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Study of protein-protein interaction in bacterial pathogenesis: PIXL (photo-induced cross-linking) methodology
Žídek, Radek ; Šulc, Miroslav (advisor) ; Ječmen, Tomáš (referee)
Gramnegativní bakterie druhu Bordetella pertussis jsou původci smrtelného lidského onemocnění označovaného jako pertuse, častěji jako černý kašel. Tyto bakterie produkují adenylátcyklázový toxin (ACT), který se váže na povrch makrofágů a umožňuje vpravit do cytosolu hostitelské buňky přes cytoplazmatickou membránu adenylátcyklázovou doménu (dAC). Abychom mohli studovat kovalentní interakce mezi proteiny pomocí fotochemického zesítění, byl náš studovaný protein exprimován s foto-methioninem (kyselinou L-2-amino-5,5- azi-hexanovou) v kultivačním médiu v bakteriálním kmenu Escherichia coli B834 (DE3). Foto- methionin je netoxickým analogem L-methioninu, takže je normálně inkorporován pomocí aminoacyl-tRNA syntház do struktury adenylátcyklázové domény. Maximální míry inkorporace foto-methioninu do struktury dAC bylo dosaženo po optimalizaci celého expresního protokolu. Celková míra inkorporace foto-methioninu do struktury proteinu po optimalizaci zjištěná hmotnostně-spektrometrickou analýzou byla až 80 %. Získaný protein s inkorporovaným foto- methioninem byl izolován. Byly provedeny síťovací experimenty s kalmodulinem a vláknitým hemaglutininem. Při těchto experimentech bylo provedeno jak fotochemické, tak i chemické zesítění. Vzniklé kovalentně zesítěné produkty byly rozděleny pomocí SDS-PAGE a...
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Expression and purification of protein photo-initiated nanoprobe: tool to study clinically relevant protein-protein interactions
Knížek, Antonín ; Šulc, Miroslav (advisor) ; Koblihová, Jitka (referee)
Cytochrome b5 is a key protein in the function and regulation of the mixed function monooxygenase (MFO) system in mammalian endoplasmic reticulum and is, therefore, a clinically relevant target for biochemical studies. To study its interactions within the MFO system using photo-initiated crosslinking, we have developed cytochrome b5 mutants with methionine in several key amino acid positions within the primary amino acid sequence, such as serine 23 and leucine 41. Also, naturally presented Met in positions 96, 126 and 131 were mutated to Leu with no effect to cytochrome b5 activity. Our protein was expressed in E. coli B834 auxotrophic type with L-2-amino-5,5-azi-hexanoic acid (photo-Met) present in the cultivation medium. This methionine analogue with photolabile diazirine ring is readily incorporated in Met positions into the primary sequence of proteins by aminoacyl-tRNA synthetases. The whole expression protocol was optimized to achieve maximal percentage of photo-Met incorporation into the expressed protein sequence. Up to 93.4% incorporation of photo-Met was achieved. The expressed protein was isolated and photo-Met incorporation was established with MALDI-TOF mass spectrometry. After reconstitution with its natural interaction partners - full-length cytochrome P450 2B4 (rabbit isoform) or...
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Interaction PI4 kinase IIalpha with VAMP3 protein
Dubánková, Anna ; Šulc, Miroslav (advisor) ; Teisinger, Jan (referee)
Phosphoinositides are very important in regulation activity of many signaling proteins not just in cellular membranes. Phosphatidylinositol - 4 - kinases (PI4K) generate phosphatidylinositol - 4 - phosphate, an emerging regulatory molecule and precursor of important regulatory phosphoinositides. PI4Ks are associated with pathogenicity of several RNA viruses including Picornaviridae (poliovirus, coxsackie virus, aichi virus, enterovirus 71) and Flaviviridae (hepatitis C virus). PI4Ks also play important role in cancer. This study strives to clarify the mechanism of regulation of PI4K type II α by its potential interaction with Vesicle - associated membrane protein 3 (VAMP 3) of the SNARE protein family (Soluble N - ethylmaleimide Sensitive Fusion Attachment Protein Receptor).
