RESUMO
Physical exercise has profound effects on quality of life and susceptibility to chronic disease; however, the regulation of skeletal muscle function at the molecular level after exercise remains unclear. We tested the hypothesis that the benefits of exercise on muscle function are linked partly to microtraumatic events that result in accumulation of circulating heme. Effective metabolism of heme is controlled by Heme Oxygenase-1 (HO-1, Hmox1), and we find that mouse skeletal muscle-specific HO-1 deletion (Tam-Cre-HSA-Hmox1fl/fl) shifts the proportion of muscle fibers from type IIA to type IIB concomitant with a disruption in mitochondrial content and function. In addition to a significant impairment in running performance and response to exercise training, Tam-Cre-HSA-Hmox1fl/fl mice show remarkable muscle atrophy compared to Hmox1fl/fl controls. Collectively, these data define a role for heme and HO-1 as central regulators in the physiologic response of skeletal muscle to exercise.
Assuntos
Heme Oxigenase-1/genética , Heme/metabolismo , Proteínas de Membrana/genética , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/genética , Condicionamento Físico Animal/fisiologia , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Ferroquelatase/genética , Ferroquelatase/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Transdução de Sinais , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Photodynamic therapy (PDT) has been used to treat certain types of non-melanoma skin cancer with promising results. However, some skin lesions have not fully responded to this treatment, suggesting a potential PDT-resistant phenotype. Therefore, novel therapeutic alternatives must be identified that improve PDT in resistant skin cancer. In this study, we analyzed the cell viability, intracellular protoporphyrin IX (PpIX) content and subcellular localization, proliferation profile, cell death, reactive oxygen species (ROS) detection and relative gene expression in PDT-resistant HSC-1 cells. PDT-resistant HSC-1 cells show a low quantity of protoporphyrin IX and low levels of ROS, and thus a low rate of death cell. Furthermore, the resistant phenotype showed a downregulation of HSPB1, SLC15A2, FECH, SOD2 and an upregulation of HMBS and BIRC5 genes. On the other hand, epigallocatechin gallate catechin enhanced the MAL-PDT effect, increasing levels of protoporphyrin IX and ROS, and killing 100% of resistant cells. The resistant MAL-PDT model of skin cancer squamous cells (HSC-1) is a reliable and useful tool to understand PDT cytotoxicity and cellular response. These resistant cells were successfully sensitized with epigallocatechin gallate catechin. The in vitro epigallocatechin gallate catechin effect as an enhancer of MAL-PDT in resistant cells is promising in the treatment of difficult skin cancer lesions.
Assuntos
Anticarcinógenos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Catequina/análogos & derivados , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Terapia Combinada/métodos , Fotoquimioterapia/métodos , Neoplasias Cutâneas/tratamento farmacológico , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/farmacologia , Carcinoma de Células Escamosas/radioterapia , Catequina/farmacologia , Morte Celular/efeitos da radiação , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Hipóxia Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ferroquelatase/genética , Ferroquelatase/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/radioterapia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Survivina/genética , Survivina/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMO
Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron-dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA-PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA-PpIX fluorescence in tumor cells with lower FECH activity (MDA-MB-231, Hs 578T) than in tumor cells with higher FECH activity (MDA-MB-453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.
