RESUMO
Disturbance of the brain homeostasis, either directly via the formation of abnormal proteins or cerebral hypo-perfusion, or indirectly via peripheral inflammation, will activate microglia to synthesise a variety of pro-inflammatory agents which may lead to inflammation and cell death. The pro-inflammatory cytokines will induce changes in the iron proteins responsible for maintaining iron homeostasis, such that increased amounts of iron will be deposited in cells in the brain. The generation of reactive oxygen and nitrogen species, which is directly involved in the inflammatory process, can significantly affect iron metabolism via their interaction with iron-regulatory proteins (IRPs). This underlies the importance of ensuring that iron is maintained in a form that can be kept under control; hence, the elegant mechanisms which have become increasingly well understood for regulating iron homeostasis. Therapeutic approaches to minimise the toxicity of iron include N-acetyl cysteine, non-steroidal anti-inflammatory compounds and iron chelation.
Assuntos
Ferro , Doenças Neuroinflamatórias , Humanos , Inflamação/metabolismo , Ferro/metabolismo , Proteínas Reguladoras de Ferro/metabolismo , Microglia/metabolismoRESUMO
Iron loading in some brain regions occurs in Parkinson's Disease (PD), and it has been considered that its removal by iron chelators could be an appropriate therapeutic approach. Since neuroinflammation with microgliosis is also a common feature of PD, it is possible that iron is sequestered within cells as a result of the "anaemia of chronic disease" and remains unavailable to the chelator. In this review, the extent of neuroinflammation in PD is discussed together with the role played by glia cells, specifically microglia and astrocytes, in controlling iron metabolism during inflammation, together with the results of MRI studies. The current use of chelators in clinical medicine is presented together with a discussion of two clinical trials of PD patients where an iron chelator was administered and showed encouraging results. It is proposed that the use of anti-inflammatory drugs combined with an iron chelator might be a better approach to increase chelator efficacy.
Assuntos
Terapia por Quelação/métodos , Inflamação , Microglia/metabolismo , Doença de Parkinson/terapia , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Quelantes/farmacologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Ferro/química , Quelantes de Ferro/uso terapêutico , Imageamento por Ressonância Magnética , Neuroglia/metabolismo , Neurônios/patologiaRESUMO
Iron chelation therapy, either subcutaneous or orally administered, has been used successfully in various clinical conditions. The removal of excess iron from various tissues, e.g., the liver spleen, heart, and the pituitary, in beta thalassemia patients, has become an essential therapy to prolong life. More recently, the use of deferiprone to chelate iron from various brain regions in Parkinson's Disease and Friederich's Ataxia has yielded encouraging results, although the side effects, in <2% of Parkinson's Disease(PD) patients, have limited its long-term use. A new class of hydroxpyridinones has recently been synthesised, which showed no adverse effects in preliminary trials. A vital question remaining is whether inflammation may influence chelation efficacy, with a recent study suggesting that high levels of inflammation may diminish the ability of the chelator to bind the excess iron.
RESUMO
Although iron is crucial for neuronal functioning, many aspects of cerebral iron biology await clarification. The ability to quantify specific iron forms in the living brain would open new avenues for diagnosis, therapeutic monitoring, and understanding pathogenesis of diseases. A modality that allows assessment of brain tissue composition in vivo, in particular of iron deposits or myelin content on a submillimeter spatial scale, is magnetic resonance imaging (MRI). Multimodal strategies combining MRI with complementary analytical techniques ex vivo have emerged, which may lead to improved specificity. Interdisciplinary collaborations will be key to advance beyond simple correlative analyses in the biological interpretation of MRI data and to gain deeper insights into key factors leading to iron accumulation and/or redistribution associated with neurodegeneration.
Assuntos
Química Encefálica , Encéfalo/fisiologia , Ferro/análise , Bainha de Mielina/química , Neurobiologia/métodos , Neuroimagem/métodos , Humanos , Neurobiologia/tendências , Neuroimagem/tendênciasRESUMO
Our understanding of the broad principles of cellular and systemic iron homeostasis in man are well established with the exception of the brain. Most of the proteins involved in mammalian iron metabolism are present in the brain, although their distribution and precise roles in iron uptake, intracellular metabolism and export are still uncertain, as is the way in which systemic iron is transferred across the blood-brain barrier. We briefly review current concepts concerning the uptake and distribution of iron in the brain, before turning to the ways in which brain iron homeostasis might be regulated. The distribution of iron between different brain regions is then discussed as is the increase in brain iron with normal aging, and the different forms in which iron is present. The increased levels of iron found in specific brain regions and their potential contribution to neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease and other polyglutamine expansion diseases, amyotrophic lateral sclerosis, Friedreich's ataxia, as well as a number of neurodegenerative diseases with iron accumulation, are discussed. The interactions between neuroinflammation and iron are presented, and the chapter concludes with a review of current clinical studies and discussion of the potential and efficacy of iron chelation therapy in the treatment of neurodegenerative diseases.
