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Heme sensor proteins are the fourth group of hemoproteins. In this group of hemoproteins heme plays an important role in signalization. Dissociation and/or association of heme detecting proteins serves as an important physiological function in regulation of enzyme activity or gene expression. In this bachelor thesis all the actual knowledge about selected forms of eukaryotic heme sensor proteins previously published in scientific articles are summarized. The experimental part of this bachelor thesis is focused on preparation of recombinant protein heme regulated inhibitor (HRI) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). Firstly the preparation of the plasmids with genes HRI and eIF2α was conducted. In the next step these proteins were prepared in prokaryotic system formed by E. coli BL-21(DE3). The final sample of HRI (7,7 μM in total volume 400 µl and 60 % of homogenity) and the final sample of eIF2α (51,3 μM in total volume 400 µl and 80 % of homogenity) were obtained by the purification process. The study of thermal stability of these samples provided important informations on appropiate storage and manipulation with them in further experiments. Key words: heme-base sensors, heme, kinase, tranduction of signal, isolation of plasmids, prokaryotic expresion,...

Protein kinases ERK1 and ERK2 are one of the most studied proteins in cell signalling. Both proteins are involved in a plethora of processes, such as phosphorylation and activation of kinases as part of signalling pathways. Enzymes ERK1 and ERK2 are part of MAPK/ERK signalling cascade, connected to many cellular including cell proliferation, cell growth or differentiation. The MAPK/ERK signalling cascade is often activated in different types of tumors, making it a candidate for developing new chemical inhibitors. One of the important questions in fundamental research of ERK1 and ERK2 protein kinases is the search for difference between these proteins. Current knowledge points to redundancy of both proteins, howver several examples suggest otherwise. Recently, the work presented in Casanova et al. 2012 indirectly suggests divergent effect of ERK1 and ERK2 on cap-independent translation initiation. In the Laboratory of RNA biochemistry we focus on HCV IRES (Hepatitis C Virus Internal Ribosome Entry Site) dependent translation initiation. This diploma thesis lead to establish RNA interference method in our laboratory and to establish reporter system to study ERK1 and ERK2 effect on HCV IRES dependent translation initiation. Based on our data acquired during our research, we present in this work...

v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...

EN Protein-protein interactions have an exceptional position among other mechanisms in the regulation of signal transduction. Their systematic investigation is very important and logical step in the process of understanding to the transduction and its mechanisms at a molecular level. During my Ph.D. I was particularly interested in three important processes. ASK1 kinase is well-known initiator of the apoptosis. Under physiological conditions it is maintained in an inactive state by its two interaction partners the 14-3-3 protein and TRX1. These two proteins dissociate in the presence of reactive oxygen species by unclear mechanism and the kinase is therefore activated. The next process is an interaction between the 14-3-3 protein and phosducin and investigation of their role in the G protein signalling especially important in the biochemistry of vision. The third process is an activation of protein Nth1 through the interaction with Bmh1, yeast analog of the 14-3-3 protein, and calcium cations. I employed various biophysical method, particularly analytical ultracentrifugation, in order to explain molecular mechanisms of described processes. These techniques were used to solve the low-resolution structures of complexes TRX1 and the 14-3-3 protein with corresponding binding domains of ASK1. These...

Protein phosphorylation is a key regulator in cellular signaling pathways. It is involved in most cellular events in which interplay between phosphatases and kinases strictly controls bio-logical processes, such as differentiation, proliferation and apoptosis. Altered or defective signaling pathways often result in various diseases, emphasizing the importance of studying the phosphoproteome. The abundance of phosphoproteins in the proteome is often very low, which requires specific and highly sensitive approaches. By using quantitative proteomics methods, we are able to analyze changes in abundance of proteins and their posttranslational modifications and then changes in signaling pathways. In this review, we describe quantitative proteomics methods, which could be used for study of phosphoproteins and their connection in signaling pathways.

Extracellular signal molecules are recognized by membrane receptors on the surface of eukaryotic cells. Receptors transmit the signal into the intracellular space where activation of the concrete enzymes occurs. Activated enzymes may be protein kinases that phosphorylate the substrate proteins corresponding to the requirements for specific recognition by a protein kinase. Substrate proteins may be structural proteins and enzymes, which in turn transmit the signal or directly affect the physiological processes of the cell. The protein kinase family accounts for ERK1 and ERK2 (Extracellular signal-regulated kinase 1 and 2), enzymes which affect cell growth, cell cycle and numerous other physiological processes of the cell. Protein kinases are dynamic molecules, which undergo a series of conformational changes during their catalytic cycle and whose stability and function are affected by conformational changes. Conserved amino acid residues carry out the function of protein kinases. These factors are also involved in interactions with protein substrates and regulatory proteins, and are responsible for specific function of protein kinase.

Protein kinase ASK1 (apoptosis signal-regulating kinase 1) is a member of the mitogen- activated protein kinase kinase kinase (MAP3K) family and plays a crucial role in immune and stress responses. Since the increased activity of ASK1 has been linked to the development of several diseases including cancer, cardiovascular and neurodegenerative diseases, this enzyme is a promising target for therapeutical intervention in these pathologies. The molecule of ASK1 consists of 1374 amino acid residues, but catalytic activity possesses only a kinase domain located approximately in the middle of the molecule. The activity of ASK1 is regulated by interactions with various proteins including the 14-3-3 protein. This protein recognizes a phosphorylated motif around Ser966 at the C-terminus of the catalytic domain of ASK1. This binding interaction inhibits ASK1 through unknown mechanism. ASK1 under stress conditions, such as oxidative stress, is dephosphorylated at Ser966 and the 14-3-3 protein dissociates. This dissociation is then one of the factors that lead to the activation of ASK1. The aim of this diploma thesis was to prepare a complex of the catalytic domain of ASK1 with the 14-3-3 protein for subsequent structural studies. Both proteins were expressed in E. coli cells and successfully purified. In...

MAP kinase signaling cascade plays an important role in the cellular response to various stress stimuli from the external environment. This signaling cascade is divided into three levels: MAP kinase kinase kinases (MAP3K) phosphorylate and thus activate MAP kinase kinases (MAP2K) and those subsequently phosphorylate and thus activate MAP kinase (MAPK) pathway, which regulates many cellular functions such as apoptosis, cell differentiation and morphogenesis. One of the important MAP3K is protein kinase ASK1 (Apoptosis signal-regulating kinase 1), which is an important regulator of cellular immune and stress responses. Given that the increased activity of ASK1 is related to the development of serious diseases such as cancer, cardiovascular and neurodegenerative diseases, ASK1 is an interesting target in the pharmacy in the development of new drugs. Human ASK1 consists of 1374 amino acids and is divided into three domains: a central Ser/Thr catalytic domain and two coiled-coil domains, of which the first is located at the N- and the second at the C-terminus of the molecule of this protein kinase. ASK1 is regulated by its binding partners, which include a small cellular redox protein thioredoxin (Trx-1), which binds to the N-terminal part of ASK1. Trx-1 is a potent antioxidant and so it protects cells...