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Analysis of mitochondrial energetic metabolism in early stages of Huntington's disease
Ďurčeková, Katarína ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. The average age of onset of HD is 40 years and affected individuals have an unfavourable prognosis. Since there is still no curative treatment available, the current treatment of HD focuses only on symptom suppression. The clinical manifestation of HD is preceded by an asymptomatic period in which continuous biochemical, structural and functional changes at the cellular level are already occurring. Our previous studies have demonstrated impaired mitochondrial parameters in lymphocytes and cultured fibroblasts of patients with advanced HD. The aim of this study was to characterize the essential components of mitochondrial energy metabolism by functional methods and to find suitable mitochondrial biomarkers to monitor progression of HD. The analysed set consisted of 12 cell lines of cultured skin fibroblasts from patients with genetically proven HD, four patients in asymptomatic, four in early and four in symptomatic stages of HD. Four lines from healthy individuals served as controls. Mitochondrial respiration and glycolytic activity were analysed by real-time monitoring of oxygen consumption and pH changes, ATP production by luminescence, and metabolic turnover...
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Mitochondrial cytochrome c oxidase: cyanide inhibition and role of assembly factor Surf1 defect
Nůsková, Hana ; Drahota, Zdeněk (advisor) ; Kalous, Martin (referee)
The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....
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The effect of endothelial cell proliferation on susceptibility to mitochondrially controlled apoptosis and oxidative stress.
Blecha, Jan ; Novák, Petr (advisor) ; Plecitá, Lydie (referee)
Mitochondria are multifunctional organelles playing a key role in energy metabolism and cell death induction. Mitochondria, and specifically their respiratory chain, are also the main producers of reactive oxygen species (ROS) in cells. Metabolism can be affected by the state of cellular proliferation and certain ROS-inducing agents have an antiangiogenic effect based on the preferential elimination of proliferating endothelial cells (EC). Therefore, in this work we investigated, whether mitochondria could be responsible for different sensitivity of proliferation and confluent EC to cell death. We mainly focused on systems that regulate ROS level and apoptosis: respiratory chain (ROS production), antioxidant defense (ROS detoxification) and Bcl-2 family of proteins (apoptosis regulation). First, we treated EC with functional and nonfunctional respiratory chain with various oxidative stress- and apoptosis-inducing agents and determined ROS production and susceptibility to apoptosis in proliferating and confluent cells. Our results show that functional respiratory chain greatly increases the susceptibility of proliferating cells to ROS induction and apoptosis, whereas in qiescent cells it protects against cell death. Given these findings, we assessed the activity of respiratory chain in proliferating...
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Complex I of mitochondrial respiratory chain a its disorders.
Rodinová, Marie ; Hansíková, Hana (advisor) ; Kalous, Martin (referee)
NADH: ubiquinone oxidoreductase (Complex I) is a multisubunit protein complex of inner mitochondrial membrane. Complex I is the biggest and most complicated part of oxidative phosphorylation system, which is responsible for the cell ATP production. It consists of 45 subunits. 7 subunits are mitochondrial encoded, remainder 38 are nuclear encoded. NADH: ubiquinone oxidoreductase has L-shaped structure, which is built of two arms: membrane arm and matrix located peripheral arm. Complex I oxidize the NADH molecule. The electron transport is coupled with proton pumping across the inner mitochondrial membrane to intermembrane space, where proton gradient developed and which is used by ATP synthase to ATP synthesis. Deficiencies of NADH: ubiquinone oxidoreductase represent extensive, clinically and genetic heterogeneous group of mitochondrial diseases. Decrease of activity and amount of complex I, decrease of ATP production, changes of membrane potential, mitochondrial morphology and mitochondrial network and increasing of production of reactive oxygen species are found in cells with defects of NADH: ubiquinone oxidoreductase. Combination of this features lead to serious illnesses, which are almost fatal and we still haven't any useful therapy. Aim of this study is to summarize present knowledge about...
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Functional characterization of LACE1 APTase and mitochondrial AAA proteases YME1L and AFG3L2 in mitochondrial protein homeostasis.
Tesařová, Jana ; Stibůrek, Lukáš (advisor) ; Kalous, Martin (referee) ; Pecina, Petr (referee)
Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...
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The effect of endothelial cell proliferation on susceptibility to mitochondrially controlled apoptosis and oxidative stress.
