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Biogenesis of mitochondria in parasitic protist Trypanosoma brucei
Mach, Jan
In last decade, investigations of mitochondria including their various reduced forms such as hydrogenosomes and mitosomes revealed unexpected diversity of this indispensable organelle. Interestingly, the single mitochondrion of parasitic protist Trypanosoma brucei is able to undergo remarkable functional and structural changes reflecting available carbon sources. Moreover, it was proposed that trypanosomes belong among the most ancient eukaryotes and as such, their mitochondria raised high attention of biologists. To contribute to the knowledge of mitochondrial biogenesis and function, we focused on studies of two key mitochondrial processes, the processing of preproteins that are imported to the mitochondria, and mechanism of pyruvate transport to these organelles. Moreover, we also investigated uptake of iron by T. brucei. This metal is essential for function of numerous proteins, particularly for iron-sulfur proteins in mitochondria. Evolutionary history of trypanosomes and their mitochondrion is a question of debates. According to some reports, mitochondrion of trypanosomes represent an ancient form of this organelle, which is supported by identification of putative "archaic" translocase of the outer mitochondrial membrane (ATOM) and finding of only a single type of translocation pore in...
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Iron-Sulfur cluster assembly in Monocercomonoides exilis
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Balk, Janneke (referee) ; Tsaousis, Anastasios (referee)
In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...
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Iron-sulphur cluster synthesis in anaerobic protists
Zelená, Marie ; Hampl, Vladimír (advisor) ; Mach, Jan (referee)
Iron-sulpur clusters are small ubiquitous cofactors of proteins present in all cells. These proteins participate in many important processes such as gene expression or respiratory chain. In vivo synthesis of iron-sulphur clusters requires complex biosynthetic pathways. In eukaryotic cells these pathways are localized inside mitochondria and plastids and in the cytosol. Cytosolic synthesis depends on a product of the mitochondrial pathway. Mitochondria of anaerobic protists in most cases went through reductive evolution, which is associated with the reduction of mitochondrial iron-sulphur cluster biosynthetic pathway. In many cases, these organisms acquired alternative pathways via lateral gene transfer, which act either as a complement to the mitochondrial pathway, or as its complete functional substitution. Replacement of the mitochondrial pathway might have been a key event for secondary loss of mitochondria. Key words: Iron-sulphur clusters, anaerobic protists, ISC, CIA, SUF, mitochondrion
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Developmental, pathobiochemical and molecular aspects of selected inborn errors of metabolism
Kolářová, Hana ; Honzík, Tomáš (advisor) ; Drahota, Zdeněk (referee) ; Morava Kozicz, Eva (referee)
Inborn errors of metabolism represent a heterogenous group of rare conditions, most having an incidence of less than 1 in 100,000 births. Because of their low prevalence, they are on the margin of attention of general research and even more so of large pharmaceutical companies. Study of rare diseases is the only way to design therapeutic options in order to improve quality of life of affected patients. Present Thesis particularly focuses on disturbances in mitochondrial energy metabolism. The main goals were the characterization of mitochondrial biogenesis within foetal development, as well as in childhood and adulthood. Another aim was to define clinical, biochemical and molecular aspects of mitochondrial optic neuropathies in childhood and adulthood. This work supported the earlier observations that gestational week 22 is the edge of viability, which has to be taken into account in upcoming discussions about guidelines on resuscitation of preterm neonates. Secondly, over last four years, we managed to examine and describe large cohort of patients with optic neuropathies based on a mitochondrial dysfunction. We have managed to characterize the biochemical and molecular-genetic background in more than 200 patients, and both selected cases (LHON/MELAS overlap syndrome) and cohort studies (MELAS,...
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Losses of mitochondria and plastids in the evolution of eukaryotes
Trokšiar, David ; Hampl, Vladimír (advisor) ; Hadariová, Lucia (referee)
- 5 - Abstract: Mitochondria and plastids were acquired by endosymbiotic event, where prokaryotic organism was engulfed by ancestors of extant eukaryotes. There are more known endosymbiotic events in plastid evolution. In primary endosymbiosis cyanobacterium cell was engulfed by heterotrophic eukaryotic organism. In following secondary, tertiary and quaternary endosymbiotic events eukaryotic cell was engulfed by another eukaryote. Mitochondria originated by engulfment of α-proteobacteria. In the evolution of eukaryotes, reduction of mitochondria occurred in many lineages, making living under anaerobic conditions possible. The least reduced form is anaerobic mitochondria, which together with aerobic mitochondria and hydrogen producing mitochondria, possess genome. Hydrogenosomes and the most reduced form mitosomes, does not possess genome. Plastid reductions led to loss of photosynthetic ability. In last years, more examples of organisms that lost entirely their semi- autonomous organelle, are coming. Loss occur at two parasitic representatives of the Alveolata group, and one endobiotic oxymonad. Parasites Cryptosporidium parvum and Hematodinium lost nonphotosyntetic plastid, whereas Monocercomoides lost its mitochondria. Semi-autonomous organelles were dispensable, because all representatives have access to...
