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1.
Plant Physiol Biochem ; 137: 14-24, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30710795

RESUMO

Glutamyl-tRNA reductase1 (HEMA1) and ferrochelatase1 (FC1) are both expressed in response to salt stress in the biosynthetic pathway of tetrapyrroles. Peanut (Arachis hypogaea L.) HEMA1 and FC1 were isolated by RT-PCR. The amino acid sequence encoded by the two genes showed high similarity with that in other plant species. The AhFC1 fusion protein was verified to function in chloroplast using Arabidopsis mesophyll protoplast. Sense and wild-type (WT) tobaccos were used to further study the physiological effects of AhHEMA1 and AhFC1. Compared with WT, the Heme contents and germination rate were higher in AhFC1 overexpressing plants under salt stress. Meanwhile, overexpressing AhHEMA1 also led to higher ALA and chlorophyll contents and multiple physiological changes under salt stress, such as higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), lower contents of reactive oxygen species (ROS) and slighter membrane damage. In addition, the activities of CAT, POD and APX in the AhFC1 overexpressing plants were significantly higher than that in WT lines under salt stress, but the activity of SOD between the WT plants and the transgenic plants did not exhibit significant differences. These results suggested that, peanut can enhance resistance to salt stress by improving the biosynthesis of tetrapyrrole biosynthetic.


Assuntos
Arachis/genética , Proteínas de Plantas/genética , Estresse Salino/genética , Tabaco/genética , Ácido Aminolevulínico/metabolismo , Membrana Celular/metabolismo , Clorofila/genética , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Heme/biossíntese , Heme/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino/fisiologia , Plântula/genética , Plântula/metabolismo , Tetrapirróis/genética , Tetrapirróis/metabolismo , Tabaco/fisiologia
2.
Trends Parasitol ; 35(3): 213-225, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30686614

RESUMO

Traditionally, host haem has been recognized as a cytotoxic molecule that parasites need to eliminate or detoxify in order to survive. However, recent evidence indicates that some lineages of parasites have lost genes that encode enzymes involved specifically in endogenous haem biosynthesis. Such lineages thus need to acquire and utilize haem originating from their host animal, making it an indispensable molecule for their survival and reproduction. In multicellular parasites, host haem needs to be systemically distributed throughout their bodies to meet the haem demands in all cell and tissue types. Host haem also gets deposited in parasite eggs, enabling embryogenesis and reproduction. Clearly, a better understanding of haem biology in multicellular parasites should elucidate organismal adaptations to obligatory blood-feeding.


Assuntos
Heme/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Adaptação Fisiológica , Animais , Heme/biossíntese , Heme/genética
3.
Mutat Res ; 777: 52-63, 2018 Jul - Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30115430

RESUMO

Anemia is defined by a deficiency of hemoglobin, an iron-rich protein that binds oxygen in the blood. It can be due to multiple causes, either acquired or genetic. Alterations of genes involved in iron metabolism may be responsible, usually at a young age, for rare forms of chronic and often severe congenital anemia. These diseases encompass a variety of sideroblastic anemias, characterized by the presence of ring sideroblasts in the bone marrow. Clinical expression of congenital sideroblastic anemia is either monosyndromic (restricted to hematological lineages) or polysyndromic (with systemic expression), depending on whether iron metabolism, and especially heme synthesis, is directly or indirectly affected. Beside sideroblastic anemias, a number of other anemias can develop due to mutations of key proteins acting either on cellular iron transport (such as the DMT1 transporter), plasma iron transport (transferrin), and iron recycling (ceruloplasmin). Contrasting with the aforementioned entities which involve compartmental, and sometimes, systemic iron excess, the iron refractory iron deficiency anemia (IRIDA) corresponds to a usually severe anemia with whole body iron deficiency related to chronic increase of plasma hepcidin, the systemic negative regulator of plasma iron. Once clinically suggested, these diseases are confirmed by genetic testing in specialized laboratories.


