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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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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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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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Study of expression and maturation of mitochondrial oxidative phosphorylation system during mammal's prenatal period
Mrhálková, Andrea ; Hůlková, Martina (advisor) ; Ješina, Pavel (referee)
Postnatal adaptation of neonate to extrauterine life is among others dependent on maturation of mitochondrial oxidative phosphorylation system (OXPHOS). It depends on effective mitochondrial biogenesis during fetal developement. The inadequate capacity of mitochondrial OXPHOS system plays an important role in the neonatal mortality and morbidity. Therefore the study of mitochondrial biogenesis on molecular and biochemical level is important to improve the care of very premature neonates, especially critically ill premature neonates. This thesis has been worked out in The laboratory for study of mitochondrial disorders (Department of Pediatrics, 1st Faculty of Medicine, Charles University in Prague). The thesis is based on molecular genetic analyses, which are focused on characterisation of ATP synthase gene expression and on changes in mitochondrial DNA content during human and rat fetal development. The results provide the better insight into mitochondrial respectively ATP synthase biogenesis during human and rat fetal development.
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Structural and Functional Interactions of Mitochondrial ADP-Phosphorylating Apparatus
Nůsková, Hana ; Houštěk, Josef (advisor) ; Kolarov, Jordan (referee) ; Kuda, Ondřej (referee) ; Panicucci Zíková, Alena (referee)
The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...
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Mechanisms of regulation of inhibitory factor IF1
Sklenář, Filip ; Dlasková, Andrea (advisor) ; Zelenka, Jaroslav (referee)
Inhibitory factor 1 (IF1) is one of the major regulators of mitochondrial ATP synthase activity, a key enzyme of energy metabolism. Its inhibitory effects are known in conditions such as hypoxia or starvation, but the hypothesis that IF1 inhibits ATP synthase activity even under physiological conditions is still not entirely accepted. Disorders of ATP synthase regulation can be fatal to the cell and have been described, for example, in carcinogenesis and ischemia. It has also been found that silencing of the IF1 gene in pancreatic β-cells increases insulin secretion, and thus, IF1 may be important in the pathogenesis of type 2 diabetes. The goal of this work is to summarize the current knowledge about the IF1 protein and to obtain new results that will help elucidate the mechanism by which this protein regulates mitochondrial ATP synthase. Specifically, this work deals with the ratio of IF1 protein to ATP synthase in pancreatic β-cells, depending on different culture conditions. It further investigates the occurrence of post-translational modifications of the IF1 protein in pancreatic β-cells (INS- 1E model cells), which may play a role in the regulation of IF1 activity. It also deals with the cellular ATP/ADP ratio, which is one of the key factors for insulin secretion by pancreatic β-cells. An...
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Structure and physiological role of the mitochondrial permeability transition pore
Eliáš, Jan ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
Mitochondrial permeability transition pore (mPTP) is Ca2+ dependent channel localised in the inner mitochondrial membrane. One of its defining characteristics is inhibition by nanomolar concentrations of immunosuppressant cyclosporine A (CsA). Together with additional interacting proteins, which regulate its opening, mPTP forms a permeability transition protein complex. Persistent opening of mPTP is accompanied by mitochondrial swelling and a subsequent collapse of organelle, which precedes release of proapoptotic proteins and programmed cell death. Channel forming unit of mPTP remains unknown, despite intense and long-lasting study. Numerous proteins were proposed to play a role of channel forming subunit of mPTP, including complex of ANT and VDAC, ANT alone, PiC or even ATP synthase. Despite the fact, that molecular structure remains elusive, mPTP seems to play a role in a range of pathophysiological processes or diseases associated with them. Among others this includes ischemia/reperfusion injury, neurological and muscle dystrophies, or tumorigenesis. Keywords: mitochondria, mitochondrial permeability transition pore, cyclosporine A, programmed cell death, ATP synthase, oxidative phosphorylation apparatus.
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