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Prediction of effects of single point mutations on protein-nucleic acids interactions
Štěpánková, Věra Tereza ; Novotný, Marian (advisor) ; Neuwirthová, Tereza (referee)
Single point mutations are the most common type of mutations and many of them can have pathogenic effects, it is therefore useful to be able to correctly and effectively predict their impact on a given protein. Proteins interacting with nucleic acids are essential for most cellular processes. Progress in computational methods and machine learning enables the development of increasingly high-quality tools for predicting the effect of mutations on proteins. An important category of these predictors are tools for predicting the effect of single point mutations on protein interactions with nucleic acids. This thesis focuses on currently available tools for prediction of missense mutation effect on protein-NA interactions, which predict quantitative changes in protein and nucleic acid affinity and estimate the severity of the mutation based on this change. Attention is also paid to methods for evaluating the quality of predictions of individual tools. Keywords: point mutation, prediction, interaction, mechanism
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Design, preparation and structural studies of biologically relevant protein variants of cancer-related Carbonic Anhydrase IX
Fejfarová, Adéla ; Maloy Řezáčová, Pavlína (advisor) ; Novotný, Marian (referee)
3 Abstract Carbonic anhydrase IX (CA IX) represents an attractive target for the development of anticancer drugs as it is overexpressed in various types of solid tumors. By its catalytic activity, CA IX assists the cancer cells to maintain the optimal intracellular pH and to acidify the extracellular milieu promoting tumor development. There are twelve enzymatically active carbonic anhydrases (CAs) present in human body, all sharing a high sequence identity and a typical β-sheet structural fold of the well-studied catalytic domain. Although, the activity of CAs is efficiently inhibited by sulfonamide-containing compounds, the design of inhibitor selective to the cancer-related CA IX has been hampered by the high sequence conservation. CA IX has several unique features compared to other members of the family, which investigation may help in the development of selective drug compounds. Namely, it is a type I transmembrane dimeric protein with unique N-terminal proteoglycan-like (PG) domain, extracellular catalytic domain, and short cytoplasmic C-terminal segment. The above denoted traits make CA IX subject of structure-based drug design efforts. However, the expression and purification in high yield as well as crystallization experiments has been challenging. Therefore, protein variant bearing six amino acid...
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Studium konformačních efektů vazby ligandu na proteinovou strukturu
Šefců, Oskar ; Novotný, Marian (advisor) ; Hexnerová, Rozálie (referee)
This bachelor thesis aims to provide a comprehensive overview of the effects of ligand binding on the 3D structure of proteins. The focus will be on examples where ligand binding causes a significant conformational change between the structure without the ligand (apo structure) and the structure with the ligand (holo-structure). The thesis will discuss various techniques used to determine the structural changes caused by ligand binding, including X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. Furthermore, the thesis will provide insight into the structural and functional implications of ligand-induced conformational changes in proteins. Overall, this thesis will serve as a resource for understanding the role of ligand binding in modulating the 3D structure of proteins. Keywords: apo, holo, ligand, conformational change, IDP
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Gene expression analysis on a subgene level
Kloda, František ; Fišer, Karel (advisor) ; Novotný, Marian (referee)
RNA sequencing allows investigation of expression of singular genes in cells. It is possible to interpret the arisen data on multiple levels, each level providing a different type of information. Apart from measuring expression of whole genes, it is possible to quantify expression of singular exons, or transcripts (gene isoforms), which allows more detailed study of regulatory mechanisms. The main difference between approaches is in determining the origin of short reads. This step is significantly more complex in analysis of expression of transcripts, as transcripts derived from the same gene have typically larger rate of sequential similarity. In this thesis, we describe eleven tools for subgene level expression analysis a as comparison we have tested three of these tools on real patient data. The results provided by all three tools proved to be very similar with the greatest difference being the time needed for the analysis.