Assuntos
Anemia/genética , Ferro/metabolismo , Mutação , Doenças Raras/genética , Anemia/etiologia , Animais , Heme/biossíntese , Humanos , Absorção Intestinal , Sobrecarga de Ferro/genética , Mitocôndrias/fisiologia , Doenças Raras/etiologia
4.
Int J Mol Sci ; 19(7)2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29958424

RESUMO

Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme, 5-aminolevulinate synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from “gain-of-function” ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer.


Assuntos
5-Aminolevulinato Sintetase/química , Anemia Sideroblástica/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , 5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/patologia , Animais , Domínio Catalítico , Biologia Computacional , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Heme/biossíntese , Heme/genética , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutação/genética , Multimerização Proteica/genética
5.
Gene ; 668: 182-189, 2018 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-29787825

RESUMO

Congenital sideroblastic anemia (CSA) is a series of rare, heterogeneous disorders, characterized by iron overload in the mitochondria of erythroblasts and ringed sideroblasts in bone marrow. In recent years, rapid development of next-generation sequencing technology brings great advance in understanding of genetic and pathophysiologic features of CSA. Based on the pathophysiology of mitochondrial iron metabolism, causative genes of CSA can be divided into three subtypes: heme biosynthesis related; iron­sulfur cluster biosynthesis and transportation related; and mitochondrial respiratory chain synthesis related. Patients with CSA present various clinical manifestation due to relevant mutation gene and require different treatment strategies. The recognition of the causative genes and evolution of pathogenicity is critical. In this review, we summarize the recent progress in mutation genes of CSA, and its potential role in the pathogenesis, diagnosis and treatment.


Assuntos
Anemia Sideroblástica/genética , Mutação , Anemia Sideroblástica/congênito , Anemia Sideroblástica/metabolismo , Heme/biossíntese , Humanos , Ferro/metabolismo , Proteínas Mitocondriais/biossíntese
6.
Environ Toxicol ; 33(6): 695-705, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29663608

RESUMO

Hexachloronaphthalenes (HxCNs) are the most toxic congeners of polychlorinated naphthalenes, a group of compounds lately included into the list of persistent organic pollutants (POPs). This study presents the effects of 90-day intragastric administration of HxCN to female Wistar rats at doses of 0.03, 0.1, and 0.3 mg/kg body weight. The study examined selected parameters of the heme synthesis pathway, oxidative stress, hepatic cytochromes level, and basic hematology indicators. A micronucleus test was also performed. The subchronic exposure of rats to HxCN resulted in disruption of heme biosynthesis, hematological disturbances, and hepatotoxicity. The highest dose of HxCN inhibited aminolevulinic acid dehydratase (ALA-D) and uroporphyrinogen decarboxylase (URO-D). Accumulation of higher carboxylated porphyrins in the liver and increased excretion of 5-aminolevulinic acid in the urine was observed after a dose of 0.1 mg/kg body weight. The most sensitive effect of HxCN in rats was very strong induction of hepatic CYP1A1 activity, which was observed after the lowest dose. The highest dose of HxCN induced significant thrombocytopenia, thymic atrophy and hepatotoxicity, expressed as hepatomegaly and hepatic steatosis.


Assuntos
Heme/biossíntese , Naftalenos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Administração Oral , Animais , Citocromo P-450 CYP1A1/metabolismo , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Naftalenos/administração & dosagem , Sintase do Porfobilinogênio/metabolismo , Ratos , Ratos Wistar , Testes de Toxicidade Crônica
7.
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
9.
J Basic Microbiol ; 58(2): 198-205, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29164655

