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Specificita interakcí protein-protein a jejich modulace
Pham, Phuong Ngoc ; Schneider, Bohdan (advisor) ; Damborský, Jiří (referee) ; Vaněk, Ondřej (referee)
(EN) Protein-protein interactions (PPI) have essential roles in life processes, and abnormal PPI are associated with many human diseases. Given their importance, PPI have received increasing attention and became drug targets. However, the design of specific PPI and their modulation is challenging. Cytokine-receptor interactions are especially important in the regulation of the immune system. Interleukin-10 (IL-10) over-production results in excessive immunosuppressive effects, tumor growth and infection. The interaction between interferon gamma receptor 2 (IFN- γR2) and interferon gamma (IFN-γ) leads to activation of downstream signaling pathways but the mechanism of such interaction is elusive. Interleukin-24 (IL-24) is another cytokine that signals through receptors sharing the interleukin-20 receptor two (IL-20R2) subunit and has important roles in autoimmunity and cancer. The aims of this Ph.D. thesis are to study PPI from several aspects emphasizing their specificity. The first goal is to develop a novel protein scaffold and subsequently evolve it into a high-affinity binder specific for human IL-10. The second goal is to understand the structural basis for receptor specificity of human IFN-γ. The third goal is to modulate the binding affinity between human IL- 24 and its receptor IL-20R2 by...
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Discovery of General Controů Nondereprssible 5 protein interactors using proximity labeling
Potapenkova, Uliana
Usually, proteins don’t act as isolated species, but they perform their function in complexes with other proteins. Identification of protein-protein interactions (PPIs) brings insights into regulation of plant developmental processes and their response to the environmental conditions. In recent years, several methods to study PPIs were developed, since they represent the key objectives of plant system biology research. This thesis is focused on the Proximity Labeling (PL) assay in order to study GCN5 protein interactors. GCN5 is a histone acetyltransferase that plays a crucial role in plants’ defense responses to various abiotic stresses. Studies of its interactors provide valuable information on the histone acetylation mechanisms and the way plants cope with adverse conditions. Here, the advantages and disadvantages of PL assay are summarized and compared with other methods of PPIs studies. In the experimental part of this work, GCN5 was cloned and plasmid for further biotin-based proximity labeling was prepared.
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Interaction of protein subunits SEC10 and SEC15 of the exocyst tethering complex
Bartáková, Anna ; Ryšlavá, Helena (advisor) ; Čermáková, Michaela (referee)
The exocyst is an evolutionarily conserved tethering complex involved in the regulation of the secretory pathway in eukaryotic cells. As an effector of Small GTPases, the exocyst contributes to efficient targeting of secretory vesicles to the sites of intense exocytosis at the plasmatic membrane through interactions with specific membrane phospholipids. The exocyst complex consists of eight subunits: SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84. Each subunit binds at least two other subunits, among them the interaction of SEC10 and SEC15 is crucial for the exocyst function. The question remains how this particular interaction is evolutionarily conserved across different plant taxonomic groups. SEC10 and SEC15 genes from the three evolutionary groups of plants (green algae - Klebsormidium nitens, mosses - Marchantia polymorpha, angiosperms - Arabidopsis thaliana) were cloned into yeast two-hybrid vectors for studying protein interactions. Testing the interaction of SEC10 and SEC15 exocyst subunits from different plant species in the yeast-two hybrid system showed that despite partially different protein sequences of the tested subunits, their interaction across different evolutionary groups of plants remains very well conserved.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš ; Schneider, Bohdan (advisor) ; Pichová, Iva (referee) ; Kukačka, Zdeněk (referee)
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Current methods in protein-protein interactions research
Křivková, Jana ; Hrdý, Ivan (advisor) ; Kučerová, Jitka (referee)
Protein-protein interactions (PPI) have a crucial role in all processes in living cells. Understanding the interactions between proteins allows us to describe cell processes in more detail and their study opens new possibilities for drug design. The importance of the question of studying PPI is shown in the recent development of various methods for their identification and description. The aim of this thesis is to give an overview of new and improved experimental methods of identification and characterization of protein-protein interactions. Methods described in this thesis are divided in four chapters - proximity-dependent labelling methods (BioID, BioID2, APEX, TurboID, MiniTurbo, PUP-IT, AirID, SPPLAT, EMARS), cross-linking methods (XS-MS), fluorescence methods for identification and visualization (BiFC, FRET, BRET) and biophysical methods for description of kinetics and thermodynamics parameters of interaction (SPR, ITC, MT).
