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Mimetics of polypeptide hormones from the insulin family
Mařík, Vojtěch ; Jiráček, Jiří (advisor) ; Zouhar, Petr (referee)
1 The family of human insulin-like peptide hormones includes insulin, insulin-like growth factors 1 and 2 (IGF-1 and 2), relaxins 1-3, and INSL3-6 polypeptides. These polypeptide hormones share similar 3D structures while retaining a similar disulfide bridge pattern and lower or higher primary sequence similarity. It is supposed that these hormones may have evolved from the same hypothetical evolutionary precursor probably already before evolution of vertebrates. These hormones have different functions from regulating glucose entry into cells and regulating the body's basal energy balance, through fetal development, growth and healing of the organism to important functions in reproduction of organisms. In addition, these peptides may be involved in the development of diseases such as diabetes mellitus, growth disorders but also cancer. Mimetics are compounds that mimic the structure of natural molecules and agonize or antagonize their biological effects. Their main advantage over natural peptide hormones may be in their greater metabolic stability, cheaper production or easier route of administration to the body, but also in altered biological activity. Hormone mimetics can therefore be used for treatment of many diseases. In this review we will focus on the known mimetics of the insulin family of hormones,...
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Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
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Study of physiological functions of betaine homocysteine S-methyltransferase and betaine homocysteine S-methyltransferase 2
Mládková, Jana ; Jiráček, Jiří (advisor)
Betaine homocysteine S-methyltransferase (BHMT) and betaine homocysteine S-methyltransferase 2 (BHMT-2) are mammalian cytosolic metalloenzymes. They both participate in the metabolism of homocysteine (Hcy), specifically Hcy remethylation, mainly in liver and kidney cells. BHMT catalyzes the transfer of a methyl group from betaine to L-Hcy, yielding L-methionine and dimethylglycine (DMG). BHMT-2 catalyzes the transfer of a methyl group from S-methylmethionine (SMM) to L-Hcy as well, yielding two molecules of L-methionine. Disorders in Hcy metabolism could lead to the so called hyper- homocysteinemia and homocystinuria, which can be connected with several pathological conditions. BHMT is already relatively well characterized enzyme. Its crystal structure and reaction mechanism have been described and a series of BHMT inhibitors have been prepared. The specific inhibitors enabled further in vivo studies and, recently, Bhmt-/- mice model has been successfully developed. In contrast, the research of BHMT-2 is still at the beginning and physiological functions of the enzyme are unknown so far. The reason is that BHMT-2 is a highly unstable enzyme and also there is a lack of selective BHMT-2 inhibitors. BHMT and BHMT-2 are very similar enzymes which have 73% amino acid identity. This thesis provides new...
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Determination of binding constants of human insulin complexes with serotonin, dopamine, arginine, and phenol by pressure assisted partial filling affinity capillary electrophoresis
Šolínová, Veronika ; Žáková, Lenka ; Jiráček, Jiří ; Kašička, Václav
A new method, pressure assisted partial filling affinity capillary electrophoresis (PF-ACE), has been developed to study noncovalent interactions of the hexamer of human insulin (HI) with cationic ligands, such as phenolic neurotransmitters serotonin and dopamine, and amino acid arginine, or with anionic ligand phenol, in alkaline aqueous solutions. The apparent binding constants, Kb, of the HI-ligand complexes were determined from the dependence of the effective migration time changes of the above ligands on the variable zone lengths of HI dissolved in the background electrolyte and hydrodynamically introduced into the bare fused silica capillary close to the UV detector. The HI interactions with the above ligands were found to be moderately strong, with Kb values in the range 385-1314 L/mol.
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Synthesis and characterization of new insulin analogs with a triazole bridge at the C-terminus of the B chain
Kuntová, Vendula ; Jiráček, Jiří (advisor) ; Ryšlavá, Helena (referee)
Insulin is a peptide hormone responsible for maintaining glucose homeostasis in the circulation. Insulin interacts with two isoforms of the insulin receptor, IR-A and IR-B, which have different tissue distribution. IR-A is supposed to have rather mitogenic function and IR-B rather metabolic function. The goal of this study was to develop insulin analog, which will be more selective for IR-B than human insulin. We prepared three new insulin analogs with a 1,2,3-triazole bridge at the positions B26 and B29. The triazole bridge was formed by Cu(I)- catalysed cycloaddition between side chains of azidopentanoic acid (N3Pent) at B26 and propargylglycine (Prg) at B29. The analogs differed in configurations on C carbons of unusual amino acids at the positions B26 and B29. Specifically, we prepared insulin analog 1 with D- N3PentB26 and D-PrgB29, insulin analog 2 with D-N3PentB26 and L-PrgB29 and insulin analog 3 with L-N3PentB26 and D-PrgB29. New analogs were tested for their binding to both isoforms (IR-A and IR-B) of the insulin receptor. Analogs 1 and 2 were less potent in binding than human insulin and had no selectivity for receptor isoforms. Analog 3 was 4-times more potent in binding to IR-B and 2-times more potent in binding to IR-A than human insulin. However, the binding selectivity of the...