Assuntos
Ácido Aminolevulínico/farmacologia , Desferroxamina/farmacologia , Ferroquelatase/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/farmacologia , Ácido Aminolevulínico/administração & dosagem , Neoplasias da Mama , Linhagem Celular Tumoral , Células Epiteliais , Feminino , Ferroquelatase/genética , Inativação Gênica , Humanos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Protoporfirinas/administração & dosagem , Sideróforos/farmacologiaRESUMO
BACKGROUND: Combined inheritance of genetic variants in ferrochelatase gene (FECH) are implicated in clinical manifestation of Erythropoietic Protoporphyria (EPP). OBJECTIVE: Identify the genetic variants in FECH gene and their associations in the expression of EPP in Argentina. Determine the allelic frequency of polymorphic variants, associations in cis and its linkage disequilibrium. METHODS: The FECH gene was PCR-amplified and sequenced. Allelic variants of intragenic polymorphisms were identified by PCR followed by sequencing or restriction digestion analysis. Residual FECH activity was determined by prokaryotic expression in Escherichia coli JM109. Data were analyzed using Haploview and Statistix 9. RESULTS: Ten mutations were identified: three novel (p.S222N; p.R298X and p.R367X) and seven already known (g.12490_18067del; p.R115X; p.I186T; c.580_584delTACAG; c.598 + 1 G>T; p.Y209X and p.W310X). The p.R115X mutation was found in two families. The p.S222N mutation expressed 5% of normal activity. Only individuals who inherited a mutation combined in trans to a low expression allele c.1-251G, c.68-23T, and c.315-48C, showed clinical symptoms. The absence of c.315-48C variant was sufficient for not triggering EPP. However, these variants showed high levels of cosegregation and GTC haplotype is over-represented in EPP patients. CONCLUSION: In the dominant inheritance form of EPP, c.315-48C variant in trans to the mutated allele is sufficient to trigger the disease. The presence of GTC haplotype in all patients with dominant EPP could be due to the high level of cosegregation of c.315-48C with c.1-251G and c.68-23T variants in our population.
Assuntos
Ferroquelatase/genética , Variação Genética , Protoporfiria Eritropoética/genética , Adolescente , Adulto , Argentina , Criança , Pré-Escolar , Humanos , Pessoa de Meia-Idade , Mutação , Polimorfismo Genético , Protoporfiria Eritropoética/diagnóstico , Adulto JovemRESUMO
Erythropoietic protoporphyria (EPP) is an inherited disorder of porphyrin metabolism in which decreased activity of ferrochelatase (FECH) leads to accumulation of protoporphyrin IX (PP IX) in red blood cells, plasma, liver, and bile, and increased PP IX excretion in feces. Clinically, EPP is characterized by photosensitivity that begins in early childhood and includes burning, swelling, itching, and painful erythema in sun-exposed areas. Chronic liver disease is an important complication in a minority of EPP patients, and in some cases liver transplantation has been performed. So far, about 110 different mutations and several polymorphisms have been characterized in the human FECH gene. The relationship between mutations, polymorphisms, and porphyria development in Argentinean patients was investigated. This is the first genetic study carried out in the Argentinean population. In five Argentinean EPP families we detected three novel mutations: a deletion (451delT) producing a stop codon located 18 codons downstream from the mutation and two splicing mutations: IVS1-2A>G leading to exon 2 skipping and IVS4-2A>G, which causes the loss of the first 48 bp of exon 5. We also found two previously described mutations: C343T and 400delA, which produce stop codons. All patients had an FECH activity 25% of normal and also had the polymorphisms -251A>G in the promoter region and IVS1-23 C>T and IVS3-48 T>C. Our findings provide supporting evidence for the concept that the inheritance of the low expression allele IVS3-48C in trans with a mutation in the FECH gene is necessary for EPP to become clinically manifest.
Assuntos
Análise Mutacional de DNA/métodos , Ferroquelatase/genética , Mutação , Protoporfiria Eritropoética/genética , Adolescente , Adulto , Alelos , Argentina , Criança , Pré-Escolar , Família , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
As the key component of many hemoproteins (heme-containing proteins), heme is involved in a broad range of biological processes. Enzymes required for heme biosynthesis and degradation pathways are evolutionarily conserved. While heme metabolism has been studied extensively, the expression of heme metabolism enzymes during development has not been described. Here, we report that all heme biosynthases and two heme oxygenases, which initiate heme degradation, are dynamically expressed during Xenopus embryonic development. All heme synthases, with the exception of aminolevulinic acid synthase 2, are maternally expressed. At neurula stage, heme synthases are expressed in the developing neural tissue and in migrating neural crest cells. At the swimming tadpole stage, expression of heme synthases can be detected in multiple lineages, including eyes, neural crest cells, developing central nervous system, ventral blood island, pronephron, and pronephric tubule. Similar to heme synthases, heme oxygenases are expressed maternally. Zygotic expression of heme oxygenases is mainly restricted to the developing neural and neural crest lineages. Unlike heme synthases, heme oxygenases are not expressed in the ventral blood island and are expressed at a very low level in the pronephron and pronephric tubule. This indicates that heme metabolism may play important roles during development.
