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Mechanism of inducible gene expression of resistance protein Vga(A)LC from Staphylococcus haemolyticus.
Novotná, Michaela ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
The staphylococcal protein VgaA belongs to ARE ABCF family, which confers resistance to ribosome binding antibiotics by the target protection mechanism. VgaA confers resistance to lincosamides, streptogramins A and pleuromutilins and thus provides the so-called LSAP resistance phenotype. The expression of resistance genes often reduces fitness in the absence of an antibiotic, therefore the expression of resistance genes is often tightly controlled and triggered only in response to the presence of an antibiotic to which the protein confers resistance. The inducible expression has also been observed for the vgaA gene, nevertheless, its mechanism has not been elucidated. In the diploma thesis, it was shown that the vgaALC gene from Staphylococcus haemolyticus is regulated by ribosome-mediated attenuation. The mechanism is based on the detection of translation inhibitors via a ribosome translating a special regulatory open reading frame (uORF), which is part of an attenuator located in the 5' untranslated region of the mRNA. The vgaALC gene is regulated at the transcriptional level in response to LSAP antibiotics. Antibiotic specificity of induction is affected not only by the nature of the peptide encoded by uORF but also by the antibiotic specificity of the resistance protein. Fluorescence microscopy...
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Analysis of Src dynamics in cellular structures
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Rozbeský, Daniel (referee)
Src kinase is a key element in many signaling pathways affecting cellular processes such as differentiation, proliferation, motility, or migration. Deregulation of its activity is associated with the promotion of cancer. Therefore, understanding its cellular function is vital. Src activity directly correlates with its structure; when Src is active, it adopts opened conformation, when inactive, it is in closed conformation stabilized by intramolecular interactions. Detection of the conformation can be used to analyze Src activity. In this thesis, conformation-sensitive FRET-based Src biosensor was improved using mNeonGreen as a new acceptor fluorophore in the existing design and the properties of the new biosensor were compared with the original Src biosensor. The new biosensor is able to detect changes in Src conformation and can be stably expressed in cells. Src activity in focal adhesion was analyzed and higher Src activity in these structures was confirmed. Although the new biosensor did not exhibit significantly better sensitivity to Src conformational changes, it still proved to be a useful tool to study Src activity, and mNeonGreens higher brightness makes it more suitable for microscopic experiments. Key words: Src, FRET, biosensor, live-cell imaging, mNeonGreen
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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The role of p130Cas substrate domain mediated signaling in cancer cell migration, invasiveness and metastasis of cancer cells
Zemanová, Kateřina ; Brábek, Jan (advisor) ; Čáslavský, Josef (referee)
p130Cas (Crk-associated substrate) was first described over 30 years ago as a protein that associates with the v-src and v-crk oncoproteins and undergoes tyrosine phosphorylation. Proteins of the CAS family are an important part of cellular biological processes in normal and pathological situations. The existence of 15 YXXP repetitive motifs is characteristics for substrate domain. p130Cas is an adapter protein that allows interactions between proteins that lead to assembly of multiprotein complexes. The p130Cas protein regulates these multiprotein complexes, which further drive chemotaxis, apoptosis, differentiation and migration. Overproduction of CAS proteins was found in connection with a poor prognosis and an increased incidence of metastases. Also, the elevated expression of proteins of the CAS family is related to resistance to some types of chemotherapeutics.
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee) ; Hejnar, Jiří (referee)
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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Analyzing the role of the p130Cas SH3 domain in p130Cas-mediated signaling
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee) ; Truksa, Jaroslav (referee)
The adaptor protein p130Cas (CAS, BCAR1) represents a nodal signaling platform for integrin and growth factor receptor signaling, and influences normal development and tissue homeostasis. Its altered expression drives many pathological conditions including tumor growth, metastasis and drug resistance in many cancer types. How p130Cas contributes to many of these pathologies is still poorly understood. Therefore, the overall aim of my PhD work was to provide new insights to p130Cas signaling and its regulation. The SH3 domain is indispensable for p130Cas signaling, but the ligand binding characteristics of the p130Cas SH3 domain, and the structural determinants of its regulation were not well understood. To be able to study various aspects of p130Cas signaling we identified an atypical binding motif in p130Cas SH3 domain by establishing collaborations with Dr Veverka (Structural biology) and Dr Lepšík (Computational biochemistry; Academy of Sciences, CZ) which gave new insight into this binding interface. Through these collaborations I generated chimeras of p130Cas SH3 domain with its ligands for structural NMR analysis and learned how to visualize and analyze structures. Furthermore, my work expanded our knowledge of p130Cas SH3 ligand binding regulation and led to a novel model of Src-p130Cas- FAK...
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Adhesion structures of leukemia cells and their regulation by Src family kinases
Obr, Adam ; Kuželová, Kateřina (advisor) ; Brdička, Tomáš (referee) ; Brábek, Jan (referee)
Adhesion signaling is a field of cell biology studied mostly on adherent cell types. However, hematopoietic cells grow in suspension, and use adhesion to the extracellular matrix (ECM) only in their early development, or - in case of differentiated cells - to perform the tasks they are specialized for. Peripheral leukemic cells are derived from more or less immature hematopoietic precursors that have, among other alterations, defects in adhesion to the bone marrow microenvironment. On the other hand, leukemic stem cells (LSC) use adhesion to the bone marrow ECM as a mean to evade chemotherapy, and are a source of the minimal residual disease, and of the disease relapses. Kinases of the Src family (SFK) are known regulators of adhesion signaling in adherent cell types, and their overexpression and/or hyperactivation is often seen in malignant diseases. They are also involved in hematooncologic disease progression and resistance to therapy, particularly in several types of leukemias. In the present work, we used a variety of methods including microimpedance measurement, fluorimetric measurement of adhered cell fraction, immunoblotting, confocal microscopy, and interference reflection microscopy. Our results indicate that active Lyn kinase, a hematopoietic SFK, is present in adhesion structures of...
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Christian Student Movements in the 20th Century
Rosický, Aleš ; Moree, Pieter (advisor) ; Filipi, Pavel (referee)
This Bachelor thesis deals with the origin, development and focus of student Christian movements, especially in the 20th century, but by description of the origins of movements, it intervenes to the 19th century as well as by chapter about the situation in the Czech Republic to the 21st century. It focuses in particular on IFES movement and its part in the Czech Republic - UKH. The thesis describes origin and orientation of YMCA, SVM, WSCF, Campus Crusade and movements which developed from these organizations in the Czech Republic as well. Even though the majority of contents of the thesis concern about historical facts and points of view of individual authors on specific movements, the thesis is also dealing with the question, why individual movements were established and what circumstances led to this. The author of this text also attempts to understand the situation of connection or splitting of particular movements. The thesis is generally trying to understand particular international movements within the context of contemporary background in the church environment. The author is also trying to look under the scope of those movements and find out what way they were participating in mission and evangelism and what activities they offered to students as well.
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The search for novel interaction partners of SH3 domain of an adaptor protein p130Cas
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Forstová, Jitka (referee)
Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...
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