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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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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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Significance of the S1 subsite of rhomboid intramembrane proteases for catalysis and inhibitor design
Kučerová, Jolana ; Stříšovský, Kvido (advisor) ; Hodek, Petr (referee)
This thesis focuses on the development of specific inhibitors of rhomboid intramembrane proteases. These inhibitors are needed for the cell-biological investigation of rhomboid proteases and their potential pharmacological targeting, as rhomboid proteases have been associated with various diseases, such as malaria, Parkinson's disease, cancer or toxoplasmosis. The thesis advisor's laboratory has recently discovered the first such group of compounds, the peptidyl ketoamides. To exploit them fully, it is necessary to examine their properties and the possibilities of their modifications. In this work, synthetic fluorogenic substrates and enzyme kinetics were used to examine the possibilities of exploiting the S1 subsite in the rhomboid active site for rhomboid inhibitor design. Furthermore, using variants of these substrates modified by unnatural amino acids in the P1 position, the mechanism of water transfer to the rhomboid active site was investigated. Comparison of cleavage rates of ten fluorogenic substrates modified in the P1 position revealed that the E. coli rhomboid protease GlpG strongly prefers side chains -CH2-CH3 and -CH3 in the P1 position (i.e. binding into the S1 subsite). This trend was apparent also with peptidyl ketoamide inhibitors. The present substrate and inhibitor study suggests...
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Intramembrane proteases and their medical significance
Deylová, Anna ; Stříšovský, Kvido (advisor) ; Macůrková, Marie (referee)
Intramembrane proteases are membrane enzymes whose active site is buried below the surface of the biological lipid membrane, and which cleave other membrane proteins within their transmembrane domains. They are divided into four families according to their catalytic residues - aspartate, serine (often called rhomboids), metalloproteases and the recently described glutamate proteases. By proteolytic cleavage inside lipid bilayer they affect many significant biological processes such as metabolism of lipids, cell proliferation and adhesion, regulation of developmental signaling, degradation of signal peptides, and membrane protein quality control. This work focusses on the role of intramembrane proteases in various diseases and biological mechanisms associated with pathological processes. These are specifically Alzheimer's disease, infection by unicellular parasites (Mycobacterium tuberculosis, Entamoeba histolytica and Plasmodium falciparum), maturation of hepatitis C virus, Bunyamwera virus and swine influenza virus, and mitochondrial dysfunction.
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Evolution of regulatory mechanisms of EGF receptor activation
Trávníčková, Květa ; Stříšovský, Kvido (advisor) ; Koudelková, Lenka (referee)
Signalling through EGF receptor is crucial both for ontogenesis and for maintaining homeostasis in adult organisms. It is involved in controlling cellular behaviours such as proliferation, migration or differentiation. This thesis provides an insight into evolution of the regulatory mechanisms of EGF receptor activation by discussing their principles in C. elegans, D. melanogaster and H. sapiens sapiens, on the basis of which conclusions about their evolutionary tendencies are made. Attention is focused on the roles of the rhomboid family of proteins, whose activity is tightly associated with EGF receptor signalling. Dysregulation of the EGF receptor unnegligibly contributes to the development of various diseases, mainly many types of cancer, but also schizophrenia, psoriasis and cardiovascular disorders. Experimental results obtained on this field of research therefore have the potential to be applied in drug design.
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Antigen cross-presentation - mechanism and biological significance
Boháčová, Šárka ; Stříšovský, Kvido (advisor) ; Černý, Jan (referee)
iii Abstract Antigen cross-presentation is a process, when dendritic cells present exogenous antigens in context of MHC-I to CD8+ T lymphocytes. Unlike classical antigen presentation, this one goes crosswise, because exogenous antigens are otherwise usually presented on MHC-II and endogenous antigens on MHC-I glycoproteins. Molecular mechanism of cross-presentation has not been well established yet. Two major pathways are considered - vacuolar and cytosolic. In the vacuolar pathway, the internalised antigens are cleaved in the endosome by proteases and then loaded onto MHC-I. In the cytosolic pathway, the internalised antigens leave the endosome to be cleaved by the proteasome in the cytosol. They are then imported into the endoplasmic reticulum (ER) to by loaded onto MHC-I as in classical antigen presentation, or they go back into the endosome where the MHC-I loading machinery is trafficked. This process is mediated by ER proteins including those participating in ERAD, by Rab GTPases regulating vesicular transport, and by structures important for endosome maturation. Cross-presentation is important in medicine, because it ensures activation of CD8+ T lymphocytes against intracellular pathogens and cancer cells, and induction of tolerance at the...
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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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