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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.
World J Gastroenterol ; 25(7): 880-887, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30809087

RESUMO

BACKGROUND: Porphyria is a rare disease with complex classification. Erythropoietic protoporphyria (EPP) is an autosomal recessively inherited disease, and most are caused by mutations in the FECH gene. EPP combined with liver injury is even rarer. CASE SUMMARY: This paper reports a case of EPP which was admitted to the hospital with abnormal liver function and diagnosed by repeated questioning of medical history, screening of common causes of severe liver injury, and second generation sequencing of the whole exon genome. We also summarize the clinical characteristics of EPP with liver injury, and put forward some suggestions on EPP to provide a reference for the diagnosis of such rare disease. CONCLUSION: A new mutation locus (c.32_35dupCCCT) which may be related to the disease was found by detecting the FECH gene in the pedigree of this case.


Assuntos
Ferroquelatase/genética , Hepatite/diagnóstico , Protoporfiria Eritropoética/diagnóstico , Doenças Raras/diagnóstico , Adulto , Biópsia , Análise Mutacional de DNA , Erros de Diagnóstico , Hepatite/etiologia , Hepatite/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Testes de Função Hepática , Masculino , Linhagem , Protoporfiria Eritropoética/complicações , Protoporfiria Eritropoética/genética , Protoporfiria Eritropoética/patologia , Doenças Raras/complicações , Pele/patologia
3.
Intern Med ; 57(17): 2505-2509, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30175727

RESUMO

A 27-year-old man bearing an erythropoietic protoporphyria (EPP)-associated ferrochelatase (FECH) mutation was admitted to our hospital for general malaise and marked elevation of the serum levels of hepatobiliary enzymes and bilirubin. Initial treatment with plasma exchange did not reduce the blood protoporphyrin or serum liver enzyme levels, so phlebotomy was started. Surprisingly, weekly phlebotomy normalized the serum levels of liver enzymes, accompanied by a marked reduction in the blood protoporphyrin levels. The clinical course of this case strongly suggests that phlebotomy may be a suitable treatment option for EPP-related hepatopathy.


Assuntos
Hepatopatias/etiologia , Hepatopatias/terapia , Flebotomia , Protoporfiria Eritropoética/complicações , Adulto , Ferroquelatase/genética , Humanos , Fígado/enzimologia , Hepatopatias/enzimologia , Masculino , Mutação , Protoporfirinas/sangue
4.
BMC Plant Biol ; 17(1): 187, 2017 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084526

RESUMO

BACKGROUND: Non-essential trance metal such as cadmium (Cd) is toxic to plants. Although some plants have developed elaborate strategies to deal with absorbed Cd through multiple pathways, the regulatory mechanisms behind the Cd tolerance are not fully understood. Ferrochelatase-1 (FC1, EC4.99.1.1) is the terminal enzyme of heme biosynthesis, catalyzing insertion of ferrous ion into protoporphyrin IX. Recent studies have shown that FC1 is involved in several physiological processes. However, its biological function associated with plant abiotic stress response is poorly understood. RESULTS: In this study, we showed that AtFC1 was transcriptionally activated by Cd exposure. AtFC1 overexpression (35S::FC1) lines accumulated more Cd and non-protein thiol compounds than wild-type, and conferred plant tolerance to Cd stress, with improved primary root elongation, biomass and chlorophyll (Chl) content, and low degree of oxidation associated with reduced H2O2, O·2- and peroxides. In contrast, the AtFC1 loss of functional mutant fc1 showed sensitivity to Cd stress. Exogenous provision of heme, the product of AtFC1, partially rescued the Cd-induced toxic phenotype of fc1 mutants by improving the growth of seedlings, generation of glutathione (GSH) and phytochelatins (PCs), and GSH/PCs-synthesized gene expression (e.g. GSH1, GSH2, PCS1, and PCS2). To investigate the mechanism leading to the AtFC1 regulating Cd stress response in Arabidopsis, a transcriptome of fc1 mutant plants under Cd stress was profiled. Our data showed that disfunction of AtFC1 led to 913 genes specifically up-regulated and 522 genes down-regulated in fc1 mutants exposed to Cd. Some of the genes are involved in metal transporters, Cd-induced oxidative stress response, and detoxification. CONCLUSION: These results indicate that AtFC1 would act as a positive regulator of plant tolerance to Cd stress. Our study will broaden our understanding of the role of FC1 in mediating plant response to Cd stress and provide a basis for further exploration of its downstream genes.


