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Expression and purification of proteins with unnatural aminoacids, and determination of their structure by combination of chemical or photochemical modification and mass spectrometry
Bortel, Tomáš ; Ječmen, Tomáš (advisor) ; Kavan, Daniel (referee)
Residue-specific non-natural amino acid (NNAA) incorporation has become a widely used approach to introduce bioorthogonal groups and therefore functionalities into proteins26 . These functionalities are harnessed through click chemistry, conjugating labelled proteins to affinity or fluorescent probes. A less utilized approach involves probing the effects of bioorthogonal groups on the structure-function relationship of labelled proteins47 . In order to investigate these relationships, there is a need to express large amounts of proteins with maximal incorporation of NNAAs. Here, we employed photo-methionine (pMet), azidohomoalanine (AHA) and homopropargylglycine (HPG) as methionine (Met) surrogates. We investigated the impact of these NNAAs on bacterial growth of prototrophic E. coli BL21 or Met- -auxotrophic E. coli B834 in Met-free MM-M9 medium. We monitored the expression of cytochrome b5 (cyt b5) and MBP-GFP. Using MS and LC-MS based approaches, we determined the NNAA incorporation in these recombinant proteins. Prototrophic E. coli BL21 expressed significantly higher amounts of cyt b5 compared to B834 with pMet and AHA, but the incorporation rates fell sharply after 4 hours. In contrast, Met-auxotrophic B834 expressed smaller amounts of protein, but with incorporation of pMet in 50 - 70% range...
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Chemical modification of glycopeptides and prediction of their retention on hydrophilic liquid chromatography
Pála, Anastasie ; Ječmen, Tomáš (advisor) ; Křížek, Tomáš (referee)
5 Abstract Glycoproteins are an integral part of many cellular processes, without which the complex life of higher organisms wouldn't be possible. Glycoproteins are also important from clinical perspective, since changes of physiological state caused by diseases lead to changes in protein glycosylation. Changes in the abundance of glycosylations or structures of synthesised glycans are associated with diseases such as Alzheimer's disease or cancer and as such can be used as biomarkers for diagnostic purposes. There are several approaches used in the study of glycoproteins, one of which is glycopeptide analysis of enzymatically cleaved glycoproteins. This approach allows analysis of glycoproteins even in complex matrices such as biological samples, as the glycan residues are still connected to their glycosylation sites. This makes the identification of glycoproteins along with their associated glycan structures possible, which oftentimes isn't possible with other analytical approaches. Glycopeptide analysis readily uses high performance liquid chromatography coupled with mass spectrometry. Hydrophilic interaction chromatography is often chosen as the separation mode for glycoproteomic studies, due to its ability to separate glycopeptides with different glycans attached. This chromatographic mode was used in...
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Study of metabolism of tyrosine kinase inhibitors and mechanism of their anticancer effects
Vavrová, Katarína ; Indra, Radek (advisor) ; Souček, Pavel (referee) ; Ječmen, Tomáš (referee)
As inhibitors of tyrosine kinases have become more widely used in anticancer therapy, detailed understanding of their pharmacokinetic profile is increasingly essential for the improvement of treatment outcomes. Herein, the metabolism in liver microsomes in animal models and humans was studied. Clarification of which liver enzymes, specifically cytochromes P450 (CYP), are involved in the metabolism of vandetanib, cabozantinib and, lenvatinib, may play a key role in oncology practice, as these anticancer drugs are administrated in combination with other drugs. This co-administration may pose a risk of drug interactions, which may result in decreased efficacy or increased drug-related toxicity. To elucidate the metabolism of our selected anticancer drugs in human liver, the same approaches were gradually used- correlation analysis using human liver microsomes from 12 different donors, inhibition studies, and the use of recombinant CYP. In addition, an approach with animal liver microsomes that were premedicated with inducers of individual CYP was used to elucidate vandetanib metabolism in rat liver. Our first studied tyrosine kinase inhibitor was vandetanib. In the first paper, we studied its metabolism in humans through various approaches, and subsequently in another publication in rat. The...
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Production and characterization of galectin-3 mutant
Dubanych, Yurii ; Vaněk, Ondřej (advisor) ; Ječmen, Tomáš (referee)
Natural killer cells are large granular lymphocytes of innate immunity that are characterized by the ability to kill cancer and virus-damaged cells without prior activation. Cytotoxic functions of NK cells are regulated on the one hand through surface receptors recognizing MHC-I molecules, on the other hand by the presence of a set of activating and inhibitory receptors that are under normal conditions in balance with each other. Therefore, the fate of the target cell depends not only on the expression of MHC-I, but also on the expression of ligands that activate NK cell receptors. One of the activating receptors of NK cells is NKp30. Three specific cellular ligands have been discovered for NKp30: human BCL-2-associated athanogen 6 (BAG-6, also known as BAT3), tumour antigen B7-H6, and the newly discovered ligand galectin-3. All these ligands are often expressed by cancer cells, where BAG-6 and Gal-3 inhibit NK cell functions, which may be a mechanism for tumour escape from the immune system. Therefore, Gal-3 is a new potential drug target that, by inhibiting Gal-3, can help the immune system defend itself against malignantly transformed cells. This bachelor's thesis includes the verification of the effect of the Cys173 - Ser173 mutation in the carbohydrate recognition domain of galectin-3 on the...
