|
| |
|
| |
|
| |
|
|
Study of the role of the B-chain N-terminus conformation of insulin in binding to the insulin receptor
Kosinová, Lucie ; Žáková, Lenka (advisor) ; Obšil, Tomáš (referee)
According to the International Diabetes Federation (IDF), there were 371 million people in the age from 20 to 79 years worldwide affected by diabetes in 2012. This means diabetes has become a global epidemic disease and, therefore, the importace of insulin research still grows. Insulin is a protein hormone that plays a key role in regulating blood glucose level which has a widespread impact on whole metabolism. Insulin acts through binding of its monomeric form to the insulin receptor. It is clear that insulin monomer has to undergo structural changes upon binding to the insulin receptor as the residues which are crucial for the interaction are burried within the native form. According to studies of highly active hormone analogs and the new information about the insulin-insulin receptor complex, there is a strong evidence that the C-terminal part of the B-chain is a dynamic element in insulin activation and receptor binding. Probably, there is also a great importance of the B-chain N- terminus and the transition between T and R conformations of insulin. However, the exact significance of the T and R states of insulin still remains unclear. In this work, several new insulin analogs AibB3-insulin, AibB5-insulin, AibB8- insulin, N-MeAlaB8-insulin and D-ProB8-insulin were prepared for the purpose of...
|
|
|
Synthesis and characterization of the new insulin analogues with the aim to clarify the interaction of insulin with its receptor
Kletvíková, Emília ; Jiráček, Jiří (advisor) ; Obšil, Tomáš (referee) ; Hrabal, Richard (referee)
The objective of this thesis is to characterize insulin analogues modified at the C-terminus of the B-chain with the aim to observe the impact of the inserted modifications on the insulin-insulin receptor (IR) interaction and the ability of the analogues to dimerize. Therefore, a series of analogues with modifications at B24-B26 positions was prepared. Using the synthetic and semisynthetic methods we inserted coded and non-coded amino acids to this part of B-chain. We studied full-length analogues and analogues truncated by three to four amino acids. Binding affinity of all analogues to the insulin receptor was determined by competition of analogue with radioactive (125I) human insulin. Dissociation constant in the dimer dissociation process of selected analogues (especially of those with N- methylation of B23-B24, B24-B25 and B25-B26 peptide bonds) was determined by isothermal titration microcalorimetry. The crystal structures of several analogues were resolved by X-ray crystallography and nuclear magnetic resonance. The structural results showed the consequences of inserted modifications to the insulin molecule. We characterized analogues with higher, equipotent and lower binding affinity to the IR. The results...
|
|
|
Molecular mechanisms and functions of 14-3-3 proteins
Šilhán, Jan ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Schneider, Bohdan (referee)
Závěr Hlavním cílem této doktorské práce bylo objasnění molekulárních mechanismů funkce 14-3-3 proteinů a vlivu na proteiny FOXO4 a tyrosinhydroxylasu. V první časti této práce (publikace I) byla potvrzena předložená hypotéza polohy Cterminálního konce molekuly 14-3-3. Bylo ukázáno, že v nepřítomnosti ligandu se Cterminální konec nachází ve vazebném místě a brání tak vstupu ligandů. Po vazbě fosforylovaných ligandů, dochází k velmi silné vazbě a vytěsnění C-terminálního konce 14-3- 3 proteinu z vazebného místa. Tyto výsledky jsou v souladu s původními pracemi, které navrhly důležitost tohoto segmentu jako inhibitoru nepatřičných ligandů. Druhá část této doktorské práce poskytuje rozsáhlý popis vlivu 14-3-3 proteinů na transkripční faktory FOXO4. S použitím stacionární a časově-rozlišené fluorescence byla studována interakce 14-3-3 proteinu s fosforylovaným transkripčním faktorem FOXO4. Navázání 14-3-3 proteinu způsobuje rozpad komplexu FOXO4:DNA. Tato část práce charakterizuje interakci 14-3-3 proteinu s DNA-vazebnou doménou FOXO4. Výsledky neprokázaly výrazné konformační změny v rámci DNA-vazebné domény. Spíše dochází ke sterickému bránění vazby DNA (publikace II). Ve třetí části se práce zabývá studiem interakcí 14-3-3 s fosforylovaným ligandem odvozeným od C-konce enzymu serotonin N-acetyltransferasa...
|
|
|
Study of the interaction between the C-terminus of DNA-binding domain of FOXO4 and DNA
Zusková, Iva ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Forkhead transcription factors are structurally similar molecules containing approximately 110-amino-acid-long DNA-binding domain known as a forkhead domain. Protein FOXO4 is a member of subgroup "O" of forkhead transcription factors. Members of this subgroup play a key role in many biologically important processes. For example, FOXO factors participate in metabolism control, cell-cycle control, apoptosis and oxidative stress resistance. The forkhead domain (DNA-binding domain) consists of three α-helices (H1, H2 and H3), three β-strands (S1, S2 and S3) and two flexible loops (called wings W1 and W2). The role of the wing W2 in FOXO binding to the target DNA is still elusive. Wing W2 probably interacts with the DNA in the region upstream of the core motif. It has been speculated that the FOXO DNA-binding affinity depends on A-T content (number of A-T pairs) in the region upstream of the core motif. In order to investigate this hypothesis, DNA- binding domain of the FOXO4 protein was expressed and purified and it was determined its binding affinity for three molecules of double stranded DNA containing different number of A-T pairs in the region upstream of the core motif using steady-state fluorescence anisotropy- based method. Our results show no significant differences between obtained FOXO4...
|
|
| |
|
|
Structure and interactions of selected forkhead transcription factors
Kohoutová, Klára ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee)
This diploma thesis is a part of a project aiming to develop and study specific inhibitors of FOXO3 transcriptional activity. FOXO3 belongs together with FOXO1, FOXO4 and FOXO6 to FOXO subfamily of forkhead family transcription factors. FOXO transcription factors are evolutionary conserved proteins playing important roles in numerous cellular processes, such as apoptosis, cell cycle regulation and metabolism. Due to their ability to induce apoptosis and to block the cell cycle they are considered tumor suppresors. However, it has been shown that increased activity of FOXO proteins is connected with many kinds of cancer. In such cases FOXO proteins function to maintain cell homeostasis. They promote tumor resistance against chemotherapy as well as they speed up its growth. The aim of this project is to develop specific inhibitors able to bind to FOXO3 DNA-binding domain (DBD, residues 156-269) and to block its interaction with target DNA. Development of specific inhibitors of FOXO3 transcriptional programme requires knowledge of solution structure of all FOXO DBDs and detailed insight into their interaction with target DNA. So far crystal structures of complexes of FOXO1, FOXO3 and FOXO4 with target DNA and solution NMR structures of apo DBDs of FOXO3 and FOXO4 have been solved. One of the goals of...
|
|
|
Biophysical characterization of the N-terminal part of protein kinase ASK1.
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...
|
guest ::
