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Theoretical study of enzymes related to carcinogenesis: DNA polymerase β and cytochromes P450
Jeřábek, Petr ; Martínek, Václav (advisor) ; Entlicher, Gustav (referee) ; Ettrich, Rüdiger (referee)
Present doctoral thesis contributed to understanding of mechanistic principles of two enzymes participating in the process of carcinogenesis; DNA polymerase (pol ) and cytochromes P450 (CYP). Pol is part of the DNA base-excision repair mechanism (BER). The primary role of pol in, the BER mechanism, is inserting a new nucleotide into a DNA strand according to Watson-Crick base pairing rules. Pol plays an important role in the process of carcinogenesis, approximately 30 % of human tumors express pol mutants. The ability of pol to discriminate between "right" and "wrong" nucleotide during the insertion process is called fidelity. We employed computational methods to elucidate molecular basis of the fidelity of pol . First, the relative free energy calculation method LRA was employed to compare differences in free energies between the "right" and "wrong" nucleotide during its insertion into DNA. The results indicated a better stabilization of transition-state of the nucleophilic substitution catalyzed by pol in the case of the "right" versus "wrong" nucleotide. This difference resulted in an 80-fold contribution to its fidelity. Further, computational methods FEP and LIE were used to examine how mutations effect fidelity of pol . Results were than correlated with experimental data...
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Biochemical changes accompanying the aging of red wine
Vacková, Tereza ; Hudeček, Jiří (advisor) ; Entlicher, Gustav (referee)
Flavonoids are plant secondary metabolites, which belong to several groups varying in their chemical structure. Anthocyanins and tannins are important flavonoid components of wine that are responsible for its color, taste and other sensory properties. The concentration of anthocyanins in wine is affected by grape variety, processing technology, and climatic conditions. In this Thesis, we studied the changes in color and in related chemical composition, using three non-commercial samples of red wine: Svatovavřinecké (year 2010 and 2012), and home-made wine (prepared without addition of SO2). These changes in color were determined using standard colorimetric method (CIELab) and also a simplified two-parametric spectrophotometric method (tint/color density). The content of anthocyanins was followed using analytical RP-HPLC method. In paralel, simplified oenologic methods for estimation of phenolic compounds were used. Generally the wine samples changed color to darker tint. Chemically, this was caused by polymerisation reactions between anthocyanins and phenolic compounds. This led to the formation of stable pigments characterised by a higher absorption maximum at longetr wavelength, hence a darker tint. Key words: anthocyanins, color, red wine, phenolic compounds, malvidin-3-glucosid, polymeric reactions,...
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Functional analysis of syntaxin 16 phosphorylation using yeast as a model
Volfová, Barbora ; Entlicher, Gustav (advisor) ; Dráber, Petr (referee)
4 Abstract Mechanism of fusion of intracellular membranes in eukaryotic cells involves several protein families including soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins and Sec1/Munc-18 related proteins (SM proteins). It is known that the transport is evolutionary conserved from yeast to man. Therefore for facilitating of the research, we can use simple eukaryotes Saccharomyces cerevisiae. Mammalian SNARE protein syntaxin 16 has a yeast homologue Tlg2p which is used in this study as a model for studying affects of phosphorylation to the syntaxin 16 function. Also their binding partners, SM proteins mVps45p (mammalian) and yeast Vps45p are homologous. Phosphorylation of SNARE proteins is known as a possible way of regulation of membrane fusion. Abolishment of one of the putative phosphorylation sites in Tlg2p protein, serine 90 leads to dominant effects on the exocytic and endocytic pathways. The work presented in this study shows some phenotypes of mutants based on this phosphorylation site of protein Tlg2p. Those mutants are S90A (cannot be phosphorylated) and S90D (phosphomimetic - acid carboxyl group mimics phosphate group). It was revealed that the phosphorylation of Tlg2p protein at serine 90 or the mutation Tlg2p-S90D may play some role in protecting Tlg2p...
