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2.
Nature ; 575(7782): 380-384, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31666695

RESUMO

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Neoplasias Pulmonares/fisiopatologia , Potencial da Membrana Mitocondrial , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Camundongos , Camundongos Transgênicos , Compostos Organofosforados , Tomografia por Emissão de Pósitrons
3.
J Biol Chem ; 297(4): 101135, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461091

RESUMO

Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.


Assuntos
Ferredoxinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ferredoxinas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
J Bone Miner Metab ; 38(4): 544-554, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31983034

RESUMO

INTRODUCTION: The purpose of this study was to compare bone mineral density (BMD) and bone turnover markers between combined oral contraceptive (COC) and non-COC users over 12 months. MATERIALS AND METHODS: COC users (n = 34, age = 19.2 ± 0.5) and non-COC users (n = 28, age = 19.3 ± 0.6) provided serum at baseline, 6 months, and 12 months. C-terminal telopepetides (CTX) and pro-collagen type 1 N-terminal propeptides (P1NP) were determined using ELISA. BMD was measured at the three time points using dual-energy x-ray absorptiometry (DXA). RESULTS: COC users had greater CTX than non-COC users at baseline (18.6 ± 8.2 vs. 13.8 ± 5.3 ng/mL, P = 0.021) and 6 months (20.4 ± 10.3 vs. 14.2 ± 8.5 ng/mL, P = 0.018). Controlling for lean mass, groups were similar in BMD. Over 12 months, non-COC users maintained BMD at the spine, while the COC users declined 2.2% in lateral spine BMD (0.773 ± 0.014 to 0.756 ± 0.014 g/cm2, P = 0.03) and 0.7% in anterior-posterior spine BMD (1.005 ± 0.015 to 0.998 ± 0.015 g/cm2, P = 0.069). Non-COC users increased in BMD of the whole body over 12 months (P < 0.001) while COC users had no change. Women who began COCs within 4 years after menarche had lower BMD at the hip and whole body. Women taking very low dose COCs (20 mcg ethinyl estradiol, EE) significantly declined in CTX, P1NP, and lateral spine BMD in comparison to participants using low dose COCs (30/35 mcg EE). CONCLUSION: College-aged women who did not use COCs increased BMD of the whole body, while COC users had elevated bone turnover, declines in spinal BMD, and lack of bone acquisition of the whole body over 12 months. Young females who initiate COC use early after menarche may experience skeletal detriments.


Assuntos
Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Anticoncepcionais Orais Combinados/farmacologia , Absorciometria de Fóton , Biomarcadores/metabolismo , Etinilestradiol/farmacologia , Feminino , Humanos , Coluna Vertebral/efeitos dos fármacos , Adulto Jovem
5.
J Biol Chem ; 290(34): 20804-20814, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26085103

RESUMO

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1-101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.


Assuntos
Elétrons , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Ascórbico/farmacologia , Dissulfetos/química , Dissulfetos/metabolismo , Glutationa/farmacologia , Metalochaperonas/genética , Metalochaperonas/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Mutação , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Plasmídeos/química , Plasmídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Substâncias Redutoras/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais
6.
J Biol Chem ; 287(37): 31258-69, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22767599

RESUMO

The Mia40 import pathway facilitates the import and oxidative folding of cysteine-rich protein substrates into the mitochondrial intermembrane space. Here we describe the in vitro and in organello oxidative folding of Cmc1, a twin CX(9)C-containing substrate, which contains an unpaired cysteine. In vitro, Cmc1 can be oxidized by the import receptor Mia40 alone when in excess or at a lower rate by only the sulfhydryl oxidase Erv1. However, physiological and efficient Cmc1 oxidation requires Erv1 and Mia40. Cmc1 forms a stable intermediate with Mia40 and is released from this interaction in the presence of Erv1. The three proteins are shown to form a ternary complex in mitochondria. Our results suggest that this mechanism facilitates efficient formation of multiple disulfides and prevents the formation of non-native disulfide bonds.


