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Role of molecular chaperones Hsp70 and Hsp90 in the replication cycle of DNA viruses
Žáčková, Sandra ; Horníková, Lenka (advisor) ; Poláková, Ingrid (referee)
Molecular chaperones are proteins which enable other proteins to assemble into native conformation and are essential for viability of the cells. Chaperones of the Hsp70 family bind to newly synthetized and denaturated proteins, prevent their aggregation and facilitate their assembly. They participate in assembly and disassembly of oligomers and also in the transport across the membranes. Chaperones of the Hsp90 family do not participate in the assembly of nascent or denaturated proteins. They bind proteins which are nearly in native conformation and enable them to assemble into conformation suitable for ligand binding or interacting with other proteins. These attributes predestinate chaperones to participate in the replication cycle of DNA viruses. A huge amount of proteins is translated during viral infection. These proteins require the chaperones to facilitate their assembly and are also required for assembly into oligomers and macromolecular structures. In addition to capsid assembly the chaperones also participate in transport of genetic information to the sites of replication, disassembly of incoming viral particles or replication of viral DNA. Therefore, the development of specific chaperone inhibitors is a promising approach. They could be used against broad spectrum of viral infections...
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Structural studies of inhibitory mechanisms of phosphatidylinositol kinases
Gregor, Jiří ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee)
+ssRNA viruses after entering the cell develop platforms for RNA replication called replication organelles. Due to the activity of phosphatidylinositol 4-kinases is in these areas a higher concentration of PI4P, which establishes suitable binding environment for the viral polymerase 3DPOL . One of these kinases is PI4KB, which is recruited to the membrane by the ACBD3 protein, which is itself recruited by giantin. Some kobuviruses and enteroviruses from the Picornaviridae family use their 3A protein to displace ACBD3 protein from the complex with giantin and transfer it from Golgi aparathus to the replication organelles. Here, PI4KB binds to ACBD3 protein and synthesizes PI4P. Recently, two proteins - TBC1D22A and TBC1D22B - were discovered to bind to the same area of ACBD3 protein as PI4KB. The goal of this project was verification of this interaction and its subsequent characterization (e.g. dissociation constant measurements). My goal was to crystallize complexes of these interaction partners and to solve three-dimensional structure. Our results suggest, that interaction of ACBD3 protein with peptides derived from TBC1D22A and TBC1D22B proteins is much lower compared to interaction between ACBD3 protein and PI4KB. I successfully prepared crystals, however, they diffracted poorly, not allowing us to solve...
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DNA damage-inducible protein: interaction partners and potential role in proteasomal degradation
Belza, Jan ; Grantz Šašková, Klára (advisor) ; Vaněk, Ondřej (referee)
Ddi1-like proteins (DNA damage-inducible) have been so far best characterized in yeast (Ddi1 protein from Saccharomyces cerevisiae), drosophila and quite recently also in human (Ddi2 protein). Based on their domain architecture they belong to the family of proteasomal shuttle proteins that transport ubiquitinated proteins for proteasomal degradation. They contain ubiquitin-associated domain (UBA) responsible for the interaction with proteasome subunits and ubiquitin-like domains (UBL) required for the interaction with ubiquitinated proteins. However, they also seem to have other functions. The yeast homolog Ddi1 is involved in cell cycle control, plays a role in the degradation of HO endonuclease and probably also functions as a negative regulator of exocytosis. The cellular functions of recently identified human homolog Ddi2 are not yet understood. In order to contribute to the understanding of the physiologic functions of human Ddi2 protein, we decided to identify potential binding partners of this protein. Therefore, we prepared DNA constructs for the expression of Ddi2 in mammalian HEK293 cells fused to two affinity tags, either FLAG or HA or Myc tag, that enable their facile visualisation and purification. Affinity chromatography coupled to mass spectrometry (AP-MS) aided in identifying...
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Microcalorimetry as a method to analyze protein interactions with ligands
Durčák, Jindřich ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The interactions of proteins with their binding partners occur in every living organisms, in almost every cell process. Therefore the exploration of protein interactions forms significant part of biochemical research. It appears that even more valuable information than the value of equilibrium constants of these interactions is determination of individual energy components - changes in enthalpy and entropy. Thermodynamic analysis by isothermal titration calorimetry (ITC) can determine the changes in entropy and enthalpy caused by formation of complex of binding partners. Microcalorimetry is also an important optimization technique in development of new drugs, for example antiretrovirotics. Despite HIV is a virus known for over 30 years, intensive research has neither brought vaccine nor drug that would permanently cure patients. Already 26 drugs were approved, most of them target viral enzymes reverse transcriptase and protease. Antiretroviral treatment prevents the propagation of HIV and maintains immune system, but long - term use leads to resistance against drugs, which is caused by mutations in the target proteins. One of relatively new targets of therapeutic intervention is capsid core formation of during assembly of new virions. During the assembly many protein - protein interactions take...
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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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Physical interactions of the splicing factor Prp45
Kratochvílová, Eliška ; Folk, Petr (advisor) ; Doubravská, Lenka (referee)
It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.
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