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Application potential of modification approaches (chemical agents, photo-nanoprobes) and mass spectrometry to study protein structure and protein-protein interaction
Ptáčková, Renata ; Šulc, Miroslav (advisor) ; Levová, Kateřina (referee) ; Osička, Radim (referee)
A comprehensive understanding of physiological role of proteins requires knowledge of their three-dimensional structure, dynamics and protein-protein interactions. Chemical cross-linking in combination with mass spectrometry represents an alternative approach to standard methods for protein structure elucidation (X-ray crystalography, NMR spectroscopy) and enables characterization of interaction interface within protein complexes in their native states. The presented thesis is mainly focused on novel cross-linking methodology based on the in vivo incorporation of methionine analog with photo-reactive functional group (photo-Met) into the sequence of studied protein (so called protein photo-nanoprobe). Interaction between two molecules of 14-3-3zeta protein was used as a model to test and optimize the protein photo-nanoprobe production. The findings confirmed usefulness of this approach for mapping the protein-protein interactions. The photo-initiated cross-linking was used to detect the heterooligomeric membrane structures of cytochromes P450 2B4 and b5 and the molar ratio of cytochromes within individual complexes was assessed. The chemical cross-linking in combination with mass spectrometry was employed to characterize the interaction of their catalytic domains and two mutual orientations of...
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Study of interactions of PI4 kinase
Eisenreichová, Andrea ; Šulc, Miroslav (advisor) ; Obšilová, Veronika (referee)
The family of 14-3-3 proteins is one of great regulatory significance, which can be found in all the eucaryotic organisms and consists of seven isoforms in human cells. The function of 14-3-3 proteins rests in the interaction with their ligands, of which several hundreds has been identified. The key role of these partners comes to pass in many cellular processes such as signalization, regulation of a cell cycle and division, apoptosis and others. This thesis deals with the interaction of 14-3-3 protein with fosfatidylinositol 4-kinase IIIβ on a molecular level using the method of X-ray crystallography. Phosphatidylinositol 4-kinase IIIβ (PtdIns4KIIIβ) situated on a cytosol side of mostly Golgi aparatus membranes catalyses the connection of a phosphate group to the fourth carbon of an inositol circle. The activity of PtdIns4KIIIβ depends upon the phosphorylation of Ser294. Not only this phosphorylation increases the kinase activity PtdIns4KIIIβ, but is the condition of 14-3-3 proteins binding as well. This interaction provides the protection of PtdIns4KIIIβ against dephosphorylation and this way it guarantees continual synthesis of phosphatidylinositol 4-phosphate, a major signalization molecule and the precursor of other phosphate derivatives of phosphatidylinositol. (In Czech)
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Mass spectrometry in proteomics: structural biology and clinical applications
Pavlásková, Kateřina ; Šulc, Miroslav (advisor) ; Obšilová, Veronika (referee) ; Hernychová, Lenka (referee)
Mass spectrometry (MS) is a rapid, specific and very sensitive analytical method with a broad spectrum of proteomic applications such as protein identification and sequencing, 3D protein structure characterization or study of protein-protein interaction. The introduction of two ionization techniques in late 1980's that are able to ionize the large biomolecules such as proteins, oligosaccharides or nucleic acids with no or low fragmentation has started the rapidly expanding field of MS-based proteomics. The presented thesis was aimed at the application of mass spectrometric approaches to answer several proteomic questions. Firstly we have employed the chemical cross-linking in combination with MS analysis to solve the 3D structure and protein-protein interactions of three model systems: (1) homodimeric human regulatory protein 14-3-3, (2) model of 14-3-3 and regulatory domain of tyrosine hydroxylase, and (3) system of two membrane proteins, cytochrome P450 2B4 and cytochrome b5, involved in xenobiotics biotransformation. This approach works in aqueous solutions under physiological conditions and thus preserves native structure of the investigated proteins. The second part of the thesis was focused on MS identification of proteins/peptides in fungal spores of Aspergillus and Pseudallescheria...
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Study of modified amino acid incorporation into cytochrome b5
Koberová, Monika ; Hodek, Petr (advisor) ; Pavlásková, Kateřina (referee)
A cytochrome b5 (cyt b5) can influence cytochrome P450 (CYP)-dependent reactions. In consequence of these reactions cytochrome b5 can participate in substance activation (for example drugs, carcinogens) or it can influence proportions of formed metabolites. A mechanism of cyt b5 action has not been fully explained yet. Elucidation of protein-protein interactions in monooxygenase system could explain of the mechanism of cyt b5 action. To study these interactions by using cross-linking techniques is necessary to prepare photolabile cyt b5, which after photoactivation generated higly reactive intermediates which can create a complex with nearby components of the monoogynesase system. This thesis describes how was developed the method for the production of recombinant cyt b5 with modified amino acids. Cyt b5 was expressed in a bacterial strain E. coli BL-21 (DE3) Gold. Before the expression induction, cells were transformed into the limiting medium (DMEM-LM) which did not contain L-leucine and L-methionine. The limiting medium was supplemented by deuterated amino acid d3-methyl-L-methionine and D,L-Leucine. Expressed cyt b5 was isolated and incorporation of d3-methyl-L-methionene has been verified by mass spectrometry. Cyt b5 was obtained mainly as the apoprotein (apo-cyt b5). That is why in this...
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