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...

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...

The goal of this diploma thesis was to find optimal conditions for expression of ASK1 kinase in prokaryotic expression system and to optimize purification protocol which enables preparing of milligram amounts of stable and soluble protein. Different conditions of expression were tested in E. coli cells including temperature of expression, cultivation medium or the length of induction. Different methods of purification were tested during the development of the purification protocol. The final protocol is based on chelate chromatography followed by gel permeation chromatography. The result of the diploma thesis is a protocol that allows preparing 1 mg of pure ASK1 kinase from 1 liter of medium.

Hippo signaling pathway represents organ size control mechanism constrained between all metazoans. Individual components of the Hippo signaling pathway were identified as key tumor-suppressors which phosphorylate and inhibit activity of several oncogenic factors and signaling pathways (such as YAP/TAZ, PI3K and mTOR). MST1 kinase is a part of central protein complex of the Hippo signaling pathway and its activation is involved in anti-cancer activity of several drugs. We have demonstrated activation of the MST1 kinase by natural compounds in leukemic cells followed by inhibition of proliferation and induction of apoptosis. Shikonin represents natural naphthoquinonic compound isolated from Lithospermum erythrorhizon which acts as inhibitor of glycolysis and mitochondrial respiratory chain in human cells. Shikonin induces fast activation of the MST1 protein in leukemic cells however mechanism of this activation remains unknown. Therefore, we tried to characterize posttranslational modifications of the MST1 kinase during shikonin treatment of leukemic cells. Firstly, we isolated MST1 kinase from control and shikonin-treated cells using immunoprecipitation. Then we characterized posttranslational modifications of the MST1 protein employing mass spectrometry. Using this approach we found out...

Calmodulin kinase cascade is a signaling pathway which is involved in the response to the increasing intracellular calcium levels. Ca2+ is a ubiquitous second messenger which promotes wide-range of cellular signaling events. Many of these signaling pathways start with the binding of Ca2+ to its primary intracellular receptor calmodulin. Calmodulin in turn binds to its downstream targets in the Ca2+ /calmodulin signaling cascade. One of the most important enzymes of this cascade is a Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). CaMKK2 is a serine/threonine protein kinase which regulates for example gene transcription or energy homeostasis by phosphorylation of its downstream targets. Catalytic domain (which provides kinase activity) is located in the middle part of the protein and possesses structure typical for kinases. CaMKK2 consists of 588 amino acids but the secondary structure is known only for the region of the kinase domain (298 residues). The rest of the protein is assumed to be unstructured as long as CaMKK2 is not bound to any interaction partner. The aim of this study was to prepare several constructs of human isoform of CaMKK2 for the further structural and activity studies. It is believed that CaMKK2 is regulated by site-specific phosphorylation. Phosphorylation of some...

The following diploma thesis titled Effects of sarcosine on calmodulin-dependent intracellular signalization is dedicated to analyzation of current findings in the area of increased sarcosine levels effects to calmodulin and calmodulin dependent kinases mediated intracellular signalization and experimental verification of these findings on malignant and non-malignant cell cultures of prostate origin. Use of sarcosine as a possible marker of prostate cancer is an assumption for elaboration of this diploma thesis. Diagnostics of prostate cancer could be simpler and more effective in case of its confirmation as a usable marker by availability of simple home-testing kits reacting on sarcosine level in urine of tested individual.

El objetivo general de este trabajo ha sido estudiar la posible implicación de diferentes cascadas de proteín kinasas en las modificaciones cardiacas que se producen tras la administración de naloxona a ratas dependientes de morfina. Los datos obtenidos indican que durante la abstinencia a morfina se produce un aumento del turnover de NA, de la actividad TH y de su fosforilación en serina 40 y 31, lo que sugiere la puesta en marcha de mecanismos post-transcripcionales. Por otra parte, la vía de la PKA estaría implicada en el incremento del turnover de NA, en el aumento de TH total y en la fosforilación y activación de TH en serina 40 durante dicho síndrome. Por último, la vía de la PKC sería una de las vías implicadas en la expresión de c-fos, así como la de las ERK, que estaría también implicada en la activación de TH en serina 31. / The main aim of this work was to study the posible involvement of different protein kinases in the cardiac adaptive changes induced during morphine withdrawal. Our results show an increase of NA turnover, TH activity and TH phosphorylation at serine 31 and 40, suggesting starting post-trascriptional mechanisms. On the other hand, PKA transduction system could be implicated in the enhanced NA turnover, in the total TH increase and in the phosphorylation and activation of TH at serine 40 during this syndrome. Finally, PKC pathway would be involved in c-Fos expression as well as ERK system which would also be responsible for TH phosphorylation at serine 31.

Polzerová, I: Use of kinase modulation in the Alzheimer's disease pathogenesis. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2016, 91 p. Data used in this Diploma Thesis have been taken from foreigner scientific literary sources. It provides the summary of the not yet explored natural compounds from marine organisms with kinase inhibitory activity. The first chapter Alzheimer's disease describes a characteristic of the disease, its etiopathogenesis, risk factors and currently available treatment. At the beginning of the second chapter are mentioned new perspective approaches to treatment of Alzheimer's disease. Most of this chapter deals with kinases as potential therapeutic targets in the treatment of Alzheimer's disease. In the chapter, physiologic and pathophysiologic functions of GSK-3β and CK-1δ are described in the organism, and also, other kinases are mentioned which are involved in the pathogenesis of the disease. Next part dedicates analytical methods suitable for testing activity and inhibition of kinases in vitro and in silico, also deals with summary of the synthetic kinase inhibitors and characterizes an their properties. In this chapter is also described main part of this work - the...

PMEG [9-(2-phosphonomethoxyethyl)guanine] and PMEDAP [9-phosphonomethoxy- ethyl)-2,6-diaminopurine] are acyclic nucleoside phosphonates possessing cytotoxic properties. Antiproliferative effect of PMEG was demonstrated in various tumor cell lines in vitro. PMEG also represents an active component of some experimental prodrugs with enhanced selectivity and efficacy (such as GS-9219). PMEDAP seems to have weaker effect in vitro compared to PMEG, however it exhibited pronounced antitumor effect in SD-rats with spontaneous lymphoma. Therefore it was included in the present study as well. The aim of this study was to describe the interactions of PMEG and PMEDAP with p38 MAP kinase signaling and its relationship to the apoptosis. We investigated the influence of these compounds on the expression of four genes encoding p38 MAPK isoforms and whether this change is translated into the protein. It was found that PMEG up-regulates p38β and γ mRNA in CCRF-CEM cells and p38 β and δ in HL-60 cells. The effect of PMEDAP was less pronounced than that of PMEG. However, total p38 protein level remained unaffected by PMEG and PMEDAP. Activation of p38 MAPK cascade was also measured in the cells exposed to these agents using phospho-specific antibodies. We found that neither PMEG nor PMEDAP activated p38 kinase...