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2.
Nat Commun ; 11(1): 5261, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067417

RESUMO

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/metabolismo , Mamíferos/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Inibidores Enzimáticos/química , Feminino , Mamíferos/genética , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Simulação de Dinâmica Molecular , Fosforilação Oxidativa , Piridinas/química , Piridinas/metabolismo
3.
Nat Commun ; 11(1): 5265, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067432

RESUMO

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.


Assuntos
Carcinoma Ductal Pancreático/imunologia , Evasão da Resposta Imune , Células-Tronco Neoplásicas/imunologia , Neoplasias Pancreáticas/imunologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Feminino , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Fosforilação Oxidativa , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia
4.
PLoS One ; 15(10): e0239804, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031404

RESUMO

Mitochondrial OXPHOS generates most of the energy required for cellular function. OXPHOS biogenesis requires the coordinated expression of the nuclear and mitochondrial genomes. This represents a unique challenge that highlights the importance of nuclear-mitochondrial genetic communication to cellular function. Here we investigated the transcriptomic and functional consequences of nuclear-mitochondrial genetic divergence in vitro and in vivo. We utilized xenomitochondrial cybrid cell lines containing nuclear DNA from the common laboratory mouse Mus musculus domesticus and mitochondrial DNA (mtDNA) from Mus musculus domesticus, or exogenous mtDNA from progressively divergent mouse species Mus spretus, Mus terricolor, Mus caroli and Mus pahari. These cybrids model a wide range of nuclear-mitochondrial genetic divergence that cannot be achieved with other research models. Furthermore, we used a xenomitochondrial mouse model generated in our laboratory that harbors wild-type, C57BL/6J Mus musculus domesticus nuclear DNA and homoplasmic mtDNA from Mus terricolor. RNA sequencing analysis of xenomitochondrial cybrids revealed an activation of interferon signaling pathways even in the absence of OXPHOS dysfunction or immune challenge. In contrast, xenomitochondrial mice displayed lower baseline interferon gene expression and an impairment in the interferon-dependent innate immune response upon immune challenge with herpes simplex virus, which resulted in decreased viral control. Our work demonstrates that nuclear-mitochondrial genetic divergence caused by the introduction of exogenous mtDNA can modulate the interferon immune response both in vitro and in vivo, even when OXPHOS function is not compromised. This work may lead to future insights into the role of mitochondrial genetic variation and the immune function in humans, as patients affected by mitochondrial disease are known to be more susceptible to immune challenges.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial , Interferons/imunologia , Mitocôndrias/genética , Animais , Linhagem Celular , Feminino , Genótipo , Imunidade Inata , Masculino , Camundongos/classificação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação Oxidativa
5.
Tumour Biol ; 42(10): 1010428320965284, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33028168

RESUMO

Glucose, as the main consuming nutrient of the body, faces different destinies in cancer cells. Glycolysis, oxidative phosphorylation, and pentose phosphate pathways produce different glucose-derived metabolites and thus affect cells' bioenergetics differently. Tumor cells' dependency to aerobic glycolysis and other cancer-specific metabolism changes are known as the cancer hallmarks, distinct cancer cells from normal cells. Therefore, these tumor-specific characteristics receive the limelight as targets for cancer therapy. Glutamine, serine, and fatty acid oxidation together with 5-lipoxygenase are main pathways that have attracted lots of attention for cancer therapy. In this review, we not only discuss different tumor metabolism aspects but also discuss the metabolism roles in the promotion of cancer cells at different stages and their difference with normal cells. Besides, we dissect the inhibitors potential in blocking the main metabolic pathways to introduce the effective and non-effective inhibitors in the field.


Assuntos
Antineoplásicos/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Medicina de Precisão , Antineoplásicos/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Metabolismo Energético/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/etiologia , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Medicina de Precisão/métodos
6.
Am J Physiol Regul Integr Comp Physiol ; 319(4): R476-R484, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32877243

RESUMO

Changes in cardiomyocyte metabolism have been heavily implicated in cardiac injury and heart failure (HF). However, there is emerging evidence that metabolism in nonmyocyte populations, including cardiac fibroblasts, immune cells, and endothelial cells, plays an important role in cardiac remodeling and adaptation to injury. Here, we discuss recent advances and insights into nonmyocyte metabolism in the healthy and injured heart. Metabolic switching from mitochondrial oxidative phosphorylation to glycolysis is critical for immune cell (macrophage and T lymphocyte) and fibroblast phenotypic switching in the inflamed and fibrotic heart. On the other hand, cardiac endothelial cells are heavily reliant on glycolytic metabolism, and thus impairments in glycolytic metabolism underlie endothelial cell dysfunction. Finally, we review current and ongoing metabolic therapies for HF and the potential implications for nonmyocyte metabolism.


