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1.
BMC Neurol ; 19(1): 153, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31279336

RESUMO

BACKGROUND: Multiple Mitochondrial Dysfunctions Syndrome 4 (MMDS4) is manifested as a result of ISCA2 mutations. ISCA2 is a vital component of 4Fe-4S clusters assembly machine. Therefore, in MMDS4 patients, deficient mitochondrial respiratory chain complexes I and II, Aconitase and Succinate dehydrogenase of Kerbs cycle and Lipoic Acid Synthetase in the biosynthesis of lipoic acid are expected. CASE PRESENTATIONS: A 7 months boy in an Iranian consanguineous family with progressive neurodegenerative problems was referred to us. Primarily, general laboratory tests, Abdomen ultrasonography and brain magnetic resonance imaging were performed. In order to find out the genetic problem in this case Whole Exome Sequencing (WES) following by Sanger sequencing was carried out. A novel variant (c.355G > A, p.Ala119Thr) in ISCA2 gene was identified by WES in the proband. Confirmation and segregation in the family for this variant was performed by Sanger sequencing. In-Silico prediction of the ISCA2 secondary structure showed that a helix motif in the Fe-S biosynthesis domain of ISCA2 protein will be eliminated as a result of this variant. CONCLUSIONS: We reported the first patient with ISCA2 variant in Iranian population and the third one in the world reported for ISCA2 gene, so far associated with early-onset mitochondrial neurodegeneration. However further functional studies on this variant or finding it in other patients with similar clinical problems are needed to confirm the pathogenicity of this variant.


Assuntos
Proteínas com Ferro-Enxofre/genética , Doenças Mitocondriais/genética , Complexo I de Transporte de Elétrons/genética , Humanos , Lactente , Irã (Geográfico) , Imagem por Ressonância Magnética , Masculino , Mitocôndrias , Doenças Mitocondriais/diagnóstico por imagem , Mutação , Doenças Neurodegenerativas/genética
2.
Biochim Biophys Acta Bioenerg ; 1860(7): 573-581, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226318

RESUMO

Respiratory complex I catalyses the reduction of ubiquinone (Q) from NADH coupled to proton pumping across the inner membrane of mitochondria. The electrical charging of the inner mitochondrial membrane drives the synthesis of ATP, which is used to power biochemical reactions of the cell. The recent surge in structural data on complex I from bacteria and mitochondria have contributed to significant understanding of its molecular architecture. However, despite these accomplishments, the role of various subdomains in redox-coupled proton pumping remains entirely unclear. In this work, we have mutated conserved residues in the loop of the PSST subunit that faces the ~30 Šlong unique Q-binding tunnel of respiratory complex I. The data show a drastic decrease in Q reductase activity upon mutating several residues despite full assembly of the complex. In-silico modeling and multiple microsecond long molecular dynamics simulations of wild-type and enzyme variants with exchanges of conserved arginine residues revealed remarkable ejection of the bound Q from the site near terminal electron donor N2. Based on experiments and long-time scale molecular simulations, we identify microscopic elements that dynamically control the diffusion of Q and are central to redox-coupled proton pumping in respiratory complex I.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Mutação , Ubiquinona/metabolismo , Yarrowia/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Proteínas Fúngicas/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Subunidades Proteicas , Homologia de Sequência , Ubiquinona/química
4.
Parasitol Res ; 118(7): 2193-2201, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31134335

RESUMO

The larval stage of the species complex Echinococcus granulosus sensu lato (s.l.) is the cause of a widespread zoonotic disease known as cystic echinococcosis (CE). The disease is highly prevalent in southern Italy and represents a serious public health issue. The main aim of this study was to characterize E. granulosus s.l. genotypes from wild boar on a continental area of Italy (Campania region), using recently developed mtDNA markers of nad2 and nad5 for reliable identification of different genotypes. Here, nad5 (680 bp) allowed for a clear identification of G1 and G3, whereas a combination of nad2 (714 bp) and nad5 (1394 bp in total) did the same for genotype G7 and its haplogroups G7a and G7b. The results of this study revealed for the first time the presence of genotype G7 in continental Italy. While haplogroup G7b was previously shown to be restricted to the islands of Corsica and Sardinia, here we demonstrate that haplogroup G7b is also present on the mainland of Italy. This work has implications in designing future strategies to reduce CE in Italy.


