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Protein-protein interaction mapping of cytochrome P-450 by methods using chemical modification and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Cytochromes P-450 (P450s) belong to haemoprotein superfamily and they are responsible for metabolism of a wide variety of compounds, among others many drugs and carcinogens. P450s serve as the terminal oxidases in the mixed function oxidase system in cooperation with a redox partner NADPH: cytochrome P450 reductase (CPR) providing input of two electrons to the reaction cycle of P450. The CPR can be substituted by other redox partner of P450, cytochrome b5 (cyt b5), to deliver the second electron. Three dimensional structure of P450 is required in order to fully understand its reaction mechanism. At the present time, a homology model of cytochrome P-450 2B4 (CYP 2B4) is available in our laboratory. In this study, the mapping of interaction domain between CYP 2B4 and cyt b5 employing a crosslinking agent EDC to form amide bonds between close complementary charged amino acid side chains was the first goal. We have identified five interacting amino acid pairs in total using mass spectrometry (MS). The second research interest was to verify and refine the CYP 2B4 model using a photoaffinity labelling with N-(p-azidobenzyl)-N-methyl-p-aminophenylamine probe. This photoreactive probe is known as CYP 2B4 ligand binding to the central iron atom of haem. After photoactivation the arginine 197 was found by MS...
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Mutagenesis and expression of protein NKp80, the activation receptor of human lymphocytes
Pazderová, Kristýna ; Vaněk, Ondřej (advisor) ; Ječmen, Tomáš (referee)
The subject of this study is a receptor NKp80, also known as killer cell lectin-like subfamily F, member 1 (KLRF1). It is an activating receptor which forms homodimers on the surface of natural killer (NK) cells. Receptor NKp80 binds to a ligand, AICL, which is naturally expressed on all myeloid cells. Upon a substantial increase in AICL expression, for example in cancer cell, the cell then becomes a target for an NK cells expressing the receptor NKp80. Ultimately, the complex NKp80:AICL is therefore a potential target for the immunotherapeutic treatment of myeloid leukaemia. The aim of the study was to the produce and purify a series of mutants of an extracellular domain of NKp80 by replacing cysteins by serines in a segment of extracellular domain called the stalk region. Here, by introducing the mutations, we studied their effect on homodimer formation. The proteins were prepared in HEK293S GnTI- cells using stable transfection. Altogether, we produced seven mutants with all possible combinations of mutations of the three cysteins in the stalk region. We then analysed the proteins using size exclusion chromatography and differential scanning fluorimetry. Lastly, we deglycosylated the proteins to verify that NKp80 is present in several glycoforms. Our results show that none of the variants of...
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Protein radical footprinting as a tool for protein therapeutcs validation.
Binar, Michal ; Novák, Petr (advisor) ; Ječmen, Tomáš (referee)
Mass spectrometry techniques are very important and useful tool in studying of protein structure. One of these techniques is the covalent labeling of proteins. The covalent labeling can be used for determination of the accessibility of protein surface and their dynamics in general. Although a lot of reagents have already been developer for this method, most of them are limited by their ability to selectively react with only some of amino acids, mostly lysine, arginine or tyrosine, and non-selective methods such as fast photochemical oxidation of proteins are very demanding and costly. Therefore, there is still a endeavor to develop new methods of covalent labeling. This new method could be trifluoromethylation, which is increasingly used in the modification of organic molecules, and whose potential we have decided to study. In this work we used a new group of formally electrophilic agents, which is formed on the basis of a cyclic hypervalent iodide nucleus. This group so-called Togni reagents was used as a tool for radical protein labeling. Since the structure was well characterized by X-ray, NMR and MS, the human carbonic anhydrase has been selected as a model protein. The modified protein was analyzed by a bottom up approach using a combination of liquid chromatography and high resolution tandem...
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Protein-protein interaction mapping of cytochrome P-450 by methods using /chemical modification and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor)
Mapping of protein-protein interactions in the system of cytochrom P-450 by chemical modification and mass spectrometry Abstract Cytochromes P-450 (P450s) belong to haemoprotein superfamily and they are responsible for metabolism of a wide variety of compounds, among others many drugs and carcinogens. P450s serve as the terminal oxidases in the mixed function oxidase system in cooperation with a redox partner NADPH: cytochrome P450 reductase (CPR) providing input of two electrons to the reaction cycle of P450. The CPR can be substituted by other redox partner of P450, cytochrome b5 (cyt b5), to deliver the second electron. Three dimensional structure of P450 is required in order to fully understand its reaction mechanism. At the present time, a homology model of cytochrome P-450 2B4 (CYP 2B4) is available in our laboratory. In this study, the mapping of interaction domain between CYP 2B4 and cyt b5 employing a crosslinking agent EDC to form amide bonds between close complementary charged amino acid side chains was the first goal. We have identified five interacting amino acid pairs in total using mass spectrometry (MS). The second research interest was to verify and refine the CYP 2B4 model using a photoaffinity labelling with N-(p-azidobenzyl)-N-methyl-p-aminophenylamine probe. This photoreactive probe is...