Assuntos
Metalochaperonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Metalochaperonas/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Ligação Proteica , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Proc Natl Acad Sci U S A ; 107(21): 9578-83, 2010 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-20457929

RESUMO

The TIM22 protein import pathway mediates the import of membrane proteins into the mitochondrial inner membrane and consists of two intermembrane space chaperone complexes, the Tim9-Tim10 and Tim8-Tim13 complexes. To facilitate mechanistic studies, we developed a chemical-genetic approach to identify small molecule agonists that caused lethality to a tim10-1 yeast mutant at the permissive temperature. One molecule, MitoBloCK-1, attenuated the import of the carrier proteins including the ADP/ATP and phosphate carriers, but not proteins that used the TIM23 or the Mia40/Erv1 translocation pathways. MitoBloCK-1 impeded binding of the Tim9-Tim10 complex to the substrate during an early stage of translocation, when the substrate was crossing the outer membrane. As a probe to determine the substrate specificity of the small Tim proteins, MitoBloCK-1 impaired the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex mediates the import of a subset of inner membrane proteins. MitoBloCK-1 also inhibited growth of mammalian cells and import of the ADP/ATP carrier, but not TIM23 substrates, confirming that MitoBloCK-1 can be used to understand mammalian mitochondrial import and dysfunction linked to inherited human disease. Our approach of screening chemical libraries for compounds causing synthetic genetic lethality to identify inhibitors of mitochondrial protein translocation in yeast validates the generation of new probes to facilitate mechanistic studies in yeast and mammalian mitochondria.


Assuntos
Hidrazonas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Tioureia/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Hidrazonas/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Estrutura Molecular , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Tioureia/química , Tioureia/farmacologia
8.
EMBO J ; 26(23): 4801-11, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17972915

RESUMO

Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.


Assuntos
Citocromo-c Peroxidase/fisiologia , Citocromos c/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Alelos , Bioquímica/métodos , Elétrons , Peróxido de Hidrogênio/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Biológicos , Modelos Genéticos , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Oxigênio/química , Oxigênio/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo
9.
J Neurosci ; 26(2): 644-54, 2006 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-16407562

RESUMO

Filamentous tau inclusions in neurons and glia are neuropathological hallmarks of tauopathies. The discovery of microtubule-associated protein tau gene mutations that are pathogenic for a heterogenous group of neurodegenerative disorders, called frontotemporal dementia and parkinsonism linked to chromosome-17 (FTDP-17), directly implicate tau abnormalities in the onset/progression of disease. Although the role of tau pathology in neurons in disease pathogenesis is well accepted, the contribution of glial pathology is essentially unknown. We recently generated a transgenic (Tg) mouse model of tau pathology in astrocytes by expressing the human tau protein under the control of the glial fibrillary acidic protein (GFAP) promoter. Both wild-type and FTDP-17 mutant GFAP/tau Tg animals manifest an age-dependent accumulation of tau inclusions in astrocytes that resembles the pathology observed in human tauopathies. We further demonstrate that both strains of Tg mice manifest compromised motor function that correlates with altered expression of the glial glutamate-aspartate transporter and occurs before the development of tau pathology. Subsequently, the Tg mice manifest additional deficits in neuromuscular strength that correlates with reduced expression of glutamate transporter-1 (GLT-1) and occurs concurrent with tau inclusion pathology. Reduced GLT-1 expression was associated with a progressive decrease in sodium-dependent glutamate transport capacity. Reductions in GLT-1 expression were also observed in corticobasal degeneration, a tauopathy with prominent pathology in astrocytes. Less robust changes were observed in Alzheimer's disease in which neuronal tau pathology predominates. Thus, these Tg mice recapitulate features of astrocytic pathology observed in tauopathies and implicate a role for altered astrocyte function in the pathogenesis of these disorders.


