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The effect of 14-3-3 protein on intradomain interaction of ubiquitin ligase Nedd4-2
Pohl, Pavel ; Obšilová, Veronika (advisor) ; Žáková, Lenka (referee) ; Pavlíček, Jiří (referee)
EN The human ubiquitin ligase Nedd4-2 (NEDD4L) ubiquitinates a wide range of membrane proteins and receptors, playing a key role in maintaining homeostasis. This enzyme is regulated by phosphorylation and subsequent interaction with 14-3-3 proteins, which primarily affects its ability to interact with various substrates. However, very little is known about the molecular basis of this protein-protein interaction. In this work, we focused on biophysical characterization of the role of individual phosphorylation sites and also on mapping the structural changes in the Nedd4- 2 protein induced by 14-3-3 protein binding. Our experiments using analytical ultracentrifugation methods revealed that two phosphorylation sites Ser342 and Ser448 are primarily required for stable binding of Nedd4-2 to 14-3-3 proteins. The crystal structure of the 14-3-3ηΔC:Nedd4-2335-455 T367A complex than revealed the simultaneous binding of both phosphorylated residues to the binding groove of 14-3-3 protein. Subsequent modeling based on small-angle X-ray scattering and chemical cross-linking data combined with mass spectrometry indicated extensive structural changes in the individual domains of the Nedd4-2 protein. Binding of 14-3-3η protein blocks the WW3 domain of Nedd4-2 in the central channel of 14-3-3 protein, while...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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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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The role of posttranslational modifications of minor proteins and acetylation of microtubules in mouse polyomavirus infection
Mariničová, Zuzana ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Mouse polyomavirus (MPyV) capsid is composed of the main capsid protein VP1 and minor capsid proteins VP2 and VP3. Minor proteins are not essential capsid assembly, but they are key for efficient viral infection. The first part of this thesis studies the modifications of VP2 and VP3, the deamidation of Asn at 253 of VP2 (137 of VP3) and N-terminal acetylation of Ala of VP3, which could be the cause of double bands for VP2 and VP3 on SDS-PAGE. Mutated genomes of MPyV N253D (Asn to Asp) and N253E (Asn to Glu) simulating deamidation and A117V (Ala to Val) with reduced acetylation were prepared previously. We prepared three isolations of the mutant viruses and we confirmed that the deamidation is the cause of the double bands. Mutant viruses were compared to the wild type in terms of efficiency of infection, but the role of deamidation could not be proven. Virus A117V is noninfectious either due to lowered acetylation or the substitution of amino acid at this position. This thesis also studies the role of -tubulin acetylation in the infection of MPyV. The role of -tubulin acetylation in viral infection is being investigated to find new antiviral strategies. Acetylation rises after MPyV infection, but this is not due to a change in mRNA expression of tubulin acetylating (TAT1) or deacetylating enzyme...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Assessing biochemical properties of PDE8A1: Design of experimental system in living cells"
Galica, Tomáš ; Černý, Jan (advisor) ; Mašek, Tomáš (referee)
4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin
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