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Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2
Kalábová, Dana ; Obšilová, Veronika (advisor) ; Pavlíček, Jiří (referee) ; Žáková, Lenka (referee)
Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2 Abstract Caspase-2 is a protease standing apically in the cascade of reactions leading to apoptosis. Properly functional apoptosis eliminates damaged cells, autoreactive lymphocytes or redundant groups of cells in ontogeny. The process of caspase-2 activation must be precisely regulated. One of the described ways of caspase-2 regulation causing its inhibition is posttranslational modification phosphorylation with subsequent binding of the regulatory scaffold protein 14-3-3. The aim of this dissertation is to explain the molecular mechanism of this regulation. To understand the interaction between the proteins, it was necessary to first identify the phosphorylation sites in the caspase-2 molecule recognized by the 14-3-3 protein and then describe the detailed structure of the binding complex. The structure was characterized by a number of biochemical and biophysical methods, such as analytical ultracentrifugation, native electrophoresis in TBE buffer, polarization-fluorescence assay, hydrogen/deuterium exchange coupled to mass spectrometry, or crystallization; and the results led to stimulating conclusions. Activation of caspase-2 begins with its binding to adaptor proteins, cleavage and dimerization of the catalytic subunits. The...
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Regulation of transcription in Streptococcus pneumoniae
Zajíčková, Adéla ; Doubravová, Linda (advisor) ; Hnilicová, Jarmila (referee)
Regulation of transcription in Streptococcus pneumoniae Streptococcus pneumoniae, an opportunistic human pathogen, encodes a single eukaryotic-like Ser/Thr kinase, StkP, which plays a crucial role in cell growth and division. In a phosphoproteomic study, StkP-dependent phosphorylation of SigAunder antibiotic- induced stress conditions was demonstrated in vivo. SigA, a housekeeping factor, in complex with RNA polymerase, is responsible for the transcription of most genes in S. pneumoniae. Therefore, phosphorylation of this important regulator might play an important role in transcriptional regulation. In this work, we confirmed that StkP phosphorylates SigA at residues T291 and T306 in vitro. However, we could not detect SigA phosphorylation in vivo, and the role of phosphorylation remains to be elucidated. We detected all RNAP subunits, including SigA, in the RNAP complex of S. pneumoniae by mass spectrometry analysis. The protein Spr0726 was identified as part of the RNAP complex. The reciprocal interaction between Spr0726 and RNAP was confirmed by immunoprecipitation experiments. In functional studies, we found that deletion of the spr0726 gene leads to a decrease in the amount of the subunits β and α in the RNAP complex. Our data suggest that the Spr0726 protein may affect the stability of the RNAP...
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The impact of post-translational modifications on TRPC5 ion channel activation and modulation
Mitro, Michal ; Zímová, Lucie (advisor) ; Dolejší, Eva (referee)
Transient Receptor Potential Canonical 5 (TRPC5), a calcium-permeable ion channel, acts as a versatile receptor in sensory neurons, kidneys, and the brain, impacting inflammatory responses and various types of pain. While post-translational modifications influence TRPC5 gating and membrane trafficking, only a few have been described so far. The identification of phosphorylation sites was based on available high-throughput bioinformatics and mass spectrometry data. Subsequently, functional characterization of these sites was conducted by introducing phospho-mimicking aspartate or phospho-null alanine mutations using site-directed mutagenesis. Utilizing patch-clamp in whole-cell configuration, membrane currents evoked by voltage or agonist stimuli were examined. The results revealed that individual substitutions at the N-terminal S193 and S195 with aspartates significantly slowed the gating kinetics. Additionally, a gain-of-function phenotype was observed with S193A. Molecular dynamics simulations provided insight into how phosphorylation at S193 induces changes in interactions among neighboring subunits. Moreover, biotinylation experiments indicated that the alterations in the activity of the S193 mutations are not due to increased targeting of the channels to the plasma membrane. Taken together,...
