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Inhibitory effect of 17α-ethinylestradiol on the activity of cytochrome P450 subfamilies 2B and 2C
Knapp, Kryštof ; Dračínská, Helena (advisor) ; Kukačka, Zdeněk (referee)
17α-ethinylestradiol (EE2) is a synthetic derivative of endogenous estrogen 17β-estradiol. It is widely used in pharmaceutics as a ingredient of female oral contraceptives and also in hormone replacement therapy for women with postmenopausal syndrome. Due to its high estrogenic potential together with resistance to the chemical degradation and tendency to bio- accumulate in environment it is considered as an important persistent organic pollutant. In organisms, EE2 is oxidatively metabolised by enzymes from a family of cyto- hromes P450. Some studies show that EE2 is also a potent inhibitor of some isoforms from cytochrome P450 family. In this work, we studied inhibitory effect of EE2 on two human isoforms CYP2B6, CYP2C9 and their rat orthologs CYP2B1 and 2C6, respectively. All studied isoforms exhibited reduced enzyme activity in the presence of EE2. The greatest decrease of enzyme activity was observed with CYP2B1 catalyzing pentoxyresoru n O-depentylation with value of IC50 = 9,6 µM. For hydroxylation of bupropion, selective reaction of CYP2B1 and CYP2B6, EE2 behaved as a more potent inhibitor of CYP2B6 with IC50 = 60 µM. The inhibitory effect of EE2 on rat CYP2B1 for similar reaction conditions was signi cantly lower. With 200µM concentration of EE2, CYP2B1 exhibited still more than 50 % of...
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The use of protein radical footprinting by Togni reagents in structural biology
Fojtík, Lukáš ; Kukačka, Zdeněk (advisor) ; Kolenko, Petr (referee)
Structural proteomic methods such as an ion mobility mass spectrometry, chemical cross- linking or covalent labeling have been established as powerful tools for structural studies of biomolecules in general. These methods have significantly contributed to the expansion of our knowledge about biomolecular functions, their dynamics and molecular interactions and therefore led to the understanding of important biological processes occurring in a cell. We decided to spread these methods and we developed a new radical labeling technique relaying on Fluor-alkyl radicals that does not require a laser dissociation pf hydrogen peroxide. We exploited the potential of Togni reagents to form Fluor-alkyl radicals by reducing agent under native conditions. The induction of Fluor-alkyl radicals was triggered by ascorbic acid and the labeling pulse was stopped by tryptophan. The modified proteins were analyzed by top down or bottom up approach using high resolution mass spectrometry. In case of top down approach, several fragmentation techniques (CID-ECD, ETD) were tested for protein analysis while in case of bottom up approach the analyzed proteins were digested by trypsin and separated on reverse phase column online coupled to mass spectrometer. As the model biomolecules we chose a 20 proteinogenic amino acids...
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Analysis of posttranslational modifications of proteins by mass spectrometry
Musil, Dominik ; Šulc, Miroslav (advisor) ; Kukačka, Zdeněk (referee)
Viruses, etiological agents of many infectious diseases, are small noncellular particles that run their replication process solely inside host cells. It is generally assumed that the posttranslational modifications of viral capsid proteins are responsible for their infectivity (e.g. phosphorylation catalysed by kinases of host cells). The appropriate model for study of the viral phosphorylation "profile" relation with its infectivity is mouse polyomavirus of the Polyomaviridae family. By comparison of virions, as well as the major capsid protein VP1 of wild type mouse polyomavirus with viral mutant created by deletion of the part of the genome coding regulatory proteins of virus and produced in two different cell lines WOP and 3T6 was found by mass spectrometry a major phosphorylation in the three specific amino acids of VP1. These are considered important for proper morphogenesis of virions and their ability to infect host cells. The qualitative representation was not affected by the cell line selection. Furthermore, in case of VP1 "dimerization" detected on SDS-PAGE electrophoretogram the double phosphorylation of VP1 pThr63, pSer66 was confirmed in our experimental in vivo approach. Therefore, posttranslational modifications, specifically phosphorylation, could probably affect structural...