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The influence of acyclic nucleotide phosphonates PMEG and PMEDAP on p38 kinase signaling in human leukemic cells
Nejedlá, Michaela ; Entlicher, Gustav (advisor) ; Slaninová, Jiřina (referee)
PMEG [9-(2-phosphonomethoxyethyl)guanine] and PMEDAP [9-phosphonomethoxy- ethyl)-2,6-diaminopurine] are acyclic nucleoside phosphonates possessing cytotoxic properties. Antiproliferative effect of PMEG was demonstrated in various tumor cell lines in vitro. PMEG also represents an active component of some experimental prodrugs with enhanced selectivity and efficacy (such as GS-9219). PMEDAP seems to have weaker effect in vitro compared to PMEG, however it exhibited pronounced antitumor effect in SD-rats with spontaneous lymphoma. Therefore it was included in the present study as well. The aim of this study was to describe the interactions of PMEG and PMEDAP with p38 MAP kinase signaling and its relationship to the apoptosis. We investigated the influence of these compounds on the expression of four genes encoding p38 MAPK isoforms and whether this change is translated into the protein. It was found that PMEG up-regulates p38β and γ mRNA in CCRF-CEM cells and p38 β and δ in HL-60 cells. The effect of PMEDAP was less pronounced than that of PMEG. However, total p38 protein level remained unaffected by PMEG and PMEDAP. Activation of p38 MAPK cascade was also measured in the cells exposed to these agents using phospho-specific antibodies. We found that neither PMEG nor PMEDAP activated p38 kinase...
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Metabolism of carcinogenic o-nitroanisol and its metabolite o-nitrophenol and two environmental pollutants 2-nitrobenzanthrone and 3-nitrobenzanthrone
Svobodová, Martina ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Souček, Pavel (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Metabolism of carcinogenic o-nitroanisole, its metabolite o-nitrophenol and environmental pollutants 2-nitrobenzanthrone and 3-nitrobenzanthrone Summary of PhD Thesis RNDr. Martina Svobodová Supervisor: Prof. RNDr. Marie Stiborová, DrSc. Prague 2010 RNDr. Martina Svobodová Introduction -1- INTRODUCTION 2-Nitroanisole 2-Nitroanisole (2-methoxynitrobenzene, 2-NA, figure 1) is an important industrial pollutant and a strong carcinogen for rodents causing neoplastic transformation in the urinary bladder and, to a lesser extent, in the spleen, liver and kidney [19, 30, 31] . 2-NA is also a toxic compound, causing anemia. 2-NA is used primarily as a precursor in the synthesis of o-anisidine (2-methoxyaniline), which is an intermediate in the production of many azo dyes. This compound is used in pharmaceutical industry as an intermediate in the synthesis of some medicaments [30, 31] . In spite of potent rodent carcinogenicity of 2-NA, this chemical is weakly mutagenic in the Ames test with the Salmonella typhimurium. This carcinogen also exhibits a low activity in cytogenetic tests. It induces a slight increase in chromosomal aberration and in sister chromatid exchanges, but only at high concentrations [31] . 2-nitroanisole may be...
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Eukaryotic and prokaryotic nitric oxide synthase - structure-function studies
Mikula, Ivan ; Martásek, Pavel (advisor) ; Entlicher, Gustav (referee) ; Král, Vladimír (referee)
Nitric oxide (NO) is an important signaling molecule in organisms. It plays a role in wide spectrum of physiological and pathophysiological processes, including vasodilatation, neurotransmission and host defense. The gaseous molecule of NO is produced by oxidative reaction catalyzed by proteins from the family of nitric oxide synthases (NOSs). Three NOS isoforms were identified in mammals, endothelial (eNOS), neuronal (nNOS) and inducible or immunologic (iNOS). Some bacteria harbor genes coding for proteins homologous to the mammalian NOS oxygenase domain and showing NO-producing activity in vitro. NO generated by pathologic organisms such as B. anthracis and S. aureus is supposed to play a critical role in the pathophysiological processes during the infection. Comparative study of bacterial NOS-like proteins and mammalian NOSs confirmed their principal similarity, but also revealed differences in the interactions of distinct bacterial proteins and mammalian NOS isoforms with different analogs of substrate L-arginine and various ligands. On the basis of the kinetics measurement of NO-rebinding a second NO-binding site in the active center of NOS was predicted. Further, the regulation of NO dynamic and release from the protein by the active site Hbonding network connecting the heme, the substrate and BH4...
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Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Entlicher, Gustav (referee)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
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