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1.
Nat Commun ; 11(1): 4639, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934238

RESUMO

The ability to detect, respond and adapt to mitochondrial stress ensures the development and survival of organisms. Caenorhabditis elegans responds to mitochondrial stress by activating the mitochondrial unfolded protein response (UPRmt) to buffer the mitochondrial folding environment, rewire the metabolic state, and promote innate immunity and lifespan extension. Here we show that HDA-1, the C. elegans ortholog of mammalian histone deacetylase (HDAC) is required for mitochondrial stress-mediated activation of UPRmt. HDA-1 interacts and coordinates with the genome organizer DVE-1 to induce the transcription of a broad spectrum of UPRmt, innate immune response and metabolic reprogramming genes. In rhesus monkey and human tissues, HDAC1/2 transcript levels correlate with the expression of UPRmt genes. Knocking down or pharmacological inhibition of HDAC1/2 disrupts the activation of the UPRmt and the mitochondrial network in mammalian cells. Our results underscore an evolutionarily conserved mechanism of HDAC1/2 in modulating mitochondrial homeostasis and regulating longevity.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Histona Desacetilases/metabolismo , Longevidade , Mitocôndrias/enzimologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Histona Desacetilases/genética , Macaca mulatta , Estresse Fisiológico , Resposta a Proteínas não Dobradas
2.
Nat Commun ; 11(1): 4509, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908151

RESUMO

Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/enzimologia , Glicólise/fisiologia , Mitocôndrias/enzimologia , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Metabolismo Energético/fisiologia , Mutação , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/fisiologia , Piruvato Quinase/genética , Piruvato Quinase/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(28): 16537-16545, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601191

RESUMO

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.


Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Klebsiella oxytoca/enzimologia , Nitrogenase/genética , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Proteínas de Bactérias/metabolismo , Klebsiella oxytoca/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Nitrogenase/metabolismo , Plantas/genética , Plantas/metabolismo , Processamento de Proteína Pós-Traducional
4.
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
5.
PLoS One ; 15(6): e0233856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492073

RESUMO

The enzyme ß-glucosidase 2 (GBA2) is clinically relevant because it is targeted by the drug miglustat (Zavesca®) and because it is involved in inherited diseases. Mutations in the GBA2 gene are associated with two neurological diseases on the ataxia-spasticity spectrum, hereditary spastic paraplegia 46 (SPG46) and Marinesco-Sjögren-like syndrome (MSS). To establish how GBA2 mutations give rise to neurological pathology, we have begun to investigate mutant forms of GBA2 encoded by disease-associated GBA2 alleles. Previously, we found that five GBA2 missense mutants and five C-terminally truncated mutants lacked enzyme activity. Here we have examined the cellular locations of wild-type (WT) and mutant forms of GBA2 by confocal and electron microscopy, using transfected cells. Similar to GBA2-WT, the D594H and M510Vfs*17 GBA2 mutants were located at the plasma membrane, whereas the C-terminally truncated mutants terminating after amino acids 233 and 339 (GBA2-233 and -339) were present in the mitochondrial matrix, induced mitochondrial fragmentation and loss of mitochondrial transmembrane potential. Deletional mutagenesis indicated that residues 161-200 are critical for the mitochondrial fragmentation of GBA2-233 and -339. Considering that the mitochondrial fragmentation induced by GBA2-233 and -339 is consistently accompanied by their localization to the mitochondrial matrix, our deletional analysis raises the possibility that that GBA2 residues 161-200 harbor an internal targeting sequence for transport to the mitochondrial matrix. Altogether, our work provides new insights into the behaviour of GBA2-WT and disease-associated forms of GBA2.


