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Activity of cytochromes P450 1A1, 1A2 and 3A4 expressed in eukaryotic and prokaryotic systems
Indra, Radek ; Stiborová, Marie (advisor) ; Mizerovská, Jana (referee)
Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...
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Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
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Effect of heme analogs on structure and conformational stability of cytochrome b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Cytochrome b5 is a component of the system of mixed function oxidases (MFO system) participating in metabolism of many endogenous and exogenous substances. Its main function in MFO system is reduction of cytochrome P450. The first aim of the thesis was to employ the pulse proteolysis to evaluate the influence of heme analogs on structure and conformational stability of cytochrome b5. This method utilizes the cleavage of protein by nonspecific protease (thermolysin) what is active even in the medium with high concentration of urea. Four forms of cytochrome b5, reconstituted with protoporphyrine IX containing Fe3+ , Mn3+ , Cr3+ or Co3+ ions, were prepared using titration of apocytochrome b5 with hemin or its analogs. The stability of individual forms of cytochrome b5 was than compared with apocytochrome b5 using pulse proteolysis. Finally, the relative amounts of residual cytochrome b5 were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Key words: electrophoresis, proteolysis, cytochrome b5, stability. (In Czech)
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The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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The mechanism of action of anticancer drug ellipticin in target tissues of its effect
Vranová, Iveta ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...
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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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Oxidation of ellipticine by human cytochromes P450 expressed in prokaryotic and eukaryotic systems
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...
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(Construction of deletion mutants of human cytochrome b5 using gene synthesis)
Kotlánová, Iveta ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochrome b5 is a small amphipathic protein. The human form is anchored to the outer membrane of the endoplasmic reticulum and mitochondria, a free form is located in red blood cells. It consists of two domains: a large hydrophilic domain binds heme, a small hydrophobic domain anchors cytochrome b5 to the microsomal membrane. Both domains are connected by linker chain of about 15 amino acids, which gives a flexibility to the protein. Its length plays an important role in transferring electrons to cytochrome P450. If the linker domain is too short, cytochrome b5 is not able to tranfer electrons to cytochrome P450 and not participates in the reactions of MFO system. Other functions are preserved. The aim of this study was to design and build 4 deletion mutants of cytochrome b5 using gene synthesis. The linker domain contains long and short deletions, which are expected to have distortion interaction with cytochrome P450. Part of this thesis was the expression of heterologous proteins by cells of Escherichia coli strain XL10-Gold and DH5α. As expression vectors for the transformation were used plasmids pET- 30a(+) and pET-22b. DNA from cells was isolated and the accuracy of the genetic code was verified using the sequencing. Keywords: cytochrome b5, heterologous expression, gene synthesis (In Czech)
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Mechanism of enzymatic activation of carcinogens and drugs by the system of cytochrome P450
Indra, Radek ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Koblihová, Jitka (referee)
13 Abstract An environmental pollutant and a human carcinogen benzo[a]pyrene (BaP) is after its activation with cytochrome P450 (CYP) able to covalently bind to DNA. In the thesis, one of the target was to investigate an influence of individual components of mixed function monooxygenase (MFO) system on metabolism of benzo[a]pyrene and generation of adducts of activated BaP with DNA. The study was particularly focused to increase our knowledge on the effect of cyt b5 on metabolism of BaP by cytochrome P450 1A1 (CYP1A1) and its potential to serve as a donor of electrons during the reaction cycle of this cytochrome P450. The effect of cyt b5 on generation of BaP metabolites and adducts of BaP with DNA was investigated. In addition the effect of two different expression systems for cytochrome P450 1A1 (prokaryotic and eukaryotic) was also studied. The influence of cyt b5 on oxidation another xenobiotic compound, a plant alkaloid ellipticine that exhibit antitumor activities, was also investigated. Its pharmacological efficiency, as well as side effects depends on its metabolic activation by cytochrome P450. CYP3A4 is very important for ellipticine activation and therefore this enzyme was used in our experiments. Furthermore, a suitability of rat as a model organism mimicking the metabolic fate of BaP...
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The mechanism of action of anticancer drug ellipticin in target tissues of its effect
Mrízová, Iveta
Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...
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