Assuntos
Humanos , Animais , Desenvolvimento Embrionário , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Ferroquelatase/genética , Ferroquelatase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Heme/genética , Heme/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Hibridização In Situ , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Xenopus/genética , Xenopus/metabolismoRESUMO
As the key component of many hemoproteins (heme-containing proteins), heme is involved in a broad range of biological processes. Enzymes required for heme biosynthesis and degradation pathways are evolutionarily conserved. While heme metabolism has been studied extensively, the expression of heme metabolism enzymes during development has not been described. Here, we report that all heme biosynthases and two heme oxygenases, which initiate heme degradation, are dynamically expressed during Xenopus embryonic development. All heme synthases, with the exception of aminolevulinic acid synthase 2, are maternally expressed. At neurula stage, heme synthases are expressed in the developing neural tissue and in migrating neural crest cells. At the swimming tadpole stage, expression of heme synthases can be detected in multiple lineages, including eyes, neural crest cells, developing central nervous system, ventral blood island, pronephron, and pronephric tubule. Similar to heme synthases, heme oxygenases are expressed maternally. Zygotic expression of heme oxygenases is mainly restricted to the developing neural and neural crest lineages. Unlike heme synthases, heme oxygenases are not expressed in the ventral blood island and are expressed at a very low level in the pronephron and pronephric tubule. This indicates that heme metabolism may play important roles during development.(AU)
Assuntos
Humanos , Animais , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Ferroquelatase/genética , Ferroquelatase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Heme/genética , Heme/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Hibridização In Situ , Xenopus/embriologia , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.
Assuntos
Ferroquelatase/genética , Hormônios Esteroides Gonadais/fisiologia , Glândula de Harder/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting/métodos , Castração , Clonagem Molecular/métodos , Cricetinae , DNA Circular/genética , Estradiol/administração & dosagem , Estradiol/fisiologia , Estro/fisiologia , Feminino , Expressão Gênica/genética , Fígado/enzimologia , Masculino , Mesocricetus , Progesterona/administração & dosagem , Progesterona/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Testosterona/administração & dosagem , Testosterona/fisiologiaRESUMO
Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well.
Assuntos
Brucella abortus/enzimologia , Ferroquelatase/fisiologia , Animais , Brucella abortus/crescimento & desenvolvimento , Brucella abortus/patogenicidade , Ferroquelatase/genética , Ferroquelatase/metabolismo , Células HeLa , Hemina , Humanos , Líquido Intracelular/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , VirulênciaRESUMO
In Saccharomyces cerevisiae, as in all eukaryotic organisms, delta-aminolevulinic acid (ALA) is a precursor of porphyrin biosynthesis, a very finely regulated pathway. ALA enters yeast cells through the gamma-aminobutyric acid (GABA) permease Uga4. The incorporation of a metabolite into the cells may be a limiting step for its intracellular metabolization. To determine the relationship between ALA transport and ALA metabolization, ALA incorporation was measured in yeast mutant strains deficient in the delta-aminolevulinic acid-synthase, uroporphyrinogen III decarboxylase, and ferrochelatase, three enzymes involved in porphyrin biosynthesis. Results presented here showed that neither intracellular ALA nor uroporphyrin or protoporphyrin regulates ALA incorporation, indicating that ALA uptake and its subsequent metabolization are not related to each other. Thus a key metabolite as it is, ALA does not have a transport system regulated according to its role.