Assuntos
Encéfalo/fisiologia , Terapia por Quelação , Ferro/fisiologia , Doenças Neurodegenerativas/tratamento farmacológico , Envelhecimento , Doença de Alzheimer , Esclerose Lateral Amiotrófica , Animais , Encéfalo/fisiopatologia , Ataxia de Friedreich , Homeostase , Humanos , Doença de Huntington , Ferro/toxicidade , Doença de ParkinsonRESUMO
Parkinson's disease (PD) is associated with increased iron levels in the substantia nigra (SNc). This study evaluated whether the iron chelator, deferiprone, is well tolerated, able to chelate iron from various brain regions and improve PD symptomology. In a randomised double-blind, placebo controlled trial, 22 early onset PD patients, were administered deferiprone, 10 or 15 mg/kg BID or placebo, for 6 months. Patients were evaluated for PD severity, cognitive function, depression rating and quality of life. Iron concentrations were assessed in the substantia nigra (SNc), dentate and caudate nucleus, red nucleus, putamen and globus pallidus by T2* MRI at baseline and after 3 and 6 months of treatment. Deferiprone therapy was well tolerated and was associated with a reduced dentate and caudate nucleus iron content compared to placebo. Reductions in iron content of the SNc occurred in only 3 patients, with no changes being detected in the putamen or globus pallidus. Although 30 mg/kg deferiprone treated patients showed a trend for improvement in motor-UPDRS scores and quality of life, this did not reach significance. Cognitive function and mood were not adversely affected by deferiprone therapy. Such data supports more extensive clinical trials into the potential benefits of iron chelation in PD.
Assuntos
Química Encefálica/efeitos dos fármacos , Quelantes de Ferro/uso terapêutico , Ferro/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Piridonas/uso terapêutico , Idoso , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Deferiprona , Método Duplo-Cego , Feminino , Humanos , Inflamação/sangue , Inflamação/induzido quimicamente , Ferro/urina , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/diagnóstico por imagem , Estudos Prospectivos , Piridonas/sangueRESUMO
During ageing, different iron complexes accumulate in specific brain regions which are associated with motor and cognitive dysfunction. In neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, changes in local iron homoeostasis result in altered cellular iron distribution and accumulation, ultimately inducing neurotoxicity. The use of iron chelators which are able to penetrate the blood brain barrier and reduce excessive iron accumulation in specific brain regions have been shown to reduce disease progression in both Parkinson's disease and Friedreich's Ataxia. Neuroinflammation often occurs in neurodegenerative diseases, which is mainly sustained by activated microglia exhibiting the M1 phenotype. Such inflammation contributes to the disease progression. Therapeutic agents which reduce such inflammation, e.g. taurine compounds, may ameliorate the inflammatory process by switching the microglia from a M1 to a M2 phenotype.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Ferro/metabolismo , Doenças Neurodegenerativas/metabolismo , Inflamação Neurogênica/metabolismo , Envelhecimento/patologia , Animais , Encéfalo/patologia , Humanos , Quelantes de Ferro , Doenças Neurodegenerativas/patologia , Inflamação Neurogênica/patologiaRESUMO
This review will summarise the current state of our knowledge concerning the involvement of iron in various neurological diseases and the potential of therapy with iron chelators to retard the progression of the disease. We first discuss briefly the role of metal ions in brain function before outlining the way by which transition metal ions, such as iron and copper, can initiate neurodegeneration through the generation of reactive oxygen and nitrogen species. This results in protein misfolding, amyloid production and formation of insoluble protein aggregates which are contained within inclusion bodies. This will activate microglia leading to neuroinflammation. Neuroinflammation plays an important role in the progression of the neurodegenerative diseases, with activated microglia releasing pro-inflammatory cytokines leading to cellular cell loss. The evidence for metal involvement in Parkinson's and Alzheimer's disease as well as Friedreich's ataxia and multiple sclerosis will be presented. Preliminary results from trials of iron chelation therapy in these neurodegenerative diseases will be reviewed.
