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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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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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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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