Assuntos
Glucosilceramidase/metabolismo , Mitocôndrias/patologia , Alelos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Glucosilceramidase/genética , Humanos , Potencial da Membrana Mitocondrial , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Mutação , Paraplegia Espástica Hereditária/genética , Degenerações Espinocerebelares/genética
6.
J Parasitol ; 106(3): 383-391, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32491171

RESUMO

The long-term fidelity of pinniped hosts to their natal rookery site suggests the genetic architecture of their Uncinaria spp. hookworms should be strongly structured by host breeding biology. However, historical events affecting host populations may also shape parasite genetic structure. Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 86 Uncinaria lucasi individuals were obtained to assess genetic variation and structure of nematodes from 2 host species (68 hookworms from northern fur seals; 18 hookworms from Steller sea lions) and rookeries from 3 widely separated geographic regions: the western Bering Sea and Sea of Okhotsk, eastern Bering Sea, and the eastern Pacific Ocean. High COI haplotype (h = 0.96-0.98) and nucleotide (π = 0.014) diversity was found. The haplotype network showed a star-shaped pattern with a large number of haplotypes separated by few substitutions. The network did not show separation of U. lucasi by geographic region or host species. Fst values between U. lucasi individuals representing geographic regions showed no differentiation, consistent with the absence of genetic structure. At face value, this lack of genetic structure in U. lucasi suggests high gene flow but could also be explained by recent (post-glacial) population expansions of northern fur seals and their hookworms.


Assuntos
Ancylostomatoidea/fisiologia , Caniformia/parasitologia , Infecções por Uncinaria/veterinária , Sequência de Aminoácidos , Ancylostomatoidea/genética , Animais , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Variação Genética , Haplótipos/genética , Infecções por Uncinaria/parasitologia , Infecções por Uncinaria/transmissão , Masculino , Mitocôndrias/enzimologia , Oceano Pacífico , Alinhamento de Sequência/veterinária
7.
Nucleic Acids Res ; 48(12): 6799-6810, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484546

RESUMO

Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.


Assuntos
Mitocôndrias/genética , Nitrosação/genética , Estresse Nitrosativo/genética , Treonina-tRNA Ligase/genética , Aminoacil-tRNA Sintetases/genética , Aminoacilação/genética , Animais , Domínio Catalítico/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Cinética , Camundongos , Mitocôndrias/enzimologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Treonina-tRNA Ligase/química
8.
Nucleic Acids Res ; 48(14): 8022-8034, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32573735

RESUMO

Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.


Assuntos
Metiltransferases/metabolismo , Mitocôndrias/enzimologia , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/enzimologia , Animais , Células Cultivadas , Metilação , Camundongos , Mitocôndrias/metabolismo , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , RNA Ribossômico/química , RNA Ribossômico 28S/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
9.
Korean J Parasitol ; 58(2): 201-204, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32418391

RESUMO

Taenia saginata infection has seldom been reported in Cambodia. In this study, we performed a survey of intestinal parasites in 1,156 residents of Preah Vihear and Stung Treng Provinces in 2018. The results revealed that 26 (2.4%) cases were positive for Taenia spp. eggs. In order to obtain the strobilae of the tapeworms, 2 patients in Preah Vihear were treated with praziquantel and purged with magnesium salts. The proglottids expelled after the medication were morphologically and molecularly analyzed to determine the species. The main uterine lateral braches in gravid proglottids were >15 in number suggesting that they are either T. saginata or Taenia asiatica. The sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and 2 nuclear loci, elongation factor-1 alpha (ef1) and ezrin-radixin-moesin-like protein (elp), were identical to the sequences of T. saginata available in GenBank but distant from Taenia solium, T. asiatica, and T. saginata-T. asiatica hybrid. This is the first report of the presence of T. saginata in the northern part of Cambodia bordering Lao PDR based on a molecular confirmation.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Taenia saginata , Teníase/diagnóstico , Animais , Camboja/epidemiologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/enzimologia , Taenia saginata/genética , Teníase/epidemiologia
10.
Nat Commun ; 11(1): 2508, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427840

RESUMO

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance.


Assuntos
Metabolismo dos Lipídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Neoplasias de Próstata Resistentes à Castração/enzimologia , Antagonistas de Receptores de Andrógenos/administração & dosagem , Progressão da Doença , Homeostase , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fosfolipídeos/metabolismo , Próstata/enzimologia , Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo
11.
PLoS Genet ; 16(4): e1008330, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324744

RESUMO

The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.


