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Proteins interacting with oxygen and its reactive species in Naegleria gruberi
Malych, Ronald ; Šuťák, Róbert (advisor) ; Rohlena, Jakub (referee)
Naegleria gruberi is a free-living non-pathogenic amoeba. It is a close relative to Naegleria fowleri, a pathogen that causes primary amoebic meningoencephalitis in humans (PAM). As a free-living organism, Naegleria gruberi is adapted to aerobic lifestyle but also possesses remarkable traits of anaerobic organism such as Fe-Fe hydrogenase capable of hydrogen production. This work focuses on three types of iron-containing proteins interacting with oxygen and its reactive species (ROS) that were uncovered in the genome of N. gruberi - hemerythrin, protoglobin and rubrerythrin. Studied proteins have been isolated and purified as recombinant proteins and antibodies have been produced against all three of them. We found a single homolog of rubrerythrin in the genome of N. gruberi and successfully localised it in the mitochondrion in contrast to hemerythrin and protoglobin that exhibit cytosolic localisation. In vitro characterization of these recombinant proteins included mainly size-exclusion chromatography and UV-vis spectrophotometry. Ability to bind oxygen was shown by spectral changes of recombinant hemerythrin purified under anaerobic conditions and recombinant protoglobin isolated aerobically. Western blot analysis revealed changes in expression levels of these proteins in N. gruberi cultivated...
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Copper metabolism in pathogenic eukaryotic microorganisms
Doležalová, Taťána ; Šuťák, Róbert (advisor) ; Pilátová, Jana (referee)
Copper is an essential trace element for almost every living organism. Its fundamental role in metabolism is caused by the redox properties of this metal. Owing to that it is a vital cofactor of many enzymes participating in cell energy metabolism as well as in the detoxification of reactive oxygen species. Nevertheless, too high copper concentration can damage the cells. The toxic effect of copper is manifested usually as the production of hydroxyl radicals distorting the cell structures. It can also damage Fe-S clusters that are essential for many enzymes. To avoid toxic manifestations the cell must balance on the edge of proper copper import and regulated export. Many specialized transporters and ATPases serve exclusively for copper transport. There are also known metallochaperones binding the potentially toxic metals and transporting them to the target protein. An interesting role in metabolism is played by the metallothioneins that bind cytosolic copper. Understanding copper homeostasis in pathogenic organisms reveals many interesting possibilities for better targeted treatment. For example, diseases caused by opportunistic fungi like Candida albicans, Aspergillus fumigatus or Cryptococcus neoformans contribute significantly to the deaths of immunodeficient patients. Some copper metabolism...
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Antiparasitic and antimycotic effect of different iron chelators
Šipková, Anna ; Šuťák, Róbert (advisor) ; Kuthan, Martin (referee)
Iron plays a key role in many metabolic pathways in the cell, but it may become toxic at higher concentrations. Therefore, the maintenance of iron homeostasis is crucial for cell viability and is strictly regulated. This element has also an important role in the host-parasite interaction. Parasites are fully dependent on iron uptake from the host environment. Iron uptake is a very difficult process thanks to effective sequestration of the host's iron supplies, which makes iron almost inaccessible to parasites. Imported iron is mostly transported to the mitochondria where it is necessary for its proper function. This work is focused on the effect of modified chelators on pathogenic fungi and the chosen kinetoplastid parasites, which cause serious human diseases. These diseases are becoming a great threat due to emerging drug resistances. Modified chelators are able to target mitochondrial functions and affect iron homeostasis, which can lead to a promising antiparasitic and antimycotic effect. Modified chelators in tested organisms can inhibit their gorwth even in nM concetrations, affect mitochondrial respiration, membrane potential and membrane permeability. Thanks to mitochondrial targeting the potency was much higher compared to the unmodified compounds. This work contains also research of new...
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Proteins interacting with oxygen and its reactive species in Naegleria gruberi
Malych, Ronald ; Šuťák, Róbert (advisor) ; Rohlena, Jakub (referee)
Naegleria gruberi is a free-living non-pathogenic amoeba. It is a close relative to Naegleria fowleri, a pathogen that causes primary amoebic meningoencephalitis in humans (PAM). As a free-living organism, Naegleria gruberi is adapted to aerobic lifestyle but also possesses remarkable traits of anaerobic organism such as Fe-Fe hydrogenase capable of hydrogen production. This work focuses on three types of iron-containing proteins interacting with oxygen and its reactive species (ROS) that were uncovered in the genome of N. gruberi - hemerythrin, protoglobin and rubrerythrin. Studied proteins have been isolated and purified as recombinant proteins and antibodies have been produced against all three of them. We found a single homolog of rubrerythrin in the genome of N. gruberi and successfully localised it in the mitochondrion in contrast to hemerythrin and protoglobin that exhibit cytosolic localisation. In vitro characterization of these recombinant proteins included mainly size-exclusion chromatography and UV-vis spectrophotometry. Ability to bind oxygen was shown by spectral changes of recombinant hemerythrin purified under anaerobic conditions and recombinant protoglobin isolated aerobically. Western blot analysis revealed changes in expression levels of these proteins in N. gruberi cultivated...
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Iron homeostasis in malaria
Wernerová, Klára ; Šuťák, Róbert (advisor) ; Voleman, Luboš (referee)
Although malaria is a well-studied infectious disease, we are still unable to fight it effectively, as evidenced by a large number of infected people. Many drugs are available against malaria. However, because of incessantly emerging resistances, new, more effective antimalarials need to be developed. One possibility is to target the parasite's iron metabolism, the essential element of all organisms. Iron participates in DNA synthesis, respiration, energy production. It acts as a cofactor of ribonucleotide reductase, and metalloproteins with FeS clusters or heme. During the infection, the parasite must compete with the host for nutrients, including iron. The mechanism of iron uptake or excretion in malaria parasite is not completely clear. Only two iron transporters are known, but it is already evident, that there must be more of them. The Plasmodium parasite digests a large amount of hemoglobin, which is degraded into free heme and denatured globin. Free heme is toxic to the cell though. Plasmodium defends itself from the toxicity of free heme by forming chemically inert hemozoin. This unique mechanism of protection against the free heme toxicity is very useful for Plasmodium and other blood parasites, but it also becomes an advantageous target for drugs because the mechanism is present only in...