Blecha, Jan ; Novák, Petr (advisor) ; Plecitá, Lydie (referee)
Mitochondria are multifunctional organelles playing a key role in energy metabolism and cell death induction. Mitochondria, and specifically their respiratory chain, are also the main producers of reactive oxygen species (ROS) in cells. Metabolism can be affected by the state of cellular proliferation and certain ROS-inducing agents have an antiangiogenic effect based on the preferential elimination of proliferating endothelial cells (EC). Therefore, in this work we investigated, whether mitochondria could be responsible for different sensitivity of proliferation and confluent EC to cell death. We mainly focused on systems that regulate ROS level and apoptosis: respiratory chain (ROS production), antioxidant defense (ROS detoxification) and Bcl-2 family of proteins (apoptosis regulation). First, we treated EC with functional and nonfunctional respiratory chain with various oxidative stress- and apoptosis-inducing agents and determined ROS production and susceptibility to apoptosis in proliferating and confluent cells. Our results show that functional respiratory chain greatly increases the susceptibility of proliferating cells to ROS induction and apoptosis, whereas in qiescent cells it protects against cell death. Given these findings, we assessed the activity of respiratory chain in proliferating...
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Impact of isolate deficiency of F1FO-ATP syntthase on other complexes of oxidative phosphorylation in skin fibroblasts depending on cullture conditions
Kedrová, Kateřina ; Stiborová, Marie (advisor) ; Poljaková, Jitka (referee)
Isolated deficiency of F1FO-ATPsynthase is a soubgroup of mitochondrial diseases caused by mutations in nuclear and mitochondrial-encoded structural subunits, or nuclear-encoded assembly factors of F1FO-ATPsynthase. The most often mutations are found in a MTATP6 gene localized in the mitochondrial DNA and a TMEM70 gene, localized in the nuclear DNA. A MTATP6 gene encodes subunit a of F1FO-ATPsynthase and its mutation usually leads to reduced phosphorylation activity of F1FO-ATPsynthase. A TMEM70 gene encodes a 21 kDa mitochondrial protein of the inner mitochondrial membrane of not completely explained function and its mutation results in the decrease in a content of fully assembled F1FO- ATPsynthase. The aim of this thesis was to investigate the impact of isolated F1FO- ATPsynthase deficiency on the oxidative phosphorylation system (complex I-IV), other selected mitochondrial proteins, and mitochondrial network in two cell lines of primary human skin fibroblasts with an isolated deficiency of F1FO-ATPsynthase (mutation m.8851T>C in MTATP6 and mutation c.317-2A>G in TMEM70) during the first days of their cultivation in media containing galactose or glucose as a carbohydrate source with a presence or absence of L-glutamine. The control cell line was found to have higher amounts of respiratory chain...
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Mitochondrial cytochrome c oxidase: cyanide inhibition and role of assembly factor Surf1 defect
Nůsková, Hana ; Kalous, Martin (referee) ; Drahota, Zdeněk (advisor)
The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....
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Complex I of mitochondrial respiratory chain a its disorders.
Rodinová, Marie ; Kalous, Martin (referee) ; Hansíková, Hana (advisor)
NADH: ubiquinone oxidoreductase (Complex I) is a multisubunit protein complex of inner mitochondrial membrane. Complex I is the biggest and most complicated part of oxidative phosphorylation system, which is responsible for the cell ATP production. It consists of 45 subunits. 7 subunits are mitochondrial encoded, remainder 38 are nuclear encoded. NADH: ubiquinone oxidoreductase has L-shaped structure, which is built of two arms: membrane arm and matrix located peripheral arm. Complex I oxidize the NADH molecule. The electron transport is coupled with proton pumping across the inner mitochondrial membrane to intermembrane space, where proton gradient developed and which is used by ATP synthase to ATP synthesis. Deficiencies of NADH: ubiquinone oxidoreductase represent extensive, clinically and genetic heterogeneous group of mitochondrial diseases. Decrease of activity and amount of complex I, decrease of ATP production, changes of membrane potential, mitochondrial morphology and mitochondrial network and increasing of production of reactive oxygen species are found in cells with defects of NADH: ubiquinone oxidoreductase. Combination of this features lead to serious illnesses, which are almost fatal and we still haven't any useful therapy. Aim of this study is to summarize present knowledge about...
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