Assuntos
Alquil e Aril Transferases/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alelos , Anticódon , Citosol/enzimologia , Teste de Complementação Genética , Humanos , Mitocôndrias/enzimologia , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Alinhamento de Sequência , Especificidade por Substrato
12.
Artigo em Inglês | MEDLINE | ID: mdl-32244096

RESUMO

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.


Assuntos
Aciltransferases , Lipoilação , Lycopersicon esculentum , Mitocôndrias , Plastídeos , Aciltransferases/genética , Aciltransferases/metabolismo , Escherichia coli/genética , Lycopersicon esculentum/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Plastídeos/enzimologia , Ácido Tióctico/metabolismo
13.
J Dairy Sci ; 103(7): 6557-6568, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32331890

RESUMO

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and ß-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.


Assuntos
Doenças dos Bovinos/metabolismo , Ácidos Graxos não Esterificados/administração & dosagem , Fígado Gorduroso/veterinária , Metabolismo dos Lipídeos/efeitos dos fármacos , Sirtuína 3/metabolismo , Ácido 3-Hidroxibutírico/sangue , Acetil-CoA Carboxilase/efeitos dos fármacos , Acil-CoA Oxidase/efeitos dos fármacos , Animais , Carnitina O-Palmitoiltransferase/efeitos dos fármacos , Bovinos , Doenças dos Bovinos/prevenção & controle , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/sangue , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Mitocôndrias/enzimologia , Sirtuína 3/genética , Triglicerídeos/metabolismo
14.
Nature ; 579(7799): 433-437, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132706

RESUMO

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies1-3. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress6. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown1,2,7. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.


Assuntos
Citosol/metabolismo , Citosol/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Genoma Humano/genética , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Ligação Proteica , Estresse Fisiológico , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
15.
Mol Cell ; 77(5): 1107-1123.e10, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142684

RESUMO

Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tioléster Hidrolases/genética , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
16.
Clin Sci (Lond) ; 134(7): 677-694, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32167139

RESUMO

Excessive mitochondrial fission has been identified as the central pathogenesis of diabetic kidney disease (DKD), but the precise mechanisms remain unclear. Disulfide-bond A oxidoreductase-like protein (DsbA-L) is highly expressed in mitochondria in tubular cells of the kidney, but its pathophysiological role in DKD is unknown. Our bioinformatics analysis showed that tubular DsbA-L mRNA levels were positively associated with eGFR but negatively associated with Scr and 24h-proteinuria in CKD patients. Furthermore, the genes that were coexpressed with DsbA-L were mainly enriched in mitochondria and were involved in oxidative phosphorylation. In vivo, knockout of DsbA-L exacerbated diabetic mice tubular cell mitochondrial fragmentation, oxidative stress and renal damage. In vitro, we found that DsbA-L was localized in the mitochondria of HK-2 cells. High glucose (HG, 30 mM) treatment decreased DsbA-L expression followed by increased mitochondrial ROS (mtROS) generation and mitochondrial fragmentation. In addition, DsbA-L knockdown exacerbated these abnormalities, but this effect was reversed by overexpression of DsbA-L. Mechanistically, under HG conditions, knockdown DsbA-L expression accentuated JNK phosphorylation in HK-2 cells. Furthermore, administration of a JNK inhibitor (SP600125) or the mtROS scavenger MitoQ significantly attenuated JNK activation and subsequent mitochondrial fragmentation in DsbA-L-knockdown HK-2 cells. Additionally, the down-regulation of DsbA-L also amplified the gene and protein expression of mitochondrial fission factor (MFF) via the JNK pathway, enhancing its ability to recruit DRP1 to mitochondria. Taken together, these results link DsbA-L to alterations in mitochondrial dynamics during tubular injury in the pathogenesis of DKD and unveil a novel mechanism by which DsbA-L modifies mtROS/JNK/MFF-related mitochondrial fission.


Assuntos
Diabetes Mellitus Experimental/enzimologia , Nefropatias Diabéticas/enzimologia , Glutationa Transferase/deficiência , Túbulos Renais/enzimologia , Mitocôndrias/enzimologia , Dinâmica Mitocondrial , Animais , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Glutationa Transferase/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Túbulos Renais/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos Knockout , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
17.
Mol Cell ; 77(5): 1124-1142.e10, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142685

RESUMO

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.


