Insulin-like Growth Factor 1 Analogs Clicked in the C Domain : Chemical Synthesis and Biological Activities

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications

Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains

Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone : Receptor Binding Specificity

Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain

Mikrobiologie snídaňových směsí a lyofilizovaného ovoce

The diploma thesis "Microbiology of breakfast cereals and lyophilized fruit" considers microorganisms and their occurrence in lyophilized fruit and in raw materials that are used to produce breakfast cereals. It focuses on the production, composition and storage options of breakfast cereals and lyophilized fruit. The thesis is focused on fruit lyophilization and the benefits of this method, compared to the classic heat drying. The practical part is focused on the microbiological analysis of selected products of breakfast cereals and lyophilized fruit. Samples were analyzed for the total number of microorganisms, fungi, yeast, coliform bacteria, and Escherichia coli. Finally, it was determined whether the microbiological limits set according to the norm. From the obtained results, it is clear, that higher number of MOs was evident in the sliced lyophilized fruit and in the chocolate-containing breakfast cereals. The results also show that the microbiological limit was not exceeded in the selected samples

Development of novel inhibitors of Zn-metalloenzyme betaine: homocysteine .I.S./I.-methyltransferase

New approach for the discovery of novel phosphinic pseudopeptide inhibitors of Zn-metalloenzyme betaine: homocysteine .I.S./I.-methyltransferase

Mikrobiologická kvalita masa

This bachelor thesis deals with microbiological quality of meat. From a chemical point of view, meat and meat products have a high content of water and other substances that create the optimal environment for the growth and multiplication of undesirable microorganisms. In the case of imperfect handling and processing of the meat, contamination with microorganisms will occur and thus the sensory properties will change, and the spoilage will gradually increase. The development of microorganisms is also undesirable in terms of foodborne diseases, which can negatively affect the human body and affect it in the form of salmonellosis, campylobacteriosis, etc. The questionnaire survey revealed whether people are familiar with the notion of foodborne diseases and the potential risk that may arise with the consumption of meat and meat products. The survey shows that primary school children are more familiar with this potential risk compared to older people. A part of the thesis is a suggested recommendation to increase familiarity with this topic