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Oxidative stress in pathogenic amoebae
Plíva, Jan ; Šuťák, Róbert (advisor) ; Smutná, Tamara (referee)
Reactive oxygen species are substances that contain an atom of oxygen with an unpaired electron. This substance is highly reactive thanks to that unpaired electron. Organisms are capable of utilizing this reactivity in a lot of reactions. Cells can create reactive oxygen species as a by-product of aerobic respiration or by the action of enzymes such as NADPH oxidase. Reactive oxygen species play a crucial role in host defense against parasitic organisms. During host defense, parasitic organisms are destroyed by immune cells utilizing oxygen species. These reactions are in place also in the case of infection caused by pathogenic amoebae. Amoebae are eukaryotic unicellular organisms that are in some cases capable of causing serious illnesses. During the infections, amoebae must be able to bypass the host immune system, thus reactive oxygen species. This is accomplished by parasitic detoxification systems which helps pathogenic amoebae to overcome oxidative stress induced by reactive oxygen species. However, pathogenic amoebae are also capable of using reactive oxygen species to overcome the host immune system. This fact only confirms how tangled and complicated is the effect of reactive oxygen species during an interaction between the host organism and parasite.
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Role of sulfhydryl oxidase 1 in cancerogenesis
Beranová, Lea Marie ; Truksa, Jaroslav (advisor) ; Šuťák, Róbert (referee)
Disulfide bridges play a significant role in protein-folding as well as en- zyme activity and thus regulate many intra- and extracellular processes. Sulfhydryl oxidase QSOX1 forms S-S bridges de novo, modulating the activity of its substrates and thus directly or indirectly influences vital cel- lular processes. The first part of this thesis focuses on characterization of the role of QSOX1 in cancerogenesis, using breast cancer cell lines (MCF7, MDA-MB-231) and pancreatic cancer cell line (Panc-1), while the second part emphasizes the regulation of QSOX1 expression by different oxygen concentrations. To study the effect of QSOX1 on proliferation of triple-negative cancer cells MDA-MB-231, two genetically modified cell lines - QSOX1-overexpressing and QSOX1 knockout cell lines - were constructed. While increased QSOX1 protein levels do not have a significant effect, the absence of QSOX1 leads to a decreased cellular growth. Lack of QSOX1 also results in visible change in cellular morphology. QSOX1 knockout cells can be mostly characterized as more round-shaped with less noticeable or completely missing lamellipo- dia. This finding is with agreement with to-date literature suggesting that QSOX1 is important not only for cellular proliferation but also for migration and invasiveness. While authenticating the theory of...
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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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Iron metabolism in Naegleria gruberi
Arbon, Dominik ; Šuťák, Róbert (advisor) ; Krijt, Matyáš (referee)
The metabolism of iron ions is a crucial process in all living organisms and its correct regulation is essential for basic life functions. Homeostasis of iron ions is closely regulated, it usually appears as a component of various proteins and plays role in many oxidation-reduction reactions. Naegleria gruberi is a non-pathogenic, free living protozoon, that serves as a laboratory model for closely related pathogenic Naegleria fowleri. This work focuses on the study of selected metabolites of N. gruberi, that were possible to detect and quantify by the means of modern metabolomic methods, and the influence on culture cultivated in environment with lack of iron ions was shown. The discovery of effect of this condition on the energetic metabolism of this protozoan is an important aspect of understanding the biological processes on cellular level. This method proved a significant influence on certain metabolites and modification of certain metabolic pathways as a direct effect of decreased availability of iron ions. Second part of this work was focused on the enzyme alcohol dehydrogenase, that was found in the genome of this protozoon. Unusual aspects of this enzyme include a N-terminal mitochondrial presequence, prompting about mitochondrial localization, and utilization of iron ion as a prosthetic...
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FeS cluster assembly in cytosol of Trichomonas vaginalis
Stojanovová, Darja ; Tachezy, Jan (advisor) ; Šuťák, Róbert (referee)
Iron-sulfur (FeS) cluster assembly is extensively studied in model organisms, e.g. Saccharomyces cerevisiae, Homo sapiens, and more recently in Trypanosoma brucei. However, little is known about FeS assembly in divergent anaerobic organisms such as Trichomonas vaginalis, which parasites in the human urogenital tract. This parasitic protist possesses anaerobic form of mitochondria, the hydrogenosome, in which some component of FeS cluster assembly machinery (ISC) has been identified, whereas the cytosolic CIA pathway has not been studied so far. Our work deals mainly with TvIscU, a component of ISC pathway, and T. vaginalis CIA pathway. We suggest that both hydrogenosomal and cytosolic FeS cluster assembly pathways of this parasite differ from typical models. We examined possible ISC-CIA relationship. Next, we found homologues for several key components involved CIA machinery, namely Nbp35, Cfd1, Nar1, Cia1 and Cia2. However, we did not identify any homologous proteins to Tah18, Dre2 and Mms19. We expressed identified proteins with HA-tag and localized them by cell fractionation and immunofluorescence microscopy in T. vaginalis. Finally, we immunoprecipitated two Cfd1 paralogues, TvCfd1A and TvCfd1B to search for their interacting partners. The results suggest that these two paralogues interact with...
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