Assuntos
Células-Tronco Embrionárias Humanas/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Mitofagia , Células-Tronco Neurais/enzimologia , Neurogênese , Neurônios/enzimologia , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células HeLa , Células-Tronco Embrionárias Humanas/patologia , Humanos , Cinética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Células-Tronco Neurais/patologia , Neurônios/patologia , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteômica , Transdução de Sinais , Tioléster Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
18.
Nat Commun ; 11(1): 698, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019928

RESUMO

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.


Assuntos
Aconitato Hidratase/metabolismo , Macrófagos/enzimologia , Óxido Nítrico/metabolismo , Quinase Piruvato Desidrogenase (Transferência de Acetil)/metabolismo , Aconitato Hidratase/genética , Animais , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Quinase Piruvato Desidrogenase (Transferência de Acetil)/genética , Ácido Pirúvico/metabolismo
19.
Biochemistry (Mosc) ; 85(Suppl 1): S56-S78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32087054

RESUMO

Oxidative stress resulting from accumulation of reactive oxygen, nitrogen, and halogen species (ROS, RNS, and RHS, respectively) causes the damage of cells and biomolecules. However, over the long evolutionary time, living organisms have developed the mechanisms for adaptation to oxidative stress conditions including the activity of the antioxidant system (AOS), which maintains low intracellular levels of RONS (ROS and RNS) and RHS. Moreover, living organisms have adapted to use low concentrations of these electrophiles for the regulation of cell functions through the reversible post-translational chemical modifications of redox-sensitive amino acid residues in intracellular effectors of signal transduction pathways (protein kinases and protein phosphatases), transcription factors, etc. An important fine-tuning mechanism that ensures involvement of RONS and RHS in the regulation of physiological processes is interconversion between different reactive species. This review focuses on the complex networks of interacting RONS and RHS types and their endogenous sources, such as NOX family of NADPH oxidases, complexes I and III of the mitochondrial electron transport chain, NO synthases, cytochrome P450-containing monooxygenase system, xanthine oxidoreductase, and myeloperoxidases. We highlight that kinetic parameters of reactions involving RONS and RHS determine the effects of these reactive species on cell functions. We also describe the functioning of enzymatic and non-enzymatic AOS components and the mechanisms of RONS and RHS scavenging under physiological conditions. We believe that analysis of interactions between RONS and relationships between different endogenous sources of these compounds will contribute to better understanding of their role in the maintenance of cell redox homeostasis as well as initiation and progression of diseases.


Assuntos
Halogênios/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Antioxidantes/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Radicais Livres/metabolismo , Halogenação , Humanos , Mitocôndrias/enzimologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/metabolismo , Oxirredução , Estresse Oxidativo , Peroxidase/metabolismo , Xantina Desidrogenase/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-32074218

RESUMO

Proinflammatory and inflammatory mediators induced by Trypanosoma cruzi infection increase the oxidative stress, generating toxicity for cells targeting mitochondria of different tissues. We studied the activity of citrate synthase and complexes I-IV of respiratory chain in mitochondria of blood lymphomonocyte fraction, from albino Swiss mice infected with different isolates of T. cruzi , during Chagas disease evolution. Complexes I-IV were modified in infected groups (p<0.05) in all the stages, and an inflammatory process of different magnitudes was detected in the heart and skeletal muscle according to the isolate. The citrate synthase activity presented modifications in the SGO Z12 and the Tulahuen group (p<0.05). Hearts showed fiber fragmentation and fibrosis; skeletal muscle presented inflammatory infiltrates and in the Tulahuen infected group, there were also amastigote nests. The inflammatory processes produced an oxidative stress that induced different alterations of mitochondrial enzymes activities in the lymphomonocyte fraction that can be detected by a simple blood extraction, suggesting that they could be used as disease markers, especially in the indeterminate phase of Chagas disease.


Assuntos
Doença de Chagas/enzimologia , Citrato (si)-Sintase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Animais , Doença de Chagas/metabolismo , Doença de Chagas/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Masculino , Mitocôndrias/parasitologia , Mitocôndrias/patologia , Parasitemia
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