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Mitochondrial protein import.
Drašnarová, Zuzana ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
Most of mitochondrial proteins are synthesized in the cytoplasm and after that transported to the outer or the inner membrane or to the intermembrane space and the mitochondrial matrix. All mitochondrial proteins cross the outer membrane via the TOM complex. From here different populations of proteins follow distinct transport routes: (i) β-barel proteins are assembled in the outer membrane with the help the SAM complex, (ii) after the passage through the TOM complex the intermembrane space proteins are bound by the MIA pathway, (iii) the mitochondrial carrier proteins of the inner mitochondrial membrane require the activity of the TIM22 complex and finally (iv) the matrix proteins as well as the small sub-population of the inner membrane proteins are transported via the TIM23 complex. Whereas the transport across the outer mitochondrial membrane does not require the additional energy, the transport across the inner membrane depends on ATP and/or the membrane potential. The transported proteins carry targeting sequences which are recognized by the outer membrane receptors. Key words: protein import, mitochondria, translocase, membrane, matrix, intermembrane space
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How Intracellular Pathogens Manipulate Cellular Trafficking.
Petrů, Markéta ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
Many intracellular single-celled organisms belong to medically important human pathogens. The selected parasites are subject of this thesis - Chlamydia spp., Legionella pneumophila, Trypanosoma cruzi and Toxoplasma gondii - as well as their interactions with the vesicular transport of the host cell. Basic pathways of vesicular transport are delineated and important participating molecules described. Furthermore, the effector proteins of pathogens that interact with these molecules are included. The special chapter is devoted to phenomenon of mimetics of SNARE proteins by bacteria. The manuscript concludes with a chapter on LpSNARE of Legionella pneumophila, which was found in our laboratory and which is a topic of my experimental work herein.
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Metabolism of iron in amoebas
Glavanakovová, Marie ; Šuťák, Róbert (advisor) ; Pyrih, Jan (referee)
Ferritins are complex protein structures for iron storage and detoxification. They are typically found in the cytoplasm of the cell; however, they were also identified in mitochondria and chloroplasts. They are distributed among all living organisms but uncommon in non-photosynthetic protists. Surprisingly, a ferritin gene was found in the genome of free-living amoeba Naegleria gruberi. Bioinformatic analysis confirms that it belongs to a group of eukaryotic ferritins. Upon creating antibodies in a laboratory rat, we localized the protein in mitochondria of N. gruberi. The native recombinant protein without the mitochondrial presequence was used for further in vitro experiments. Using growth experiments, we established that the expression of ferritin is dependent on a concentration of iron in cultivation media; however, we were not able to directly prove its ability to bind iron in vitro. We concluded that the function of the ferritin of N. gruberi is most likely related to metabolism of iron. Side experiments were focused on a different protein from the ferritin family - Dps protein in Acanthamoeba castellanii. Despite of a successful creation of antibodies, we were not able to detect the expression of protein neither in total cell lysates nor in subcellular fractions of acanthamoebas under...
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Iron metabolism of parasitic protist Trypanosoma brucei
Krejbichová, Lucie ; Mach, Jan (advisor) ; Pyrih, Jan (referee)
Trypanosoma brucei is a parasite most frequently occurring in Sub-Saharan Africa that causes sleeping sickness in humans and various similar illnesses in animals. The bloodsucking tsetse flies (Glossina) transfer the parasite to humans, their final hosts. Throughout its complex life cycle, Trypanosoma occurs in different environments and undergoes various morphological and metabolic changes. Iron is an important element for all living organisms, including Trypanosoma. The metal plays a crucial role in the host-parasite interaction since trypanosomes are dependent on the iron they acquire from the host or vector. Trypanosomes use iron in metabolic reactions, such as energy metabolism, respiration, nucleic acid synthesis, detoxification, and cellular homeostasis. It is an important element in the synthesis of iron-sulfur clusters which function as cofactors during the above-mentioned reactions. The understanding of iron metabolism in the cell can facilitate the development of new medicaments, an example being iron chelators.
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Investigation of newly discovered protein GL50803_16424 in Giardia intestinalis.
Pelc, Josef ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
The anaerobic unicellular eukaryotic organism Giardia intestinalis is a worldwide parasite. Giardiasis, the intestinal disease caused by Giardia, is one of the most common parasitic disease in the developed part of the world, that causes health problems not only to humans but also to animals. This organism is also interesting for its many unique cellular features. One of them is the presence of mitosomes - the organelles derived from mitochondria. Analogously to mitochondria, mitosome is limited by two membranes and shares the mode of the protein transport. However, mitosome does not have its own genome and as far as we know, there is only one pathway of the iron-sulfur cluster biosynthesis in this organelle. Using the in vivo enzymatic tagging technique, several novel mitosomal proteins were identified, including GL50803_16424. The protein GL50803_16424 attracted our attention by interacting with components of all mitosomal subcompartments: the outer membrane, the membrane and the matrix. In addition, the expression of HA-tagged GL50803_16424 resulted in the formation of peculiar structures near the mitosomes never seen before in G. intestinalis. Bioinformatic approaches revealed that the GL50803_16424 has domain similar to the myelodysplasia- myeloid leukemia factor 1-interacting protein. Our...
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Copper metabolism of Naegleria gruberi
Ženíšková, Kateřina ; Šuťák, Róbert (advisor) ; Pyrih, Jan (referee)
Copper is an important trace element, essential for all organisms. As a cofactor, it takes part in key biochemical reactions. For example, it conveys electron transfer in complex IV in the respiratory chain, functions in detoxification of hydroxyl radicals and participates on import of iron ions into the cell. Homeostasis of copper ions must be strictly regulated, to prevent their accumulation in the organism. Excess of these ions is highly toxic and can lead to cell death. Copper ions take part in reactions leading to creation of hydroxide radicals, a dangerous member of reactive oxygen species, that damages the structure of lipids, proteins and DNA. Many studies also describe the importance of copper ions in virulence, where these ions induce the immune response of the host organism. Effect of availability of copper ions on metabolism was studied on the organism Naegleria gruberi. The sequenced genome of this organism reveals, that metabolic pathways of N. gruberi are very universal. Possible flexibility of metabolism of this organism, in relation with copper ion availability, was confirmed in this work by proteomic analysis and by determining the activities of the respiration chain. Furthermore, the dependence of uptake of iron ions on copper was confirmed. Second part of this work focuses on...
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Structure and function of mitochondrial secretin.
Klápšťová, Veronika ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...
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