Assuntos
Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Tauopatias/metabolismo , Substituição de Aminoácidos , Animais , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Transportador 1 de Aminoácido Excitatório/deficiência , Transportador 1 de Aminoácido Excitatório/fisiologia , Transportador 2 de Aminoácido Excitatório/deficiência , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Debilidade Muscular/genética , Debilidade Muscular/fisiopatologia , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Proteínas tau
10.
Anticancer Res ; 37(7): 3379-3383, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28668825

RESUMO

BACKGROUND/AIM: Hepatocellular carcinoma is a substantial healthcare burden with high prevalence and poor prognosis. As such, efforts are continually made to uncover molecules relevant in cancer biology, that are exploitable as targets for therapy. The mitochondrion is the powerhouse of the cell and exhibits altered functionality in the malignant state, including aberrant regulation of apoptosis and cellular respiration. Augmenter of liver regeneration (ALR) is a multifunctional mitochondrial protein that demonstrates anti-oxidative and anti-apoptotic properties and plays a key role in liver regeneration. MATERIALS AND METHODS: The present study systematically reviews the available literature on the role of ALR in cancer. RESULTS: Systematic search of PubMed resulted in 12 studies discussing ALR in multiple types of cancer. More specifically, ALR appears to be up-regulated in malignant cells and tissues. Furthermore, treatment of cells with exogenous ALR shows an anti-apoptotic effect while silencing or inhibiting ALR decreases cell and tumor survival. CONCLUSION: ALR clearly plays a role in cancer biology and demonstrates potential as a therapeutic target.


Assuntos
Redutases do Citocromo/metabolismo , Regeneração Hepática/fisiologia , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Apoptose/fisiologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Regulação para Cima/fisiologia
11.
Mol Biol Cell ; 28(21): 2773-2785, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28814504

RESUMO

Prokaryotes have aerobic and anaerobic electron acceptors for oxidative folding of periplasmic proteins. The mitochondrial intermembrane space has an analogous pathway with the oxidoreductase Mia40 and sulfhydryl oxidase Erv1, termed the mitochondrial intermembrane space assembly (MIA) pathway. The aerobic electron acceptors include oxygen and cytochrome c, but an acceptor that can function under anaerobic conditions has not been identified. Here we show that the fumarate reductase Osm1, which facilitates electron transfer from fumarate to succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the mitochondrial intermembrane space and assembles with Erv1 in a complex. In reconstitution studies with reduced Tim13, Mia40, and Erv1, the addition of Osm1 and fumarate completes the disulfide exchange pathway that results in Tim13 oxidation. From in vitro import assays, mitochondria lacking Osm1 display decreased import of MIA substrates, Cmc1 and Tim10. Comparative reconstitution assays support that the Osm1/fumarate couple accepts electrons with similar efficiency to cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus Osm1/fumarate is a new electron acceptor couple in the mitochondrial intermembrane space that seems to function in both aerobic and anaerobic conditions.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/metabolismo , Citocromos c/metabolismo , Dissulfetos/metabolismo , Transporte de Elétrons , Elétrons , Fumaratos/metabolismo , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Microssomos/enzimologia , Microssomos/metabolismo , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Dobramento de Proteína , Transporte Proteico , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Succinato Desidrogenase/genética
12.
J Neurosci ; 25(14): 3539-50, 2005 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-15814784