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Functional analysis of the ERK signaling pathway in epithelial cells
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee)
The MAPK/ERK pathway, which is evolutionarily conserved in eukaryotes is one of the most intensively studied signaling pathways and consists of a three-tier cascade of Raf- MEK-ERK protein kinases. A variety of extracellular signals are transduced from receptors to hundreds of substrates by a series of sequential phosphorylations leading from Raf to MEK to ERK. The ERK pathway regulates a plethora of cell- and extracellular signal- specific responses such as gene expression, proliferation, differentiation, migration, and apoptosis. The proper execution of these physiological processes requires a precise temporal and spatial regulation of the pathway and disruption of the regulatory mechanisms leads to pathological consequence such as tumor transformation. Specificity and regulation of signal transduction are provided in part by the presence of isoforms at each level of the ERK signaling pathway. The functional differences between the effector protein kinases ERK1 and ERK2 have been controversial for a long time, but it is still unclear how important they are in achieving an appropriate cellular response. In this work, we focused on the functional characterization of ERK1 and ERK2 isoforms in MDCK epithelial cells. Specifically, we examined the effects of ERK2 inactivation on cell morphology and...
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Role of the WIP1 phosphatase in the nucleolus
Palková, Natálie ; Macůrek, Libor (advisor) ; Sztacho, Martin (referee)
Protein phosphatase 2C delta (known as WIP1) is an important negative regulator of the DNA damage response (DDR) signalling. As a chromatin-bound protein, it dephosphorylates and thus inactivates ATM kinase and the transcription factor p53. Increased expression of WIP1 suppresses the function of the tumour suppressor p53 and contributes to the development of several cancer types, including breast and brain tumours. In recent years, it has been shown that WIP1 can also regulate cellular processes that are not directly linked to DDR, such as ensuring telomere stability. However, alternative functions of the WIP1 protein have not yet been fully understood. In this work, we described a novel role of the WIP1 phosphatase in the nucleolus, the organelle responsible for ribosome biogenesis. We found that WIP1 associates with many nuclear proteins, and using deletion mutants, we identified a nucleolar localisation sequence (NoLS) at the C-terminus of the protein. Using super-resolution microscopy, we detected the localisation of WIP1 phosphatase in the fibrillar centres of the nucleolus. We employed an inducible Cas9 system for generating double-strand breaks in ribosomal DNA and found that WIP1 has an impact on recruitment of DNA repair factors to the nucleolar caps. Analysis based on quantitative...
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Hypothetical protein Spr1962 as a new substrate of the signalling pathway of the Ser/Thr protein kinase StkP and phosphatase PhpP.
Mašková, Kateřina ; Ulrych, Aleš (advisor) ; Fišer, Radovan (referee)
Hypothetical protein Spr1962 as a new substrate of the signalling pathway of the Ser/Thr protein kinase StkP and phosphatase PhpP The extracellular human pathogen Streptococcus pneumoniae encodes in its genome only a eukaryotic-type serine/threonine protein kinase (StkP) and the corresponding PP2C phosphatase (PhpP) in its genome. This makes it a unique model organism for the studying of signaling pathways in bacteria. To date, several substrates of this signaling protein have been identified and characterized, including DivIVA, KhpB, FtsA, FtsZ, MacP, GlmM, GpsB, ComE and others. These proteins are involved in various cellular processes, including cell division and peptidoglycan biosynthesis. In a global phosphoproteome study based on the LC- MS approach, one of the potential new substrates was the hypothetical protein Spr1962, whose phosphorylation was detected only in the hyperphosphorylated strain with deleted PhpP phosphatase. The aim of this work was to characterize this new substrate Spr1962 and to clarify its possible function. Based on the significant structural similarity to Spr1962 the FloT flotillin protein of Bacillus subtilis, it was suggested that it might be a protein with an analogous function in pneumococci. Deletion of this gene in different genetic backrounds was found to cause an...
