RESUMO
Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Galactosemias , Galactosefosfatos , Mitocôndrias , Galactosemias/metabolismo , Galactosemias/patologia , Galactosemias/genética , Galactosefosfatos/metabolismo , Humanos , Animais , Ratos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fosforilação Oxidativa/efeitos dos fármacos , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Galactose/metabolismoRESUMO
The efficiency of the DNA barcoding relies on sequencing fragment of the Cytochrome C Subunit I (COI) gene, which has been claimed as a tool to biodiversity identification from distinct groups. Accordingly, the goal of this study was to identify juvenile fish species along an estuary of Caeté River in the Brazilian Blue Amazon based on. For this purpose, we applied the DNA barcoding and discuss this approach as a tool for discrimination of species in early ontogenetic stages. A 500-bp fragment was obtained from 74 individuals, belonging to 23 species, 20 genera, 13 families and seven orders. About 70% of the 46 haplotypes revealed congruence between morphological and molecular species identification, while 8% of them failed in identification of taxa and 22% demonstrated morphological misidentification. These results proved that COI fragments were effective to diagnose fish species at early life stages, allowing identifying all samples to a species-specific status, except for some taxa whose COI sequences remain unavailable in public databases. Therefore, we recommend the incorporation of DNA barcoding to provide additional support to traditional identification, especially in morphologically controversial groups. In addition, periodic updates and comparative analyses in public COI datasets are encouraged.
Assuntos
Código de Barras de DNA Taxonômico , Estuários , Humanos , Animais , Código de Barras de DNA Taxonômico/métodos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Filogenia , Peixes , DNA/genéticaRESUMO
The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Oxigênio , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Oxigênio/metabolismo , Methanosarcina/genética , Methanosarcina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredutases/metabolismo , Metano/metabolismo , Citocromos/metabolismo , Acetatos , Lactatos/metabolismoRESUMO
Mitochondrial translation is a highly regulated process, and newly synthesized mitochondrial products must first associate with several nuclear-encoded auxiliary factors to form oxidative phosphorylation complexes. The output of mitochondrial products should therefore be in stoichiometric equilibrium with the nuclear-encoded products to prevent unnecessary energy expense or the accumulation of pro-oxidant assembly modules. In the mitochondrial DNA of Saccharomyces cerevisiae, COX1 encodes subunit 1 of the cytochrome c oxidase and COB the central core of the cytochrome bc1 electron transfer complex; however, factors regulating the expression of these mitochondrial products are not completely described. Here, we identified Mrx9p as a new factor that controls COX1 and COB expression. We isolated MRX9 in a screen for mitochondrial factors that cause poor accumulation of newly synthesized Cox1p and compromised transition to the respiratory metabolism. Northern analyses indicated lower levels of COX1 and COB mature mRNAs accompanied by an accumulation of unprocessed transcripts in the presence of excess Mrx9p. In a strain devoid of mitochondrial introns, MRX9 overexpression did not affect COX1 and COB translation or respiratory adaptation, implying Mrx9p regulates processing of COX1 and COB RNAs. In addition, we found Mrx9p was localized in the mitochondrial inner membrane, facing the matrix, as a portion of it cosedimented with mitoribosome subunits and its removal or overexpression altered Mss51p sedimentation. Finally, we showed accumulation of newly synthesized Cox1p in the absence of Mrx9p was diminished in cox14 null mutants. Taken together, these data indicate a regulatory role of Mrx9p in COX1 RNA processing.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.
Assuntos
Mitofagia , NADP Trans-Hidrogenases , Trifosfato de Adenosina , Adulto , Animais , Apolipoproteínas/metabolismo , Apolipoproteínas B/metabolismo , Autofagia/genética , Dióxido de Carbono/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona , Citocromos b/metabolismo , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Ferro/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais , NAD/metabolismo , NADP Trans-Hidrogenases/metabolismo , PPAR alfa/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Sequestossoma-1/metabolismo , Succinato Desidrogenase/metabolismo , Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Ubiquinona , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismoRESUMO
KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Flavoproteínas Transferidoras de Elétrons , Aminoácidos de Cadeia Ramificada/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Técnicas de Cultura de Células , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas , Isovaleril-CoA Desidrogenase/genética , Isovaleril-CoA Desidrogenase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , MutaçãoRESUMO
More than 100 years since the first description of Chagas Disease and with over 29,000 new cases annually due to vector transmission (in 2010), American Trypanosomiasis remains a Neglected Tropical Disease (NTD). This study presents the most comprehensive Trypanosoma cruzi sampling in terms of geographic locations and triatomine species analyzed to date and includes both nuclear and mitochondrial genomes. This addresses the gap of information from North and Central America. We incorporate new and previously published DNA sequence data from two mitochondrial genes, Cytochrome oxidase II (COII) and NADH dehydrogenase subunit 1 (ND1). These T. cruzi samples were collected over a broad geographic range including 111 parasite DNA samples extracted from triatomines newly collected across North and Central America, all of which were infected with T. cruzi in their natural environment. In addition, we present parasite reduced representation (Restriction site Associated DNA markers, RAD-tag) genomic nuclear data combined with the mitochondrial gene sequences for a subset of the triatomines (27 specimens) collected from Guatemala and El Salvador. Our mitochondrial phylogenetic reconstruction revealed two of the major mitochondrial lineages circulating across North and Central America, as well as the first ever mitochondrial data for TcBat from a triatomine collected in Central America. Our data also show that within mtTcIII, North and Central America represent an independent, distinct clade from South America, named here as mtTcIIINA-CA, geographically restricted to North and Central America. Lastly, the most frequent lineage detected across North and Central America, mtTcI, was also an independent, distinct clade from South America, noted as mtTcINA-CA. Furthermore, nuclear genome data based on Single Nucleotide Polymorphism (SNP) showed genetic structure of lineage TcI from specimens collected in Guatemala and El Salvador supporting the hypothesis that genetic diversity at a local scale has a geographical component. Our multiscale analysis contributes to the understanding of the independent and distinct evolution of T. cruzi lineages in North and Central America regions.
