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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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Ddi1-like proteins: a novel family of retroviral-like aspartyl proteases
Šmilauerová, Kristýna ; Grantz Šašková, Klára (advisor) ; Šmahel, Michal (referee)
Ubiquitin-proteasome system is one of the key pathways which maintain cell homeostasis. Its purpose is to degrade damaged, misfolded or unnecessary proteins. It is also involved in multiple other processes such as DNA damage repair, cell cycle control or signaling. The entire system consists of multiple components, which are mutually strictly regulated. Important part of this system is group of so called proteasome adaptor proteins. Their role is to recognize and bind targeted substrates and transport them to the proteasome for degradation. Ddi1-like (abbrev. from DNA damage-inducible protein 1) protein family, a group of proteins with retroviral aspartyl protease-like domain, belongs to proteasome adaptor proteins. Global biological role of this protein family is only partially understood the most studied member is Ddi1 protein from Saccharomyces cerevisiae, and it is thus a subject of active research. This thesis summarizes published information about this protein family, describes its general characteristics and known functions, situates them in the context of cell processes and thereby might suggest the course of further study.
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