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The use of mass spectrometer for aminoacid analysis
Matoušková, Zuzana ; Kavan, Daniel (advisor) ; Kukačka, Zdeněk (referee)
Amino acid analysis is used across many disciplines. However, it carries certain problems such as missing asparagine, glutamine and tryptophan, due to the primary purpose for which it was developer. Nevertheless, there are many applications in which these missing amino acids need to be quantified as well. A faster but less sensitive alternative would be the mass spectrometry detection, which also makes it possible to analyze other metabolites and non-amino compounds. It is necessary to know all the possibilities and limits of the newly developed method to be applicable to the full benefit. This bachelor thesis involves both a method in which individual amino acids are separated using hydrophilic interaction chromatography and subsequently detected by mass spectrometry as well as its basic characterization and comparison with a commonly used fluorescence method commercially available from Waters.
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Structure-functional study of electrotransport protein systems
Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kukačka, Zdeněk (referee)
Electron transport processes are an extremely important field of study in modern biochemistry and structural/functional proteomics. Azurin is one of the most utilised model systems for study of redox and electron transport processes in proteins. We have utilised photo-induced crosslinking (PIXL) to study oligomerization of azurin in solution and the effect of L-2-amino-5,5-azi-hexanoic acid (photo-Met) - a structural photoinducible analogue of canonical amino acid Met - on electron transport processes in azurin. The optimisation of expression conditions of recombinant azurin in auxotrophic E. coli B834 cells was done to maximise photo-Met incorporation percentage in azurin sequence (70% incorporation was measured via MALDI-TOF mass spectrometry). Through the optimisation of purification protocol (example: cell disintegration, acid precipitation of proteins, adding metallic ligand during cell sonication) we have increased the purity and yield of final product and reduced the purification time. Final preparations (wild-type azurin (WT) with Met, WT with photo-Met and "All-Phe" mutant (all Trp/Tyr replaced by Phe) with photo-Met) were exposed to intense UV-light (PIXL) and evaluated via UV-VIS spectroscopy and SDS-PAGE. During PIXL experiment some photo-Mets incorporated into azurin were able to: (i)...
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Preparation of cytochrome b5 mutants containing photoreactive amino acids, and their crosslinking with the interaction partners
Landl, David ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Cytochrome b5 is an electron transport protein of a clinically prominent mixed-function oxygenase (MFO) system. This system participates in the first stage of xenobiotic biotransformation. The hydrofility of xenobiotics is increased by introduction of an oxygen atom into their structure. The MFO system also activates or deactivates certain drugs and carcinogens. Cytochrome b5 affects reactions catalyzed by the terminal oxygenases of the system - cytochromes P450. Electrons are donated to cytochrome b5 by redox partners NADH:cytochrome b5 reductase and NADPH:cytochrome P450 reductase. The aim of this work was to demonstrate capability of photo-crosslinking approach to fixate transient interactions within MFO system. Covalent complexes obtained by this technique could be further analyzed by mass spectrometry, which can provide structural information about the binding sites of the proteins. We prepared a mutant cytochrome b5 comprising photo-reactive methionine analogue in the position 41 of the sequence. We expressed the protein employing E. coli B834 (DE3) auxotrophic strain in 300 ml of the limit medium supplemented with L-2- amino-5,5-azi-hexanoic acid (photo-methionine). The rate of the unnatural amino acid incorporation was determined by mass spectrometry and it was about 40 % after 16 hours of...
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Characterization of protein structures using chemical cross-linking and mass spectrometry.
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee) ; Hernychová, Lenka (referee)
Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.
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Investigation of the β-N-Acetylhexosaminidase Stucture from Aspergillus oryzae
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Ryšlavá, Helena (referee)
in English β-N-acetylhexosaminidase (EC 3.2.1.52) belongs to exoglycosidase, and is one of the most abundant enzymes found in organisms from bacteria to human. The fungal β-N-acetylhexosaminidase from Aspergillus oryzae is composed of propeptide and catalytic domain. The propeptide is noncovalently associated with the catalytic domain of the enzyme. Propeptide is essential for the enzyme activity. While the structure of the catalytic domain was desidned by homology modeling, the structure of the propeptide has not been resolved yet. In this study, the position where the propeptide is associated with the catalytic domain, was uncovered. Presented work consists of two parts. First part deals with optimization of production conditions, purification and crystallization of β-N-acetylhexosaminidase from the filamentous fungi Aspergillus oryzae. Second part is devoted to the study of interaction between propeptide and catalytic domain, which was characterized by chemical cross-linking and high-resolution mass spectrometry. It was found that the structural changes of the catalytic domain depend on the presence of the propeptide molecule. Moreover the region of propeptide-catalytic domain interaction was revealed.
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