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Ubiquitin-proteasome system in studies of its inhibition and its utilization in the cell-based assay measuring viral protease activity
Fürst, Eliška ; Majerová, Taťána (advisor) ; Čermák, Lukáš (referee)
and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...
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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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The role of Fbxo38 ubiquitin ligase in mouse spermatogenesis
Zobalová, Eliška ; Čermák, Lukáš (advisor) ; Stopka, Pavel (referee)
Cullin-dependent ubiquitin ligases are responsible for the regulation of most cellular processes. Despite their mutated forms being the cause of many human diseases, their physiological roles are not sufficiently described. In the presented results, we focused on the physiological role of ubiquitin ligase SCFFBXO38 (SKP1-CULLIN1-FBXO38), whose mutated forms are responsible for the progression of distal neuropathy. Preparation of mouse model deficient in FBXO38 revealed that homozygous pups were born in a lower than expected ratio. Animals were growth-retarded, both at the level of the whole organism and individual organs, especially the liver and testes. Males with a deletion in the Fbxo38 gene had significantly lower reproductive capacity, which was associated with lower production of mature sperm and pathological changes in the structure of seminiferous tubules. We found that the FBXO38 protein is functionally expressed in Sertoli cells responsible for regulating spermatogenesis and seminiferous tubules integrity. Detailed analysis of spermatogenic populations revealed a defect at the level of spermatocyte differentiation. The dynamics of this differentiation depend on the hematotesticular barrier functional integrity formed by the intercellular junctions of Sertoli cells. We confirmed that the...
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Ubiquitin-proteasome system in studies of its inhibition and its utilization in the cell-based assay measuring viral protease activity
Fürst, Eliška ; Majerová, Taťána (advisor) ; Čermák, Lukáš (referee)
and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...
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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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The ubiquitin–proteasome system is involved in the regulation of activity of spermadhesin aqn1 and acrosin inhibitor, the two sperm surface proteins, during porcine fertilization
Jonáková, Věra ; Yi, Y.J. ; Postlerová, Pavla ; Pěknicová, Jana
The spermadhesin AQN1and acrosin inhibitor (AI/SPINK2) proteins bind to the sperm plasma membrane at ejaculation. The AQN1 has been implicated in sperm binding to zona pellucida (ZP) of the oocyte as well as in sperm interactions with the epithelium of the oviductal sperm reservoir. The SPINK2 protects spermatozoa from proteolytic degradation during their trip up the female genital tract toward the oocyte. This study examined the role of two components of the 19S proteasome regulatory complex, the ubiquitin C-terminal hydrolase UCHL3 and PSMD8 in the AQN1-mediated boar sperm binding to zona pellucida. Interaction of PSMD4 subunit with the acrosomal surface-associated acrosin inhibitor AI/SPINK2 provided another line of evidence for the presence of 26S proteasomes on the sperm surface. Detection of the ubiquitinated forms of SPINK2 supports the hypothesis that SPINK2 activity is controlled by ubiquitin-proteasome system (UPS). The activity of the porcine AQN1, and thus the efficiency of sperm-oocyte recognition/binding, may be controlled by elements of the sperm surface-bound UPS, in particular by UCHL3, and by proteasomal regulatory complex subunit PSMD8. Ubiquitinated isoforms of AQN1 were also detected in boar sperm extracts. The UCHL inhibitor ubiquitin aldehyde and the antibodies against UCHL3 or PSMD8 increased the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). In contrast, the addition of recombinant UCHL3 to fertilization medium significantly reduced polyspermy rates, while maintaining satisfactory rate of monospermic fertilization (~50%). These results are significant for production agriculture. The high level of polyspermy that hinders porcine IVF for commercial embryo transfer could be mitigated by the modulation of the UCHL3 and/or PSMD8 activity.
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