Assuntos
Proteínas de Arabidopsis/genética , Cádmio/toxicidade , Ferroquelatase/genética , Genes de Plantas/fisiologia , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Ferroquelatase/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Estresse Fisiológico/genética
5.
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
6.
Clin Chem ; 63(10): 1614-1623, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28784691

RESUMO

BACKGROUND: There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. METHODS: Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. RESULTS: Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by ß-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. CONCLUSIONS: Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.


Assuntos
Anemia/sangue , Anemia/genética , Metilação de DNA , DNA/sangue , DNA/genética , Eritroblastos/patologia , Anemia/diagnóstico , Anemia/patologia , Anemia Aplástica/sangue , Anemia Aplástica/diagnóstico , Anemia Aplástica/genética , Anemia Aplástica/patologia , Anemia Ferropriva/sangue , Anemia Ferropriva/diagnóstico , Anemia Ferropriva/genética , Anemia Ferropriva/patologia , Diagnóstico Diferencial , Eritroblastos/metabolismo , Eritropoese , Ferroquelatase/genética , Humanos , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Talassemia beta/sangue , Talassemia beta/diagnóstico , Talassemia beta/genética , Talassemia beta/patologia
7.
JAMA Dermatol ; 153(8): 789-796, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28614581

RESUMO

Importance: Autosomal recessive erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are rare photodermatoses presenting with variable degrees of painful phototoxicity that markedly affects quality of life. The clinical variability, determinants of severity, and genotype/phenotype correlations of these diseases are not well characterized. Objective: To describe the baseline clinical characteristics, genotypes, and determinants of disease severity in a large patient cohort with EPP or XLP. Design, Setting, and Participants: A prospective observational study was conducted among patients with confirmed diagnoses of EPP or XLP from November 1, 2010, to December 6, 2015, at 6 academic medical centers of the Porphyrias Consortium of the National Institutes of Health Rare Diseases Clinical Research Network. Detailed medical histories, including history of phototoxicity and treatment, were collected on standardized case report forms. Patients underwent baseline laboratory testing, total erythrocyte protoporphyrin (ePPIX) testing, and molecular genetic testing. Data were entered into a centralized database. Main Outcomes and Measures: Results of biochemical and genetic tests were explored for association with clinical phenotype in patients with EPP or XLP. Results: Of the 226 patients in the study (113 female and 113 male patients; mean [SD] age, 36.7 [17.0] years), 186 (82.3%) had EPP with a FECH (OMIM 612386) mutation and the common low-expression FECH allele IVS3-48T>C, and only 1 patient had 2 FECH mutations. Twenty-two patients had XLP (9.7%; 10 male and 12 female patients), and 9 patients (4.0%) had elevated ePPIX levels and symptoms consistent with protoporphyria but no detectable mutation in the FECH or ALAS2 (OMIM 301300) gene. Samples of DNA could not be obtained from 8 patients. Patients' mean (SD) age at symptom onset was 4.4 (4.4) years. Anemia (107 [47.3%]), history of liver dysfunction (62 [27.4%]), and gallstones (53 [23.5%]) were commonly reported. Higher ePPIX levels were associated with earlier age of symptom onset (median ePPIX levels for those who developed symptoms before vs after 1 year of age, 1744 vs 1567 µg/dL; P = .02), less sun tolerance (median ePPIX levels for those reporting symptoms before vs after 10 minutes of sun exposure, 2233 vs 1524 µg/dL; P ≤ .001), and increased risk of liver dysfunction (median ePPIX levels for those with liver dysfunction vs normal liver function, 2016 vs 1510 µg/dL; P = .003). Patients with EPP and FECH missense mutations had significantly lower ePPIX levels than those with other mutations (1462 vs 1702 µg/dL; P = .01). Male patients with XLP had significantly higher ePPIX levels, on average, than did patients with EPP (3574 vs 1669 µg/dL; P < .001). Marked clinical variability was seen in female patients with XLP owing to random X-chromosomal inactivation. Conclusions and Relevance: These data suggest that higher ePPIX levels are a major determinant of disease severity and risk of liver dysfunction in patients with EPP or XLP. These findings provide a framework for clinical monitoring and management of these disorders.


