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The preparation and characterization of IGF2 prohormones
Potalitsyn, Pavlo ; Žáková, Lenka (advisor) ; Obšilová, Veronika (referee) ; Hudeček, Jiří (referee)
Insulin-like growth factors 1 and 2 are protein hormones that share similar structural and functional features with insulin. They are commonly abbreviated as IGF1 and IGF2. They play important roles in prenatal development and throughout lifespan. Stimulation of growth of all cell types and significant metabolic effects also belong among their known functions. Dysregulation of their activity or elevated levels were observed in a number of diseases such as diabetes mellitus type 2, cancer, hyperthyroidism, amyotrophic lateral sclerosis, muscle dystrophy, acromegaly, Laron dwarfism and other disorders. Functions of IGF2 are less studied than functions of the other members of the family and even less is known about precursors of IGF2. To address these issues we started with production of IGF2 analogues with enhanced specificity to IR for better understanding the mechanism of receptor activation by IGF2. We produced site-specific analogues modified at sites important for receptor binding. IGF2 analogue with Phe48His mutation is the strongest IGF2-derived binder for IR-A reported ever and it is able to activate IR-A to about 60% of insulin ability. We developed new rapid, robust, and sensitive binding assays for testing of ligands of the receptor for IGF2 (IGF2R). First, we immobilized domain 11 of...
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Preparation of preptin, a peptide secreted from pancreatic islet beta cells, and its effect on insulin secretion
Zrubecká, Denisa ; Žáková, Lenka (advisor) ; Dračínská, Helena (referee)
in English Pancreas is an endocrine tissue which affects many physiological processes in organism. It has been known for a long lime that the pancreatic beta cells secret insulin. However, there are more hormones and substances secreted from the beta cells, mainly peptides such as insulin-like growth factors IGF, shorter peptides like amylin and many more. Some of them have not been even identified. Among these pancreatic products Ch. Buchanan's group from Auckland University, New Zeland identified 34 amino acids long peptide called preptin in 2001. Its amino acid sequence is identical with part of the preform of insulin-like growth factor 2 chain. It has been discovered that preptin has effect on osteoblast proliferation and insulin secretion. Nowadays preptin is being studied like a potential osteoporosis cure thanks to its increasing effect on osteoblasts proliferation. This thesis describes preparation and characterization of human and rat preptin and their two fragments (which contain amino acids 1-16 and 17-34) using solid-phase synthesis. The aim of the thesis is to test effects of rat preptin and its two fragments on insulin secretion in pancreatic beta cell line INS-1E. Part of the thesis is focused on binding assays of human preptin and its two fragments to IGF-1 receptor and also to...
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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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IGFBP3 expressing rekombinant vaccinia virus used for tumor therapy
Musil, Jan ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...
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