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1.
Chembiochem ; 25(5): e202300738, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38141230

RESUMO

Adrenodoxin reductase (AdxR) plays a pivotal role in electron transfer, shuttling electrons between NADPH and iron/sulfur adrenodoxin proteins in mitochondria. This electron transport system is essential for P450 enzymes involved in various endogenous biomolecules biosynthesis. Here, we present an in-depth examination of the kinetics governing the reduction of human AdxR by NADH or NADPH. Our results highlight the efficiency of human AdxR when utilizing NADPH as a flavin reducing agent. Nevertheless, akin to related flavoenzymes such as cytochrome P450 reductase, we observe that low NADPH concentrations hinder flavin reduction due to intricate equilibrium reactions between the enzyme and its substrate/product. Remarkably, the presence of MgCl2 suppresses this complex kinetic behavior by decreasing NADPH binding to oxidized AdxR, effectively transforming AdxR into a classical Michaelis-Menten enzyme. We propose that the addition of MgCl2 may be adapted for studying the reductive half-reactions of other flavoenzymes with NADPH. Furthermore, in vitro experiments provide evidence that the reduction of the yeast flavin monooxygenase Coq6p relies on an electron transfer chain comprising NADPH-AdxR-Yah1p-Coq6p, where Yah1p shuttles electrons between AdxR and Coq6p. This discovery explains the previous in vivo observation that Yah1p and the AdxR homolog, Arh1p, are required for the biosynthesis of coenzyme Q in yeast.


Assuntos
Ferredoxina-NADP Redutase , Ferredoxinas , Humanos , Ferredoxina-NADP Redutase/metabolismo , NADP/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquinona , Flavinas/metabolismo
2.
PLoS Comput Biol ; 12(1): e1004690, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26808124

RESUMO

Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.


Assuntos
Sítios de Ligação/fisiologia , Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Ubiquinona/química , Ubiquinona/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Biologia Computacional , Simulação por Computador , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Ubiquinona/genética
3.
Biochem J ; 472(1): 97-109, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26371374

RESUMO

The ERα (oestrogen receptor α)-derived peptide ERα17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the Gαi protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of ß-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ERα is down-regulated by prolonged treatment with E2 (oestradiol) or specific ERα siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ERα is required for the action of ERα17p. In addition, its PLM sequence, which interferes with the formation of the ERα-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ERα17p is, to our knowledge, the first known peptide targeting ERα-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oligopeptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/metabolismo , Western Blotting , Calmodulina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Ratos , Receptor Cross-Talk/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , beta-Arrestinas
4.
J Asthma ; 48(6): 640-6, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21609306

RESUMO

RATIONALE: The association between asthma and anxiety disorders in teenagers is well documented, but data about the association with mood disorders are scarce. METHODS: We analyzed data from a cross-sectional study conducted among ninth grade schoolchildren in France in 2003-2004. The teenagers were selected by two-stage sampling and interviewed by school doctors/nurses using a standardized questionnaire including questions about asthma and asthma-like symptoms. They also completed a self-administered questionnaire in which the occurrence of major depressive episodes (MDEs) during the past 12 months was assessed by the Composite International Diagnostic Interview-Short Form. RESULTS: A total of 7000 teenagers (mean age 15.1 years) were included. The prevalence of wheezing in the past 12 months was 10.0% and that of current asthma (wheezing in the past 12 months in children who had already had asthma attacks, or treatment for wheezing or asthma in the past 12 months) was 8.5%. The prevalence of MDE during the past year was 14.2% in teenagers with current asthma versus 9.2% among the others. The association between current asthma and past-year MDE remained significant after adjustment for age, gender, family structure, and the father's employment status. Asthma was uncontrolled (at least four attacks of wheezing, one awakening per week due to wheezing, one severe wheezing, four unplanned medical visits, or one hospitalization for a wheezing attack in the past year) in more than half (58.3%) of asthmatic teenagers with an MDE in the past year versus 35.3% of those without an MDE. CONCLUSION: Asthma is associated with a higher prevalence of MDE. Among adolescents with asthma, MDE is associated with poorer asthma control. These findings highlight the need for a comprehensive care management of asthma in France that takes the psychological dimension into account.


Assuntos
Asma/complicações , Asma/epidemiologia , Transtorno Depressivo Maior/complicações , Transtorno Depressivo Maior/epidemiologia , Adolescente , Fatores Etários , Asma/terapia , Peso Corporal , Criança , Estudos Transversais , Emprego/estatística & dados numéricos , Características da Família , Feminino , França/epidemiologia , Inquéritos Epidemiológicos , Humanos , Masculino , Pais , Prevalência , Fatores de Risco , Fatores Sexuais , Inquéritos e Questionários
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