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Structural studies of metal-dependent hydrolases: Histone Deacetylase 6 and Glutamate Carboxypeptidase II
Shukla, Shivam ; Bařinka, Cyril (advisor) ; Stříšovský, Kvido (referee) ; Kolenko, Petr (referee)
Zinc-dependent hydrolases are a class of metalloenzymes that require zinc ions to catalyse hydrolytic reactions. Structural studies of these enzymes shall provide detailed information about the processing of their natural substrates, domain organization, and overall structural fold. This thesis describes the structural properties of two different metallohydrolases 1) human histone deacetylase 6 (HDAC6) and 2) glutamate carboxypeptidase II (GCPII) by utilizing a different set of biophysical techniques. HDAC6 is a structurally unique multidomain enzyme comprised of unstructured and globular domains. It regulates the plethora of cellular processes by removing an acetyl group from lysine side chains of target proteins. It has been known to deacetylate non-histone substrates such as tubulin, Hsp90, cortactin, and peroxiredoxins. Given its structural complexity, complete structural information of full-length HDAC6 is missing and available information is limited to its globular domains only. Hence, the integrative approach was employed in combining experimental data from several orthogonal biophysical techniques to build an in-solution structural model of HDAC6. The study reports that HDAC6 adopts multiple conformations due to its unstructured regions and exists as an ensemble of conformers in solution....
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Inhibitors of rhomboid proteases as tools for cell biology
Kuzmík, Ján ; Stříšovský, Kvido (advisor) ; Hlouchová, Klára (referee)
Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...
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Proximity proteome of intramembrane serine protease RHBDL4
Boháčová, Šárka ; Stříšovský, Kvido (advisor) ; Brábek, Jan (referee)
Regulated intramembrane proteolysis is an interesting process involved in a multitude of cellular pathways. Enzymes which catalyse this are termed intramembrane proteases (IMPRs), cleaving proteins passing through the membrane within their transmembrane domain. Rhomboid proteases are serine IMPRs. They are widely distributed among organisms and evolutionarily conserved, but despite many efforts, their physiological roles are largely unexplored. RHBDL4 is a mammalian rhomboid protease localised to the endoplasmic reticulum. It is involved in the development of colorectal cancer, which makes it an important focus of research, but its physiological function is not well understood. In order to explore it, I established and employed a proximity proteomics approach, termed APEX2. It is based on biotinylation of proteins in the spatial proximity of the target in the physiological environment of intact living cells. Labelled proteins are subsequently purified, identified and quantified by mass spectrometry. Exploring the physiological vicinity of RHBDL4, its interaction partners and substrates can be revealed and the detailed subcellular compartment, where RHBDL4 resides, can thus be inferred. During three independent experiments in HCT116 cell line, three proteins emerged repeatedly in the RHBDL4...
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Development of inhibitors of rhomboid proteases as tools for the study of their biological functions
Tichá, Anežka ; Stříšovský, Kvido (advisor) ; Šedo, Aleksi (referee) ; Konvalinka, Jan (referee)
Rhomboids are intramembrane serine proteases that belong to the evolutionarily widespread rhomboid superfamily. Rhomboids developed a slightly different catalytic mechanism compared to classical serine proteases; they utilise a catalytic dyad (Ser/His) instead of the common triad (Ser/His/Asp), and the rhomboid active site is buried in the membrane. This, coupled with their hydrophobicity, makes them quite difficult to study. Therefore, even though they are known to be involved in several important biological processes it is still not clear how exactly most of them are involved in the regulation of or in the pathologies of diseases related to these processes (such as malaria, Parkinson's disease or cancer). Our understanding is hindered by the lack of tools for their characterisation both in vitro and in vivo. In my thesis I present new fluorogenic substrates based on the LacYTM2 sequence, which is hydrolysed by several different rhomboid proteases. Using Förster resonance energy transfer (FRET)-based methods, these substrates are suitable for continuous monitoring of rhomboid activity in vitro. Modifications in the P5-P1 residues can improve selectivity for a specific rhomboid, the choice of FRET pair of fluorophores that absorbes light of longer wavelengths makes them suitable for high throughput...
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Mechanism of regulation of EGFR receptor ligand activation via the intramembrane pseudoprotease iRhom and cell surface metalloprotease ADAM17
Trávníčková, Květa ; Stříšovský, Kvido (advisor) ; Vomastek, Tomáš (referee)
Signalling through the EGF receptor is subject to a complex and multilayered regulation. One such mode of regulation is through control of ligand production which plays an important role in fine- tuning EGF receptor activation. In mammals, the production of soluble, biologically active forms of EGF receptor ligands relies on ADAM metalloproteases, predominantly ADAM10 and ADAM17. Recently, a pseudoprotease from the rhomboid-like family of intramembrane proteases, iRhom, emerged as a key positive regulator of ADAM17. However, Drosophila iRhom has also been implicated in the negative regulation of EGF receptor signalling by promoting the degradation of precursors of its ligands. Cell culture based assays suggest that mammalian iRhoms might also be involved in a similar process. In this thesis, the effect of mammalian iRhom overexpression on the levels of EGF receptor ligands has been investigated. Contrary to previous findings, the data presented in this thesis suggest that the observed effect might not be entirely iRhom specific, for the inactive mutants of rhomboid proteases also diminish the levels of EGF receptor ligands. Nor do we find the effect to be specific to EGF receptor ligands, as unrelated transmembrane proteins were also depleted by iRhom overexpression. The coexpression of ADAM17 was...
