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1.
Biochim Biophys Acta Mol Basis Dis ; 1870(7): 167340, 2024 10.
Artigo em Inglês | MEDLINE | ID: mdl-38986816

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/metabolismo
2.
Biochim Biophys Acta Mol Basis Dis ; 1868(6): 166389, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35301088

RESUMO

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.


Assuntos
Galactosemias , Galactose/metabolismo , Galactose/farmacologia , Galactosemias/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo
3.
J Biol Chem ; 295(12): 3773-3782, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-31996377

RESUMO

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.


Assuntos
Galactose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lítio/farmacologia , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Retículo Endoplasmático/metabolismo , Galactose/metabolismo , Galactosefosfatos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
4.
Biochim Biophys Acta Mol Basis Dis ; 1863(6): 1403-1409, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28213126

RESUMO

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/metabolismo
5.
Dis Model Mech ; 7(1): 55-61, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24077966

RESUMO

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.


Assuntos
Galactosemias/enzimologia , Galactosemias/genética , Regulação Fúngica da Expressão Gênica , Resposta a Proteínas não Dobradas , Processamento Alternativo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Galactoquinase/metabolismo , Galactose/metabolismo , Galactosefosfatos/química , Glicoproteínas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Mutação/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Dobramento de Proteína , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
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