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Development of chomatography-mass spectrometry technique for separation and identification of glycopeptides applicable in clinical practice
Petroušek, Filip ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Glycoproteins are commonly found in organisms and play an important role in both physiological and pathological conditions. In glycoproteins, different glycans may be present at the same glycosylation site and altered glycosylation of proteins may be a manifestation of disease. As a result, they can be used as markers in diagnostics. For the analysis of glycoproteins, their cleavage into glycopeptides, which are then identified, is often used. Liquid chromatography coupled to mass spectrometry is a widely used method for the study of glycopeptides and hydrophilic interaction chromatography (HILIC) can be used for the separation of glycopeptides. This type of chromatography allows separation of glycopeptides based on the properties of both the peptide and the bound glycan. The first aim of this work was to determine the extent to which the charge of the peptide backbone of glycopeptides affects their separation by HILIC. Penta-HILIC and HILIC-B columns were used for this purpose. Penta-HILIC contains a polyhydroxyl stationary phase, while the stationary phase of HILIC-B consists of silica gel modified with positively charged aminopropyl groups. IgG1 and IgG2 glycopeptides were separated on both columns. To determine the extent of the effect of charge and hydrophobicity during separation on HILIC-B,...
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Preparation and characterization of protein constructs for structural analysis of acylate domain of adenylate cyclase toxin from bacteria B. pertussis
Ryvola, Vojtěch ; Ječmen, Tomáš (advisor) ; Chmelík, Josef (referee)
Whooping cough or pertussis is a contagious bacterial airways diasease caused by B. pertussis. Despite the high vaccination coverage of the population in developed countries, whooping cough is one of the most common diseases that can be prevented by vaccination. Bacteria B. pertussis produces a variety of toxins that facilitate the colonization and proliferation of bacterial cells in the host ciliary epithelium of the upper respiratory tract. One of these toxins is adenylate cyclase toxin, which is released from bacterial cells by a type I secretory apparatus and, after it's calcium-induced packaging, is able to transport its N-terminal adenylate cyclase domain across the cytoplasmic membrane of host cells. This domain able to catalyse conversion of adenosine triphosphate (ATP) to a cyclic adenosine monophosphate (cAMP) after the interaction with cytoplasmatic calmodulin. High levels of cAMP subsequently lead to impaired cell signaling and to a fundamental reduction in the bactericidal functions of phagocytic cells. The aim of this bachelor thesis was to prepare and characterize 'hybrid' protein constructs, which consist of a C-terminal part of the acylation domain and an N-terminal part of the RTX domain (at positions 881-1038 and 881-1047) linked to the C-terminal part of the RTX domain (at...
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The preparation and characterization of polymeric, enzymatically cleavable carriers for cancerostatic drugs
Zelený, Jan ; Etrych, Tomáš (advisor) ; Ječmen, Tomáš (referee)
As the development of cancerostatic drugs progresses it is becoming necessary to contemplate the question of how to deliver these drugs, which themselves tend to exhibit carcinogenic properties, effectively and accurately to the affected tissues and thus to circumvent their destructive effects upon the healthy parts of the organism. One approach to delivering drugs selectively to cancerous tissues is to make use of some of the specific properties which these tissues tend to possess, one of which being the so-called enhanced permeability and retention effect (EPR effect). This effect, which will be further discussed within this thesis, allows for macromolecules that are too massive to pass from the bloodstream into healthy tissue, to exit the blood vessels of cancerous tissue and to accumulate there. Therefore, a drug molecule can specifically enter cancerous tissue along with a suitable macromolecule, to which it is conveniently attached. If, moreover, the given drug is connected to the carrier molecule via an enzymatically cleavable spacer, it is possible to make use of lysosomal proteases (such as cathepsin B, which is overexpressed in some cancer cells) in order to attain its detachment from the carrier molecule and its subsequent activation. This bachelor thesis focuses on describing the...
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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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Cooperativity of cytochrome P450 monooxygenase system in view of the modulation of drug and carcinogen metabolism
Dědič, Jan ; Hodek, Petr (advisor) ; Ječmen, Tomáš (referee)
System of mixed function oxidases and oxygenases is very much involved in metabolism of xenobiotics and endogenous compounds. System consists of several components: cytochrome P450, NADPH: cytochrome P450 oxidoreductase (CPR), cytochrome b5 and NADH: cytochrome b5 oxidoreductase. It was found, that all these components interact with each other, thus ensuring operation of the entire system. Cooperativity of the system is then dependent on many factors, notably the nature of the interactions between the components. Apparently the most frequently discussed are the interactions between CPR, cytochrome b5 and cytochrome P450. The main redox partner of cytochrome P450 is CPR that during the electron transport undergoes a significant conformational change. Cytochrome b5 may have both inhibitory and stimulatory effects on the enzymatic reaction and its mechanism of action has not been fully elucidated. Cytochrome P450 can interact among themselves to create complexes which presence may have significant influence on enzymatic reaction. However cooperativity of the system in terms of character of enzymatic reaction does not depend only on quantitative effects, such as inhibition and stimulation. It turns out, that also depends on qualitative effects, because it has been shown, that certain changes in the...
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