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Substrate specificity, mechanism and activity regulation of the rhomboid family intramembrane proteases
Škerle, Jan ; Stříšovský, Kvido (advisor) ; Hof, Martin (referee) ; Heidingsfeld, Olga (referee)
Intramembrane proteases from the rhomboid-like superfamily are enzymes widely distributed and conserved in all domains of life. They participate in many important processes such as membrane protein quality control or mitochondrial dynamics. Their activity is also linked with diseases like Parkinson's disease or cancer. This makes them potential therapeutic targets. In this work we tried to elucidate in more detail the mechanism of action of the main model intramembrane protease, GlpG from E. coli. We also focused on the mechanism of eukaryotic rhomboid RHBDL2, one of the four mammalian rhomboids, function of which is poorly understood. To acquire more detailed information about substrate-enzyme interaction, we synthesized a series of novel peptidyl-chloromethylketone inhibitors derived from natural rhomboid substrate TatA from P. stuartii. Crystal structure of the complex of GlpG with these inhibitors revealed four substrate binding subsites (S1 to S4) of the enzyme and explained its observed substrate specificity structurally. This study showed that substrate cleavage rate can be dramatically modified by changing the substrate sequence in positions P1 to P5. This helped us develop fluorogenic transmembrane peptide substrates for rhomboid proteases, which are usable in detergent and liposomes, and...
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The role of ADAM17 and other metalloproteases in liver pathological processes
Žbodáková, Oľga ; Sedláček, Radislav (advisor) ; Muchová, Lucie (referee) ; Stříšovský, Kvido (referee)
1 Abstract Liver fibrosis is a condition described by extensive accumulation of scar tissue in the liver. With further progression, it leads to cirrhosis or even to hepatocellular carcinoma. Liver fibrosis accompanies every chronic liver disease and its prevalence in adult European population is estimated to be around 4%. During my dissertation work, I studied the function of three members of Metzincin family of metalloproteinases - ADAM17, ADAM10 and MMP-19, in liver fibrosis and liver regeneration using mouse genetic models. ADAM17 and ADAM10 are important regulators of signalling pathways which are involved in immune response as well as differentiation. Both proteases are able to cleave ectodomains of their substrates from cell membrane, affecting bioavailability of ligands and functionality of receptors. Several of their substrates are involved in liver pathologies. MMP-19 on the other hand, is a metalloprotease mainly involved in extracellular matrix cleavage, important process in fibrosis development, as well as resolution of fibrosis. Our results demonstrate that ablation of ADAM10 results in increased susceptibility to liver fibrosis in mice, both spontaneous and toxin induced. ADAM10 deficiency affected biliary epithelium, as we detected higher markers of biliary damage in serum of ADAM10 deficient...
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Protein quality control in the secretory pathway of eukaryotic cells
Bařinková, Markéta ; Stříšovský, Kvido (advisor) ; Černý, Jan (referee)
More than 30 % of the cellular proteome enters the secretory pathway during biogenesis in eukaryotic cells. The secretory pathway then ensures that these proteins are correctly folded, undergo necessary post- translational modifications, and reach their target site in membrane organelles or outside of the cell. Since a significant number of the nascent proteins in the pathway are or become dysfunctional, the cell must possess quality control mechanisms by which to weed them out. As proteins travel through the secretory pathway they may be degraded by various pathways in the endoplasmic reticulum, Golgi apparatus, endosomes, or at the plasma membrane. These degradatory pathways utilize a number of molecules including chaperones, ubiquitin ligases, and many others. They are coordinated by a unifying principle - the unfolded protein response, which acts as a support mechanism in case the degradation pathways are overwhelmed. The study of protein quality control mechanisms is necessary as they help us understand the production of a significant portion of the cellular proteome. Furthermore, defects in these degradation pathways are linked to several human diseases such as cystic fibrosis or some neurodegenerative diseases. These protein degradation pathways have been studied for decades, but thanks to...
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Rhomboid family intramembrane proteases in prokaryotes: mechanism, substrate repertoires and biological functions in the Gram-positive bacterium Bacillus subtilis.
Began, Jakub ; Stříšovský, Kvido (advisor) ; Bařinka, Cyril (referee) ; Krásný, Libor (referee)
Rhomboid proteases are a class of serine intramembrane proteases, a large family of enzymes that catalyze the proteolytic cleavage of membrane proteins within their transmembrane regions, in the hydrophobic environment of cellular lipid membranes. Rhomboid proteases were discovered in 2001 in Drosophila. In their pioneering study, Lee et al. identified the essential role of Rhomboid-1 protein (Rhom-1), which proteolytically activates the epidermal growth factor (EGF) receptor signaling pathway, in the early stages of fly eye development. Members of the rhomboid superfamily - active proteases (rhomboids) as well as their catalytically-dead counterparts (rhomboid-like proteins, including iRhoms and Derlins) - are widely conserved, implying their biological significance. Rhomboids are present in all kingdoms of life from archea to humans, while proteolytically inactive rhomboid-like proteins are present in eukaryotes only. Rhomboid superfamily proteins play roles in a wide range of processes, as diverse as signaling in metazoan development, mitochondrial biogenesis in yeast, host- cell invasion by protozoan parasites, protein quality control in the endoplasmic reticulum (ER) or bacterial quorum sensing. Rhomboids are the best understood intramembrane proteases from a structural and mechanistic points...
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Inhibitors of rhomboid proteases as tools for cell biology
Kuzmík, Ján ; Stříšovský, Kvido (advisor) ; Hlouchová, Klára (referee)
Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...
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