RESUMO

Analysis of the Amycolatopsis orientalis genome revealed that two genes, hemA1 and hemA2, belonging to divergent pathways, were involved in the biosynthesis of 5-aminolevulinic acid. The roles of hemA1 and hemA2 were elucidated via genetic manipulation and metabolite analysis. The disruption of hemA1, encoding the glutamyl-tRNAGlu reductase of the C5 pathway, was essential for cell growth and is used for heme synthesis. Overexpression of hemA1 resulted in elevated vancomycin and ECO-0501 production in Amycolatopsis orientalis, and it was also effective in increasing the production of daptomycin and natamycin in other Streptomycetes. The disruption of hemA2 indicated that it encodes the 5-aminolevulinic acid synthase of the Shemin pathway, serving as a key enzyme for the synthesis of the precursor aminohydroxycyclopentenone unit of ECO-0501. However, hemA2 disruption could not be complemented by the addition of 5-aminolevulinic acid or by the expression of hemA2 outside of the ECO-0501 gene cluster. The synthesis of ECO-0501 was only restored by the insertion of hemA2 at its original locus. The hemA2 gene could partly complement the hemA1 deficiency. Overexpression of hemA1, a key gene from the heme biosynthetic pathway, is proposed here as a new approach to improve the production of secondary metabolites in bacteria, whereas hemA2 plays different roles depending on its pattern of expression.


Assuntos
5-Aminolevulinato Sintetase/metabolismo , Actinobacteria/enzimologia , Actinobacteria/metabolismo , Aldeído Oxirredutases/metabolismo , Ácido Aminolevulínico/metabolismo , Vias Biossintéticas/genética , Heme/biossíntese , 5-Aminolevulinato Sintetase/genética , Actinobacteria/genética , Aldeído Oxirredutases/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética
10.
Int J Hematol ; 107(1): 44-54, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29139060

RESUMO

Sideroblastic anemias are anemic disorders characterized by the presence of ring sideroblasts in a patient's bone marrow. These disorders are typically divided into two types, congenital or acquired sideroblastic anemia. Recently, several genes were reported as responsible for congenital sideroblastic anemia; however, the relationship between the function of the gene products and ring sideroblasts is largely unclear. In this review article, we will focus on the iron metabolism in erythroid cells as well as in patients with congenital sideroblastic anemia.


Assuntos
Anemia Sideroblástica/congênito , Anemia Sideroblástica/genética , Células Eritroides/metabolismo , Ferro/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Anemia Sideroblástica/sangue , Anemia Sideroblástica/metabolismo , Ataxia Cerebelar , Cromossomos Humanos X/genética , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Feminino , Glutarredoxinas/genética , Proteínas de Choque Térmico HSP70/genética , Heme/biossíntese , Humanos , Erros Inatos do Metabolismo Lipídico , Síndrome MELAS , Masculino , Doenças Mitocondriais , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Doenças Musculares , Mutação
11.
FEBS J ; 284(24): 4314-4327, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29076625

RESUMO

Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying bacteria. In the course of heme d1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18-didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S-adenosyl-l-methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron-sulfur clusters. While the N-terminal [4Fe-4S] cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18-didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.


Assuntos
Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Proteínas com Ferro-Enxofre/metabolismo , Nitrato Redutase/metabolismo , Propionatos/metabolismo , Rhodobacteraceae/enzimologia , S-Adenosilmetionina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Catálise , Cromatografia Líquida de Alta Pressão , Ditionita/farmacologia , Heme/biossíntese , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/isolamento & purificação , Modelos Químicos , Estrutura Molecular , Mutagênese Sítio-Dirigida , Nitrato Redutase/genética , Nitrato Redutase/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Substâncias Redutoras/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato , Tetrapirróis/metabolismo
13.
Enzyme Microb Technol ; 106: 55-59, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28859810

RESUMO

Global substitution of canonical amino acids (cAAs) with noncanonical (ncAAs) counterparts in proteins whose function is dependent on post-translational events such as cofactor binding is still a methodically challenging and difficult task as ncAA insertion generally interferes with the cofactor biosynthesis machinery. Here, we report a technology for the expression of fully substituted and functionally active cofactor-containing hemeproteins. The maturation process which yields an intact cofactor is timely separated from cAA→ncAA substitutions. This is achieved by an optimised expression and fermentation procedure which includes pre-induction of the heme cofactor biosynthesis followed by an incorporation experiment at multiple positions in the protein sequence. This simple strategy can be potentially applied for engineering of other cofactor-containing enzymes.


