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Expression, characterisation and biological role of Ddi II, putative protein partner of proteasomal complex
Sivá, Monika ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Cell homeostasis is maintained via strictly regulated processes. One of the important regulation systems is ubiquitin-proteasome proteolytic pathway. Proteins to be degraded are posttranslationally modified with polyubiquitin chains and targeted to the proteasome for degradation. Ubiquitin-proteasome system consists of several processes: ubiquitination of target substrates via set of enzymes, substrate transfer and degradation in the 26S proteasome. There are two ways of ubiquitinated substrate recognition via proteasome. It is either directly by proteasomal receptors or by protein shuttles. Shuttling factors bind polyubiquitinated target substrate and transfer it to the entrance of proteasomal cavity thanks to their typical domain architecture. The N-terminal ubiquitin-like domain binds to regulatory particle of the proteasome and the C-terminal ubiquitin-associated domain binds polyubiqitinated chains on substrates. This thesis focuses on the human DNA damage-inducible protein homolog 2 (Ddi2), a potential member of protein shuttles of humans, and on the interaction of its ubiquitin-like domain with its putative interaction partner, a proteasomal subunit PSMD2. PSMD2 has been cloned, expressed and purified in sufficient yields for further experiments. "Cold" as well as isotopically labeled UBL domain of...
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Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
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Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
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Expression, characterisation and biological role of Ddi II, putative protein partner of proteasomal complex
Sivá, Monika ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Cell homeostasis is maintained via strictly regulated processes. One of the important regulation systems is ubiquitin-proteasome proteolytic pathway. Proteins to be degraded are posttranslationally modified with polyubiquitin chains and targeted to the proteasome for degradation. Ubiquitin-proteasome system consists of several processes: ubiquitination of target substrates via set of enzymes, substrate transfer and degradation in the 26S proteasome. There are two ways of ubiquitinated substrate recognition via proteasome. It is either directly by proteasomal receptors or by protein shuttles. Shuttling factors bind polyubiquitinated target substrate and transfer it to the entrance of proteasomal cavity thanks to their typical domain architecture. The N-terminal ubiquitin-like domain binds to regulatory particle of the proteasome and the C-terminal ubiquitin-associated domain binds polyubiqitinated chains on substrates. This thesis focuses on the human DNA damage-inducible protein homolog 2 (Ddi2), a potential member of protein shuttles of humans, and on the interaction of its ubiquitin-like domain with its putative interaction partner, a proteasomal subunit PSMD2. PSMD2 has been cloned, expressed and purified in sufficient yields for further experiments. "Cold" as well as isotopically labeled UBL domain of...
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Expression and characterization of recombinant capsid protein from HIV and its mutants: towards inhibition of virus assembly
Sivá, Monika ; Konvalinka, Jan (advisor) ; Maloy Řezáčová, Pavlína (referee)
Human immunodeficiency virus infection has been a threat to the world for the last thirty years. It causes a condition called acquired immunodeficiency syndrome leading to complete collapse of immune system and death if not treated. There are many anti-HIV drugs that are part of the combined antiretroviral treatment but they only slow down the progress of the infection. Although the success of all the 31 antiviral agents is remarkable, the cure is not efficient enough. The research of potencial new HIV drugs is now focusing on new targets of viral inhibition. The capsid protein is a potential target of virion assembly and maturation inhibitors due to its multimerization features. The N-terminal domains of six capsid proteins create hexamers. These are connected to each other by dimers of the C-terminal domains according to X-ray and NMR studies. There are inhibitors that bind to the C-terminal domain, alter its conformation and weaken the protein-protein interaction of the dimer. Protein calorimetry is a method that could detect and quantify protein-protein interactions and thus capsid protein dimerization and its inhibition. We expressed and purified recombinant wild-type capsid protein and its C-terminal domain that both dimerize in solution and crystals. Their dimerization constant was determined by...
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