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Structure and function of ubiquitin-activating enzyme UBA1
Pánska, Dominika ; Vávra, Jiří (advisor) ; Onhajzer, Jakub (referee)
Ubiquitin-activating enzyme, also known as UBA1, is an essential enzyme in the process of ubiquitin activation in all eukaryotic cells, and its loss, as well as complete disfunction, leads inevitably to death of an organism. In humans, nuclear (UBA1a) and cytoplasmic (UBA1b) isoforms are known. Ubiquitination, the process at the beginning of which UBA1 stands as a key player, is important for proteasomal degradation of proteins, cell cycle progression, DNA damage repairs, fertilisation, as well as antiviral response of an organism. Its impaired function is the cause of many oncological and neurodegenerative diseases or just recently discovered autoinflammatory syndrome VEXAS. This thesis includes basic and current knowledge about UBA1, its structure, functions in cells and pathologies. Key words: UBA1, ubiquitin, catalytic cysteine, proteasomal degradation, VEXAS
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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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