Assuntos
Remodelamento Atrial/fisiologia , Fibroblastos/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Remodelação Ventricular/fisiologia , Células Endoteliais/metabolismo , Glicólise/fisiologia , Humanos , Fosforilação Oxidativa
7.
Mol Cell ; 79(6): 1051-1065.e10, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32877643

RESUMO

Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genética
8.
Mol Cell ; 80(1): 43-58.e7, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32937100

RESUMO

Immune cell function depends on specific metabolic programs dictated by mitochondria, including nutrient oxidation, macromolecule synthesis, and post-translational modifications. Mitochondrial adaptations have been linked to acute and chronic inflammation, but the metabolic cues and precise mechanisms remain unclear. Here we reveal that histone deacetylase 3 (HDAC3) is essential for shaping mitochondrial adaptations for IL-1ß production in macrophages through non-histone deacetylation. In vivo, HDAC3 promoted lipopolysaccharide-induced acute inflammation and high-fat diet-induced chronic inflammation by enhancing NLRP3-dependent caspase-1 activation. HDAC3 configured the lipid profile in stimulated macrophages and restricted fatty acid oxidation (FAO) supported by exogenous fatty acids for mitochondria to acquire their adaptations and depolarization. Rather than affecting nuclear gene expression, HDAC3 translocated to mitochondria to deacetylate and inactivate an FAO enzyme, mitochondrial trifunctional enzyme subunit α. HDAC3 may serve as a controlling node that balances between acquiring mitochondrial adaptations and sustaining their fitness for IL-1ß-dependent inflammation.


Assuntos
Ácidos Graxos/metabolismo , Histona Desacetilases/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Mitocôndrias/metabolismo , Adulto , Animais , Caspase 1/metabolismo , Feminino , Humanos , Inflamação/patologia , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Células Mieloides/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Oxirredução , Fosforilação Oxidativa , Adulto Jovem
9.
Nat Commun ; 11(1): 4056, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792483

RESUMO

Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid ß-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.


Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia/metabolismo , Mitocôndrias/metabolismo , Animais , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citometria de Fluxo , Humanos , Leucemia/genética , Leucemia Mieloide Aguda/patologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipogênese/genética , Lipogênese/fisiologia , Camundongos , Mitocôndrias/genética , Oxirredução , Fosforilação Oxidativa
10.
Free Radic Res ; 54(7): 477-496, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32842814

RESUMO

Smokeless tobacco (SLT) or chewing tobacco has been a highly addictive practice in India across ages, posing major threat to the systemic health and possibly neurodegeneration. Earlier studies showed components of SLT could be harmful to neuronal health. However, mechanism of SLT in neurodegeneration remained unexplored. This study investigated the detrimental role of SLT on differentiated neuronal cell lines, PC12 and SH-SY5Y by using graded doses of water soluble lyophilised SLT. Reduced cell viability, compromised mitochondrial structure and functions were observed when neuronal cell lines were treated with SLT (6 mg/mL) for 24 h. There was reduction of oxidative phosphorylation and aerobic glycolysis as determined by diminution of ATP production (2.5X) and basal respiration (1.9X). Mitochondrial membrane potential was dropped by 3.5 times. Bid, a pro-apoptotic Bcl-2 family protein, has imperative role in regulating mitochondrial outer membrane permeabilization and subsequent cytochrome c release leading to apoptosis. This article for the first time indicated the involvement of Bid in SLT mediated neurotoxicity and possibly neurodegeneration. SLT treatment enhanced expression of cleaved-Bid in time dependent manner. The involvement of Bid was further confirmed by using Bid specific shRNA which reversed the effects of SLT and conferred significant protection from apoptosis up to 72 h. Thus, our results clearly indicated that SLT induced neuronal cell death occurred via production of ROS, alteration of mitochondrial morphology, membrane potential and oxidative phosphorylation, inactivation of survival pathway and activation of apoptotic markers mediated by Bid. Therefore, Bid could be a potential future therapeutic target for SLT induced neurodegeneration.


Assuntos
Neurônios/patologia , Tabaco sem Fumaça/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , Dano ao DNA , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação Oxidativa , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Proteína Supressora de Tumor p53/metabolismo
11.
Toxicol Lett ; 333: 232-241, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32835834

RESUMO

Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, hepatotoxic and immunotoxic effects. We previously demonstrated that OTA could cause mitochondrial function disturbance in GES-1 cells in vitro, which lead to the presumption that the glucose metabolism of GES-1 cells will be altered by OTA. Therefore in the present study, we explored the toxicity of OTA on glucose metabolism of GES-1 cells and the molecular mechanism. We found that OTA could induce aerobic glycolysis, evidenced shown by increase of glucose consumption, lactate production and cellular ATP concentration. We further detected expressions of GLUT1 and glycolytic enzymes including HK2, PFK1, PKM2 and LDHA as well as tricarboxylic acid (TCA) cycle-associated enzymes including IDH1, OGDH and CS. The results showed that expression of GLUT1 as well as the activities and expressions of HK2, PFK1 and LDHA were significantly increased while IDH1 and OGDH were reduced by OTA. As to PKM2, western blot showed that OTA could elevated the phospho-PKM2 Ser37 protein level and induce the nuclear accumulation of PKM2, which was further supported by immunofluorescence analyses, in addition, pyruvate kinase activity was reduced by OTA. In conclusion, these findings suggest that OTA exposure induces the metabolic shift from oxidative phosphorylation to aerobic glycolysis via regulating the activities and expressions of glycolysis and TCA-cycle associated molecules in GES-1 cells.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Glucose/metabolismo , Ocratoxinas/toxicidade , Linhagem Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Fosforilação Oxidativa/efeitos dos fármacos
12.
Nat Commun ; 11(1): 4205, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826891

RESUMO

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.


