|
|
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...
|
|
|
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...
|
|
|
Functional characterisation of new components of mitochondrial proteome.
Kovalčíková, Jana ; Vrbacký, Marek (advisor) ; Červinková, Zuzana (referee) ; Ješina, Pavel (referee)
1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...
|
|
|
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...
|
|
|
Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
|
|
|
Functional characterisation of new components of mitochondrial proteome.
Kovalčíková, Jana ; Vrbacký, Marek (advisor) ; Červinková, Zuzana (referee) ; Ješina, Pavel (referee)
1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...
|
|
|
Use of Canistherapy for Stimulation of Persons with Rare Diseases
Prokopová, Zuzana ; Hájková, Vanda (advisor) ; Šumníková, Pavlína (referee)
The aim of this thesis was to assess the influence of canistherapeutic intervention on the level of fine motor skills in patients with rare diseases, namely mitochondrial encephalocardiomyopathy caused by TMEM70 gene mutation and Leigh syndrome. Canistherapeutic intervention in these patients is based on activation of motoric functions during programme reasonably designed according to the needs of specific patients. The assistance of the dog is used to achieve this activation. The dog acts as an important motivational factor and the direct contact with it contributes to development of sensomotoric skills of these patients. The thesis summarizes general information about rare diseases and focuses on one of the subgroups of mitochondrial disorders. It presents knowledge about the Leigh syndrome and mitochondrial encephalocardiomyopathy caused by TMEM70 gene mutation. Subsequently the thesis presents information about canistherapy and fine motor skills. The thesis further contains case reports of patients, data from research units and their evaluation. The aim of the thesis was to find out whether patients with mitochondrial disease show improvement of quality and effectivity of selected grasp forms and thus faster and more precise execution assigned tasks focusing on object manipulations. Partial...
|
|
|
Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
|
|
|
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...
|
|
|
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...
|
guest ::
