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Supercomplexes in the respiratory chain of mitochondria
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Holzerová, Kristýna (referee)
Mitochondria are very important organelles of eukaryotic cell. In mitochondria, there are located many metabolic reactions including oxidative phosphorylation (OXPHOS). In this process, respiratory chain enzyme complexes couple the oxidation of NADH and FADH2 to vectorial proton transport across the inner mitochondrial membrane. ATP synthase then uses the resulting electrochemical potential to synthesize ATP from ADP and phosphate. Recent studies of the OXPHOS demonstrate higher structures of complexes so-called supercomplexes which facilitate substrate channeling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain.
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Sites and Consequences of Mitochondreal ROS Production
Mráček, Tomáš ; Houštěk, Josef (advisor) ; Pelouch, Václav (referee) ; Kolarov, Jordan (referee)
The thesis consists of 8 articles (6 published and 2 submitted), which may be divided into two major lines. The first (articles 1-4) deals with mitochondrial glycerophosphate dehydrogenase. We have shown that this enzyme is capable of massive ROS production and in the presented works we studied in more detail this ROS production as well as some aspects of enzyme biogenesis, so as to better understand its physiological significance. The second line (articles 5-8) represents our studies on the potential involvement of ROS in pathogenesis of mitochondrial diseases. 1. Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide; Drahota Z., Chowdhury SKR., Floryk D., Mráček T., Wilhelm J., Rauchová H., Lenaz G., Houštěk J.; J Bioenerg Biomembr. 2002; 34(2):105-13. Indeed, this was first article to demonstrate ROS production by mammalian mGPDH. We studied oxidation of glycerophosphate by brown adipose tissue mitochondria, which are known to contain high amounts of mGPDH. We found significant GP-dependent AA- stimulated production of H2O2. To further verify our findings, we used three independent methods for H2O2 measurement - fluorescence detection with p-hydroxyphenylacetic acid, antimycine A - insensitive oxygen consumption, and luminometric...
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Mitochondrial ATP synthase deficiencies of a nuclear genetic origin
Karbanová, Vendula ; Houštěk, Josef (advisor) ; Kalous, Martin (referee) ; Rossmeisl, Martin (referee)
ATP synthase represents the key enzyme of cellular energy provision and ATP synthase disorders belong to the most deleterious mitochondrial diseases affecting pediatric population. The aim of this thesis was to identify nuclear genetic defects and describe the pathogenic mechanism of altered biosynthesis of ATP synthase that leads to isolated deficiency of this enzyme manifesting as an early onset mitochondrial encephalo-cardiomyopathy. Studies in the group of 25 patients enabled identification of two new disease-causing nuclear genes responsible for ATP synthase deficiency. The first affected gene was TMEM70 that encodes an unknown mitochondrial protein. This protein was identified as a novel assembly factor of ATP synthase, first one specific for higher eukaryotes. TMEM70 protein of 21 kDa is located in mitochondrial inner membrane and it is absent in patient tissues. TMEM70 mutation was found in 23 patients and turned to be the most frequent cause of ATP synthase deficiency. Cell culture studies also revealed that enzyme defect leads to compensatory-adaptive upregulation of respiratory chain complexes III and IV due to posttranscriptional events. The second affected gene was ATP5E that encodes small structural epsilon subunit of ATP synthase. Replacement of conserved Tyr12 with Cys caused...
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The content of components of ATP synthasome in different rat tissues and in patients with defects in ATP synthase
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Kalous, Martin (referee)
The complexes of oxidative phosphorylation (OXPHOS) are situated in the inner mitochondrial membrane in higher structural and functional complexes, so-called supercomplexes, which facilitates substrate channeling. ATP synthase is also able to organize in higher structures. In mammalian mitochondria, ATP synthase is usually present in a dimeric form. There is evidence of its trimerization and even tetramerization. Furthermore, it seems that ATP synthase catalyzing the phosphorylation of ADP to ATP, adenine nucleotide translocator (ANT) ensuring the exchange of ADP for newly synthesized ATP across the inner mitochondrial membrane and phosphate carrier (PiC) allowing the import of inorganic phosphate (Pi) into the matrix of mitochondria are assembled in a supercomplex called ATP synthasome. This association among the components of phosphorylative apparatus seems to increase the efficiency of processes leading to the ATP synthesis. First, we studied amounts of the components of phosphorylative apparatus in connection with various ATP synthase contents among mitochondria isolated from nine rat tissues. Mitochondrial proteins were separated by denaturing electrophoresis (SDS-PAGE) and their content was analyzed using specific antibodies. In agreement with our expectations, the highest content of...
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