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Analogues of IGF-1 for the study of interactions of the hormone with the receptors for IGF-1 and insulin
Macháčková, Kateřina ; Jiráček, Jiří (advisor) ; Obšilová, Veronika (referee) ; Šulc, Miroslav (referee)
Insulin/IGF system is a complex network of three similar hormones (insulin, IGF-1 and IGF-2) and their three similar receptors (IR-A, IR-B and IGF-1R,), which play important roles in maintaining basal energy homeostasis of the organism, in growth, development, life-span but also in development of diseases such as diabetes mellitus, cancer, acromegaly or Laron dwarfism. Despite structural similarities between family members, each member have its unique role in the system. Identification of structural determinants in insulin and IGFs that trigger their specific signalling pathways is important for rational drug design for safer treatment of diabetes or for more efficient combating of cancer or growth-related disorders. In this thesis, we focused on identification of such structural determinants in IGF-1. Comparison of our data with parallel studies with IGF-2 and insulin could give a more complex picture of the problem. First of all, we developed necessary methodologies for the preparation of IGF-1 analogues. We developed a new methodology for the total chemical synthesis of IGF-1 analogues based on the solid-phase synthesis of fragments and their ligation by a CuI -catalyzed cycloaddition of azides and alkynes. In parallel, we developed a procedure for a recombinant production of IGF- 1 and its...
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Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Jiráček, Jiří (referee) ; Schneider, Bohdan (referee)
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
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Preparation of insulin analogs for the study of interactions with insulin receptors.
Hanková, Kateřina ; Jiráček, Jiří (advisor) ; Dračínská, Helena (referee)
Insulin jako jeden z důležitých hormonů lidského organismu se podílí na řadě metabolických procesů. Jednou z funkcí insulinu je pak regulace hladiny glukosy v krvi a jejího vstupu do tkání, ale i stimulace růstu buněk. Insulin má velmi podobnou primární i terciální strukturu s růstovými faktory IGF-1 a IGF-2, jejichž primární funkcí je regulace růstových procesů organismu. Zprostředkování účinku těchto tří hormonů na buněčné úrovni je zajištěno díky jejich specifickým receptorům (mitogenní isoformě receptoru insulinu IR-A, metabolické isoformě IR-B a receptoru pro IGF-1), což může, díky podobnosti těchto receptorů i hormonů, vést ke křížovým interakcím mezi nimi. Poruchy tohoto systému mohou vést k závažným onemocněním. Nejrozšířenějším z těchto nemocí je diabetes mellitus, ale závažné jsou i poruchy růstu a rakovinné bujení. Vytvoření takového analogu insulinu, který bude selektivní pouze pro isoformu IR-B receptoru insulinu, by mohlo vést zejména k bezpečnější léčbě diabetu. Tato práce se zaměřuje na lepší pochopení významu jednotlivých aminokyselin na daných pozicích v A-řetězci insulinu, u kterých je předpokládáno, že jsou součástí vazebného místa hormonu pro receptory. Tyto znalosti by mohly vést k následnému lepšímu porozumění významu těchto aminokyselin ve vazbě na receptory IR-A, IR-B a...
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Synthesis and characterization of new insulin derivatives with altered selectivity for insulin and IGF-1 receptors.
Halamová, Tereza ; Jiráček, Jiří (advisor) ; Dračínská, Helena (referee)
Insulin receptor (IR) exists in two isoforms (IR-A and IR-B), which differ in the tissue distribution and probably also in their function, i.e. in their response to insulin binding. It is supposed that IR-A activates mainly mitogenic processes and that IR-B triggers mainly metabolic effects resulting in the uptake of glucose by muscle and fat cells. Insulin can also weakly bind to the receptor for IGF-1 (IGF-1R), a growth factor involved in the regulation of growth and development. Insulin derivatives selectively binding only to one of the receptors would be interesting for the study of the receptors but also potentially for the treatment of diseases such as diabetes or cancer. Here we used our experience in the structure-activity studies of insulin for the design, synthesis and biological characterization of 4 new insulin derivatives in order to modify their selectivity towards the individual receptors. We systematically modified insulin by amidation of the C-terminus of its B-chain or by prolongation of the B-chain by 1-3 carboxyamidated glycine residues. Binding affinities of all new analogues for IR-A and IR-B were determined and for some of the analogues binding affinities for IGF-1R as well. Finally, abilities of analogues to activate autophosphorylation of intracellular subunits of IR-A and...
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