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Investigation of the acyltransferase RtxC interaction with the Kingella kingae toxin RtxA
Lichvárová, Michaela ; Osičková, Adriana (advisor) ; Černá, Věra (referee)
The bacterium Kingella kingae was first isolated in 1960 by microbiologist Elizabeth O. King and until recently, it was considered a rare cause of human disease. However, over the past 30 years, an increasing number of papers have shown that this bacterium is an important paediatric pathogen, mainly affecting children aged 6 months to 3 years, causing mainly septic arthritis, osteomyelitis, infective endocarditis, and bacteraemia. K. kingae displays a strong cytotoxic effect against a variety of host cell types, which is caused by the secreted cytolysin RtxA, a member of the RTX (Repeats in ToXin) family. RtxA binds to glycosylated structures of the host cell, subsequently inserts into its cytoplasmic membrane, and forms cation-selective pores, leading to disruption of ion homeostasis and lysis of the attacked cell. RtxA is produced as an inactive protoxin proRtxA. Its activation is mediated by the acyltransferase RtxC, which transfers acyl chains to conserved lysine residues K558 and K689 in the protoxin. It uses the acyl carrier protein ACP as the acyl donor. Currently, it is unclear how the RtxC, acyl-ACP, and proRtxA proteins interact with each other and which amino acid residues are responsible for these interactions. The aim of this thesis was to identify the residues responsible for these...
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Identification of residues of acylated domain of RTX toxins involved in acyltransferase binding
Grobarčíková, Michaela ; Mašín, Jiří (advisor) ; Černý, Ondřej (referee)
Both adenylate cyclase toxin (CyaA) and α-hemolysin (HlyA) are members of Repeats in ToXins (RTX) cytolysins that play key roles in the virulence of Bordetella pertussis and Escherichia coli, respectively. Bacterial RTX toxins represent a growing group of proteins produced by gram- negative bacteria. These pore-forming RTX toxins share several notable common features: (1) they require post-translational activation by attachment of fatty acid chains to two lysine residues; (2) they contain a hydrophobic domain that forms cation-selective pores in target cell membranes; (3) they are secreted by a type I secretion system; (4) after secretion, they become biologically active by binding of Ca2+ to the nonapeptide glycine- and aspartate-rich repeats. CyaA translocates a unique AC enzyme to the cytosol of phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. CyaA and HlyA also permeabilize the cell membrane of eukaryotic cells through cation-selective pores. Both toxins preferentially bind to cells expressing β2 integrins but can also interact with a variety of cells that do not express integrins or with naked lipid membranes. Both toxins are activated from protoxin form by post- translational acylation mediated by a specific acyltransferase. CyaA is activated by...
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The role of fatty acylation in activity of proteins
Grobarčíková, Michaela ; Mašín, Jiří (advisor) ; Petráčková, Denisa (referee)
Post-translational modifications are covalent and generally enzyme-mediated modifications of proteins. These modifications can serve to create active forms of proteins and they can also expand the cellular repertoire of proteins derived from standard amino acids. Known post-translational modifications include for example phosphorylation, glycosylation, ubiquitination, proteolysis and also acylation discussed in more detail in this thesis. Acylation of proteins, the covalent attachment of an acyl group, is a very frequent protein modification. This reaction is typically mediated by specific acyl transferases and involves transport of an acyl group from a donor to an amino acid residue. A diverse spectrum of cellular proteins is post-translationally acylated and therefore become biologically active. This phenomenon occurs in bacterial toxins, which are important factors of the virulence of pathogenic bacteria. Protein lipidation regulates numerous biological pathways such as membrane targeting, protein secretion, cell signaling, and apoptosis. Posttranslational acylation is also required for Ras activity and many other cancer-causing proteins. Therefore, inhibitors of acyltransferases of these proteins are being tested as targets for antitumor drugs. In this work, findings about individual types of...
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