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Trk1 Potassium Importers - key transport systems for yeast cell fitness and stress tolerance
Masaryk, Jakub ; Sychrová, Hana (advisor) ; Heidingsfeld, Olga (referee) ; Malínský, Jan (referee)
One of the key prerequisites for yeast cell growth is the uptake of essential compounds, such as potassium. Potassium is a vital monovalent cation and its sufficient intracellular concentration is crucial for various processes, for instance: regulation of membrane potential and cell turgor, enzymatic activity, and protein synthesis. A sufficient internal concentration of potassium is also one of the pivotal signals for cell division. However, as also excess of potassium might lead to unfavourable physiological consequences in yeast, such as deacidification of vacuoles and depolarization of plasma membrane, it is imperative for the yeast cells that the whole process of potassium acquisition is a tightly regulated affair, in order to maintain proper potassium homeostasis. In yeast Saccharomyces cerevisiae, uniporter Trk1 is considered a key player in potassium uptake. The presented thesis aimed to provide novel knowledge regarding Trk1, more specifically to study its ability to modify its capacity for potassium uptake, putative regulation by phosphorylation, and involvement in the survival of glucose-induced cell death (GICD). Additionally, potassium-uptake systems in selected non-conventional species were characterized as well. The most distinctive feature of Trk1 is its alleged ability to switch...
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The effect of 14-3-3 protein on intradomain interaction of ubiquitin ligase Nedd4-2
Pohl, Pavel ; Obšilová, Veronika (advisor) ; Žáková, Lenka (referee) ; Pavlíček, Jiří (referee)
EN The human ubiquitin ligase Nedd4-2 (NEDD4L) ubiquitinates a wide range of membrane proteins and receptors, playing a key role in maintaining homeostasis. This enzyme is regulated by phosphorylation and subsequent interaction with 14-3-3 proteins, which primarily affects its ability to interact with various substrates. However, very little is known about the molecular basis of this protein-protein interaction. In this work, we focused on biophysical characterization of the role of individual phosphorylation sites and also on mapping the structural changes in the Nedd4- 2 protein induced by 14-3-3 protein binding. Our experiments using analytical ultracentrifugation methods revealed that two phosphorylation sites Ser342 and Ser448 are primarily required for stable binding of Nedd4-2 to 14-3-3 proteins. The crystal structure of the 14-3-3ηΔC:Nedd4-2335-455 T367A complex than revealed the simultaneous binding of both phosphorylated residues to the binding groove of 14-3-3 protein. Subsequent modeling based on small-angle X-ray scattering and chemical cross-linking data combined with mass spectrometry indicated extensive structural changes in the individual domains of the Nedd4-2 protein. Binding of 14-3-3η protein blocks the WW3 domain of Nedd4-2 in the central channel of 14-3-3 protein, while...
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The effect of phosphorylation in the regulation of Rho-GTPases
Novotná, Michaela ; Rösel, Daniel (advisor) ; Hála, Michal (referee)
This bachelor thesis is focused on regulating of the activity of the Rho GTPases. Rho GTPases are important signalling proteins that are involved in regulation of many cellular processes, for example dynamic cytoskeleton changes (especially actin fibres) associated with cell adhesion and migration, vesicle trafficking or cell cycle. The activity of the Rho GTPases is primarily regulated by other proteins affecting the presence GTP/GDP or by posttranslational modifications. This thesis is focused on a narrow section of these modifications, namely phosphorylations. Phosphorylation of the Rho GTPases affects for example their activity, localization, or degradation. Understanding the mechanism of regulation of Rho GTPase activity is important for comprehension of cellular processes. Rho GTPases are involved, for example, in tumour cell invasiveness. By studying Rho GTPases and their regulation, it is possible to better target potential treatment or even prevent the occurrence of the metastases.
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Non-conventional bacterial signaling pathways
Krupička, Jiří ; Branny, Pavel (advisor) ; Beranová, Jana (referee)
Two component systems were traditionally considered as main phosphorylation systems of bacteria involved in cell signalling. Recently, attention focuses increasingly on bacterial eukaryote-like Ser/Thr protein kinases (eSTKs). These protein kinases are structurally similar to their eukaryotic counterparts. Some eSTKs possess additional domains such as extracellular PASTA domains that were discovered in a variety of gram-positive bacteria. It has been proved that these domains can act as sensors for unlinked peptidoglycan fragments. However, majority of environmental signal molecules still remains unknown. eSTKs phosphorylate a broad spectrum of substrates including proteins involved in various cell processes such as virulence, cell wall biosynthesis, cell division, and central and secondary metabolism. Cross talk between eSTKs and two component systems also occurs. In this thesis, the current knowledge about eSTKs and their significant substrates in different bacterial species is discussed.
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