Q59555514
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Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms of metabolic disturbances. Recent linkage analysis in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), identified two closely linked quantitative trait loci (QTL) associated with glucose oxidation and glucose incorporation into BAT lipids in the vicinity of Wars2 (tryptophanyl tRNA synthetase 2 (mitochondrial)) gene on chromosome 2. The SHR harbors L53F WARS2 protein variant that was associated with reduced angiogenesis and Wars2 thus represents a prominent positional candidate gene. In the current study, we validated this candidate as a quantitative trait gene (QTG) using transgenic rescue experiment. SHR-Wars2 transgenic rats with wild type Wars2 gene when compared to SHR, showed more efficient mitochondrial proteosynthesis and increased mitochondrial respiration, which was associated with increased glucose oxidation and incorporation into BAT lipids, and with reduced weight of visceral fat. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance in muscle and white adipose tissue. In summary, these results demonstrate important role of Wars2 gene in regulating BAT function and consequently lipid and glucose metabolism.
- MeSH
- energetický metabolismus * genetika MeSH
- fenotyp MeSH
- genetická predispozice k nemoci MeSH
- genetické asociační studie MeSH
- glukosa metabolismus MeSH
- hnědá tuková tkáň metabolismus patologie MeSH
- kultivované buňky MeSH
- lokus kvantitativního znaku MeSH
- metabolismus lipidů MeSH
- mitochondrie metabolismus MeSH
- mutace * MeSH
- nitrobřišní tuk metabolismus patofyziologie MeSH
- obezita genetika metabolismus patofyziologie MeSH
- potkani inbrední SHR MeSH
- tryptofan-tRNA-ligasa genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
l-asparaginase (ASNase), a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), hydrolyzes plasma asparagine and glutamine and thereby disturbs metabolic homeostasis of leukemic cells. The efficacy of such therapeutic strategy will depend on the capacity of cancer cells to adapt to the metabolic challenge, which could relate to the activation of compensatory metabolic routes. Therefore, we studied the impact of ASNase on the main metabolic pathways in leukemic cells. Treating leukemic cells with ASNase increased fatty-acid oxidation (FAO) and cell respiration and inhibited glycolysis. FAO, together with the decrease in protein translation and pyrimidine synthesis, was positively regulated through inhibition of the RagB-mTORC1 pathway, whereas the effect on glycolysis was RagB-mTORC1 independent. As FAO has been suggested to have a pro-survival function in leukemic cells, we tested its contribution to cell survival following ASNase treatment. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, constitutive activation of the mammalian target of rapamycin pathway increased apoptosis in leukemic cells treated with ASNase, but did not increase FAO. Our study uncovers a novel therapeutic option based on the combination of ASNase and FAO inhibitors.
- MeSH
- akutní lymfatická leukemie farmakoterapie metabolismus patologie MeSH
- asparaginasa terapeutické užití MeSH
- autofagie účinky léků MeSH
- lidé MeSH
- mastné kyseliny metabolismus MeSH
- monomerní proteiny vázající GTP fyziologie MeSH
- multiproteinové komplexy fyziologie MeSH
- nádorové buněčné linie MeSH
- oxidace-redukce MeSH
- pyrimidiny biosyntéza MeSH
- TOR serin-threoninkinasy fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.
- MeSH
- druhová specificita MeSH
- fibroblasty metabolismus patologie MeSH
- Leighova nemoc genetika metabolismus patologie MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- orgánová specificita MeSH
- respirační komplex IV genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. METHODS: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses. RESULTS: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p 50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations. CONCLUSION: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes. GENERAL SIGNIFICANCE: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.
- Publikační typ
- časopisecké články MeSH
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis.
- MeSH
- genetická predispozice k nemoci genetika MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé MeSH
- mitochondriální nemoci enzymologie genetika MeSH
- mitochondriální protonové ATPasy genetika MeSH
- mitochondrie enzymologie genetika patologie MeSH
- modely genetické MeSH
- mutace genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
F1Fo-ATP synthase is a key enzyme of mitochondrial energy provision producing most of cellular ATP. So far, mitochondrial diseases caused by isolated disorders of the ATP synthase have been shown to result from mutations in mtDNA genes for the subunits ATP6 and ATP8 or in nuclear genes encoding the biogenesis factors TMEM70 and ATPAF2. Here, we describe a patient with a homozygous p.Tyr12Cys mutation in the epsilon subunit encoded by the nuclear gene ATP5E. The 22-year-old woman presented with neonatal onset, lactic acidosis, 3-methylglutaconic aciduria, mild mental retardation and developed peripheral neuropathy. Patient fibroblasts showed 60-70% decrease in both oligomycin-sensitive ATPase activity and mitochondrial ATP synthesis. The mitochondrial content of the ATP synthase complex was equally reduced, but its size was normal and it contained the mutated epsilon subunit. A similar reduction was found in all investigated F1 and Fo subunits with the exception of Fo subunit c, which was found to accumulate in a detergent-insoluble form. This is the first case of a mitochondrial disease due to a mutation in a nuclear encoded structural subunit of the ATP synthase. Our results indicate an essential role of the epsilon subunit in the biosynthesis and assembly of the F1 part of the ATP synthase. Furthermore, the epsilon subunit seems to be involved in the incorporation of subunit c to the rotor structure of the mammalian enzyme.
- MeSH
- fibroblasty enzymologie chemie MeSH
- kultivované buňky MeSH
- lidé MeSH
- missense mutace * MeSH
- mitochondriální nemoci * enzymologie genetika MeSH
- mitochondriální protonové ATPasy * genetika nedostatek MeSH
- mladý dospělý MeSH
- molekulární sekvence - údaje MeSH
- proteiny genetika chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- Check Tag
- lidé MeSH
- mladý dospělý MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- práce podpořená grantem MeSH
The subunit epsilon of mitochondrial ATP synthase is the only F1 subunit without a homolog in bacteria and chloroplasts and represents the least characterized F1 subunit of the mammalian enzyme. Silencing of the ATP5E gene in HEK293 cells resulted in downregulation of the activity and content of the mitochondrial ATP synthase complex and of ADP-stimulated respiration to approximately 40% of the control. The decreased content of the epsilon subunit was paralleled by a decrease in the F1 subunits alpha and beta and in the Fo subunits a and d while the content of the subunit c was not affected. The subunit c was present in the full-size ATP synthase complex and in subcomplexes of 200-400 kDa that neither contained the F1 subunits, nor the Fo subunits. The results indicate that the epsilon subunit is essential for the assembly of F1 and plays an important role in the incorporation of the hydrophobic subunit c into the F1-c oligomer rotor of the mitochondrial ATP synthase complex. Copyright 2009 Elsevier B.V. All rights reserved.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- genový knockdown MeSH
- HEK293 buňky MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- mitochondriální protonové ATPasy * antagonisté a inhibitory genetika chemie metabolismus MeSH
- mitochondrie * enzymologie MeSH
- oxidativní fosforylace MeSH
- podjednotky proteinů MeSH
- proteiny * antagonisté a inhibitory genetika chemie metabolismus MeSH
- RNA interference MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
