Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Sci Rep ; 8(1): 6577, 2018 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-29700325

RESUMO

A deficient activity of one or more of the mitochondrial oxidative phosphorylation (OXPHOS) enzyme complexes leads to devastating diseases, with high unmet medical needs. Mitochondria, and more specifically the OXPHOS system, are the main cellular production sites of Reactive Oxygen Species (ROS). Increased ROS production, ultimately leading to irreversible oxidative damage of macromolecules or to more selective and reversible redox modulation of cell signalling, is a causative hallmark of mitochondrial diseases. Here we report on the development of a new clinical-stage drug KH176 acting as a ROS-Redox modulator. Patient-derived primary skin fibroblasts were used to assess the potency of a new library of chromanyl-based compounds to reduce ROS levels and protect cells against redox-stress. The lead compound KH176 was studied in cell-based and enzymatic assays and in silico. Additionally, the metabolism, pharmacokinetics and toxicokinetics of KH176 were assessed in vivo in different animal species. We demonstrate that KH176 can effectively reduce increased cellular ROS levels and protect OXPHOS deficient primary cells against redox perturbation by targeting the Thioredoxin/Peroxiredoxin system. Due to its dual activity as antioxidant and redox modulator, KH176 offers a novel approach to the treatment of mitochondrial (-related) diseases. KH176 efficacy and safety are currently being evaluated in a Phase 2 clinical trial.


Assuntos
Cromanos/farmacologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Cromanos/química , Relação Dose-Resposta a Droga , Complexo I de Transporte de Elétrons/deficiência , Glutationa/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/etiologia , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosforilação Oxidativa , Peroxirredoxinas/química , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
2.
Int J Biochem Cell Biol ; 63: 66-70, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25668473

RESUMO

Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These "mitochondrial dynamics" are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δψ). The latter is considered a functional readout of mitochondrial "health" and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.


Assuntos
Metabolismo Energético , Potencial da Membrana Mitocondrial , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Oxirredução
3.
Sci Rep ; 5: 8035, 2015 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-25620325

RESUMO

In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Doença de Leigh/genética , Aprendizado de Máquina , Doenças Mitocondriais/genética , Cromanos/administração & dosagem , Citrato (si)-Sintase/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Doença de Leigh/tratamento farmacológico , Doença de Leigh/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
4.
Biochim Biophys Acta ; 1787(5): 296-302, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19366608

RESUMO

The mammalian MTERF family of proteins has four members, named MTERF1 to MTERF4, which were identified in homology searches using the mitochondrial transcription termination factor, mTERF (here denoted MTERF1) as query. MTERF1 and MTERF3 are known to participate in the control of mitochondrial DNA transcription, but the function of the other two proteins is not known. We here investigate the structure and function of MTERF2. Protein import experiments using isolated organelles confirm that MTERF2 is a mitochondrial protein. Edman degradation of MTERF2 isolated from stably transfected HeLa cells demonstrates that mature MTERF2 lacks a targeting peptide (amino acids 1-35) present in the precursor form of the protein. MTERF2 is a monomer in isolation and displays a non sequence-specific DNA-binding activity. In vivo quantification experiments demonstrate that MTERF2 is relatively abundant, with one monomer present per approximately 265 bp of mtDNA. In comparison, the mtDNA packaging factor TFAM is present at a ratio of one molecule per approximately 10-12 bp of mtDNA. Using formaldehyde cross-linking we demonstrate that MTERF2 is present in nucleoids, and therefore must be located in close proximity to mtDNA. Taken together, our work provides a basic biochemical characterization of MTERF2, paving the way for future functional studies.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Mitocôndrias/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/isolamento & purificação , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cromatografia em Gel , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mamíferos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais , Dados de Sequência Molecular
5.
Cell ; 130(2): 273-85, 2007 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-17662942

RESUMO

Regulation of mammalian mtDNA gene expression is critical for altering oxidative phosphorylation capacity in response to physiological demands and disease processes. The basal machinery for initiation of mtDNA transcription has been molecularly defined, but the mechanisms regulating its activity are poorly understood. In this study, we show that MTERF3 is a negative regulator of mtDNA transcription initiation. The MTERF3 gene is essential because homozygous knockout mouse embryos die in midgestation. Tissue-specific inactivation of MTERF3 in the heart causes aberrant mtDNA transcription and severe respiratory chain deficiency. MTERF3 binds the mtDNA promoter region and depletion of MTERF3 increases transcription initiation on both mtDNA strands. This increased transcription initiation leads to decreased expression of critical promoter-distal tRNA genes, which is possibly explained by transcriptional collision on the circular mtDNA molecule. To our knowledge, MTERF3 is the first example of a mitochondrial protein that acts as a specific repressor of mammalian mtDNA transcription initiation in vivo.


Assuntos
DNA Mitocondrial/genética , Regulação para Baixo/genética , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Transporte de Elétrons , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Marcação de Genes , Genes Essenciais , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Miocárdio/patologia , Miocárdio/ultraestrutura , Especificidade de Órgãos , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Curr Genet ; 48(4): 265-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16193327

RESUMO

The human mitochondrial transcription termination factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.


Assuntos
Filogenia , Plantas/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Mamíferos , Proteínas Mitocondriais , Dados de Sequência Molecular , Alinhamento de Sequência
7.
EMBO J ; 23(12): 2423-9, 2004 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-15167897

RESUMO

We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Sequência de Bases , DNA Mitocondrial/biossíntese , Técnicas In Vitro , Dados de Sequência Molecular , Moldes Genéticos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...