Assuntos
Equinococose/veterinária , Echinococcus granulosus/isolamento & purificação , Doenças dos Suínos/parasitologia , Animais , Echinococcus granulosus/genética , Complexo I de Transporte de Elétrons/genética , França , Genótipo , Itália , Mitocôndrias/genética , Suínos , Zoonoses/parasitologia
5.
Mol Cell ; 74(5): 877-890.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31023583

RESUMO

Endoplasmic reticulum (ER) stress and unfolded protein response are energetically challenging under nutrient stress conditions. However, the regulatory mechanisms that control the energetic demand under nutrient and ER stress are largely unknown. Here we show that ER stress and glucose deprivation stimulate mitochondrial bioenergetics and formation of respiratory supercomplexes (SCs) through protein kinase R-like ER kinase (PERK). Genetic ablation or pharmacological inhibition of PERK suppresses nutrient and ER stress-mediated increases in SC levels and reduces oxidative phosphorylation-dependent ATP production. Conversely, PERK activation augments respiratory SCs. The PERK-eIF2α-ATF4 axis increases supercomplex assembly factor 1 (SCAF1 or COX7A2L), promoting SCs and enhanced mitochondrial respiration. PERK activation is sufficient to rescue bioenergetic defects caused by complex I missense mutations derived from mitochondrial disease patients. These studies have identified an energetic communication between ER and mitochondria, with implications in cell survival and diseases associated with mitochondrial failures.


Assuntos
Fator 4 Ativador da Transcrição/genética , Metabolismo Energético/genética , Fator de Iniciação 2 em Eucariotos/genética , Mitocôndrias/genética , eIF-2 Quinase/genética , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Glucose/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mutação de Sentido Incorreto/genética , Nutrientes/metabolismo , Fosforilação , Fatores de Processamento de Serina-Arginina/genética , Transdução de Sinais
6.
Biomolecules ; 9(4)2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934776

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient's brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.


Assuntos
Técnicas de Cocultura , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Doença de Parkinson/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Linhagem Celular Tumoral , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Mol Cell ; 74(3): 452-465.e7, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30879903

RESUMO

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.


Assuntos
Mitocôndrias/metabolismo , NADH Desidrogenase/genética , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Animais , Apoptose/genética , Respiração Celular/genética , Citosol/metabolismo , Drosophila melanogaster/genética , Complexo I de Transporte de Elétrons/genética , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Transdução de Sinais/genética
8.
Nucleic Acids Res ; 47(5): 2472-2486, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30753671

RESUMO

The RNA-binding protein GRSF1 (G-rich RNA sequence-binding factor 1) critically maintains mitochondrial homeostasis. Accordingly, loss of GRSF1 impaired mitochondrial respiration and increased the levels of reactive oxygen species (ROS), triggering DNA damage, growth suppression, and a senescent phenotype characterized by elevated production and secretion of interleukin (IL)6. Here, we characterize the pathways that govern IL6 production in response to mitochondrial dysfunction in GRSF1-depleted cells. We report that loss of GRSF1 broadly altered protein expression programs, impairing the function of respiratory complexes I and IV. The rise in oxidative stress led to increased DNA damage and activation of mTOR, which in turn activated NF-κB to induce IL6 gene transcription and orchestrate a pro-inflammatory program. Collectively, our results indicate that GRSF1 helps preserve mitochondrial homeostasis, in turn preventing oxidative DNA damage and the activation of mTOR and NF-κB, and suppressing a transcriptional pro-inflammatory program leading to increased IL6 production.


