Hana Nůsková
Academy of Sciences of the Czech Republic
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Hana Nůsková.
Human Molecular Genetics | 2010
Johannes A. Mayr; Vendula Havlíčková; Franz A. Zimmermann; Iris Magler; Vilma Kaplanová; Pavel Ješina; Alena Pecinová; Hana Nůsková; Johannes Koch; Wolfgang Sperl; Josef Houštěk
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.
Mitochondrion | 2011
Alena Pecinová; Zdeněk Drahota; Hana Nůsková; Petr Pecina; Josef Houštěk
The primary attempt in diagnostic and experimental studies of numerous pathological states associated with mitochondrial dysfunction is a precise evaluation of changes in function, content and structure of mitochondrial OXPHOS system. The analysis of rat heart, liver, brain and kidney by oxygraphy, enzyme activities, membrane potential, and BN/SDS-PAGE western blotting demonstrated that tissue homogenates can substitute for isolated mitochondria, providing comparable qualitative mitochondrial parameters. The use of homogenate avoids the loss of the majority of mitochondria during their isolation. Only 50-100mg of the tissue is required for the complex OXPHOS analysis, i.e. five times less as compared with isolated mitochondria.
Biochimica et Biophysica Acta | 2010
Vendula Havlíčková; Vilma Kaplanová; Hana Nůsková; Zdeněk Drahota; Josef Houštěk
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.
Biochimica et Biophysica Acta | 2012
Vendula Karbanová; Alena Čížková Vrbacká; Kateřina Hejzlarová; Hana Nůsková; Viktor Stránecký; Andrea Potocká; Stanislav Kmoch; Josef Houštěk
Early onset mitochondrial encephalo-cardiomyopathy due to isolated deficiency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding enzyme-specific ancillary factor. Diminished ATP synthase results in low ATP production, elevated mitochondrial membrane potential and increased ROS production. To test whether the patient cells may react to metabolic disbalance by changes in oxidative phosphorylation system, we performed a quantitative analysis of respiratory chain complexes and intramitochondrial proteases involved in their turnover. SDS- and BN-PAGE Western blot analysis of fibroblasts from 10 patients with TMEM70 317-2A>G homozygous mutation showed a significant 82-89% decrease of ATP synthase and 50-162% increase of respiratory chain complex IV and 22-53% increase of complex III. The content of Lon protease, paraplegin and prohibitins 1 and 2 was not significantly changed. Whole genome expression profiling revealed a generalized upregulation of transcriptional activity, but did not show any consistent changes in mRNA levels of structural subunits, specific assembly factors of respiratory chain complexes, or in regulatory genes of mitochondrial biogenesis which would parallel the protein data. The mtDNA content in patient cells was also not changed. The results indicate involvement of posttranscriptional events in the adaptive regulation of mitochondrial biogenesis that allows for the compensatory increase of respiratory chain complexes III and IV in response to deficiency of ATP synthase.
BBA clinical | 2014
Petr Pecina; Hana Houšťková; Tomáš Mráček; Alena Pecinová; Hana Nůsková; Markéta Tesařová; Hana Hansikova; Jan Janota; Jiří Zeman; Josef Houštěk
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 p50 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.
International Journal of Developmental Neuroscience | 2013
Jaroslava Folbergrová; Pavel Ješina; Hana Nůsková; Josef Houštěk
We have recently demonstrated the evidence of oxidative stress in brain of immature rats during seizures induced by dl‐homocysteic acid (dl‐HCA). The aim of the present study was to investigate the antioxidant defense mechanisms under these conditions. Seizures were induced in immature 12‐day‐old rats by bilateral icv infusion of dl‐HCA (600 nmol/side), and the activities of the main antioxidant enzymes were examined in supernatants of the cerebral cortex during the acute phase of seizures and at several periods of survival, up to 5 weeks, following these seizures. In control animals individual antioxidant enzymes revealed different changes during the studied postnatal period (PD 12 till PD 47). Total superoxide dismutase (SOD), CuZn SOD (SOD1), Mn SOD (SOD2) and glutathione peroxidase (GPX) activities were increasing while, catalase activity decreased and the activity of glutathione reductase (GR) remained unchanged. In HCA‐treated animals, the activity of total SOD, SOD1 and particularly SOD2 significantly increased at 20 h and 6 days of survival. Importantly, upregulation of SOD2 was also confirmed in mitochondria at the protein level by immunoblotting. The activities of other antioxidant enzymes including catalase and GPX did not significantly differ upon HCA treatment from the appropriate controls at any of the studied time intervals. The pronounced and selective upregulation of SOD2 points to enhanced ROS levels in the mitochondrial matrix. This may be associated with inhibition of respiratory chain complex I that we have demonstrated in our previous studies. The present findings suggest that oxidative stress occurring in the brain of immature rats during and following the seizures induced by dl‐HCA is apparently due to both the increased free radical production and the limited antioxidant defense.
Biochimica et Biophysica Acta | 2016
Nikola Kovářová; Petr Pecina; Hana Nůsková; Marek Vrbacký; Massimo Zeviani; Tomáš Mráček; Carlo Viscomi; Josef Houštěk
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.
PLOS ONE | 2013
Nikola Kovářová; Tomáš Mráček; Hana Nůsková; Eliška Holzerová; Marek Vrbacký; Petr Pecina; Kateřina Hejzlarová; Katarína Kľučková; Jakub Rohlena; Jiri Neuzil; Josef Houštěk
Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.
Biochemical and Biophysical Research Communications | 2010
Zdeněk Drahota; Marek Vrbacký; Hana Nůsková; Ludmila Kazdova; Vaclav Zidek; Vladimír Landa; Michal Pravenec; Josef Houštěk
The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC(50) 4microM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.
Human Molecular Genetics | 2016
Marek Vrbacký; Jana Kovalčíková; Kallayanee Chawengsaksophak; Inken M. Beck; Tomáš Mráček; Hana Nůsková; David Sedmera; František Papoušek; František Kolář; Margarita Sobol; Pavel Hozák; Radislav Sedlacek; Josef Houštěk
TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.