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1.
Pestic Biochem Physiol ; 159: 1-8, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400771

RESUMO

We examined the molecular regulation of porphyrin biosynthesis and protective responses in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum Fe-chelatase (BjFeCh) after treatment with acifluorfen (AF). During the photodynamic stress imposed by AF, transcript levels of BjFeCh in transgenic plants increased greatly; moreover, transcript levels of OsFeCh2 remained almost constant, whereas in wild type (WT) plants they were considerably down-regulated. In the heme branch, transgenic plants exhibited greater levels of OsFC and HO transcripts than WT plants in the untreated stems as well as in the AF-treated leaves and stems. Both WT and transgenic plants treated with AF substantially decreased transcript levels for all the genes in the chlorophyll branch, with less decline in transgenic plants. After AF treatment, ascorbate (Asc) content and the redox Asc state greatly decreased in leaves of WT plants; however, in transgenic plants both parameters remained constant in leaves and the Asc redox state increased by 20% in stems. In response to AF, the leaves of WT plants greatly up-regulated CatA, CatB, and GST compared to those of transgenic plants, whereas, in the stems, transgenic plants showed higher levels of CatA, CatC, APXb, BCH, and VDE. Photochemical quenching, qP, was considerably dropped by 31% and 18% in WT and transgenic plants, respectively in response to AF, whereas non-radiative energy dissipation through non-photochemical quenching increased by 77% and 38% in WT and transgenic plants, respectively. Transgenic plants treated with AF exhibited higher transcript levels of nucleus-encoded photosynthetic genes, Lhcb1 and Lhcb6, as well as levels of Lhcb6 protein compared to those of WT plants. Our study demonstrates that expression of BjFeCh in transgenic plants influences not only the regulation of porphyrin biosynthesis through maintaining higher levels of gene expression in the heme branch, but also the Asc redox function during photodynamic stress caused by AF.


Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/enzimologia , Ferroquelatase/metabolismo , Nitrobenzoatos/farmacologia , Oryza/metabolismo , Porfirinas/biossíntese , Proteínas de Bactérias/genética , Ferroquelatase/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Plantas Geneticamente Modificadas
2.
Food Chem ; 256: 25-30, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29606445

RESUMO

The post-mortem accumulation of the heme biosynthesis metabolite zinc protoporphyrin IX (ZnPP) in porcine muscle is associated with both a meat-inherent and a bacterial enzymatic reaction during meat storage. To estimate the bacterial impact on ZnPP formation, meat and meat-like media were investigated by HPLC-FLD (and MALDI-TOF-MS) after inoculation with a representative microorganism (P. fluorescens). Results indicate the principal ability of meat-inherent bacteria to form ZnPP in meat extracts and meat-like media, but not on the meat muscle. Thus it was concluded that the ZnPP formation in meat is due to a meat-inherent enzymatic reaction induced by porcine ferrochelatase (FECH), while the bacterial (FECH) induced reaction seems to be not significant.


Assuntos
Carne/análise , Protoporfirinas/análise , Pseudomonas fluorescens/metabolismo , Animais , Músculos do Dorso/química , Músculos do Dorso/metabolismo , Ferroquelatase/metabolismo , Heme/biossíntese , Pseudomonas fluorescens/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
3.
Mol Microbiol ; 106(6): 961-975, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29030914

RESUMO

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large-scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe2+ or Mg2+ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen-regulated pufQBALMX operon encoding the reaction centre-light-harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG-immunoprecipitation experiments retrieve a ferrochelatase-PufQ-carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen-limiting conditions. The co-location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light-harvesting polypeptides.


Assuntos
Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Ferroquelatase/metabolismo , Processos Heterotróficos , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Processos Fototróficos , Rhodobacter sphaeroides/metabolismo , Aerobiose , Anaerobiose , Proteínas de Bactérias/genética , Carotenoides/metabolismo , Coproporfirinogênios/metabolismo , Ferroquelatase/genética , Heme/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Liases/metabolismo , Mutação , Óperon , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Protoporfirinas/metabolismo , Rhodobacter sphaeroides/genética , Tetrapirróis/biossíntese
4.
Biochem J ; 474(20): 3513-3522, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28864672

RESUMO

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.