Assuntos
5-Aminolevulinato Sintetase/deficiência , Ferroquelatase/genética , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Protoporfiria Eritropoética/fisiopatologia , Qualidade de Vida , 5-Aminolevulinato Sintetase/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Genótipo , Humanos , Hepatopatias/genética , Hepatopatias/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Estudos Prospectivos , Protoporfiria Eritropoética/genética , Índice de Gravidade de Doença , Adulto Jovem
8.
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
9.
Int J Dermatol ; 56(3): 272-276, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28054335

RESUMO

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare inherited disorder of heme biosynthesis caused by decreased activity of the enzyme ferrochelatase (FECH ). The frequency of the hypomorphic c.333-48C allele in a population directly contributes to the prevalence of EPP in the same population. This study sought to identify the molecular basis of EPP in a Chinese patient from Singapore and the c.333-48C allele frequency among the Chinese population in Singapore. MATERIALS AND METHODS: FECH gene was screened for mutation in the patient's DNA sample by polymerase chain reaction amplification and DNA sequencing. To validate the identified mutation, the FECH region harboring the mutation was screened in DNA samples from all healthy controls. One patient and 46 ethnically matched healthy controls were included in the study. RESULTS: A novel c.474dupC which leads to a frameshift and premature stop codon was identified in one allele, while the other allele showed to carry c.333-48C and c.337C>T variants in the patient's FECH. The frequency of the c.333-48C hypomorphic allele is 27% among Chinese population in Singapore. CONCLUSIONS: c.474dupC in one allele trans to hypomorphic c.333-48C and c.337C>T allele in FECH gene may be the underlying cause of the clinical EPP of the studied patient. The FECH hypomorphic c.333-48C allele frequency in Singapore is lower than the Han Chinese (41.3%) and Japanese (43%) populations but nearly the same as the Southeast Asian (31%) population and higher than the European (2.7-11%) population.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Ferroquelatase/genética , Protoporfiria Eritropoética/genética , Estudos de Casos e Controles , Criança , Análise Mutacional de DNA , Mutação da Fase de Leitura , Frequência do Gene , Humanos , Masculino , Singapura
10.
Dis Model Mech ; 10(3): 225-233, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28093505

RESUMO

Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3' splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing.


Assuntos
Ferroquelatase/genética , Mutação/genética , Protoporfiria Eritropoética/enzimologia , Protoporfiria Eritropoética/genética , Alelos , Processamento Alternativo/genética , Animais , Sequência de Bases , Células Sanguíneas/patologia , Cruzamento , Modelos Animais de Doenças , Éxons/genética , Genótipo , Recombinação Homóloga/genética , Humanos , Luz , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Protoporfiria Eritropoética/sangue , Protoporfiria Eritropoética/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pele/patologia , Pele/efeitos da radiação
11.
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
12.
J Dermatol ; 44(6): 651-655, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28026050

RESUMO

We report the case of a 42-year-old man with a 5-year history of myelodysplastic syndrome and photosensitivity who had developed painful erythema and blisters on sun-exposed sites. Histological examination of a mildly lichenified lesion on the dorsal finger revealed extensive deposits of a hyaline-like, periodic acid-Schiff-positive material around superficial dermal blood vessels. Laboratory tests showed elevated erythrocyte protoporphyrin and normal urinary porphyrins, suggesting a diagnosis of erythropoietic protoporphyria. Late-onset erythropoietic protoporphyria is rare and is usually associated with an acquired somatic mutation of the ferrochelatase gene secondary to a hematological malignancy such as myelodysplastic syndrome. DNA analysis revealed that our patient has the homozygous IVS3-48C polymorphism that is a low-expression variant of wild-type ferrochelatase allele.


Assuntos
Ferroquelatase/genética , Síndromes Mielodisplásicas/complicações , Transtornos de Fotossensibilidade/etiologia , Protoporfiria Eritropoética/genética , Adulto , Idade de Início , Humanos , Masculino , Protoporfiria Eritropoética/complicações
13.
Chem Commun (Camb) ; 53(3): 541-544, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27966701

RESUMO

Stereochemically-pure 2'-O-(2-methoxyethyl)-phosphorothioate (PS-MOE) oligonucleotides were synthesized from new chiral oxazaphospholidine-containing nucleosides. Thermal stability studies showed that the incorporation of Rp-PS linkages increased RNA-binding affinity. In cells, a full Rp-PS-MOE splice-switching oligonucleotide targeting part of the ferrochelatase gene was more potent than its Sp-PS counterpart, but of similar potency to the stereorandom PS-parent sequence.


Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/farmacologia , Animais , Apolipoproteínas B/genética , Sequência de Bases , Células COS , Cercopithecus aethiops , Ferroquelatase/genética , Humanos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Fosforotioatos/síntese química , RNA Mensageiro/genética , Estereoisomerismo
14.
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
15.
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
16.
J Zhejiang Univ Sci B ; 17(10): 813-820, 2016 Oct..
Artigo em Inglês | MEDLINE | ID: mdl-27704751

RESUMO

Erythropoietic protoporphyria (EPP), an autosomal dominant disease, is caused by partial deficiency of ferrochelatase (FECH), which catalyzes the terminal step of heme biosynthesis because of loss-of-function mutations in the FECH gene. To date, only a few cases have been described in Asia. In this study, we describe the clinical features of two Chinese patients with EPP, with diagnosis confirmed by the increase of free protoporphyrin in erythrocytes, detection of plasma fluorescence peak at 630-634 nm, and analysis of FECH gene mutations. Using gene scanning, we identified a small deletion in the FECH gene (c.973 delA) in one proband (patient A) and a pathogenic FECH mutation (c.1232 G>T) in the other (patient B) and also observed some nucleotide variations (c.798 C>G, c.921 A>G, IVS1-23 C>T, IVS3+23 A>G, IVS9+35 C>T, and IVS3-48 T>C) in these patients. The family pedigree of patient A was then established by characterization of the genotype of the patient's relatives. We also analyzed the potential perniciousness of the missense mutation with bioinformatic software, Polyphen and Sift. In summary, Chinese EPP patients have similar manifestations to those of Caucasians, and identification of the Chinese FECH gene mutations expands the FECH genotypic spectrum and may contribute to genetic counseling.


Assuntos
Ferroquelatase/genética , Mutação , Protoporfiria Eritropoética/genética , Adulto , Humanos , Masculino
17.
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
18.
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
19.
Br J Dermatol ; 174(1): 172-5, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26280465

RESUMO

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria caused by both the partial deficiency of ferrochelatase (FECH) and the existence of cytosine at IVS3-48 in trans to a mutated FECH allele. However, physicians occasionally encounter patients with EPP with a mild phenotype associated with a slight increase in the erythrocyte-free protoporphyrin concentration and no FECH gene mutations. In this study, genetic analyses were performed on three patients with a mild phenotype of EPP, with photosensitivity, slightly increased erythrocyte-free protoporphyrin concentrations and only a few fluorocytes in the peripheral blood. After obtaining the patients' and their parents' informed consent, a direct sequence analysis of the FECH gene and a restriction fragment length polymorphism analysis were performed on samples from the patients. The FECH gene mutation was not detected in the direct sequence analyses in any of the patients. However, all three patients had the homozygous IVS3-48C polymorphism. These findings suggest that homozygous IVS3-48C polymorphism of the FECH gene is associated with a slight elevation of the protoporphyrin level in erythrocytes, resulting in a mild EPP phenotype.


Assuntos
Ferroquelatase/genética , Mutação/genética , Polimorfismo Genético/genética , Protoporfiria Eritropoética/genética , Criança , DNA Recombinante/genética , Feminino , Homozigoto , Humanos , Masculino , Recidiva
20.
J Dermatol ; 43(4): 414-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26387792

RESUMO

A 12-year-old boy with photosensitivity since 3 years of age presented with small concavities on both cheeks, the nasal root and the dorsal surface of both hands. According to the clinical features, erythropoietic protoporphyria (EPP) was suspected. Urine and blood samples were tested for porphyrin derivatives, which revealed a markedly elevated level of erythrocyte protoporphyrin (EP) and a diagnosis of EPP was made. The patient's mother had no photosensitivity, however, lesions appearing slightly as small scars were found on the dorsum of her right hand; his elder sister and father showed no rash. The EP levels were elevated in samples from his mother and mildly elevated in those from his elder sister and father. To obtain a definitive diagnosis, genetic analyses were performed using samples from all family members, which revealed no mutations in the ferrochelatase-encoding gene (FECH), which is responsible for EPP. Instead, a pathological mutation of the 5-aminolevulinic acid synthase-encoding gene (ALAS2) was identified in samples from the patient, his mother and his elder sister, confirming a definitive diagnosis of X-linked dominant protoporphyria (XLDPP). This is the first Japanese family reported to have XLDPP, demonstrating evidence of the condition in Japan. In addition, because XLDPP is very similar to EPP in its clinical aspects and laboratory findings, a genetic analysis is required for the differential diagnosis.


Assuntos
5-Aminolevulinato Sintetase/deficiência , Ferroquelatase/genética , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Protoporfiria Eritropoética/diagnóstico , Protoporfiria Eritropoética/genética , Protoporfirinas/análise , 5-Aminolevulinato Sintetase/análise , 5-Aminolevulinato Sintetase/genética , Alelos , Bochecha , Criança , Diagnóstico Diferencial , Mãos , Humanos , Japão , Masculino , Mutação , Linhagem
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