Assuntos
Quelantes de Ferro/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/metabolismo , Humanos , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismoRESUMO
SUMMARY: In the CNS, iron in several proteins is involved in many important processes such as oxygen transportation, oxidative phosphorylation, myelin production, and the synthesis and metabolism of neurotransmitters. Abnormal iron homoeostasis can induce cellular damage through hydroxyl radical production, which can cause the oxidation and modification of lipids, proteins, carbohydrates, and DNA. During ageing, different iron complexes accumulate in brain regions associated with motor and cognitive impairment. In various neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, changes in iron homoeostasis result in altered cellular iron distribution and accumulation. MRI can often identify these changes, thus providing a potential diagnostic biomarker of neurodegenerative diseases. An important avenue to reduce iron accumulation is the use of iron chelators that are able to cross the blood-brain barrier, penetrate cells, and reduce excessive iron accumulation, thereby affording neuroprotection.
Assuntos
Envelhecimento , Encéfalo/crescimento & desenvolvimento , Quelantes de Ferro/uso terapêutico , Ferro/metabolismo , Doenças Neurodegenerativas/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Animais , Biomarcadores , Química Encefálica , Humanos , Quelantes de Ferro/farmacologia , Imageamento por Ressonância Magnética/métodos , Doenças Neurodegenerativas/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Ferroproteínas não Heme/metabolismoRESUMO
There are extensive structural similarities between eukaryotic and prokaryotic ferritins. However, there is one essential difference between these two types of ferritins: bacterioferritins contain haem whereas eukaryotic ferritins are considered to be non-haem proteins. In vitro experiments had shown that horse spleen apoferritin or recombinant horse L chain apoferritins, when co-crystallised with haemin, undergoes demetallation of the porphyrin. In the present study a cofactor has been isolated directly from horse spleen apoferritin and from crystals of the mutant horse L chain apoferritin (E53Q, E56Q, E57Q, E60Q and R59M) which had been co-crystallised with haemin. In both cases the HPLC/ESI-MS results confirm that the cofactor is a N-ethylprotoporphyrin IX. Crystal structures of wild type L chain horse apoferritin and its three mutants co-crystallised with haemin have been determined to high resolution and in all cases a metal-free molecule derived from haemin was found in the hydrophobic pocket, close to the two-fold axis. The X-ray structure of the E53Q, E56Q, E57Q, E60Q+R59M recombinant horse L-chain apoferritin has been obtained at a higher resolution (1.16Å) than previously reported for any mammalian apoferritins. Similar evidence for a metal-free molecule derived from haemin was found in the electron density map of horse spleen apoferritin (at a resolution of 1.5Å). The out-of-plane distortion of the observed porphyrin is clearly compatible with an N-alkyl porphyrin. We conclude that L-chain ferritins are capable of binding and demetallating haemin, generating in the process N-ethylprotoporphyrin IX both in vivo and in vitro.
Assuntos
Apoferritinas/química , Hemina/química , Protoporfirinas/química , Animais , Apoferritinas/genética , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cristalização , Cristalografia por Raios X , Cavalos , Metais/química , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Protoporfirinas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização por Electrospray , Baço/química , Baço/metabolismoRESUMO
The ability of a taurine prodrug, ethane ß-sultam, to reduce cellular inflammation has been investigated, in vitro, in primary cultures of alveolar macrophages and an immortilised N9 microglial cell line and in vivo in an animal model of inflammation and control rats. Ethane ß-sultam showed enhanced ability to reduce the inflammatory response in alveolar macrophages, as assayed by the lipopolysaccharide-stimulated-nitric oxide release, (LPS stimulated-NO), in comparison to taurine both in vitro (10 nM, 50 nM) and in vivo (0.15 mmol/kg/day by gavage). In addition, ethane ß-sultam, (50, 100 and 1000 nM) significantly reduced LPS-stimulated glutamate release from N9 microglial cells to a greater extent than taurine. The anti-inflammatory response of taurine was shown to be mediated via stabilisation of IkBα. The use of a taurine prodrug as therapeutic agents, for the treatment of neurological conditions, such as Parkinson's and Alzheimer's disease and alcoholic brain damage, where activated phagocytic cells contribute to the pathogenesis, may be of great potential.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Etano/farmacologia , Fagócitos/efeitos dos fármacos , Sulfonamidas/farmacologia , Taurina/análogos & derivados , Taurina/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Linhagem Celular Transformada , Células Cultivadas , Etano/análogos & derivados , Mediadores da Inflamação/química , Mediadores da Inflamação/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Fagócitos/metabolismo , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/químicaRESUMO
The iron content of the substantia nigra pars compacta increases in the brains of Parkinson's disease patients. Hence, its removal by iron chelators may retard the progression of the disease. However, information on the ability of clinically available iron chelators to cross the blood brain barrier and be neuroprotective is limited. In this present study three iron chelators, which are currently approved for clinical use, namely the hexadendate, deferrioxamine, the bidentate deferiprone and the tridendate chelator deferasirox have been investigated for their efficacy to induce neuroprotection. Previous studies have shown that both deferiprone and deferrioxamine exert neuroprotection in the 6-hydroxy dopamine (6-OHDA) model but no such studies have investigated deferasirox. Focal administration of deferasirox (0.5, 2 and 10 µg) into the substantia nigra pars compacta of rats significantly attenuated the loss of dopaminergic neurons and striatal dopamine content resulting from 6-OHDA toxicity. Systemic administration of deferasirox (20 mg/kg), deferiprone (10 mg/kg) or deferrioxamine (30 mg/kg), to the 6-OHDA rat model of Parkinson's disease, significantly attenuated the loss of dopaminergic neurons and striatal dopamine content. Further studies to comprehend the action of these chelators showed that local application of either 0.4 mM deferrioxamine, or 1 mM deferasirox, via a microdialysis probe into the striatum, prior to that of 200 µM 6-OHDA, prevented the generation of hydroxyl radicals. Our results confirm that the administration of these chelators show therapeutic efficacy and should be considered as therapeutic agents for the treatment of Parkinson's disease.
