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1.
Int J Food Microbiol ; 348: 109226, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-33964807

RESUMO

The "prise de mousse" stage during sparkling wine elaboration by the traditional method (Champenoise) involves a second fermentation in a sealed bottle followed by a prolonged aging period, known to contribute significantly to the unique organoleptic properties of these wines. During this stage, CO2 overpressure, nutrient starvation and high ethanol concentrations are stress factors that affect yeast cells viability and metabolism. Since mitochondria are responsible for energy generation and are required for cell aging and response to numerous stresses, we hypothesized that these organelles may play an essential role during the prise de mousse. The objective of this study is to characterize the mitochondrial response of a Saccharomyces cerevisiae strain traditionally used in sparkling wine production along the "prise de mousse" and study the effect of CO2 overpressure through a proteomic analysis. We observed that pressure negatively affects the content of mitochondrion-related proteome, especially to those proteins involved in tricarboxylic acid cycle. However, proteins required for the branched-amino acid synthesis, implied in wine aromas, and respiratory chain, also previously reported by transcriptomic analyses, were found over-represented in the sealed bottles. Multivariate analysis of proteins required for tricarboxylic cycle, respiratory chain and amino acid metabolism revealed differences in concentrations, allowing the wine samples to group depending on the time and CO2 overpressure parameters. Ethanol content along the second fermentation could be the main reason for this changing behavior observed at proteomic level. Further research including genetic studies, determination of ROS, characterization of mitochondrial activity and targeted metabolomics analyses is required. The list of mitochondrial proteins provided in this work will lead to a better understanding of the yeast behavior under these conditions of special interest in the wine industry.


Assuntos
Dióxido de Carbono/análise , Dióxido de Carbono/farmacologia , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia , Pressão do Ar , Etanol/metabolismo , Fermentação , Odorantes/análise , Proteoma/análise , Proteômica , Estresse Fisiológico/fisiologia , Vinho/análise , Fermento Seco/metabolismo
2.
Nat Commun ; 12(1): 3239, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050165

RESUMO

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.


Assuntos
Proteases Dependentes de ATP/ultraestrutura , Proteínas Mitocondriais/ultraestrutura , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Envelhecimento/metabolismo , Bortezomib/farmacologia , Domínio Catalítico/efeitos dos fármacos , Microscopia Crioeletrônica , Ensaios Enzimáticos , Humanos , Hidrólise , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Proteólise , Proteostase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
3.
Nat Commun ; 12(1): 3210, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050192

RESUMO

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.


Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA Mitocondrial/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Doenças Mitocondriais/terapia , Animais , Enzimas de Restrição do DNA/genética , DNA Mitocondrial/genética , Dependovirus/genética , Modelos Animais de Doenças , Fibroblastos , Edição de Genes/métodos , Vetores Genéticos/genética , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Mutação Puntual , Cultura Primária de Células , RNA de Transferência de Alanina/genética
4.
Adv Exp Med Biol ; 1269: 253-258, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33966226

RESUMO

Triple-negative breast cancer (TNBC) is a highly diverse group of cancers with limited treatment options, responsible for about 15% of all breast cancers. TNBC cells differ from each other in many ways such as gene expression, metabolic activity, tumorigenicity, and invasiveness. Recently, many research and clinical efforts have focused on metabolically targeted therapy for TNBC. Metabolic characterization of TNBC cell lines can facilitate the assessment of therapeutic effects and assist in metabolic drug development. Herein, we used optical redox imaging (ORI) techniques to characterize TNBC subtypes metabolically. We found that various TNBC cell lines had differing redox statuses (levels of reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and the redox ratio (FAD/(NADH+FAD)). We then metabolically perturbed the cells with mitochondrial inhibitors and an uncoupler and performed ORI accordingly. As expected, we observed that these TNBC cell lines had similar response patterns to the metabolic perturbations. However, they exhibited differing redox plasticity. These results suggest that subtypes of TNBC cells are different metabolically and that ORI can serve as a sensitive technique for the metabolic profiling of TNBC cells.


Assuntos
Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , NAD/metabolismo , Imagem Óptica , Oxirredução , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/genética
5.
Science ; 372(6543): 716-721, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33986176

RESUMO

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.


