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Subunit c of mammalian F1Fo ATP synthase - from molecular mechanisms of assembly to potential therapies
Marković, Aleksandra ; Mráček, Tomáš (advisor) ; Trnka, Jan (referee) ; Rohlena, Jakub (referee)
Mammalian F1Fo ATP synthase plays a crucial role in ATP production through the process of oxidative phosphorylation. Assembly of this multisubunit protein complex requires specific assembly factors. Notably, the assembling of subunit c in mammals requires several factors, yet despite decades of research, the import of subunit c, its incorporation into the inner mitochondrial membrane, and the assembly of monomers towards octameric c-ring remain enigmatic. To shed new light on this process, we first screened for interacting partners of subunit c using a mass spectrometry-based approach. Our screen identified three proteins as the most prominent interactors of subunit c- TMEM70, TMEM242, and c15orf61, which we subsequently characterized. For the initial characterization of the TMEM242 function, we generated both knockdown and knockout HEK293 models. TMEM242 knockdown led to impaired biogenesis and decreased levels of assembled ATP synthase without affecting the content of other OXPHOS complexes. On the contrary, complete deficiency of TMEM242 lead also to the downregulation of complexes I and IV, which indicates that the primary target of TMEM242 is ATP synthase. While other studies suggested that both TMEM242 and TMEM70 interact with mitochondria complex I intermediate assembly (MCIA), our...
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Study of mitochondrial morphology in pancreatic β-cells depending on the presence of different types of secretagogues
Lorenc, David ; Dlasková, Andrea (advisor) ; Mráček, Tomáš (referee)
Glucose homeostasis is crucial for the proper functioning of the organism. The pancreatic β-cells, which serve as a sensor of changes in blood glucose concentration and are responsible for the adequate release of the hormone insulin, play a crucial role in its maintenance. Increased glucose concentration activates oxidative phosphorylation and subsequently increases the concentration of cellular ATP, which then indirectly stimulates insulin secretion. The process of oxidative phosphorylation is localized in the inner mitochondrial membrane, where the final stage of processing of substrate energy into ATP occurs. To make the oxidative phosphorylation process as efficient as possible, the mitochondrial network undergoes a series of morphological changes. In this work, we aimed to elucidate the effect of changes in nutrient concentration on mitochondrial morphology in a pancreatic β-cell model, the INS1E tissue line. We used as experimental conditions: 1) a high glucose concentration at which insulin secretion is maximal, 2) a low glucose concentration at which insulin secretion does not occur, and 3) the addition of α-ketoisocaproate, a leucine metabolite that amplifies insulin secretion. We first characterized the bioenergetic parameters that influence mitochondrial morphology. A decrease in glucose...
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Molecular basis of deficit of F1Fo-ATP synthase and its impact on energy metabolism of a cell
Štufková, Hana ; Tesařová, Markéta (advisor) ; Kuncová, Jitka (referee) ; Janovská, Petra (referee)
Mitochondria's primary function is to produce energy through the process of oxidative phosphorylation. ATP synthase is a macromolecular rotary machine located in the inner mitochondrial membrane that catalyzes the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). The mitochondrial disorders due to ATP synthase deficiency represent a heterogeneous group of diseases characterized by variable severity of the phenotype with onset at birth or later in life till adulthood. Mutations in both, mitochondrial or nucelar DNA encoded genes, may result in ATP synthase impairment, either isolated or combined with deficits of other complexes of oxidative phosphorylation. The aims of the thesis were to characterize TMEM70 protein, an ATP synthase assembly factor, and to analyze the impact of novel disease variants leading to ATP synthase deficiency in patients' derived samples. TMEM70 is a 21 kDa hairpin structure protein localized in the inner mitochondrial membrane, with both termini oriented into the matrix, which forms higher oligomer structures. Our results demonstrated that the absence of TMEM70 protein leads to an isolated deficiency of complex V followed in some stage by adaptive/compensatory effect of respiratory chain complexes. Different severities...
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The Mitochondrial Contact Site and Cristae Organization System and F1FO-ATP Synthase Crosstalk is a Fundamental Property of Mitochondrial Cristae
CADENA, Lawrence Rudy
The acquisition of mitochondria from an endosymbiont closely related to extant alphaproteobacteria occurred prior to the divergence of modern eukaryotes. Since then, diverse eukaryotes have not only developed a number of different mechanisms to adapt to their environment regarding growth and proliferation, but perpetuated certain traits that have persisted for eons. This thesis postulates an ancestral mechanism for cristae development in mitochondria involving interplay between two cristae shaping protein complexes, the Mitochondrial Contact Site and Cristae Organization System and F1FO-ATP Synthase, that has remained conserved throughout eukaryotic diversification for over 2 billion years.
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Post-transcriptional regulation of TbIF1 in life cycle of Trypanosoma brucei
GRATZL, Sascha
TbIF1, a protein Inhibitor of F1-ATPase in Trypanosoma brucei, is expressed exclusively in the insect stage of the parasite. In the bloodstream form, TbIF1 is switched off, because its activity interferes with the essential role of the ATP synthase in the maintenance of the mitochondrial membrane potential. Here, we employ a series of reporter genes to study the impact of 3'UTR of TbIF1 on mRNA stability and translatability to get insight into the tight post-transcriptional control of TbIF1. We provide evidence that developmentally regulated RNA binding protein Rbp10 is critical for downregulation of TbIF1 on translation level in bloodstream-form trypanosomes.
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Biogenesis, structure and physiological functions of mitochondrial ATP synthase
Eliáš, Jan ; Mráček, Tomáš (advisor) ; Doležal, Pavel (referee)
Mammalian mitochondrial ATP synthase is an enzyme composed of 18 protein subunits, which is localised in the inner mitochondrial membrane. Its main function is to utilise proton gradient, produced by respiratory chain complexes (RCC), for the synthesis of ATP. Aside from the creation of ATP it is known that its dimers contribute to the correct mitochondrial morphology through the formation of cristae apexes. Furthermore, ATP synthase was proposed to have a role in the mitochondrial permeability transition phenomenon, which is important for regulation of programmed cell death. Over the recent years, our understanding of mammalian ATP synthase biogenesis has been tremendously improved. Its assembly process is now clarified, however the knowledge about assembly intermediates of its peripheral stalk and of subunit c are still not sufficient. We focused precisely on those unsolved questions in the fields of ATP synthase biogenesis and its secondary functions, by the production of a KO model of catalytic β subunit of mammalian ATP synthase F1 domain (βKO). This model was successfully prepared on the background of HEK293 cell line. Its characterisation revealed that disruption of the F1 structure resulted in the inability to assemble functional monomer and resulted in a decay of individual subunits. The only...
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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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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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