Assuntos
Coproporfirinas/metabolismo , Ferroquelatase/metabolismo , Ferro/metabolismo , Staphylococcus aureus/enzimologia , Sítios de Ligação/fisiologia , Coproporfirinas/química , Ferroquelatase/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
5.
Protein Sci ; 26(12): 2458-2462, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28905435

RESUMO

Isatin (indol-2,3-dione) is an endogenous non-peptide regulator exhibiting a wide range of biological and pharmacological activities, which are poorly characterized in terms of their molecular mechanisms. Identification of many isatin-binding proteins in the mammalian brain and liver suggests that isatin may influence their functions. We have hypothesized that besides direct action on particular protein targets, isatin can act as a regulator of protein-protein interactions (PPIs). In this surface plasmon resonance-based biosensor study we have found that physiologically relevant concentrations of isatin (25-100 µM) increase affinity of interactions between human recombinant ferrochelatase (FECH) and NADPH-dependent adrenodoxin reductase (ADR). In the presence of increasing concentrations of isatin the Kd values demonstrated a significant (up to 6-fold) decrease. It is especially important that the interaction of isatin with each individual protein (FECH, ADR) was basically negligible and therefore could not contribute to the observed effect. This effect was specific only for the FECH/ADR complex formation and was not observed for other protein complexes studied: FECH/cytochrome b5(CYB5A) and FECH/SMAD4.


Assuntos
Ferredoxina-NADP Redutase , Ferroquelatase , Isatina/farmacologia , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/metabolismo , Ferroquelatase/química , Ferroquelatase/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Isatina/química , Cinética , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície
6.
Gene ; 621: 5-11, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28412459

RESUMO

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported.


Assuntos
Células Precursoras Eritroides/metabolismo , Eritropoese , Ataxia de Friedreich/metabolismo , Heme/biossíntese , Estudos de Casos e Controles , Células Cultivadas , Células Precursoras Eritroides/citologia , Ferroquelatase/metabolismo , Ataxia de Friedreich/sangue , Hemoglobinas/metabolismo , Humanos , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Estresse Oxidativo , Protoporfirinas/metabolismo
7.
EMBO Mol Med ; 9(6): 786-801, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28377496

RESUMO

Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.


Assuntos
Ferroquelatase/metabolismo , Degeneração Macular/patologia , Neovascularização Patológica/fisiopatologia , Neovascularização Retiniana/fisiopatologia , Animais , Humanos , Camundongos
8.
J Inherit Metab Dis ; 40(3): 433-441, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28185024

RESUMO

Patients with erythropoietic protoporphyria (EPP) have reduced activity of the enzyme ferrochelatase that catalyzes the insertion of iron into protoporphyrin IX (PPIX) to form heme. As the result of ferrochelatase deficiency, PPIX accumulates and causes severe photosensitivity. Among different patients, the concentration of PPIX varies considerably. In addition to photosensitivity, patients frequently exhibit low serum iron and a microcytic hypochromic anemia. The aims of this study were to (1) search for factors related to PPIX concentration in EPP, and (2) characterize anemia in EPP, i.e., whether it is the result of an absolute iron deficiency or the anemia of chronic disease (ACD). Blood samples from 67 EPP patients (51 Italian and 16 Swiss) and 21 healthy volunteers were analyzed. EPP patients had lower ferritin (p = 0.021) and hepcidin (p = 0.031) concentrations and higher zinc-protoporphyrin (p < 0.0001) and soluble-transferrin-receptor (p = 0.0007) concentrations compared with controls. This indicated that anemia in EPP resulted from an absolute iron deficiency. Among EPP patients, PPIX concentrations correlated with both growth differentiation factor (GDF) 15 (p = 0.012) and male gender (p = 0.015). Among a subgroup of patients who were iron replete, hemoglobin levels were normal, which suggested that iron but not ferrochelatase is the limiting factor in heme synthesis of individuals with EPP.