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Mitochondrial energy generating capacity in cultured skin fibroblasts
Daňhelovská, Tereza ; Tesařová, Markéta (advisor) ; Ješina, Pavel (referee)
Mitochondrial disorders, with incidence 1:5000 live births children, are one of the most common metabolic diseases. Clinically, it is heterogeneous group of disorders caused by mutations in more than 250 genes. Diagnostic of patients with suspected mitochondrial disorder relies on broad spectrum of biochemical analysis. One of them is a measurement of Mitochondrial Energy Generating Capacity (MEGC). The principle of MEGC analysis is measuring oxidations rate of 14 C - labeled substrates in 10 different incubations. These incubations contain [1-14 C]pyruvate, [U-14 C]malate or [1,4-14 C]succinate, donors and acceptors of Acetyl-CoA and inhibitors of TCA cycle. The results of MEGC analysis provide a variety of information about mitochondrial energy metabolism (MEM) of individual in particular tissue. In diagnostic of patients with suspected mitochondrial disorder is MEGC routinely determined in skeletal muscle. The aim of this study is to optimize MEGC analysis for its use in cultures skin fibroblasts. In sum, MEGC analysis was performed in 23 patients with primary deficiency of oxidative phosphorylation (OXPHOS), in 7 patients with secondary deficiency of OXPHOS and in 15 controls cell lines. The results of MEGC in cultured skin fibroblasts were then compared with results of spectrophotometric...
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Biogenesis of mitochondria in parasitic protist Trypanosoma brucei
Mach, Jan ; Tachezy, Jan (advisor) ; Yurchenko, Vyacheslav (referee) ; Hashimi, Hassan (referee)
In last decade, investigations of mitochondria including their various reduced forms such as hydrogenosomes and mitosomes revealed unexpected diversity of this indispensable organelle. Interestingly, the single mitochondrion of parasitic protist Trypanosoma brucei is able to undergo remarkable functional and structural changes reflecting available carbon sources. Moreover, it was proposed that trypanosomes belong among the most ancient eukaryotes and as such, their mitochondria raised high attention of biologists. To contribute to the knowledge of mitochondrial biogenesis and function, we focused on studies of two key mitochondrial processes, the processing of preproteins that are imported to the mitochondria, and mechanism of pyruvate transport to these organelles. Moreover, we also investigated uptake of iron by T. brucei. This metal is essential for function of numerous proteins, particularly for iron-sulfur proteins in mitochondria. Evolutionary history of trypanosomes and their mitochondrion is a question of debates. According to some reports, mitochondrion of trypanosomes represent an ancient form of this organelle, which is supported by identification of putative "archaic" translocase of the outer mitochondrial membrane (ATOM) and finding of only a single type of translocation pore in...
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Biogenesis of mitochondria in parasitic protist Trypanosoma brucei
Mach, Jan
In last decade, investigations of mitochondria including their various reduced forms such as hydrogenosomes and mitosomes revealed unexpected diversity of this indispensable organelle. Interestingly, the single mitochondrion of parasitic protist Trypanosoma brucei is able to undergo remarkable functional and structural changes reflecting available carbon sources. Moreover, it was proposed that trypanosomes belong among the most ancient eukaryotes and as such, their mitochondria raised high attention of biologists. To contribute to the knowledge of mitochondrial biogenesis and function, we focused on studies of two key mitochondrial processes, the processing of preproteins that are imported to the mitochondria, and mechanism of pyruvate transport to these organelles. Moreover, we also investigated uptake of iron by T. brucei. This metal is essential for function of numerous proteins, particularly for iron-sulfur proteins in mitochondria. Evolutionary history of trypanosomes and their mitochondrion is a question of debates. According to some reports, mitochondrion of trypanosomes represent an ancient form of this organelle, which is supported by identification of putative "archaic" translocase of the outer mitochondrial membrane (ATOM) and finding of only a single type of translocation pore in...
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Impact of the glycine-rich loop on the function of processing peptidases of the mitochondrial type
Kučera, Tomáš ; Janata, Jiří (advisor) ; Bařinka, Cyril (referee) ; Ettrich, Rüdiger (referee)
The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...
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