Assuntos
Inflamação/genética , Interleucina-6/genética , Proteínas de Ligação a Poli(A)/genética , Serina-Treonina Quinases TOR/genética , Dano ao DNA/genética , Complexo I de Transporte de Elétrons/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , NF-kappa B/genética , Estresse Oxidativo/genética , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transcrição Genética
9.
Nat Commun ; 10(1): 903, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796225

RESUMO

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.


Assuntos
Adenoma Oxífilo/tratamento farmacológico , Adenoma Oxífilo/genética , Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metformina/farmacologia , Pirróis/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Drosophila , Feminino , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Camundongos Nus , NADH Desidrogenase/genética , Neovascularização Patológica/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Magn Reson Imaging ; 58: 38-43, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30668983

RESUMO

PURPOSE: To establish a brain proton magnetic resonance spectroscopy (1H MRS) experimental system using a mouse model of Leigh syndrome for monitoring intracerebral lactate levels as a biomarker of mitochondrial disease progression. MATERIALS AND METHODS: Brain 1H MRS was performed in the Ndufs4 homozygous knockout (KO) mice, a mouse model of Leigh syndrome, and control mice on a horizontal 7.0-T magnetic resonance imaging system at age 5-9 weeks. In a subset of KO mice, survival analysis was performed according to the median of the intracerebral lactate levels. In addition, in KO mice alive until 9 weeks of age, both 1H MRS and T2-weighted imaging (T2WI) were longitudinally performed in the same individuals at 5, 7, and 9 weeks of age. RESULTS: Brain 1H MRS demonstrated increased lactate levels in KO mice compared with control mice (6.4 ±â€¯1.2 mM vs. 3.3 ±â€¯0.8 mM, p < 0.0001). The increased intracerebral lactate levels were already observed at 5 weeks of age, while no obvious abnormal findings were detected in T2WI. Notably, an increased lactate level of >5.94 mM at week 5 was associated with a poor prognosis (median survival days: 24.5 vs. 42 days, log-rank p = 0.03). Longitudinal 1H MRS experiments revealed temporal increase of intracerebral lactate levels, peaking at week 7 (mean change: 2.6 ±â€¯0.7 mM, p = 0.001), followed by decrease at week 9 (mean change: -3.8 ±â€¯2.5 mM, p = 0.03), along with further disease progression, with brain lesions being detected on T2WI. CONCLUSION: Using brain 1H MRS, we demonstrated significant increase in intracerebral lactate levels in a mouse model of Leigh syndrome. Additionally, we demonstrated that intracerebral lactate is a useful biomarker of mitochondrial disease progression at stages preceding the development of brain lesions.


Assuntos
Encéfalo/diagnóstico por imagem , Ácido Láctico/análise , Doença de Leigh/diagnóstico por imagem , Espectroscopia de Prótons por Ressonância Magnética , Alelos , Animais , Biomarcadores/análise , Encéfalo/patologia , Modelos Animais de Doenças , Progressão da Doença , Complexo I de Transporte de Elétrons/genética , Feminino , Doença de Leigh/genética , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Doenças Mitocondriais/diagnóstico por imagem
11.
Mol Genet Metab ; 126(3): 250-258, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30642748

RESUMO

AIM: To perform a deep phenotype characterisation in a pedigree of 3 siblings with Leigh syndrome and compound heterozygous NDUFAF6 mutations. METHOD: A multi-gene panel of childhood-onset basal ganglia neurodegeneration inherited conditions was analysed followed by functional studies in fibroblasts. RESULTS: Three siblings developed gait dystonia in infancy followed by rapid progression to generalised dystonia and psychomotor regression. Brain magnetic resonance showed symmetric and bilateral cytotoxic lesions in the putamen and proliferation of the lenticular-striate arteries, latter spreading to the caudate and progressing to cavitation and volume loss. We identified a frameshift novel change (c.554_558delTTCTT; p.Tyr187AsnfsTer65) and a pathogenic missense change (c.371T>C; p.Ile124Thr) in the NDUFAF6 gene, which segregated with an autosomal recessive inheritance within the family. Patient mutations were associated with the absence of the NDUFAF6 protein and reduced activity and assembly of mature complex I in fibroblasts. By functional complementation assay, the mutant phenotype was rescued by the canonical version of the NDUFAF6. A literature review of 14 NDUFAF6 patients showed a consistent phenotype of an early childhood insidious onset neurological regression with prominent dystonia associated with basal ganglia degeneration and long survival. INTERPRETATION: NDUFAF6-related Leigh syndrome is a relevant cause of childhood onset dystonia and isolated bilateral striatal necrosis. By genetic complementation, we could demonstrate the pathogenicity of novel genetic variants in NDUFAF6.