Assuntos
Fator 15 de Diferenciação de Crescimento/metabolismo , Ferro/metabolismo , Protoporfiria Eritropoética/metabolismo , Anemia Hipocrômica/metabolismo , Estudos de Casos e Controles , Eritrócitos/metabolismo , Feminino , Ferritinas/metabolismo , Ferroquelatase/metabolismo , Hemoglobinas/metabolismo , Hepcidinas/metabolismo , Humanos , Masculino , Transtornos de Fotossensibilidade/metabolismo , Protoporfirinas/metabolismo , Índice de Gravidade de Doença
9.
Clin Genet ; 92(5): 495-502, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28075030

RESUMO

Erythropoietic protoporphyria (EPP) is a rare cutaneous and systemic disease caused by mutations in the ferrochelatase gene (FECH). The molecular underpinnings of EPP in Middle Eastern populations and relative to other ethnic groups secondary to increased consanguinity are unknown. To understand the molecular pathogenesis of Middle Eastern EPP, we surveyed clinicopathological and molecular features in 6 large consanguineous families from Lebanon and Syria presenting with cutaneous and systemic features consistent with EPP. We observed 30% increased liver disease and 20% elevated end-stage liver complications in our EPP cohort compared to EPP patients previously reported elsewhere. In addition, Middle Eastern EPP patients in our cohort exhibited uniquely an increased incidence of colon cancer. Sequence analysis revealed 2 novel non-synonymous FECH mutations in the studied families designated p.M294T and p.I230M. In addition, FECH activity was significantly decreased (6%) in fibroblasts obtained from sun-exposed sites in a patient with p.M294T mutation, whereas in sharp contrast, protected sites from the same patient exhibited 54% activity for the gene. We also found that sun-exposed fibroblasts, relative to sun-protected and control fibroblasts, exhibited suppressed growth and atypical morphology in vitro, and that these effects were alleviated when the cells were co-cultured with sun-protected fibroblasts. Our findings on the increased incidence of colon cancer in EPP patients prompted us to survey FECH expression patterns in cancer. Using publicly available microarray datasets we found that FECH mRNA was largely significantly decreased in colon adenocarcinomas relative to normal colon tissues. Our findings suggest that families with autosomal recessive EPP should be screened more extensively for systemic involvement including liver diseases and colon cancer, and point to a previously unknown yet plausible tumor suppressor role for FECH in colon malignancy.


Assuntos
Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Ferroquelatase/genética , Genes Supressores de Tumor , Protoporfiria Eritropoética/enzimologia , Protoporfiria Eritropoética/genética , Adolescente , Sequência de Bases , Criança , Técnicas de Cocultura , Consanguinidade , Família , Feminino , Ferroquelatase/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Líbano , Hepatopatias/complicações , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Protoporfiria Eritropoética/mortalidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
10.
J Biol Chem ; 292(5): 1815-1825, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-27998984

RESUMO

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for Cox15 oligomerization and function. Pet117 is shown to reside in the mitochondrial matrix, where it is associated with the inner membrane. Pet117 functions at the later maturation stages of the core CcO subunit Cox1 that precede Cox1 hemylation. Pet117 also physically interacts with Cox15 and specifically mediates the stability of Cox15 oligomeric complexes. This Cox15-Pet117 interaction observed by co-immunoprecipitation persists in the absence of heme a synthase activity, is dependent upon Cox1 synthesis and early maturation steps, and is further dependent upon the presence of the matrix-exposed, unstructured linker region of Cox15 needed for Cox15 oligomerization, suggesting that this region mediates the interaction or that the interaction is lost when Cox15 is unable to oligomerize. Based on these findings, it was concluded that Pet117 mediates coupling of heme a synthesis to the CcO assembly process in eukaryotes.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ferroquelatase/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Ferroquelatase/genética , Proteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Environ Microbiol ; 19(1): 106-118, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27486032

RESUMO

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC , that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP , which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter.


Assuntos
Proteínas de Bactérias/genética , Desulfovibrio vulgaris/enzimologia , Desulfovibrio vulgaris/genética , Heme/análogos & derivados , Liases/genética , Tetrapirróis/metabolismo , Uroporfirinas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Desulfovibrio vulgaris/metabolismo , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/metabolismo , Hemeproteínas/genética , Histidina/metabolismo
12.
J Chem Inf Model ; 56(12): 2421-2433, 2016 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-27801584

RESUMO

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX, the terminal step in heme biosynthesis. Some disputes in its mechanism remain unsolved, especially for human ferrochelatase. In this paper, high-level quantum mechanical/molecular mechanics (QM/MM) and free-energy studies were performed to address these controversial issues including the iron-binding site, the optimal reaction path, the substrate porphyrin distortion, and the presence of the sitting-atop (SAT) complex. Our results reveal that the ferrous iron is probably at the binding site coordinating with Met76, and His263 plays the role of proton acceptor. The rate-determining step is either the first proton removed by His263 or the proton transition within the porphyrin with an energy barrier of 14.99 or 14.87 kcal/mol by the quantum mechanical thermodynamic cycle perturbation (QTCP) calculations, respectively. The fast deprotonation step with the conservative residues rather than porphyrin deformation found in solution provides the driving force for biochelation. The SAT complex is not a necessity for the catalysis though it induces a modest distortion on the porphyrin ring.