Assuntos
Doença de Chagas/parasitologia , Mitocôndrias/genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/isolamento & purificação , América Central , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/metabolismo , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , América do Sul , Trypanosoma cruzi/genéticaRESUMO
The species of the genus Potamolithus inhabiting the southwestern basin of the Andes are difficult to distinguish due to small size and similar shell morphology. Only Potamolithus australis and Potamolithus santiagensis have been traditionally recognized in this region, but the occurrence of several morphologically similar undescribed populations could increase the regional richness. Here we delimit described and potentially undescribed cryptic species of the genus using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis and diversity indices inferred six highly differentiated haplogroups, many of them sympatric and widespread in the study area. Phylogeographic analyses suggest a scenario of recent diversification and the occurrence of multiple refuges during the successive Pleistocene glaciations. Phylogenetic analysis also recovered six major clades that showed no relationship with physiography. Species delimitation analyses consistently recognized three or four candidate species apart from P. australis and P. santiagensis. Divergence times indicate that speciation of Chilean Potamolithus began at the end of the Pliocene, probably driven by climatic rather than geographic events. Considering the high inter- and intra-basin genetic diversity, conservation efforts should be focused on protecting sympatric taxa in the basins with the highest species richness.
Assuntos
Biodiversidade , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Gastrópodes/fisiologia , Especiação Genética , Variação Genética , Filogeografia , Animais , Teorema de Bayes , Chile , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Gastrópodes/classificação , Mitocôndrias/enzimologia , Mitocôndrias/genéticaRESUMO
We investigated the effects of acute and chronic exercise, prescribed in different intensity zones, but with total load-matched on mitochondrial markers (cytochrome C oxidase subunit IV (COX-IV), mitochondrial transcription factor A (Tfam), and citrate synthase (CS) activity in skeletal muscles, heart, and liver), glycogen stores, aerobic capacity, and anaerobic index in swimming rats. For this, 2 experimental designs were performed (acute and chronic efforts). Load-matched exercises were prescribed below, above, and on the anaerobic threshold (AnT), determined by the lactate minimum test. In chronic programs, 2 training prescription strategies were assessed (monotonous and linear periodized model). Results show changes in glycogen stores but no modification in the COX-IV and Tfam contents after acute exercises. In the chronic protocols, COX-IV and Tfam proteins and CS adaptations were intensity- and tissue-dependent. Monotonous training promoted better adaptations than the periodized model. Training at 80% of the AnT improved both performance variables, emphasizing the anaerobic index, concomitant to CS and COX-IV improvement (soleus muscle). The aerobic capacity and CS activity (gastrocnemius) were increased after 120% AnT training. In conclusion, acute exercise protocol did not promote responses in mitochondrial target proteins. An intensity and tissue dependence were reported in the chronic protocols, highlighting training at 80 and 120% of the AnT. Novelty: Load-matched acute exercise did not enhance COX-IV and Tfam contents in skeletal muscles, heart, and liver. In chronic exercise, COX-IV, Tfam, and CS activity adaptations were intensity- and tissue-dependent. Monotonous training was more efficient than the periodized linear model in adaptations of target proteins and enzymatic activity.
Assuntos
Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Biogênese de Organelas , Condicionamento Físico Animal , Adaptação Fisiológica , Limiar Anaeróbio , Animais , Citrato (si)-Sintase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glicogênio/metabolismo , Ácido Láctico/sangue , Masculino , Proteínas Mitocondriais/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição/metabolismoRESUMO
AIMS: Homocysteine has been linked to neurodegeneration and motor function impairments. In the present study, we evaluate the effect of chronic mild hyperhomocysteinemia on the motor behavior (motor coordination, functional performance, and muscular force) and biochemical parameters (oxidative stress, energy metabolism, gene expression and/or protein abundance of cytokine related to the inflammatory pathways and acetylcholinesterase) in the striatum and cerebellum of Wistar male rats. MAIN METHODS: Rodents were submitted to one injection of homocysteine (0.03 µmol Hcy/g of body weight) between 30th and 60th postnatal days twice a day. After hyperhomocysteinemia induction, rats were submitted to horizontal ladder walking, beam balance, suspension, and vertical pole tests and/or euthanized to brain dissection for biochemical and molecular assays. KEY FINDINGS: Chronic mild hyperhomocysteinemia did not alter motor function, but induced oxidative stress and impaired mitochondrial complex IV activity in both structures. In the striatum, hyperhomocysteinemia decreased TNF-α gene expression and increased IL-1ß gene expression and acetylcholinesterase activity. In the cerebellum, hyperhomocysteinemia increased gene expression of TNF-α, IL-1ß, IL-10, and TGF-ß, while the acetylcholinesterase activity was decreased. In both structures, hyperhomocysteinemia decreased acetylcholinesterase protein abundance without altering total p-NF-κB, NF-κB, Nrf-2, and cleaved caspase-3. SIGNIFICANCE: Chronic mild hyperhomocysteinemia compromises several biochemical/molecular parameters, signaling pathways, oxidative stress, and chronic inflammation in the striatum and cerebellum of rats without impairing motor function. These alterations may be related to the mechanisms in which hyperhomocysteinemia has been linked to movement disorders later in life and neurodegeneration.