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Structural studies of an abasic site DNA damage repair and DNA interstrand cross-link formation
Landová, Barbora ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Schneider, Bohdan (referee)
DNA damage refers to any alteration or modification in the DNA structure that deviates from its natural state. Abasic site (Ap site) is one of the most common DNA lesions resulting from spontaneous depurination/depyrimidination or enzymatic base excision. When left unrepaired it can lead to a cascade of genetic mutations, potentially causing diseases like cancer. Understanding DNA repair mechanisms is vital for medical research and applications. Bacterial MutM is a DNA repair glycosylase, removing DNA damage generated by oxidative stress and preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of procaryotic enzymes, sharing structural and functional similarities with their eukaryotic counterparts, such as NEIL1-NEIL3. Here, I present two crystal structures of MutM from pathogenic Neisseria meningitidis: MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. One of the DNA lesion repaired by MutM is the Ap site, which, if not repaired, may spontaneously lead to the formation of an abasic site interstrand crosslink (Ap-ICL) with an adjacent adenine in the opposite strand. NEIL3 glycosylase is known to remove Ap-ICL. With a...
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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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Assay for testing inhibitors of α-synuclein amyloid fibril formation in cells
Galkin, Maksym ; Yushchenko, Dmytro (advisor) ; Hudeček, Jiří (referee) ; Schneider, Bohdan (referee)
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and has no cure yet. During PD progressive neuronal loss is accompanied by the formation of Lewy Bodies - abnormal intracellular aggregates, which are mostly formed by fibrils of neuronal protein α-synuclein (αSyn). Aggregation of αSyn was shown to correlate with the pathology progression during PD. Therefore, inhibition of αSyn aggregation and subsequent pathology spreading is a promising approach to the disease treatment. In previous years, a high variety of such inhibitors were developed including small molecules, antibodies, and fibril end-targeting proteins. While these inhibitors were profoundly tested in solutions, their performance in cell- based assays remained unclear. The main reason for this was the absence of a reliable high-throughput cell-based assay for αSyn aggregation evaluation. The application of such an assay is particularly important in the case of molecules that target amyloid fibrils because they can lack specificity and interact with other β-sheet rich proteins present in cells. Moreover, the application of the inhibitors can be severely limited due to poor membrane penetration, intracellular degradation or off-target interactions. The main goal of my work is the development of an assay...
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Development of inhibitors of amyloid fibril formation
Priss, Anastasiia ; Yushchenko, Dmytro (advisor) ; Schneider, Bohdan (referee) ; Hudeček, Jiří (referee)
α-Synuclein (α-Syn) is a small neuronal protein that is present in synapses, and, presumably, regulates the vesicle-mediated neurotransmitter release. Misfolding of α-Syn into amyloid fibrils is linked to the progression of synucleinopathies, including Parkinson's disease, the second most common neurodegenerative disorder. To date, no Parkinson's disease treatment that stops or retards neurodegeneration was established. Several direct and indirect approaches of influence on the α-Syn aggregation are extensively developed in search of potential drug candidates. Recently proposed inhibition of the α-Syn fibrils growth by blocking their ends is one of the most promising known strategies, as it opens an opportunity to design fibrillization inhibitors that act at very low concentrations. However, rational design of inhibitors of this type was not performed yet. The ultimate goal of this work was the development of highly efficient and specific inhibitors of α-Syn fibrillization. Therefore, careful structure-activity relationship analysis of the fibril end blocking inhibitors was performed and the affinity to fibril end was defined as the crucial factor for their inhibition activity. Using the recent data on the fibril structure, we designed inhibitors that are able to bind to the fibril ends with...
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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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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...
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Study of receptor-ligand pair NKR-P1F and Clrg
Kotýnková, Kristýna ; Man, Petr (advisor) ; Schneider, Bohdan (referee)
Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...
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Studies on DCL-1, receptor of dendritic cells, using NMR techniques
Pospíšilová, Eliška ; Kavan, Daniel (advisor) ; Schneider, Bohdan (referee)
(EN) The DCL-1 receptor (CD302) is predominantly expressed on the surface of dendritic cells and according to its sequence similarity DCL-1 is classified as a C-type lectin. Since its extracellular domain lacks single motives for carbohydrate binding in coordination with calcium ions, it is probable that the process of carbohydrate binding does not occur through the classical pathway as described in the case of mannose receptor or the DEC-205 receptor. Due to its colocalization with F-actin there is a presumption, that DCL-1 plays a role in cell adhesion and migration. Another role of DCL-1 could be the participation in endocytosis and subsequent targeting to lysosomes. DCL-1 was also put in connections with various pathologies in last few years Experiments described in this work can be divided into three sections. In the first part I dealt with production of a protein construct based on the extracellular domain of DCL-1. The protein was produced in M9 minimal medium with the only source of nitrogen 15 NH4Cl and the only source of carbon 13 C glucose. The result was 15 N,13 C labelled protein, used for NMR measurements. The second part is dedicated to the analysis of NMR spectra, which enabled us to assign frequencies of protein backbone and aliphatic side chains atoms. On the base of the backbone...
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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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Bioinformatic analysis of protein/DNA interactions
Božíková, Paulína ; Schneider, Bohdan (advisor) ; Hašek, Jindřich (referee)
In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.
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