RESUMO
Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Galactosemias , Galactosefosfatos , Mitocôndrias , Galactosemias/metabolismo , Galactosemias/patologia , Galactosemias/genética , Galactosefosfatos/metabolismo , Humanos , Animais , Ratos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fosforilação Oxidativa/efeitos dos fármacos , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Galactose/metabolismoRESUMO
Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.
Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/química , Composição de Medicamentos/métodos , UTP-Hexose-1-Fosfato Uridililtransferase/química , Animais , Proteínas do Capsídeo/isolamento & purificação , Linhagem Celular , Endocitose , Fluoresceína-5-Isotiocianato/química , Galactosemias/tratamento farmacológico , Galactosemias/patologia , Humanos , Cinética , Camundongos , Nanotecnologia , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/uso terapêuticoRESUMO
Classic galactosemia is an inborn error of metabolism caused by deleterious mutations in the GALT gene. A number of evidences indicate that the galactose-1-phosphate accumulation observed in patient cells is a cause of toxicity in this disease. Nevertheless, the consequent molecular events caused by the galactose-1-phosphate accumulation remain elusive. Here we show that intracellular inorganic phosphate levels decreased when yeast models of classic galactosemia were exposed to galactose. The decrease in phosphate levels is probably due to the trapping of phosphate in the accumulated galactose-1-phosphate since the deletion of the galactokinase encoding gene GAL1 suppressed this phenotype. Galactose-induced phosphate depletion caused an increase in glycogen content, an expected result since glycogen breakdown by the enzyme glycogen phosphorylase is dependent on inorganic phosphate. Accordingly, an increase in intracellular phosphate levels suppressed the galactose effect on glycogen content and conferred galactose tolerance to yeast models of galactosemia. These results support the hypothesis that the galactose-induced decrease in phosphate levels leads to toxicity in galactosemia and opens new possibilities for the development of better treatments for this disease.
Assuntos
Galactose , Galactosemias/metabolismo , Modelos Biológicos , Fosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Galactose/metabolismo , Galactose/farmacologia , Galactosemias/genética , Glicogênio/genética , Glicogênio/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes. RESULTS: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification. CONCLUSIONS: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides. GENERAL SIGNIFICANCE: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.
Assuntos
Galactosefosfatos/metabolismo , Giardia lamblia/enzimologia , Proteínas de Protozoários/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Giardia lamblia/genética , Glucofosfatos/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredução , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiorredoxinas/metabolismo , Fatores de Tempo , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
OBJECTIVE: Monogenic congenital cataract is one of the most genetically heterogeneous ocular conditions with almost 30 different genes involved in its etiology. In adult patients, genotype-phenotype correlations are troubled by eye surgery during infancy and/or long-term ocular complications. Here, we describe the molecular diagnosis of GALK1 deficiency as the cause of autosomal recessive congenital cataract in a family from Costa Rica. METHODS: Four affected siblings were included in the study. All of them underwent eye surgery during the first decade but medical records were not available. Congenital cataract was diagnosed by report. Molecular analysis included genome wide homozygosity mapping using a 250K SNP Affymetrix microarray followed by PCR amplification and direct nucleotide sequencing of candidate gene. RESULTS: Genome wide homozygosity mapping revealed a 6Mb region of homozygosity shared by two affected siblings at 17q25. The GALK1 gene was included in this interval and direct sequencing of this gene revealed a homozygous c.1144C>T mutation (p.Q382) in all four affected subjects. CONCLUSIONS: This work demonstrates the utility of homozygosity mapping in the retrospective diagnosis of a family with congenital cataracts in which ocular surgery at early age, the lack of medical records, and the presence of long term eye complications, impeded a clear clinical diagnosis during the initial phases of evaluation.
Assuntos
Catarata/congênito , Catarata/genética , Galactoquinase/genética , Genes Recessivos , Mutação , Idoso , Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Olho , Feminino , Galactoquinase/deficiência , Ligação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Patologia Molecular/métodos , Linhagem , Estudos Retrospectivos , IrmãosRESUMO
The author relates experimental work which lead him to conclude for the presence of galactokinase in animal tissues, explaining in this way that the first step of the galactose intermediary metabolism is its esterification, in the presence of ATP and the enzyme in question, activated by magnesium to galactose-1-phosphate.
O autor relata trabalhos experimentais que o levaram a concluir pela existência da galactoquinase em tecidos animais, explicando desta maneira, que a primeira fase do metabolismo intermediário da galactose nos mesmos é a sua esterificação, em presença do ATP e da enzima em questão, ativada pelo magnésio, para galactose-1-fosfato.