Expressão Gênica , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Homologia de Sequência
12.
Res Microbiol ; 154(8): 587-92, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14527660

RESUMO

Bacteria of the genus Klebsiella are important opportunistic pathogens responsible for nosocomial infections that are increasingly resistant to antimicrobial agents. Distinctive identification of the species K. oxytoca, K. pneumoniae, K. planticola, K. ornithinolytica and K. terrigena is difficult based on phenotypic tests and misidentifications are frequent in routine clinical microbiology. We developed a specific method to discriminate K. oxytoca from the other species of the genus Klebsiella, based on the PCR amplification of the polygalacturonase (pehX) gene. A PCR amplicon of 344 bp was obtained in all 35 K. oxytoca strains tested, but in none of the 29 K. pneumoniae, 12 K. planticola/K. ornithinolytica and 7 K. terrigena strains tested. The test was also negative for polygalacturonate-degrading species of the genus Erwinia. Analysis of 24 strains designated as K. pneumoniae from international collections (NCTC, PZH) revealed previous misidentification of six K. oxytoca strains. Key biochemical tests fully confirmed the pehX PCR results. The new K. oxytoca identification assay should be useful for both clinical and ecological monitoring of K. oxytoca strains, as well as for controlling the previous identification of collection strains.


Assuntos
Klebsiella oxytoca/classificação , Klebsiella oxytoca/isolamento & purificação , Poligalacturonase/genética , Reação em Cadeia da Polimerase/métodos , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Ágar , Genes Bacterianos , Klebsiella oxytoca/enzimologia , Klebsiella oxytoca/genética , Sensibilidade e Especificidade
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