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Alzheimer's disease-related changes in phosphorylation of collapsin response mediator protein 2 (CRMP2) and their effect on neuronal function
Škrlantová, Alice ; Balaštík, Martin (advisor) ; Balík, Aleš (referee)
CRMP2 was first identified in 1995 as a mediator of Sema3A signalization pathway which leads to axon growth cone collapse. Since then CRMP2 was designated as an essential cue during neuronal polarity estabilishment and neuronal growth in embryonic life. CRMP2 was also found hyperphosphorylated in NFT's and this finding led to further research of CRMP2 function in the pathogenesis of AD. The activity and proper function of CRMP2 is regulated by phosphorylation and a deeper look into the mechanism of this modification is necessary for understanding how CRMP2 influences the function of neural cells. In this thesis I focus on signaling pathways, kinases and interaction partners of CRMP2 and describe how aberrant regulation of these interactions leads to Alzheimerʼs disease development.
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Postranslation modifications affecting function of nuclear localization signal
Šebrle, Erik ; Sedláček, Radislav (advisor) ; Venit, Tomáš (referee)
Transport of proteins to the nucleus through a nuclear envelope is controlled mostly via nuclear localization signal (NLS). Nuclear localization signal is rich in positively charged amino acids arginine and lysine. It was observed that activity of this NLS could be regulated through a phosphorylation of serine in its close proximity. Either a phosphorylation of serine or phosphomimetic changes of these "presequences" could represent an important mechanism regulating a localization of protein in cells in relation to a cellular activation. In our laboratory was identified protein - Fragile X mental retardation syndrome 1 neighbor (Fmr1nb), whose cellular localization could be driven by this posttranslational modification.
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Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
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Role of phosphorylation in nuclear import of viral proteins and complexes
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Roučová, Kristina (referee)
Replication of many different viruses occurs in the nucleus of the host cell. These viruses discovered ways how to overcome the nuclear membrane and often use cell transport machinery to transport their proteins and genome into the nucleus. For many viral proteins the timing of their nuclear import in order to secure correct viral replication is important. Regulated nuclear import also allows these proteins to perform several functions depending on their localization. Nuclear import of viral proteins and complexes can be regulated by phosphorylation. Phosphorylation can, for example, modulate affinity of proteins for importins or other cellular proteins. Phosphorylation can also cause conformational change, which can lead to unmasking of localization sequence.
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Study of the unique signaling pathway of Ser/Thr protein kinase StkP and phosphatase PhpP in Streptococcus pneumoniae
Keil, Jan ; Ulrych, Aleš (advisor) ; Bobková, Šárka (referee)
The major human pathogen Streptococcus pneumoniae is a unique model for the study of eukaryotic-type serine/threonine protein kinases and its cognate phosphatases in bacteria, since it encodes only a single signaling pair composed of the StkP protein kinase and PhpP phosphatase. This signaling pair plays a role in several cellular processes, mainly in cell wall biosynthesis and cell division. StkP and PhpP proteins with a pleiotropic effect appear to regulate a complex signaling cascade by phosphorylation of many substrates. However, only a few have been characterized so far. Using MS analysis, we have identified about 90 phosphopeptides that are potential substrates for the StkP kinase and PhpP phosphatase. This diploma thesis is focused on the characterization of the new substrate Spr0929 and its role in pneumococcal physiology. One of the objectives was to investigate cell morphology of strains carrying deletion of the spr0929 gene in different genetic backgrounds. It turned out that the role of Spr0929 in cell morphology is strain specific. The growth curves of strains with this deletion were compared to that of the wild type in various physiological conditions as well. As Spr0929 contains a nucleoid-associated domain called NdpA, determination of its cell localization was an important...