Assuntos
Substituição de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Hemeproteínas/biossíntese , Hemeproteínas/genética , Sequência de Aminoácidos , Fermentação , Heme/biossíntese , Hemeproteínas/química , Engenharia de Proteínas/métodos , Modificação Traducional de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
14.
Rev. lab. clín ; 10(3): 162-170, jul.-sept. 2017. ilus
Artigo em Espanhol | IBECS | ID: ibc-164885

RESUMO

Las porfirias hepáticas agudas son 4 enfermedades raras causadas por deficiencias enzimáticas en la vía biosintética del grupo hemo. Se caracterizan por presentar síntomas neuroviscerales agudos potencialmente letales ante la presencia de factores inductores de la ALAS1. Estos factores pueden ser endógenos o exógenos tales como hormonas sexuales, ayuno, medicamentos, alcohol y tabaco, entre otros. La fisiopatología de los ataques involucra el incremento en la función de la ALAS1, la producción excesiva de precursores de porfirina y la alteración en la síntesis de hemoproteínas por la deficiencia relativa de hemo. En este artículo se revisa la interacción de esos mecanismos con algunos factores inductores, su papel en el origen de los síntomas neurológicos y cómo los tratamientos disponibles bloquean estos procesos (AU)


The acute hepatic porphyrias are a group of 4 rare diseases caused by enzymatic deficiencies in the haem biosynthetic pathway. They are characterized by presenting acute attacks of neurovisceral symptoms in presence of factors that increase the ALAS1 activity. Those factors could be endogenous or exogenous, such as sexual hormones, fasting, drugs, alcohol, tobacco, among other. The physiopathology of the attacks involves an increasing in ALAS1 function, excessive production of porphyrin precursors, and disturbances in hemoproteins synthesis due to the relative haem deficiency. The present paper is a review of the interaction of those mechanisms with some factors that induce ALAS1, their role in the origin of neurovisceral symptoms, and how the available treatments interfere with those processes (AU)


Assuntos
Humanos , Porfirias/diagnóstico , Porfirias/fisiopatologia , Porfiria Aguda Intermitente/fisiopatologia , Porfirias Hepáticas/diagnóstico , Ácido Aminolevulínico/administração & dosagem , Hemeproteínas/administração & dosagem , Heme/biossíntese , Porfirias Hepáticas/fisiopatologia , Ácido Aminolevulínico/uso terapêutico , Heme/administração & dosagem , Heme/análise
15.
J Biol Chem ; 292(41): 16942-16954, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28830930

RESUMO

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1, which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level.


Assuntos
Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Heme/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Glucose/metabolismo , Heme/genética , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
17.
J Biol Chem ; 292(39): 16284-16299, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28808058

RESUMO

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10's ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação Enzimológica da Expressão Gênica , Heme/biossíntese , Proteínas de Peixe-Zebra/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Embrião não Mamífero/enzimologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microinjeções , Morfolinos/metabolismo , Mutação , Interferência de RNA , RNA Interferente Pequeno , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
18.
Mol Microbiol ; 105(6): 825-838, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28657694