Assuntos
Distúrbios Distônicos/genética , Complexo I de Transporte de Elétrons/genética , Doença de Leigh/genética , Proteínas Mitocondriais/genética , Degeneração Estriatonigral/congênito , Biópsia , Criança , Estudos de Coortes , Feminino , Fibroblastos , Expressão Gênica , Variação Genética , Humanos , Doença de Leigh/complicações , Masculino , Músculos/patologia , Mutação , Linhagem , Irmãos , Degeneração Estriatonigral/genética
12.
PLoS One ; 14(1): e0208904, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30699114

RESUMO

Lumbricidae taxonomy is vastly restricted by the morphological simplicity of earthworms and their lack of complex appendices. This has led to confusing results in the Lumbricidae classifications, which in turn, has hindered our ability to identify and assign new and cryptic species to the family. Here we propose the addition of a new Lumbricidae genus from the Zagros and Elburz Mountains of Iran, i.e. Philomontanus gen. nov, including three new species. Our taxonomic inferences were based on the phylogenetic analysis of two nuclear gene regions (28S rDNA and 18S rDNA) and 11 mitochondrial gene regions (16S rDNA, 12S rDNA, NADH dehydrogenase I, cytochrome oxidase subunits I and II and tRNAs Asn, Asp, Val, Leu, Ala and Ser). Philomontanus gen. nov comprises the earthworm species Philomontanus sarii sp. nov., Philomontanus mahmoudi sp. nov. and Philomontanus baloutchi sp. nov. These three species are morphologically similar to each other with only a few characters separating them (e.g. size, pigmentation and position of clitellum). Our findings support the adoption of an integrative approach including molecular information (e.g., DNA sequences) to aid earthworm classification and develop a robust taxonomy.


Assuntos
Oligoquetos/genética , Animais , DNA Ribossômico/genética , Complexo I de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Evolução Molecular , Oligoquetos/classificação , Filogenia
13.
Anatol J Cardiol ; 21(1): 18-24, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30587702

RESUMO

OBJECTIVE: The aim of the present study was to determine whether pathogenic mutations were present in families with mitochondrial cardiomyopathy that presented during adolescence. METHODS: The proband was a 21-year-old man who presented clinically with palpitations, chest tightness, pulmonary hypertension, and limited exercise tolerance. Cardiac magnetic resonance imaging studies showed biventricular cardiac hypertrophy. We determine whether pathogenic mutations were present by whole-exome sequencing (WES) in families. RESULTS: Screening of the family using tandem mass spectrometry showed elevated lactic acid levels, glutaric aciduria, a mildly increased glutarylcarnitine-to-octanoylcarnitine ratio, and normal blood α-glucosidase, which was consistent with a respiratory chain complex 1 metabolic disorder. We identified a novel mutation of MT-ND5, c.1315A>G (p.Thr439Ala). Skeletal muscle biopsy histology showed predominantly ragged red fibers and few ragged blue fibers, which was consistent with mitochondrial myopathy. CONCLUSION: In the present study, we identified a novel mutation of MT-ND5, c.1315A>G (p.Thr439Ala), in a family pedigree using WES.