Assuntos
Eritropoese , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Ciclo do Ácido Cítrico , Metilação de DNA , Transporte de Elétrons , Embrião não Mamífero/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Histonas/metabolismo , Leflunomida/farmacologia , Redes e Vias Metabólicas , Metilação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Consumo de Oxigênio , Fatores de Transcrição/genética , Ubiquinona/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
6.
Biochem Biophys Res Commun ; 561: 14-18, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34000512

RESUMO

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674-685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674-685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674-685 competes with the antibody against 179-190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H2O2 but does not protect intact U373 cells against apoptogenic effect of H2O2. Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.


Assuntos
Apoptose , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Feminino , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , /crescimento & desenvolvimento , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
7.
Oxid Med Cell Longev ; 2021: 8832863, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936388

RESUMO

Reactive oxygen species (ROS) production has been associated with neuronal death. ROS are also involved in mitochondrial fission, which is mediated by Dynamin-related protein 1 (Drp1). The regulation of mitochondrial fragmentation mediated by Drp1 and its relationship to mitochondrial ROS (mtROS) in neuronal death have not been completely clarified. The aim of this study is to evaluate the role of mtROS in cell death and their involvement in the activation of Drp1 and mitochondrial fission in a model of cell death of cultured cerebellar granule neurons (CGN). Neuronal death of CGN induced by potassium deprivation (K5) and staurosporine (ST) triggers mitochondrial ROS production and mitochondrial fragmentation. K5 condition evoked an increase of Drp1 phosphorylation at Ser616, but ST treatment led to a decrease of Drp1 phosphorylation. Moreover, the death of CGN induced by both K5 and ST was markedly reduced in the presence of MitoTEMPO; however, mitochondrial morphology was not recovered. Here, we show that the mitochondria are the initial source of ROS involved in the neuronal death of CGN and that mitochondrial fragmentation is a common event in cell death; however, this process is not mediated by Drp1 phosphorylation at Ser616.


Assuntos
Dinaminas/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Animais , Morte Celular , Humanos , Fosforilação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio
8.
Nat Commun ; 12(1): 2665, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976125

RESUMO

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.


Assuntos
Envelhecimento , Células-Tronco Hematopoéticas/efeitos dos fármacos , NAD/metabolismo , Niacinamida/análogos & derivados , Compostos de Piridínio/farmacologia , Fatores Etários , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Niacinamida/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos
9.
Nat Commun ; 12(1): 2947, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011950

RESUMO

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.


Assuntos
Evolução Molecular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Modelos Moleculares , Naegleria/classificação , Naegleria/genética , Naegleria/metabolismo , Peroxissomos/metabolismo , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/classificação
10.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946854

RESUMO

Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.


Assuntos
Hexoquinase/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Autofagia/fisiologia , Sinalização do Cálcio/fisiologia , Hipóxia Celular , Peptídeos Penetradores de Células/uso terapêutico , Indução Enzimática , Regulação Neoplásica da Expressão Gênica , Glicólise/fisiologia , Hexoquinase/antagonistas & inibidores , Humanos , Membranas Intracelulares/enzimologia , Camundongos , MicroRNAs/genética , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/terapia , Neoplasias Experimentais/enzimologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional , Ratos , Ubiquitinação
11.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946872

RESUMO

Studying the proteomes of tissue-derived extracellular vesicles (EVs) can lead to the identification of biomarkers of disease and can provide a better understanding of cell-to-cell communication in both healthy and diseased tissue. The aim of this study was to apply our previously established tissue-derived EV isolation protocol to mouse lungs in order to determine the changes in the proteomes of lung tissue-derived EVs during allergen-induced eosinophilic airway inflammation. A mouse model for allergic airway inflammation was used by sensitizing the mice intraperitoneal with ovalbumin (OVA), and one week after the final sensitization, the mice were challenged intranasal with OVA or PBS. The animals were sacrificed 24 h after the final challenge, and their lungs were removed and sliced into smaller pieces that were incubated in culture media with DNase I and Collagenase D for 30 min at 37 °C. Vesicles were isolated from the medium by ultracentrifugation and bottom-loaded iodixanol density cushions, and the proteomes were determined using quantitative mass spectrometry. More EVs were present in the lungs of the OVA-challenged mice compared to the PBS-challenged control mice. In total, 4510 proteins were quantified in all samples. Among them, over 1000 proteins were significantly altered (fold change >2), with 614 proteins being increased and 425 proteins being decreased in the EVs from OVA-challenged mice compared to EVs from PBS-challenged animals. The associated cellular components and biological processes were analyzed for the altered EV proteins, and the proteins enriched during allergen-induced airway inflammation were mainly associated with gene ontology (GO) terms related to immune responses. In conclusion, EVs can be isolated from mouse lung tissue, and the EVs' proteomes undergo changes in response to allergen-induced airway inflammation. This suggests that the composition of lung-derived EVs is altered in diseases associated with inflammation of the lung, which may have implications in type-2 driven eosinophilic asthma pathogenesis.