RESUMO

Filamentous tau inclusions in neurons and glia are neuropathological hallmarks of sporadic and familial tauopathies. Because tau gene mutations are pathogenic for the autosomal dominant tauopathy "frontotemporal dementia and parkinsonism linked to chromosome 17," tau abnormalities are implicated directly in the onset and/or progression of disease. Although filamentous tau aggregates are acknowledged to play roles in degenerative mechanisms resulting in neuron loss, the contributions of glial tau pathology to neurodegeneration remain essentially unexplored. To begin to elucidate the role of glial pathology in tauopathies, we generated a transgenic (Tg) mouse model of astrocytic tau pathology by expressing the human tau protein driven by the glial fibrillary acidic protein (GFAP) promoter. Whereas endogenous tau was not detected in astrocytes of control mice, in GFAP/tau Tg mice there was robust astrocytic tau expression that was associated with a redistribution of the GFAP network. Subsequently, there was an age-dependent accumulation of tau pathology in astrocytes that was Gallyas and variably thioflavine S positive as observed in many tauopathies. The tau pathology in these Tg mice was abnormally phosphorylated, ubiquitinated, and filamentous, and the emergence of this pathology coincided with accumulation of insoluble tau protein. Furthermore, in regions with robust astrocytic tau pathology, there was mild blood- brain barrier disruption, induction of low-molecular-weight heat shock proteins, and focal neuron degeneration. Thus, these Tg mice recapitulate key features of astrocytic pathology observed in human tauopathies and demonstrate functional consequences of this pathology including neuron degeneration in the absence of neuronal tau inclusions.


Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Proteínas tau/metabolismo , Fatores Etários , Animais , Axônios/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Barreira Hematoencefálica/patologia , Western Blotting/métodos , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica/métodos , Degeneração Neural/genética , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , Ubiquitina/metabolismo , Proteínas tau/genética
13.
Dev Cell ; 25(1): 81-92, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23597483

RESUMO

The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.


Assuntos
Redutases do Citocromo/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Mitocôndrias/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/metabolismo , Animais , Apoptose , Diferenciação Celular , Respiração Celular , Sobrevivência Celular , Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Edema Cardíaco/induzido quimicamente , Edema Cardíaco/genética , Edema Cardíaco/patologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Embrião não Mamífero/patologia , Células HEK293 , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Morfolinos/farmacologia , Oxirredução , Oxigênio/metabolismo , Transporte Proteico , Especificidade por Substrato , Leveduras/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Mol Biol Cell ; 20(15): 3481-90, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19477928

RESUMO

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of -310 mV via Mia40 with an intermediate midpoint potential of -290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of -150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX(9)C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas Mitocondriais/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais , Dicroísmo Circular , Dissulfetos/química , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tolueno/análogos & derivados , Tolueno/química , Tolueno/metabolismo
15.
Am J Pathol ; 164(1): 155-66, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14695329

RESUMO

Intracellular accumulations of filamentous material composed of tau proteins are defining features of sporadic and familial neurodegenerative disorders termed "tauopathies." In Alzheimer's disease, the most common tauopathy, tau pathology is predominantly localized within neurons; however, robust glial pathology occurs in other tauopathies. Although the pathogenesis of tauopathies remains primarily unknown, molecular chaperones such as heat-shock proteins (HSPs) are implicated in these tau disorders as well as other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates such as alpha-synuclein in Parkinson's disease and polyglutamine in Huntington's disease. We analyzed a variety of tauopathies with antibodies to a panel of HSPs to determine their role in the pathogenesis of these disorders. Although HSPs are not found in neuronal tau inclusions, we demonstrate increased expression of the small HSP alphaB-crystallin in glial inclusions of both sporadic and familial tauopathies. alphaB-crystallin was observed in a subset of astrocytic and oligodendrocytic tau inclusions as well as the neuropil thread pathology in cellular processes, but the co-expression of alphaB-crystallin with tau inclusions was relatively specific to tauopathies with extensive glial pathology. Thus, increased alphaB-crystallin expression in glial tau inclusions may represent a response by glia to the accumulation of misfolded or aggregated tau protein that is linked to the pathogenesis of the glial pathology and distinct from mechanisms underlying neuronal tau pathology in neurodegenerative disease.


Assuntos
Encéfalo/patologia , Corpos de Inclusão/metabolismo , Neuroglia/patologia , Tauopatias/metabolismo , Cadeia B de alfa-Cristalina/biossíntese , Astrócitos/metabolismo , Astrócitos/patologia , Western Blotting , Encéfalo/metabolismo , Humanos , Imuno-Histoquímica , Corpos de Inclusão/patologia , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Tauopatias/patologia , Proteínas tau/metabolismo
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