|
|
Role of RecQ helicases in maintenance of genomic stability during mitosis
Černoch, Marek ; Janščák, Pavel (advisor) ; Půta, František (referee)
Helicases are proteins capable of unwinding nucleic acids, their malfunction can be dangerous for genome stability of the cell. Five RecQ-family helicases identified in human cells participate in many cellular events during the whole cell cycle, including mitosis, and therefore are very important for correct functioning. The mutations in RecQ helicases can cause them to malfunction and seriously damage various cell processes, for example DNA replication, DNA damage control or sister chromatids separation. The mutations can also lead to dangerous syndromes, with the hallmark symptom of increased risk of cancer.
|
|
| |
|
|
Noncanonical human eIF4Es in and out of the RNA granules
Frydrýšková, Klára ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Valášek, Leoš (referee)
Eukaryotic translation initiation factor eIF4E1 (eIF4E1) plays a pivotal role in the control of cap-dependent translation initiation, occurs in P- bodies and is important for the formation of stress granules (SG). Human cells encompass two other non-canonical translation initiation factors capable of cap binding although with a lower affinity for the cap: eIF4E2 and eIF4E3. Here, I investigated the ability of individual eIF4E family members and their variants to localize to SGs and P-bodies in stress-free, arsenite and heat shock conditions. Under all tested conditions, both eIF4E1 and eIF4E2 proteins and all their variants localized to P-bodies unlike eIF4E3 protein variants. Under both arsenite and heat stress conditions all tested variants of eIF4E1 and the variant eIF4E3-A localized to SGs albeit with different abilities. Protein eIF4E2 and all its investigated variants localized specifically to a major part of heat stress-induced stress granules. Further analysis showed that approximately 75% of heat stress-induced stress granules contain all three eIF4Es, while in 25% of them eIF4E2 is missing. Large ribosomal subunit protein L22 was found specifically enriched in arsenite induced SGs. Heat stress-induced re- localization of several proteins typical for P-bodies such as eIF4E2, DCP-1, AGO-2...
|
|
|
Preparation of rat NK cell receptors using HEK293T expression system.
Celadová, Petra ; Bezouška, Karel (advisor) ; Půta, František (referee)
Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying within the same family. This is also the case of rat Clr-b as a cognitive ligand for rat NKRP1B. It has been shown that in rat, this inhibitory NKRP1B-Clr-b mutual receptor system is subverted by rat cytomegalovirus protein RCTL, a viral version of Clr-b, which serves as a decoy ligand for NK cells. The aim of my diploma thesis was cloning and production of the above mentioned C-type lectin-like proteins based on transient transfection of HEK293T cell line in a suspension culture. This expression system allows us not only to obtain proteins of our interest with a satisfactory yield but also in their native conformation, removing the need for time consuming and often fruitless refolding procedures required in case of using the E. coli expression system. Success was achieved in case of Clr-b and NKRP1B receptors from both WAG and SD strains. Proteins were purified using IMAC followed by gel filtration, identified by mass spectrometry and characterized by disulfide...
|
|
|
Organelle proteomics of parasitic protists
Jedelský, Petr ; Tachezy, Jan (advisor) ; Kolářová, Libuše (referee) ; Půta, František (referee)
Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles in G. intestinalis mitosomes, notparticipating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one and twodimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...
|
|
| |
|
|
The role of cyclophilins in splicing
Cit, Zdeněk ; Půta, František (advisor) ; Novotný, Ivan (referee)
Splicing is a process essential for the proper functioning of eukaryotic cells. It is a complicated and highly dynamic process, participated by large numbers of proteins which perform diverse functions, either directly within the splicing complex, or sometimes outside of it. Among the proteins, which play an important role in splicing, are cyclophilins. Cyclophilins probably cause conformational changes in the other components of splicing complex. They can also maintain them in the desire conformation thereby they contribute to the dynamics of the spliceosome. This work provides an overview of cyclophilins, which were confirmed to participate on pre-mRNA splicing, and summarizes suggestions of possible functions that these proteins may perform.
|
|
| |
|
|
CUG Codon in Pathogenic Yeasts of the Genus Candida
Marečková, Lucie ; Heidingsfeld, Olga (advisor) ; Půta, František (referee)
2. Abstract In many Candida species the standard leucine CUG codon is translated as a serine, although not in 100% cases. This dual specifity of the CUG codon has evolved through a mechanism that required codon ambiguity mediated by a unique tRNACAG, which is in vitro aminoacylated more often by serine than by leucine. This codon ambiguity has been tolerated for more than 170 million years. The explanation at least for now is that the CUG codon reassignment could have generated genetic diversity that facilitated occurrence of new phenotypes resistant to stress. Beside this, an important step was to reduce negative impact of the codon ambiguity by crucial mutations in the structure of the ser-tRNACAG. Candida species became a valuable experimental model for elucidation of the genetic code changes. While consequences of the CUG codon reassignment have been extensively studied in Candida albicans, this topic has not yet been addressed in Candida parapsilosis. Solving the structure of C. parapsilosis secreted proteinase Sapp1p provided a tool to carry out a "case study" of possible effects of the CUG codon ambiguity. The SAPP1 gene contains one CUG codon, and the respective serine is located on the loop in the close proximity of the active site of the proteinase.
|
|
| |
guest ::