Assuntos
Vesículas Extracelulares/imunologia , Pulmão/imunologia , Proteoma , Eosinofilia Pulmonar/imunologia , Hipersensibilidade Respiratória/imunologia , Alérgenos/toxicidade , Animais , Asma , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Ontologia Genética , Pulmão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nanopartículas , Ovalbumina/toxicidade , Eosinofilia Pulmonar/etiologia , Eosinofilia Pulmonar/metabolismo , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/metabolismo
12.
Nat Commun ; 12(1): 2616, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972548

RESUMO

FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca2+ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Cálcio/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno , Retina/metabolismo , Fator de Resposta Sérica/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946753

RESUMO

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFß2) suppressed PGC-1α expression. Correspondingly, TGFß2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFß2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFß2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFß2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFß2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidores , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Epitélio Pigmentado da Retina/citologia
14.
Nat Commun ; 12(1): 2529, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953175

RESUMO

In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.


Assuntos
Dano ao DNA/fisiologia , Insuficiência Cardíaca/metabolismo , Biogênese de Organelas , RNA Longo não Codificante/metabolismo , Animais , Núcleo Celular , Metabolismo Energético , Fibroblastos , Insuficiência Cardíaca/patologia , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Células-Tronco Embrionárias Murinas , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo
15.
Cell Prolif ; 54(5): e13034, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33931895

RESUMO

OBJECTIVES: Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs. MATERIALS AND METHODS: Parkin+/+ and parkin-/- ESCs were established from E3.5 blastocysts of parkin+/- x parkin+/- mating mice. The pink1-/- , optn-/- and ndp52-/- ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness. RESULTS: PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1-/- ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N). CONCLUSIONS: The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitofagia , Proteínas Quinases/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Reprogramação Celular , Edição de Genes , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ubiquitina/metabolismo
16.
Int J Nanomedicine ; 16: 3073-3089, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953556

RESUMO

Introduction: Hyperoside (HYP), a flavonol glycoside compound, has been shown to significantly inhibit the proliferation of malignant tumors. Mitochondria serve as both "energy factories" and "suicide weapon stores" of cells. Targeted delivery of cytotoxic drugs to the mitochondria of tumor cells and tumor vascular cells is a promising strategy to improve the efficacy of chemotherapy. Objective: We report a novel dual-functional liposome system possessing both extracellular charge reversal and mitochondrial targeting properties to enhance drug accumulation in mitochondria and trigger apoptosis of cancer cells. Methods: L-lysine was used as a linker to connect 2,3-dimethylmaleic anhydride (DMA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to yield a new compound, DSPE-Lys-DMA (DLD). Then, DLD was mixed with other commercially available lipids to form charge reversed and mitochondria-targeted liposomes (DLD-Lip). The size, morphology, zeta potential, serum stability, and protein adsorption of the HYP loaded DLD-Lip (HYP/DLD-Lip) were measured. The release profile, cellular uptake, in vitro and in vivo toxicity, and anticancer activity of HYP/DLD-Lip were investigated. Results: The results showed that the mean diameter of the liposomes was less than 200 nm. The zeta potential of the liposomes was negative at pH 7.4. However, the zeta potential was positive at weak acidic pH values with the cleavage of the DMA amide. The charge reversion of HYP/DLD-Lip facilitated the cellular internalization and mitochondrial accumulation for enhanced antitumor effect. The strongest tumor growth inhibition (TGI 88.79%) without systemic toxicity was observed in DLD/HYP-Lips-treated CBRH-7919 tumor xenograft BALB/C mice. Conclusion: The charge reversed and mitochondria-targeted liposomes represented a promising anticancer drug delivery system for enhanced anticancer therapeutic efficacy.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Lipossomos/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Quercetina/análogos & derivados , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidiletanolaminas/química , Quercetina/administração & dosagem , Quercetina/química , Quercetina/farmacologia
17.
Nat Commun ; 12(1): 2804, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990571

RESUMO

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Mitocôndrias/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Feminino , Proteínas de Choque Térmico HSP40/deficiência , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos
18.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947138

RESUMO

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells; however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs.