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Modulation of interactions of cytokines and their receptors
Kolářová, Lucie ; Schneider, Bohdan (advisor) ; Rozbeský, Daniel (referee) ; Osička, Radim (referee)
Protein-protein interactions and interactions with other molecules including DNA and RNA, play an important role in a range of biological activities and processes in all living cells. Understanding of protein-protein interactions, new approaches, and tools for their modulations are valuable for medicine, biotechnology, and drug development. We used the interleukin-10 family of cytokines as a model system for our research of biological interactions and modulation of their functions. A key prerequisite to study biological processes and a detailed understanding of biomolecular interactions is a recombinant protein that is stable under a broad range of conditions. Recombinant protein expression in sufficient yield and quality is often a challenging task. Therefore, we aimed at developing new approaches in protein design and production. In the first part of our study, we modified IL-24, a member of the IL-10 family to increase its expression and stability. We demonstrated that protein engineering is a powerful tool in research of difficult protein targets. In the second part of our study, we adopted new approaches in designing new protein scaffolds suitable for use in the ribosome and yeast display techniques. Protein scaffolds have become promising alternatives to antibodies in protein drug...
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Targeted modifications of the protein-protein interactions: Ternary complex of interferon-γ as a model system
Zahradník, Jiří ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee) ; Vaněk, Ondřej (referee)
A key prerequisite for a deeper understanding of biological processes at molecular level is a detailed description of the three-dimensional structure of interaction partners and their complexes. We adopted the IFN-γ complex as our model system. Even though IFN-γ is one of the key modulators of the immunity response, which has been studied intensively for more than 60 years, the structure of the accessory receptor chain and the understanding of the IFN-γ complex is still lacking. In this work we firstly discussed the binary system between IFN-γ and its high affinity receptor R1 which is structurally known. Using a new innovative methodology we focused on the modulation of the affinity between IFN-γ and its receptor R1. Our approach was based on the modulation of protein - protein stability by mutating cavities in the proteins' structure and increasing the affinity about seven-fold. Secondly, we crystallized and solved the structure of the IFN-γ receptor 2, the accessory receptor molecule. Our analysis of variable residues on the surface of the structures of type II family receptors, to which receptor 2 belongs, revealed the putative binding site for IFN-γ. In the third part of our work, we crystallized IFN-γ from olive flounder Paralichthys olivaceus and solved its structure at 2.3 Å resolution (PDB...
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Targeted modifications of the protein-protein interactions: Ternary complex of interferon-γ as a model system
Zahradník, Jiří
A key prerequisite for a deeper understanding of biological processes at molecular level is a detailed description of the three-dimensional structure of interaction partners and their complexes. We adopted the IFN-γ complex as our model system. Even though IFN-γ is one of the key modulators of the immunity response, which has been studied intensively for more than 60 years, the structure of the accessory receptor chain and the understanding of the IFN-γ complex is still lacking. In this work we firstly discussed the binary system between IFN-γ and its high affinity receptor R1 which is structurally known. Using a new innovative methodology we focused on the modulation of the affinity between IFN-γ and its receptor R1. Our approach was based on the modulation of protein - protein stability by mutating cavities in the proteins' structure and increasing the affinity about seven-fold. Secondly, we crystallized and solved the structure of the IFN-γ receptor 2, the accessory receptor molecule. Our analysis of variable residues on the surface of the structures of type II family receptors, to which receptor 2 belongs, revealed the putative binding site for IFN-γ. In the third part of our work, we crystallized IFN-γ from olive flounder Paralichthys olivaceus and solved its structure at 2.3 Å resolution (PDB...
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Targeted modifications of the protein-protein interactions: Ternary complex of interferon-γ as a model system
Zahradník, Jiří
A key prerequisite for a deeper understanding of biological processes at molecular level is a detailed description of the three-dimensional structure of interaction partners and their complexes. We adopted the IFN-γ complex as our model system. Even though IFN-γ is one of the key modulators of the immunity response, which has been studied intensively for more than 60 years, the structure of the accessory receptor chain and the understanding of the IFN-γ complex is still lacking. In this work we firstly discussed the binary system between IFN-γ and its high affinity receptor R1 which is structurally known. Using a new innovative methodology we focused on the modulation of the affinity between IFN-γ and its receptor R1. Our approach was based on the modulation of protein - protein stability by mutating cavities in the proteins' structure and increasing the affinity about seven-fold. Secondly, we crystallized and solved the structure of the IFN-γ receptor 2, the accessory receptor molecule. Our analysis of variable residues on the surface of the structures of type II family receptors, to which receptor 2 belongs, revealed the putative binding site for IFN-γ. In the third part of our work, we crystallized IFN-γ from olive flounder Paralichthys olivaceus and solved its structure at 2.3 Å resolution (PDB...
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