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Regulation of penicillin-binding protein Pbp2a in Streptococcus penumoniae
Kubeša, Bohumil ; Doubravová, Linda (advisor) ; Pospíšil, Jiří (referee)
Regulation of penicillin-binding protein Pbp2a in Streptococcus pneumoniae Streptococcus pneumoniae is an extracellular human pathogen that encodes a unique eukaryotic-type Ser/Thr protein kinase StkP in its genome. This enzyme is involved in other cellular processes, such as cell division and cell wall synthesis, through phosphorylation with its substrates. A transmembrane protein MacP has been identified as a substrate of StkP. It is an activator of penicillin-binding protein PBP2a, which is involved in the synthesis of peptidoglycan with its transpeptidase and transglycosylase activities. We found that MacP is phosphorylated by the protein kinase StkP at positions T32 and T56. We confirmed that proteins MacP and PBP2a interact with each other and that phosphoablative and phosphomimetic mutations of the major phosphorylated residues of the MacP protein do not affect the interaction with PBP2a and do not fundamentally affect the function of MacP in vivo. Furthermore, we showed that the ∆macP mutation is synthethically lethal with the ∆pbp1a mutation, confirming that MacP is an activator of the PBP2a protein. MacP is located in the cell septum and interacts with a number of S. pneumoniae cell division proteins. Keywords: Streptococcus pneumoniae, cell division, MacP, PBP2a, phosphorylation
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Ser/Thr protein kinases in mycobacteria
Borovcová, Taťána ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
The Mycobacterium tuberculosis genome encodes 11 Ser/Thr protein kinases. These protein kinases are structurally related to eukaryotic protein kinases. The phosphoproteome contains hundreds of proteins phosphorylated on Ser/Thr residues that influence all aspects of cell biology, which supports the critical role of phosphorylation in the regulation of physiology. Particularly important role in regulation belongs to protein kinases PknA, PknB, PknG and PknL, these protein kinases occur in all species of mycobacteria. Although only PknA and PknB are essencial for the M. tuberculosis, they regulate cell shape through the regulation of cell wall synthesis and cell division. Another important protein kinase is PknG, although not essential for growth it is necessary for virulence, because it promotes the survival of pathogen inside macrophages of the host. As a result, Ser/Thr protein kinases represent an interesting target for inhibitor development that could be used as drugs against tuberculosis.
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Virulence of Bordetella pertussis from an Omics Perspective
Novák, Jakub ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Novák, Petr (referee)
The Gram-negative aerobic coccobacillus Bordetella pertussis is one of the few exclusively human pathogens and the main causative agent of the respiratory infectious disease called pertussis, or whooping cough. Despite global vaccination programs, pertussis remains an important public-health burden and still accounts for over 100,000 infant deaths and over a dozen of millions of whooping cough cases every year. Substantial effort is devoted to studies on the mechanisms of action of virulence factors of B. pertussis, but the biology of interactions of B. pertussis with its human host remains largely underexplored. Evolution, genetics and adaptation of B. pertussis to the complex environment of human nasopharynx and the mechanisms enabling B. pertussis to overcome host innate and adaptive mucosal immune defenses, remain poorly understood. In such situations, unbiased exploratory omics approaches represent valuable tools for uncovering of unknown aspects of host-pathogen interactions and open the path to detailed analysis of virulence-underlying processes by mechanistic studies. In this thesis, I am presenting the results of three omics projects on B. pertussis biology that involved high-throughput proteomics. In the inital phosphoprotemics project, we analyzed the kinase signaling pathways hijacked...
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The role of ERK1 and ERK2 protein kinases in the MAPK/ERK signaling
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Dráber, Peter (referee)
The MAPK/ERK cascade is highly conserved signalling pathway regulating cellular processes which are necessary for cell life, such as proliferation, differentiation, apoptosis or cell migration. All these cellular responses are the result of the processing of extracellular signals through three-tier ERK cascade consisting of protein kinases Raf, MEK and ERK. The signal is transmitted by sequential phosphorylation where RAF phosphorylates MEK and MEK phosphorylates and activates ERK. Protein kinase ERK then phosphorylates and regulates a wide range of substrates at different locations in the cell. This affects the cellular response to the extracellular signal. Regulation of this pathway on every level is very important and is modulated by interaction partners and adaptor proteins. Deregulation of the pathway as well as mutations of individual protein kinases can lead to severe pathological consequences. At the level of ERK, there are two isoforms, ERK1 and ERK2, which are more than 80 % identical at the amino acid level. Their high sequence similarity has triggered the interest of many authors for more detailed examination of both isoforms in respect of their evolutionary conservation and whether they are functionally redundant or whether they have specific functions. The aim of this work is to...
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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