Assuntos
Encéfalo/efeitos dos fármacos , Quelantes de Ferro/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Transtornos Parkinsonianos/tratamento farmacológico , Animais , Benzoatos/administração & dosagem , Deferasirox , Deferiprona , Desferroxamina/administração & dosagem , Radicais Livres/análise , Imuno-Histoquímica , Injeções Intraventriculares , Masculino , Microdiálise , Piridonas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Ácido Sórbico/administração & dosagem , Triazóis/administração & dosagemRESUMO
Metal ions are of particular importance in brain function, notably iron. A broad overview of iron metabolism and its homeostasis both at the cellular level (involving regulation at the level of mRNA translation) and the systemic level (involving the peptide 'hormone' hepcidin) is presented. The mechanisms of iron transport both across the blood-brain barrier and within the brain are then examined. The importance of iron in the developing foetus and in early life is underlined. We then review the growing corpus of evidence that many neurodegenerative diseases (NDs) are the consequence of dysregulation of brain iron homeostasis. This results in the production of reactive oxygen species, generating reactive aldehydes, which, together with further oxidative insults, causes oxidative modification of proteins, manifested by carbonyl formation. These misfolded and damaged proteins overwhelm the ubiquitin/proteasome system, accumulating the characteristic inclusion bodies found in many NDs. The involvement of iron in Alzheimer's disease and Parkinson's disease is then examined, with emphasis on recent data linking in particular interactions between iron homeostasis and key disease proteins. We conclude that there is overwhelming evidence for a direct involvement of iron in NDs.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Ferro/metabolismo , Doença de Parkinson/metabolismo , Doença de Alzheimer/fisiopatologia , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Encéfalo/fisiopatologia , Homeostase/fisiologia , Humanos , Doença de Parkinson/fisiopatologiaRESUMO
Iron and immunity are closely linked: firstly by the fact that many of the genes/proteins involved in iron homoeostasis play a vital role in controlling iron fluxes such that bacteria are prevented from utilising iron for growth; secondly, cells of the innate immune system, monocytes, macrophages, microglia and lymphocytes, are able to combat bacterial insults by carefully controlling their iron fluxes, which are mediated by hepcidin and ferroportin. In addition, lymphocytes play an important role in adaptive immunity. Thirdly, a variety of effector molecules, e.g. toll-like receptors, NF-κB, hypoxia factor-1, haem oxygenase, will orchestrate the inflammatory response by mobilising a variety of cytokines, neurotrophic factors, chemokines, and reactive oxygen and nitrogen species. Pathologies, where iron loading and depletion occur, may adversely affect the ability of the cell to respond to the bacterial insult.
