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Rutinosidase from Aspergillus niger - analysis of active site and its mutagenesis
Šidáková, Anna ; Bojarová, Pavla (advisor) ; Palyzová, Andrea (referee)
Rutinosidases (α-L-rhamnosyl-β-D-glucosidases) from Aspergillus niger (AnRut) are glycosidases (EC 3.2.1) that catalyze the hydrolysis of the glycosidic bond between the aglycone and the disaccharide residue rutinose. The dual substrate specificity of this enzyme group describes the parallel activity towards the substrates rutin (carrying a rutinosyl disaccharide residue) and isoquercitrin (carrying a glucosyl residue). The active site of AnRut is more complex than that of other glycosidases and is composed of the catalytic amino acids Glu210 and Glu319 in the active-site cleft and a side tunnel. This untraditional structure with distinct interactions in the tunnel and active-site cleft is the probable reason for the enzyme exceptional substrate specificity. Through point or multiple mutations of the enzyme, we can modify its primary and secondary structure, thus causing a significant shift in substrate specificity. The main goal of this thesis is the analysis of three distinct mutant variants of AnRut rutinosidase; their production, purification, and the study of the influence of the mutations on the substrate specificity of the enzymes. All variants were designed based on molecular modeling. The substrate specificity was determined by reactions of the mutant variants with previously unstudied...
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Pathogenesis of nephrotic syndrome in children and predictors of corticosteroid treatment response
Bezdíčka, Martin ; Souček, Ondřej (advisor) ; Doležel, Zdeněk (referee) ; Reiterová, Jana (referee)
Nephrotic syndrome is a kidney disease caused by injury of the podocytes. It can be secondary due to infection, systemic disease or certain drugs, but it may also present as sudden primary nephrotic syndrome without obvious inducer. Current standard treatment has many severe adverse effects. In some patients that are resistant to the initial several-week-long glucocorticoid treatment it is possible to reveal the causative genetic aetiology of the disease, whereas in the rest of them aetiology remains unknown. Those who respond well to initial glucocorticoid treatment and achieve remission may later on develop repeated relapses requiring long-term glucocorticoid therapy. This work describes our original research studies focusing on the improvement of genetic diagnostics of nephrotic syndrome, on the exploration of molecular mechanisms of the second most common genetic cause of the steroid-resistant nephrotic syndrome (transcription factor WT1 mutants) and on the search of clinical and laboratory factors that could predict the resistence to glucocorticoid treatment. By combining Sanger and next-generation sequencing (NGS) we were the first to identify monogenic cause in 38 % of Czech and Slovak children with steroid-resistant nephrotic syndrome whose samples had been collected for 18 years. The most...
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Příprava mutantního serpinu z klíštěte \kur{Ixodes ricinus}
EDEROVÁ, Monika
Point mutation altering arginin for tryptophan amino acid residue in P1 site of tick salivary serpin Iripin-1 was created using specific primers. Recombinant protein with this mutation in nucleotide sequence was then expressed in chemically competent Escherichia coli cells, extracted from them and purified by affinity and size-exclusion chromatography. To see the impact of the mutation on inhibitory function of Iripin-1, its ability to bind trypsin and form covalent complexes was evaluated.
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The role of APOBEC proteins in HPV-induced carcinogenesis
Frolíková, Daniela ; Šmahelová, Jana (advisor) ; Šroller, Vojtěch (referee)
Apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) are a family of evolutionarily conserved cytidine deaminases with the ability to bind and modify RNA and/or ssDNA. APOBEC1-4 have a number of functions in cells. Members of the APOBEC3 subfamily cause restriction of foreign nucleic acids, retrotransposons and viruses, including human papillomaviruses (HPV), and may contribute to the clearance of infection. Certain HPVs are referred to as oncogenic viruses because of their ability to induce immortalization and transformation of epithelial cells via E5, E6 and E7 oncoproteins. E6 and E7 can also induce transcription or inhibit degradation of some APOBEC3. This results in an increase in their levels in cells. APOBEC3 also act as cellular mutators, as they can catalyze deaminations on transiently produced ssDNA during replication or transcription. Deregulation of APOBEC3 caused by oncoproteins may contribute to mutagenesis. This bachelor thesis focuses on APOBEC proteins, their activation and function during HPV-induced carcinogenesis, and in particular the extent and consequences of APOBEC3 mutations. Keywords: APOBEC, mutagenesis, papillomavirus, oncoproteins, carcinogenesis
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Structure-function relationship of Kingella kingae RtxA toxin.
Růžičková, Eliška ; Osička, Radim (advisor) ; Šulc, Miroslav (referee)
Kingella kingae is a pediatrically significant, facultative pathogen. It asymptomatically colonizes the oropharynx of young children, where it is a part of the normal microflora. However, if it penetrates the respiratory epithelial barrier and begins to spread throughout the body, it can cause serious infectious diseases. Thanks to today's advanced diagnostic methods, K. kingae is included among important human pathogens, and in pediatric patients, K. kingae is reported as a frequent cause of osteoarticular infections, such as osteomyelitis and septic arthritis, bacteremia, and endocarditis. The key virulence factor of this bacterium, the cytotoxin RtxA, belongs to the RTX (Repeats in ToXin) toxin family. This family of toxins shares several characteristic features: (i) the presence of a hydrophobic pore-forming domain in the N-terminal part of the molecule containing several predicted transmembrane α-helices (ii) the inactive protoxin is activated by different types of fatty acids bound to specific lysine residues in the acylated domain, (iii) the presence of nonapeptide repeat sequences, rich in glycine and aspartate residues, that are important for the binding of calcium ions, (iv) the presence of a C-terminal secretion signal that is recognized by the type I secretion system (T1SS), and (v)...
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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Konvalinka, Jan (advisor) ; Brynda, Jiří (referee)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Analysis of essentiality of glmM gene coding for phosphoglucosamine mutase of Streptococcus pneumoniae.
Krupička, Jiří ; Branny, Pavel (advisor) ; Čáp, Michal (referee)
Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...
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Mutagenesis in Danio rerio using CRISPR technology
Nickl, Petr ; Machoň, Ondřej (advisor) ; Soukup, Vladimír (referee)
CRISPR/Cas9 technology is currently one of the most important tools of genome engineering. This technology allows a precise site-specific gene editing and such ability was applied to study the role of TALE (TALE - three amino acids loop extension) homeodomain transcription factors during neural crest cells development. The main genes of interest, belonging to sub-family of TALE proteins, are Meis1 transcription factors that are present in the zebrafish genome as two paralogous genes, meis1a and meis1b. Their function was assessed by mutating their DNA-binding domain - homeodomain to abrogate the ability of transcription factor to bind DNA and by that disturb regulatory network, in which Meis1 proteins operate in. Phenotype analysis of mutant fish would reveal a potential role of Meis1 proteins in regulation of neural crest cells development and outline the functional significance of the homeodomain in regulatory operations. To determine the regulatory relationship between meis1a and meis1b genes morpholino-based knock-down of the genes was performed. Preliminary results suggest a dominant role of Meis1b in neural crest cells regulation and importance of its homeodomain. Furthermore, knock-down of Meis1a indicates its contribution to regulation of craniofacial development. However, a detailed...
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Analysis of essentiality of glmM gene coding for phosphoglucosamine mutase of Streptococcus pneumoniae.
Krupička, Jiří ; Branny, Pavel (advisor) ; Čáp, Michal (referee)
Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...
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Role of variable chains at the interface between subunits in forming ATP-binding pocket and function of P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Novotný, Jiří (referee) ; Vlachová, Viktorie (referee)
7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...
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