|
| |
|
|
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
|
|
|
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
|
|
|
Searching for a physiological partner of Ddi2 (DNA damage-inducible protein homolog 2) protein
Kurfürst, Jaroslav ; Grantz Šašková, Klára (advisor) ; Vaněk, Ondřej (referee)
One of the most important cellular processes, essential not only for protein degradation, is the so called ubiquitin-proteasome system. A key player in this system is ubiquitin, a small protein with unique ability to form various chains. Cellular proteins marked for degradation via ubiquitin, are recruited to the proteasome either by a direct interaction with one of the intrinsic proteasomal receptors, or by adaptor proteins. These proteins typically possess ubiquitin-like domain and ubiquitin associated domain that predispose them for delivering ubiquinated proteins to the proteasome. Adaptor protein called Ddi2 differs from other members of this family by possessing additional domain called the retroviral protease like domain. This domain is structurally similar to HIV protease and its proteolytic function has been discovered only recently. Due to the presence of this proteolytic domain one could expect that Ddi2 might be a deubiquitinase. Here we therefore tested the possible cleavage of diubiquitin chains by recombinantly prepared Ddi2 protein. We can conclude that Ddi2 did not show any deubiquitinating activity in given conditions.
|
|
|
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.
|
|
|
Narušení metabolismu proteinů a jeho efekt na signalizaci cytokininů
Dufek, Martin
Cytokinins are N6 substituted adenine derivatives that affect many aspects of plant growth and development. A multistep phosphorelay systém, including hybrid sensor kinases, histidinecontaining phosphotransfer proteins and two sets of response regulators, is the key part of cytokinin signaling. However, a recent evidence indicates a crucial role for the proteasomeubiquitin systém (UPS) in the cytokinin response. Here, in this thesis entitled 'Protein metabolism disruption and its effect on cytokinin signaling' the major protein degradation mechanisms are outlined and the present-day model of cytokinin metabolism and signaling is discussed. In the experimental part, the UPS-cytokinin interaction is probed in a growth response experiment, an LC-MS proteome analysis and by the datamining of previously published proteomics data. The results indicate an interesting dosage-dependent balance between cytokinin- and proteasome-mediated signaling, and a huge impact of proteasome inhibition on cytokinin response proteins. Key words: proteasome, ubiquitin, growth response, protein degradation, LC-MS, proteome
|
|
|
Proteasomes and DNA virus infection
Vinšová, Barbora ; Drda Morávková, Alena (advisor) ; Motlová, Lucia (referee)
The development of virus infection depends on virus - host interactions. Millions of years of ongoing virus - host coevolution led to formation of many antiviral defense mechanisms as same as virus evasion strategies. Viruses have learned to intervene in the various cellular processes, modify it and take advantage of particular cellular components. One of those cellular components widely utilised by viruses is the ubiquitin-proteasome system. Proteasomes are multisubunit protein structures that under normal conditions provide degradation of damaged, missfolded or redundant cellular proteins. With their function proteasomes contribute to regulation of various cellular processes and maintain balance of proteins ratio. Viruses utilise those structures for protein degradation in order to evade host immunity system and deregulate cell cycle, to entry and unpacking of virions or in order to favor virus replication. This thesis is conceived to briefly summarize interactions of cellular ubiquitin-proteasome system and DNA viruses.
|
guest ::
