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Identification of antimicrobila peptides in spider venom
Benýšek, Jakub ; Tichá, Marie (advisor) ; Liberda, Jiří (referee)
Still increasing resistance to antibiotics leads to the need to find new active compounds with antimicrobial properties. This work is focused on the occurrence, chemical and physical description, mechanism of action and biological activity of such substances, found in spider venom. The second part is focused on isolation and identification of compounds with these properties from the venom of wild bees and a one spider. A novel peptide was isolated and identified from venom of bee Trachusa byssina. This novel peptide possess antimicrobial properties and low hemolytic activity. Molecular weight was estimated to 1749,9 ? 0,1contains 16 amino acids and is amidated on its C-terminus. Its primary structure GILSVLKNLLKKHMAS-NH2 was determined by using Edman degradation and ESI-QTOF mass spektrometry.
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Regulation of cathepsin K activity by reactive inhibitory molecules applicable in biomedicine
Benýšek, Jakub ; Mareš, Michael (advisor) ; Obšilová, Veronika (referee) ; Kutil, Zsófia (referee)
3 Abstract Human cathepsin K (KatK) is a lysosomal cysteine protease expressed predominantly in osteoclasts. It is the most effective enzyme for collagen breakdown and its physiological function lies in the degradation of the extracellular bone matrix. Increased enzymatic activity of KatK is associated with osteoporosis, rheumatoid arthritis, and osteoarthritis. This makes KatK a target for the treatment of pathologies, and chemotherapeutics based on protease inhibitors are being developed for its regulation. This work deals with reactive peptidomimetic and low molecular weight inhibitors of KatK namely thiadiazoles, vinyl ketones, and cyanohydrazides. It focuses mainly on the determination of the binding mode of selective inhibitors and characterization of their key interactions with the active site of KatK. Crystallographic structural analysis was combined with approaches of computational chemistry, enzymological analysis, and cell-based assays to elucidate the relationship between structure and biochemical activity of the investigated inhibitors. The obtained results provide important information for the design and optimization of new highly effective and selective KatK inhibitors as potential drugs and diagnostic probes. Keywords: cathepsin K, protease, inhibitor, 3D structure, osteoporosis
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Preparation of chicken antibodies against phosphoserine residues of phosphoproteins
Benýšek, Jakub ; Tichá, Marie (advisor) ; Entlicher, Gustav (referee)
New type of immunogen for the preparation of chicken antibodies specific for O- phosphorylated amino acid residues of phosphoproteins was prepared: the α-casein phosphopeptide mixture was coupled to maleinylated derivative of bovine serum albumin. Affinity chromatography on immobilized Fe(III) ions (Fe(III)-IDA-Sepharose) was used for the separation of the α-casein phosphopeptide fraction from the α-casein peptide mixture obtained by the proteolytic digestion using trypsin. The presence of coupled phosphopeptide was shown by means of MALDI-TOF MS analysis. After chicken immunisaion with the prepared immunogen the immunoglobulin fraction was isolated from egg yolks that was further purified using affinity chromatography on α-casein-Sepharose and bovine serum albumin immobilized on Sepharose (BSA-Sepharose). The specificity of obtaine immunoglobulin fractions was tested by means of ELISA tests. The obtained results showed, that the prepared chicken antibodies and their fractions obtained by affinity chromatography on α-casein-Sepharose do not recognize the O-fosforyl-L-serine residues of phosphoproteins (α-casein, phosvitin, ovalbumin). Using the prepared different antigens it has been shown, that prepared chicken antibodies interact with maleinylated chain coupled to carrier proteins. .
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Identification of antimicrobila peptides in spider venom
Benýšek, Jakub ; Liberda, Jiří (referee) ; Tichá, Marie (advisor)
Still increasing resistance to antibiotics leads to the need to find new active compounds with antimicrobial properties. This work is focused on the occurrence, chemical and physical description, mechanism of action and biological activity of such substances, found in spider venom. The second part is focused on isolation and identification of compounds with these properties from the venom of wild bees and a one spider. A novel peptide was isolated and identified from venom of bee Trachusa byssina. This novel peptide possess antimicrobial properties and low hemolytic activity. Molecular weight was estimated to 1749,9 ? 0,1contains 16 amino acids and is amidated on its C-terminus. Its primary structure GILSVLKNLLKKHMAS-NH2 was determined by using Edman degradation and ESI-QTOF mass spektrometry.
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