Assuntos
Adenocarcinoma de Células Claras/tratamento farmacológico , Antineoplásicos/uso terapêutico , Proteínas de Ligação a DNA/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Oxidiazóis/uso terapêutico , Piperidinas/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Oxidiazóis/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Piperidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Esferoides Celulares , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Oxid Med Cell Longev ; 2021: 6655685, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953835

RESUMO

Methods and Results: Herein, a comprehensive proteomic analysis was conducted on proliferative endometria from sows with low and normal reproductive performance (LRP and NRP, respectively). Enrichment analysis of differentially expressed proteins revealed alterations in endometrial remodeling, substance metabolism (mainly lipid, nitrogen, and retinol metabolism), immunological modulation, and insulin signaling in LRP sows. Importantly, aberrant lipid metabolite accumulation and dysregulation of insulin signaling were coincidently confirmed in endometria of LPR sows, proving an impaired insulin sensitivity. Furthermore, established high-fat diet- (HFD-) induced insulin-resistant mouse models revealed that uterine insulin resistance beginning before pregnancy deteriorated uterine receptivity and decreased implantation sites and fetal numbers. Mitochondrial biogenesis and fusion were decreased, and reactive oxygen species was overproduced in uteri from the HFD group during the implantation period. Ishikawa and JAR cells directly demonstrated that oxidative stress compromised implantation in vitro. Conclusions: This study demonstrated that uterine insulin sensitivity impairment beginning before pregnancy resulted in implantation and fetal loss associated with oxidative stress induced by mitochondrial dysfunction.


Assuntos
Implantação do Embrião/fisiologia , Insulina/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Útero/metabolismo , Animais , Feminino , Camundongos , Gravidez
20.
Oxid Med Cell Longev ; 2021: 5512322, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959215

RESUMO

Ketogenic diet (KD) is popular in diabetic patients but its cardiac safety and efficiency on the heart are unknown. The aim of the present study is to determine the effects and the underlined mechanisms of KD on cardiac function in diabetic cardiomyopathy (DCM). We used db/db mice to model DCM, and different diets (regular or KD) were used. Cardiac function and interstitial fibrosis were determined. T-regulatory cell (Treg) number and functions were evaluated. The effects of ketone body (KB) on fatty acid (FA) and glucose metabolism, mitochondria-associated endoplasmic reticulum membranes (MAMs), and mitochondrial respiration were assessed. The mechanisms via which KB regulated MAMs and Tregs were addressed. KD improved metabolic indices in db/db mice. However, KD impaired cardiac diastolic function and exacerbated ventricular fibrosis. Proportions of circulatory CD4+CD25+Foxp3+ cells in whole blood cells and serum levels of IL-4 and IL-10 were reduced in mice fed with KD. KB suppressed the differentiation to Tregs from naive CD4+ T cells. Cultured medium from KB-treated Tregs synergically activated cardiac fibroblasts. Meanwhile, KB inhibited Treg proliferation and productions of IL-4 and IL-10. Treg MAMs, mitochondrial respiration and respiratory complexes, and FA synthesis and oxidation were all suppressed by KB while glycolytic levels were increased. L-carnitine reversed Treg proliferation and function inhibited by KB. Proportions of ST2L+ cells in Tregs were reduced by KB, as well as the production of ST2L ligand, IL-33. Reinforcement expressions of ST2L in Tregs counteracted the reductions in MAMs, mitochondrial respiration, and Treg proliferations and productions of Treg cytokines IL-4 and IL-10. Therefore, despite the improvement of metabolic indices, KD impaired Treg expansion and function and promoted cardiac fibroblast activation and interstitial fibrosis. This could be mainly mediated by the suppression of MAMs and fatty acid metabolism inhibition via blunting IL-33/ST2L signaling.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Dieta Cetogênica/efeitos adversos , Fibrose/fisiopatologia , Mitocôndrias/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Humanos , Masculino , Camundongos
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