Assuntos
Sistema Imunitário/imunologia , Ferro/metabolismo , Macrófagos/imunologia , Microglia/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Homeostase/fisiologia , Humanos , Sistema Imunitário/metabolismo , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
Ferritins are members of a much larger superfamily of proteins, which are characterised by a structural motif consisting of a bundle of four parallel and anti-parallel alpha helices. The ferritin superfamily itself is widely distributed across all three living kingdoms, in both aerobic and anaerobic organisms, and a considerable number of X-ray structures are available, some at extremely high resolution. We describe first of all the subunit structure of mammalian H and L chain ferritins and then discuss intersubunit interactions in the 24-subunit quaternary structure of these ferritins. Bacteria contain two types of ferritins, FTNs, which like mammalian ferritins do not contain haem, and the haem-containing BFRs. The characteristic carboxylate-bridged di-iron ferroxidase sites of H chain ferritins, FTNs and BFRs are compared, as are the potential entry sites for iron and the 'nucleation' site of L chain ferritins. Finally we discuss the three-dimensional structures of the 12-subunit bacterial Dps (DNA-binding protein from starved cells) proteins as well as their intersubunit di-iron ferroxidase site.
Assuntos
Ferritinas/química , Proteínas de Ligação ao Ferro/química , Animais , Apoferritinas/química , Cristalografia por Raios X/métodos , Ferritinas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica/fisiologia , Subunidades Proteicas/química , Homologia de SequênciaRESUMO
The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.
Assuntos
Ferro/toxicidade , Macrófagos/citologia , Alvéolos Pulmonares/metabolismo , Animais , Feminino , Homeostase , Sistema Imunitário , Inflamação , Macrófagos/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Gravidez , Prenhez , Ratos , Ratos WistarRESUMO
Structural similarities between ferritins and bacterioferritins have been extensively demonstrated. However, there is an essential difference between these two types of ferritins: whereas bacterioferritins bind haem, in-vivo, as Fe(II)-protoporphyrin IX (this haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two axial Met 52 residues), eukaryotic ferritins are non-haem iron proteins. However, in in-vivo studies, a cofactor has been isolated from horse spleen apoferritin similar to protoporphyrin IX; in in-vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haemin (Fe(III)-protoporphyrin IX). Studies of haemin incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a pocket similar to that which binds haem in bacterioferritins (Précigoux et al. 1994 Acta Cryst D50, 739-743). A mechanism of demetallation of haemin by L-chain apoferritins was subsequently proposed (Crichton et al. 1997 Biochem 36, 15049-15054) which involved four Glu residues (E 53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, these four Glu have been mutated to Gln in recombinant horse L-chain apoferritin. We report here the EPR spectra of recombinant horse L-chain apoferritin and its mutant with haemin in basic and acidic conditions. These studies confirm the ability of recombinant L-chain apoferritin and its mutant to incorporate and demetallate the haemin in acidic and basic conditions.
Assuntos
Apoferritinas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hemina/metabolismo , Proteínas Mutantes/metabolismo , Animais , Apoferritinas/química , Apoferritinas/genética , Sítios de Ligação , Hemina/química , Cavalos , Proteínas Mutantes/química , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Ferritins are a family of proteins distributed widely in nature. In bacterial, plant, and animal cells, ferritin appears to serve as a soluble, bioavailable, and non-toxic form of iron provider. Ferritins from animal sources are heteropolymers composed of two types of subunit, H and L, which differ mainly by the presence (H) or absence (L) of active ferroxidase centres. We report the crystallographic structures of four human H apoferritin variants at a resolution of up to 1.5 Angstrom. Crystal derivatives using Zn(II) as redox-stable alternative for Fe(II), allows us to characterize the different metal-binding sites. The ferroxidase centre, which is composed of sites A and B, binds metal with a preference for the A site. In addition, distinct Zn(II)-binding sites were found in the 3-fold axes, 4-fold axes and on the cavity surface near the ferroxidase centre. To study the importance of the distance of the two metal atoms in the ferroxidase centre, single and double replacement of glutamate 27 (site A) and glutamate 107 (site B), the two axial ligands, by aspartate residues have been carried out. The consequences for metal binding and the correlation with Fe(II) oxidation rates are discussed.
Assuntos
Apoferritinas/química , Cristalografia por Raios X , Ferro/química , Mutação , Zinco/química , Substituição de Aminoácidos , Apoferritinas/genética , Sítios de Ligação , Catálise , Ceruloplasmina/química , Cristalização , Ferritinas/genética , Glicerol/metabolismo , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Subunidades Proteicas , Relação Estrutura-AtividadeRESUMO
An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].
Assuntos
Apoferritinas/química , Apoferritinas/genética , Hemina/química , Espectrometria de Massas , Metais/química , Animais , Cristalografia por Raios X , Cavalos , Concentração de Íons de Hidrogênio , Mutação de Sentido Incorreto , Ligação Proteica , Protoporfirinas/química , Proteínas Recombinantes , BaçoRESUMO
Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d'Estaintot, Acta Cryst. D50 (1994) 739-743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049-15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV-visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g=4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g=6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g=1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)-Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation.