|
|
Structural studies of selected protein complexes involved in signal transduction
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Bouřa, Evžen (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions are critical for most physiological and pathophysiological processes. Detailed characterization of these interactions is therefore essential not only to understand the nature of these events, but also to design strategies to target these interactions. This work focuses on the study of the structure and interactions of several proteins and their complexes. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the p38/JNK protein kinase pathways, thereby directing cells toward an inflammatory response or apoptosis. ASK1 interacts with thioredoxin (TRX), a small dithiol oxidoreductase, which inhibits ASK1, but the mechanism of this inhibition has not been clarified. CaMKK1 and CaMKK2 are Ca2+ /calmodulin (CaM)-dependent protein kinases that regulate cellular energy balance, memory, and inflammation, among others. Both are inhibited by 14-3-3 proteins, but despite their domain and sequence similarities, the extent of 14-3-3 protein- mediated inhibition is different. Estrogen receptor alpha (ERα) is a nuclear receptor involved in breast cancer. Tamoxifen, an ERα antagonist, is used to treat this disease, but resistance often develops. 14-3-3 proteins interact with ERα and inhibit its transcriptional activity,...
|
|
|
Biophysical characterization of the N-terminal part of protein kinase ASK1.
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...
|
|
|
Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
|
|
|
Structural and functional characterization of a flaviviral methyltransferase.
Todd, Matthew Zagorey ; Bouřa, Evžen (advisor) ; Honzejková, Karolína (referee)
Genus flavivirus, member of the Flaviviridae family, is a wide-spread group of viral pathogens that pose a global threat to human health. Infections result in neurological and other diseases such as encephalitis, meningitis, microcephaly or haemorrhagic fever with direct anti-viral treatment restricted to only a few members including Yellow Fever Virus (YFV) and Japanese Encephalitis Virus (JEV). The flaviviral genome consists of a positive sense single-strand RNA, translated into a long viral polyprotein cleaved by host and viral proteases into structural and non-structural proteins. Non-structural protein 5 (NS5) is a highly conserved viral protein consisting of a C-terminal RNA-dependent RNA-polymerase (RdRp) and N-terminal methyltransferase (MTase) domain. Flaviviral MTases catalyse the final step of genomic RNA cap formation, that plays a crucial role in viral translation, replication and host immune system evasion. Due to its conservative nature, the flaviviral MTase is a promising drug target. It's structural analysis is needed for the development of broad-spectrum inhibitors and anti-viral treatment. Key words Flavivirus, St. Louis Encephalitis Virus, Flaviviral methyltransferase (MTase), N-7 methylation, 2'-O methylation.
|
|
|
Biophysical characterization of the N-terminal part of protein kinase ASK1.
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...
|
|
|
Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
|
guest ::
RSS feed.