Assuntos
Cardiomiopatias/genética , Complexo I de Transporte de Elétrons/genética , Proteínas Mitocondriais/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Cardiomegalia/complicações , Cardiomiopatias/complicações , Cardiomiopatias/diagnóstico , Cardiomiopatias/diagnóstico por imagem , Diagnóstico Diferencial , Ecocardiografia , Eletrocardiografia , Tolerância ao Exercício , Feminino , Humanos , Hipertensão Pulmonar/complicações , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
14.
Biochim Biophys Acta Mol Basis Dis ; 1865(1): 98-106, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391276

RESUMO

Leigh syndrome is one of the most common childhood-onset neurometabolic disorders resulting from a primary oxidative phosphorylation dysfunction and affecting mostly brain tissues. Ndufs4-/- mice have been widely used to study the neurological responses in this syndrome, however the reason why these animals do not display strong muscle involvement remains elusive. We combined biochemical strategies and multi-platform metabolomics to gain insight into the metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4-/- mice. Enzyme assays confirmed severely reduced (80%) CI activity in both Ndufs4-/- muscle types, compared to WTs. No significant alterations were evident in other respiratory chain enzyme activities; however, Ndufs4-/- solei displayed moderate decreases in citrate synthase (12%) and CIII (18%) activities. Through hypothesis-generating metabolic profiling, we provide the first evidence of adaptive responses to CI dysfunction involving non-classical pathways fueling the ubiquinone (Q) cycle. We report a respective 48 and 34 discriminatory metabolites between Ndufs4-/- and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, electron transfer flavoprotein system, CII, and proline cycle in fueling the Q cycle. By restoring the electron flux to CIII via the Q cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency. Taken together, our results shed light on the underlying pathogenic mechanisms of CI dysfunction in skeletal muscle. Upon further investigation, these pathways could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metabolômica , Músculo Esquelético/metabolismo , Ubiquinona/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Complexo I de Transporte de Elétrons/genética , Flavoproteínas/metabolismo , Glicólise , Doença de Leigh , Masculino , Camundongos , Camundongos Knockout , Doenças Mitocondriais , Modelos Animais , Oxirredução , Fosforilação Oxidativa
15.
Biomolecules ; 8(4)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558250

RESUMO

Cyclophilin D (CyPD) is an important mitochondrial chaperone protein whose mechanism of action remains a mystery. It is well known for regulating mitochondrial function and coupling of the electron transport chain and ATP synthesis by controlling the mitochondrial permeability transition pore (PTP), but more recent evidence suggests that it may regulate electron transport chain activity. Given its identification as a peptidyl-prolyl, cis-trans isomerase (PPIase), CyPD, is thought to be involved in mitochondrial protein folding, but very few reports demonstrate the presence of this activity. By contrast, CyPD may also perform a scaffolding function, as it binds to a number of important proteins in the mitochondrial matrix and inner mitochondrial membrane. From a clinical perspective, inhibiting CyPD to inhibit PTP opening protects against ischemia⁻reperfusion injury, making modulation of CyPD activity a potentially important therapeutic goal, but the lack of knowledge about the mechanisms of CyPD's actions remains problematic for such therapies. Thus, the important yet enigmatic nature of CyPD somehow makes it a master regulator, yet a troublemaker, for mitochondrial function.


Assuntos
Ciclofilinas/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Traumatismo por Reperfusão/tratamento farmacológico , Trifosfato de Adenosina/biossíntese , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/biossíntese , Complexo I de Transporte de Elétrons/genética , Humanos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Dobramento de Proteína , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
16.
Int J Mol Sci ; 19(7)2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018222

RESUMO

Type I endometrial cancer (EC) is the most common form of EC, displaying less aggressive behavior than type II. The development of type I endometrial cancer is considered a multistep process, with slow progression from normal endometrium to hyperplasia, the premalignant form, and endometrial cancer as a result of an unopposed estrogenic stimulation. The role of mitochondria in type I EC tumor progression and prognosis is currently emerging. This review aims to explore mitochondrial alterations in this cancer and in endometrial hyperplasia focusing on mitochondrial DNA mutations, respiratory complex I deficiency, and the activation of mitochondrial quality control systems. A deeper understanding of altered mitochondrial pathways in type I EC could provide novel opportunities to discover new diagnostic and prognostic markers as well as potential therapeutic targets.