Assuntos
Ferroquelatase/metabolismo , Ferro/metabolismo , Protoporfirinas/metabolismo , Sítios de Ligação , Ferroquelatase/química , Humanos , Ferro/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Prótons , Protoporfirinas/química , Termodinâmica
13.
Plant Cell Physiol ; 57(12): 2576-2585, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27818378

RESUMO

In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 down-regulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to re-examine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.


Assuntos
Ferroquelatase/genética , Regulação da Expressão Gênica de Plantas , Protoporfirinas/metabolismo , Tabaco/enzimologia , Regulação para Baixo , Ferroquelatase/metabolismo , Heme/metabolismo , Mitocôndrias/enzimologia , Especificidade de Órgãos , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico , RNA Antissenso/genética , Tabaco/genética
14.
Biochim Biophys Acta ; 1863(12): 2859-2867, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27627839

RESUMO

In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59Fe by reticulocytes and diminishes 59Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles.


Assuntos
Endossomos/metabolismo , Ferroquelatase/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , Protoporfirinas/metabolismo , Reticulócitos/metabolismo , Transferrina/metabolismo , Animais , Transporte Biológico , Diferenciação Celular , Endocitose/fisiologia , Feto , Corantes Fluorescentes/química , Heme/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Camundongos , Mutação , Cultura Primária de Células , Reticulócitos/citologia , Coloração e Rotulagem/métodos
15.
Biochemistry ; 55(37): 5204-17, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27599036

RESUMO

Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome b5. In the presence of PGRMC1, in vitro measured FECH activity decreased in a dose-dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release, suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor.


Assuntos
Ferroquelatase/metabolismo , Proteínas de Membrana/fisiologia , Receptores de Progesterona/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos
16.
J Biol Chem ; 291(33): 17417-26, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27317660

RESUMO

The cellular transport of the cofactor heme and its biosynthetic intermediates such as protoporphyrin IX is a complex and highly coordinated process. To investigate the molecular details of this trafficking pathway, we created a synthetic lesion in the heme biosynthetic pathway by deleting the gene HEM15 encoding the enzyme ferrochelatase in S. cerevisiae and performed a genetic suppressor screen. Cells lacking Hem15 are respiratory-defective because of an inefficient heme delivery to the mitochondria. Thus, the biogenesis of mitochondrial cytochromes is negatively affected. The suppressor screen resulted in the isolation of respiratory-competent colonies containing two distinct missense mutations in Nce102, a protein that localizes to plasma membrane invaginations designated as eisosomes. The presence of the Nce102 mutant alleles enabled formation of the mitochondrial respiratory complexes and respiratory growth in hem15Δ cells cultured in supplemental hemin. Respiratory function in hem15Δ cells can also be restored by the presence of a heterologous plasma membrane heme permease (HRG-4), but the mode of suppression mediated by the Nce102 mutant is more efficient. Attenuation of the endocytic pathway through deletion of the gene END3 impaired the Nce102-mediated rescue, suggesting that the Nce102 mutants lead to suppression through the yeast endocytic pathway.


Assuntos
Endossomos/metabolismo , Heme/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Ativo/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endossomos/genética , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/genética , Mitocôndrias/genética , Mutação de Sentido Incorreto , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
17.
Appl Environ Microbiol ; 82(17): 5077-88, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27287322