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Studies of precursor forms of SARS-CoV-2 3-CL protease
Nováková, Veronika ; Majerová, Taťána (advisor) ; Novotný, Marian (referee)
The SARS-CoV-2 virus (Severe acute respiratory syndrome coronavirus 2), the causative agent of the disease COVID-19 (coronavirus disease 2019), is an enveloped virus with a positive RNA genome. After the binding of the virus to the cell receptor and the release of the genome into the cytoplasm, the viral genome is immediately translated. It is translated into a polyprotein consisting of non-structural proteins necessary for viral replication. A part of this polyprotein is also protease 3-CL, which autocatalytically cleaves itself out from this polyprotein and further releases (and thus activates) individual proteins. Due to this key function in virus replication, 3-CL protease has become an important target for the design of specific antiviral drugs. As part of this thesis, we focused on the study of the precursor form of this protease, i.e. the protease that is still embedded in the polyprotein. The precursor form of the 3-CL protease was mimicked by attaching a 100 amino acid extramembrane C-terminal fragment of the nsp4 protein to the N-terminus of the mature form of the protease. The aim was to find out, how the sequence added to the N-terminus of the matured protease affects its enzymatic characteristics. As a part of this work, mutations of the N-terminal autoprocessing site were identified,...
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Recognition by intrinsically-disordered proteins
Hub, Michal ; Novotný, Marian (advisor) ; Del Conte, Alessio (referee)
Intrinsic disorder is one of the many traits that can affect the functionality of multiple naturally occurring proteins in biological systems. This thesis reports on the latest findings on mechanisms that intrinsically disordered proteins or intrinsically disordered regions utilize in specific recognition at the molecular level. Here, the general characteristics of intrinsically disordered proteins are summarized, along with the extent of their abundance throughout different lifeforms and the variety of their molecular recognition mechanisms depicted on specific examples. Furthermore, this thesis focuses on protein transitions between ordered and disordered states induced by interaction with its' binding partner. In the last two chapters, characteristic features of intrinsically disordered proteins are described, and attention is paid to the way these features influence cellular signaling pathways such as interactional promiscuity, the role of signaling hubs, alternative splicing, and post- translational modification.
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Tools for microbial community analysis using high-throughput amplicon sequencing data
Prášilová, Alžběta ; Větrovský, Tomáš (advisor) ; Novotný, Marian (referee)
Amplicon high-throughput sequencing is currently the most widely used technique to investigate complex microbial communities. In order to analyze amplicon sequencing data, specialized software and algorithms are required to turn raw sequencing data into biologically meaningful information. This thesis serves as an overview of the current pos- sibilities and an introduction to microbial community analysis. It provides a comparison of the most widely used bioinformatics pipelines as well as a newer pipeline SEED2 and a foundation for users to make a decision based on their preferences. 1
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Framework for retrieval and analysis of proteins apo and holo forms from PDB
Král, Adam ; Hoksza, David (advisor) ; Novotný, Marian (referee)
We developed a software framework that allows the analysis of ligand-free (apo) and ligand-bound (holo) forms of proteins that are accessible in PDB. The software downloads the current version of the PDB, divides the structures into groups of the same molecules, and these into apo and holo forms. Finally, it is possible to analyze pairs of apo and holo structures with respect to their different structural characteristics. In addition to the software work itself, we present the results of selected analyses of the current version of the data in the PDB. We also verify the results against previous work.
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Comparative genomic prediction of novel transmembrane adaptor proteins and their expression analysis.
Pitule, Pavel ; Drbal, Karel (advisor) ; Novotný, Marian (referee)
Transmembrane adaptor proteins play an important role in signal transduction cascades emanating from immune cell receptors leading to cellular effector mechanisms. Despite the lack of enzymatic function, their sequence contains interaction motifs which organize other proteins in time and space and initiates building of the signaling complexes. Functionally the most important and for the longest time known TRAPs are the ones associated with immune receptors. Their deficiency has severe impacts on the signal transduction and knock-out mice show dramatic phenotypes. The deficiency of Lat - the first discovered TRAP, which is not constitutively associated with immune receptors - has comparable effects on signaling in lymphocytes. The difference between the first group of TRAPs and Lat is not only the type of association with receptors, but also the lack of tyrosine - based activation motifs (ITAMs) in Lat, but Lat contains several other binding motifs also based on the phosphorylation of tyrosine. The group of TRAPs similar to Lat up today contains eight members, but all of them, except of Lat, has only very mild or undetectable phenotype in the knock-out mice. In this work I searched the protein databases for proteins with the characteristics of TRAP using the bioinformatic tools. Based on our...
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