Assuntos
DNA Mitocondrial/genética , Neoplasias do Endométrio/genética , Mitocôndrias/genética , Mutação , Lesões Pré-Cancerosas/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Prognóstico
17.
Redox Biol ; 17: 367-376, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29857311

RESUMO

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia.


Assuntos
Fadiga Muscular/efeitos dos fármacos , Músculo Esquelético/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas/genética , Animais , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Glutarredoxinas/genética , Glicosilação , Peróxido de Hidrogênio/química , Camundongos , Contração Muscular/efeitos dos fármacos , Contração Muscular/genética , Fadiga Muscular/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Oxirredução , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas/metabolismo , Proteômica , Compostos de Sulfidrila/metabolismo
18.
Redox Biol ; 17: 207-212, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29704825

RESUMO

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at -80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.


Assuntos
Criopreservação , Fígado/metabolismo , Mitocôndrias Hepáticas/genética , Consumo de Oxigênio/genética , Animais , Biópsia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/fisiologia , Fígado/fisiologia , Mitocôndrias Hepáticas/fisiologia , Membranas Mitocondriais/metabolismo , Consumo de Oxigênio/fisiologia , Ratos
19.
Redox Biol ; 17: 192-199, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29702406

RESUMO

The initial rates of superoxide plus hydrogen peroxide (ROS) generation by intact or permeabilized rat heart mitochondria and coupled inside-out bovine heart submitochondrial particles (SMP) oxidizing NAD-dependent substrates, NADH, and succinate were measured by detecting resorufin formation in the Amplex Red assay at various oxygen concentrations. Linear dependences of the initial rates on oxygen concentration within the range of ~125-750 µM were found for all significant mitochondrial generators, i.e. the respiratory complexes and ammonium-stimulated dihydrolipoamide dehydrogenase. At lower oxygen concentrations upon its decrease from air saturation level to zero, the time-course of resorufin formation by SMP catalyzing coupled oxidation of succinate (the total ROS production by respiratory complexes II and III and by the reverse electron transfer (RET)-mediated by complex I) also corresponds to the linear dependence on oxygen with the same first-order rate constant determined in the initial rate studies. Prolonged incubation of SMP generating succinate-supported complex I-mediated ROS affected neither their NADH oxidase nor ROS generating activity. In contrast to SMP significant deviation from the first-order oxygen dependence in the time-course kinetics during coupled oxidation of succinate by intact mitochondria was evident. Complex I catalyzes the NADH:resorufin oxidoreductase reaction resulting in formation of colorless reduced resorufin. Hydrogen peroxide oxidizes reduced resorufin in the presence of peroxidase, thus showing its dihydroresorufin peroxidase activity. Combined NADH:resorufin reductase and dihydroresorufin peroxidase activities result in underestimation of the amount of hydrogen peroxide generated by mitochondria. We conclude that only initial rates of the mitochondrial ROS production, not the amount of resorufin accumulated, should be taken as the reliable measure of the mitochondrial ROS-generating activity, because of the cycling of the oxidized and reduced resorufin during Amplex Red assays fed by NADH and other possible reductant(s) present in mitochondria.


Assuntos
Transporte de Elétrons/genética , Mitocôndrias Cardíacas/metabolismo , Oxazinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/química , NAD/metabolismo , Oxazinas/química , Oxazinas/isolamento & purificação , Oxirredução , Oxigênio/metabolismo , Ratos , Ácido Succínico/metabolismo , Superóxidos/química
20.
Toxicol Lett ; 289: 1-13, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29501571

RESUMO

We performed a multiple 'omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III-V genes. Persistent transcripts and proteins were related to citric acid cycle and ß-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2-FN1-MYC-CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation.


Assuntos
Anticonvulsivantes/efeitos adversos , DNA Mitocondrial/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ácido Valproico/efeitos adversos , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Epigenômica , Perfilação da Expressão Gênica , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Cinética , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/agonistas , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteômica
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