RESUMO

UNLABELLED: Determining the function and regulation of paralogues is important in understanding microbial functional genomics and environmental adaptation. Heme homeostasis is crucial for the survival of environmental microorganisms. Most Shewanella species encode two paralogues of ferrochelatase, the terminal enzyme in the heme biosynthesis pathway. The function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, were investigated in Shewanella loihica PV-4. The disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), the precursor to heme, and decreased intracellular heme levels. hemH1 was constitutively expressed, and the expression of hemH2 increased when hemH1 was disrupted. The transcription of hemH1 was regulated by the housekeeping sigma factor RpoD and potentially regulated by OxyR, while hemH2 appeared to be regulated by the oxidative stress-associated sigma factor RpoE2. When an oxidative stress condition was mimicked by adding H2O2 to the medium or exposing the culture to light, PPIX accumulation was suppressed in the ΔhemH1 mutant. Consistently, transcriptome analysis indicated enhanced iron uptake and suppressed heme synthesis in the ΔhemH1 mutant. These data indicate that the two paralogues are functional in the heme synthesis pathway but regulated by environmental conditions, providing insights into the understanding of bacterial response to environmental stresses and a great potential to commercially produce porphyrin compounds. IMPORTANCE: Shewanella is capable of utilizing a variety of electron acceptors for anaerobic respiration because of the existence of multiple c-type cytochromes in which heme is an essential component. The cytochrome-mediated electron transfer across cellular membranes could potentially be used for biotechnological purposes, such as electricity generation in microbial fuel cells and dye decolorization. However, the mechanism underlying the regulation of biosynthesis of heme and cytochromes is poorly understood. Our study has demonstrated that two ferrochelatase genes involved in heme biosynthesis are differentially regulated in response to environmental stresses, including light and reactive oxygen species. This is an excellent example showing how bacteria have evolved to maintain cellular heme homeostasis. More interestingly, the high yields of extracellular protoporphyrin IX by the Shewanella loihica PV-4 mutants could be utilized for commercial production of this valuable chemical via bacterial fermentation.


Assuntos
Proteínas de Bactérias/genética , Ferroquelatase/genética , Regulação Enzimológica da Expressão Gênica , Shewanella/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ferroquelatase/química , Ferroquelatase/metabolismo , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Ferro/metabolismo , Protoporfirinas/metabolismo , Shewanella/genética , Shewanella/fisiologia , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico
18.
Food Chem ; 210: 491-9, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27211675

RESUMO

Recombinant ferrochelatase (BsFECH) from Bacillus subtilis expressed in Escherichia coli BL21(DE3) was found by UV-visible spectroscopy to bind the model substrate tetraphenylporphyrin-sulfonate, TPPS, with Ka=3.8 10(5)mol/L in aqueous phosphate buffer pH 5.7 at 30°C, and to interact with metmyoglobin with Ka=1.07±0.13 10(5)mol/L at 30°C. The iron/zinc exchange in myoglobin occurring during maturation of Parma hams seems to depend on such substrate binding to BsFECH and was facilitated by limited pepsin proteolysis of myoglobin to open a reaction channel for metal exchange still with BsFECH associated to globin. BsFECH increased rate of zinc insertion in TPPS significantly and showed saturation kinetics with an apparent binding constant of Zn(II) to the [enzyme-TPPS] complex of 1.3 10(4)mol/L and a first-order rate constant of 6.6 10(-1)s(-1) for dissociation of the tertiary complex, a similar pattern was found for zinc/iron transmetallation in myoglobin.


Assuntos
Ferroquelatase/metabolismo , Globinas/metabolismo , Ferro/química , Mioglobina/metabolismo , Zinco/química , Cinética , Metamioglobina/metabolismo , Mioglobina/química , Porfirinas/metabolismo , Proteólise , Proteínas Recombinantes/metabolismo
19.
Am J Physiol Heart Circ Physiol ; 310(11): H1439-47, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27037373

RESUMO

Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 µM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide.


Assuntos
Angiotensina II/farmacologia , Vasos Coronários/efeitos dos fármacos , Ferroquelatase/metabolismo , Heme/metabolismo , Mitocôndrias/efeitos dos fármacos , Guanilil Ciclase Solúvel/metabolismo , Superóxidos/metabolismo , Animais , Bovinos , Vasos Coronários/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação para Baixo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ativadores de Enzimas/farmacologia , Depuradores de Radicais Livres/farmacologia , Mitocôndrias/enzimologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Organoides , Superóxido Dismutase/metabolismo , Técnicas de Cultura de Tecidos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
20.
J Biol Chem ; 291(22): 11887-98, 2016 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-27026703

RESUMO

Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.


Assuntos
Ferroquelatase/química , Ferroquelatase/metabolismo , Heme/biossíntese , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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