Assuntos
Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Humanos , Camundongos , Fosforilação Oxidativa , Proteômica , Pirazóis/farmacologia , Piridinas/farmacologia , Quinolinas , RNA Mensageiro/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Ubiquitina-Proteína Ligases/genética
13.
Nat Commun ; 11(1): 4269, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859890

RESUMO

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.


Assuntos
Processamento Pós-Transcricional do RNA , RNA Mitocondrial/química , RNA de Transferência/química , Feminino , Código Genético , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Estrutura Molecular , Nucleosídeo Q/biossíntese , Nucleosídeo Q/química , Fosforilação Oxidativa , Placenta , Gravidez , RNA Mitocondrial/isolamento & purificação , RNA Mitocondrial/metabolismo , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , RNA-Seq
14.
Nature ; 585(7824): 288-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32641834

RESUMO

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciona intestinalis/enzimologia , Ciclo do Ácido Cítrico , Citosol/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Lactobacillus brevis/enzimologia , Masculino , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neoplasias/enzimologia , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquinona/metabolismo
15.
Nucleic Acids Res ; 48(14): 7924-7943, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652011

RESUMO

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.


Assuntos
Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/enzimologia , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Fosforilação Oxidativa , Biossíntese de Proteínas , RNA Ribossômico 16S/química , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Fatores de Transcrição/metabolismo
16.
Autophagy ; 16(10): 1915-1917, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32686580

RESUMO

Hepatocellular carcinoma (HCC) is the consequence of chronic liver damage caused by the excessive generation of reactive oxygen species (ROS). To mitigate the deleterious effects of ROS, cells activate the transcription factor NFE2L2/NRF2, which is constitutively degraded through its partner KEAP1. The inactivation of KEAP1 by ROS results in the upregulation of NFE2L2, which leads to the upregulation of critical detoxifying molecules that serve to keep ROS at tolerable levels in order to maintain cell viability. It is thought that this mechanism allows cells to accumulate mutations, which together with the additional pro-tumorigenic and pro-survival effects of NFE2L2 activation, promote cancer initiation and progression. Germane to this phenomenon is macroautophagy/autophagy, which under homeostatic conditions has also been proposed to serve as a detoxifying mechanism by clearing up toxic aggregates and damaged organelles. Our recent data establish a new paradigm for the role that autophagy plays in HCC development.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Autofagia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo , Proteína Sequestossoma-1/metabolismo
17.
Am J Respir Cell Mol Biol ; 63(4): 519-530, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32628869

RESUMO

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Pulmão/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Anexina A5/metabolismo , Movimento Celular/efeitos dos fármacos , Desoxiglucose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicólise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Pulmão/metabolismo , Masculino , Fosforilação Oxidativa/efeitos dos fármacos , Propídio/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
18.
Nature ; 583(7817): 631-637, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641830

RESUMO

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.


Assuntos
Toxinas Bacterianas/metabolismo , Citidina Desaminase/metabolismo , DNA Mitocondrial/genética , Edição de Genes/métodos , Genes Mitocondriais/genética , Mitocôndrias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sequência de Bases , Burkholderia cenocepacia/enzimologia , Burkholderia cenocepacia/genética , Respiração Celular/genética , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Genoma Mitocondrial/genética , Células HEK293 , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/terapia , Mutação , Fosforilação Oxidativa , Engenharia de Proteínas , RNA Guia/genética , Especificidade por Substrato , Sistemas de Secreção Tipo VI/metabolismo
19.
Nat Rev Drug Discov ; 19(9): 609-633, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32709961

RESUMO

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner - a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes.


Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Metabolismo Energético/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Animais , Glicólise/fisiologia , Humanos , Fosforilação Oxidativa
20.
Mol Med ; 26(1): 69, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641037

RESUMO

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.


Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Cloroquina/farmacologia , Infecções por Coronavirus/patologia , Isquemia/patologia , Músculo Esquelético/patologia , Pneumonia Viral/patologia , Animais , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Glicólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Neovascularização Fisiológica , Fosforilação Oxidativa , Pandemias , Doença Arterial Periférica/patologia , Pneumonia Viral/tratamento farmacológico , Regeneração , Transdução de Sinais
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