RESUMO

Filamentous fungi are able to differentiate morphologically and adapt the metabolism to internal and external cues. One major regulator is the so-called velvet protein, VeA, best studied in Aspergillus nidulans. The protein interacts with several other proteins to regulate light sensing, the balance between asexual and sexual development, penicillin biosynthesis or mycotoxin production. Here, we characterized a novel VeA-interacting protein, VipA. The 334 amino acid long protein comprises a FAR1-like DNA-binding domain, known from plant transcription factors like FHY3 (Far-red elongated hypocotyl 3). VipA interacted not only with VeA, but also with the WC orthologue LreA in the nuclei and with the phytochrome FphA in the cytoplasm. Conidia and cleistothecia formation was similarly affected in a vipA-deletion strain as in an fphA mutant. However, the effect was less pronounced, suggesting a modulating and not an essential role in light sensing. In addition, VipA modulated heme biosynthesis in response to light through association with the hemB promoter, the gene encoding 5-aminolevulinic acid dehydratase. After illumination of A. nidulans mycelia with white light the intracellular heme concentration increased by 30% in comparison to a vipA-deletion mutant. Hence, VipA couples heme biosynthesis to the illumination conditions.


Assuntos
Aspergillus nidulans/genética , Heme/biossíntese , Aspergillus nidulans/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Heme/metabolismo , Luz , Micotoxinas/metabolismo , Fitocromo/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo
19.
Cell Chem Biol ; 24(6): 758-766.e3, 2017 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-28602760

RESUMO

Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher's choice rather difficult. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells. For instance, in common HeLa cells miRFP703, mIFP, and smURFP were 2-, 9-, and 53-fold dimmer than miRFP670. Either co-expression of heme oxygenase or incubation of cells with heme precursor weakly affected NIR fluorescence, however, in the latter case elevated cellular autofluorescence. Exogenously added chromophore substantially increased smURFP brightness but only slightly enhanced brightness of other NIR FPs. mIFP showed intermediate, while monomeric miRFP670 and miRFP703 exhibited high binding efficiency of endogenous biliverdin chromophore. This feature makes them easy to use as GFP-like proteins for spectral multiplexing with FPs of visible range.


Assuntos
Raios Infravermelhos , Luminescência , Proteínas Luminescentes , Ácido Aminolevulínico/farmacologia , Animais , Linhagem Celular , Heme/biossíntese , Heme Oxigenase-1/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo
20.
Biochem J ; 474(14): 2315-2332, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28588043

RESUMO

Trypanosoma cruzi, the causative agent of Chagas disease, presents a complex life cycle and adapts its metabolism to nutrients' availability. Although T. cruzi is an aerobic organism, it does not produce heme. This cofactor is acquired from the host and is distributed and inserted into different heme-proteins such as respiratory complexes in the parasite's mitochondrion. It has been proposed that T. cruzi's energy metabolism relies on a branched respiratory chain with a cytochrome c oxidase-type aa3 (CcO) as the main terminal oxidase. Heme A, the cofactor for all eukaryotic CcO, is synthesized via two sequential enzymatic reactions catalyzed by heme O synthase (HOS) and heme A synthase (HAS). Previously, TcCox10 and TcCox15 (Trypanosoma cruzi Cox10 and Cox15 proteins) were identified in T. cruzi They presented HOS and HAS activity, respectively, when they were expressed in yeast. Here, we present the first characterization of TcCox15 in T. cruzi, confirming its role as HAS. It was differentially detected in the different T. cruzi stages, being more abundant in the replicative forms. This regulation could reflect the necessity of more heme A synthesis, and therefore more CcO activity at the replicative stages. Overexpression of a non-functional mutant caused a reduction in heme A content. Moreover, our results clearly showed that this hindrance in the heme A synthesis provoked a reduction on CcO activity and, in consequence, an impairment on T. cruzi survival, proliferation and infectivity. This evidence supports that T. cruzi depends on the respiratory chain activity along its life cycle, being CcO an essential terminal oxidase.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Substituição de Aminoácidos , Animais , Proliferação de Células , Cercopithecus aethiops , Biologia Computacional , Bases de Dados de Proteínas , Sistemas Especialistas , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heme/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Estágios do Ciclo de Vida , Mutagênese Sítio-Dirigida , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimento , Células Vero
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