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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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Protein radical footprinting as a tool for protein therapeutcs validation.
Binar, Michal ; Novák, Petr (advisor) ; Ječmen, Tomáš (referee)
Mass spectrometry techniques are very important and useful tool in studying of protein structure. One of these techniques is the covalent labeling of proteins. The covalent labeling can be used for determination of the accessibility of protein surface and their dynamics in general. Although a lot of reagents have already been developer for this method, most of them are limited by their ability to selectively react with only some of amino acids, mostly lysine, arginine or tyrosine, and non-selective methods such as fast photochemical oxidation of proteins are very demanding and costly. Therefore, there is still a endeavor to develop new methods of covalent labeling. This new method could be trifluoromethylation, which is increasingly used in the modification of organic molecules, and whose potential we have decided to study. In this work we used a new group of formally electrophilic agents, which is formed on the basis of a cyclic hypervalent iodide nucleus. This group so-called Togni reagents was used as a tool for radical protein labeling. Since the structure was well characterized by X-ray, NMR and MS, the human carbonic anhydrase has been selected as a model protein. The modified protein was analyzed by a bottom up approach using a combination of liquid chromatography and high resolution tandem...
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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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The influence of endocrine disruptors on the expression of cytochrome P450 1A2
Orlovská, Ľubica ; Dračínská, Helena (advisor) ; Ječmen, Tomáš (referee)
The term endocrine disruptor is used for chemical compounds which imitate or antagonize the effects of endogenic hormones, alter hormone synthesis and metabolism or modify levels of hormonal receptors. Synthetic estrogen 17α-ethinylestradiol (EE2) and carcinogenic substance benzo[a]pyrene (BaP) belong to the group of chemicals described as exogenic compounds with endocrine destruction, while estrogenic hormone 17β- estradiol (EST) figures as natural endogenic endocrine disruptor. The bachelor thesis focuses on study of the influence of these endocrine disruptors and their combinations on expression and specific activity of CYP1A2. RNA was isolated from the lungs of rats treated with the endocrine disruptors and from untreated rats. RNA was converted to cDNA by reverse transcription. Relative amount of CYP1A2 in livers, kidneys and lungs was quantified by real-time PCR. The protein expression of CYP1A2 was studied using the Western blot with consecutive immunodetection. Finally, the specific activity of hepatic CYP1A2 was determined by measuring 7-methoxyresorufin O- demethylation. It was confirmed that BaP induces gene expression of CYP1A2 in livers, kidneys and lungs, even in combination with EE2 and EST. However, both estrogens decrease the induction potential of BaP. When given individually,...
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Study of protein-protein interaction in bacterial pathogenesis: PIXL (photo-induced cross-linking) methodology
Žídek, Radek ; Šulc, Miroslav (advisor) ; Ječmen, Tomáš (referee)
Gramnegativní bakterie druhu Bordetella pertussis jsou původci smrtelného lidského onemocnění označovaného jako pertuse, častěji jako černý kašel. Tyto bakterie produkují adenylátcyklázový toxin (ACT), který se váže na povrch makrofágů a umožňuje vpravit do cytosolu hostitelské buňky přes cytoplazmatickou membránu adenylátcyklázovou doménu (dAC). Abychom mohli studovat kovalentní interakce mezi proteiny pomocí fotochemického zesítění, byl náš studovaný protein exprimován s foto-methioninem (kyselinou L-2-amino-5,5- azi-hexanovou) v kultivačním médiu v bakteriálním kmenu Escherichia coli B834 (DE3). Foto- methionin je netoxickým analogem L-methioninu, takže je normálně inkorporován pomocí aminoacyl-tRNA syntház do struktury adenylátcyklázové domény. Maximální míry inkorporace foto-methioninu do struktury dAC bylo dosaženo po optimalizaci celého expresního protokolu. Celková míra inkorporace foto-methioninu do struktury proteinu po optimalizaci zjištěná hmotnostně-spektrometrickou analýzou byla až 80 %. Získaný protein s inkorporovaným foto- methioninem byl izolován. Byly provedeny síťovací experimenty s kalmodulinem a vláknitým hemaglutininem. Při těchto experimentech bylo provedeno jak fotochemické, tak i chemické zesítění. Vzniklé kovalentně zesítěné produkty byly rozděleny pomocí SDS-PAGE a...
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