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Fluorescence studies of bacterial membrane proteins and cell signalling.
Fišer, Radovan ; Konopásek, Ivo (advisor) ; Hof, Martin (referee) ; Forstová, Jitka (referee)
(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cationselective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; αhemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic αhelix(502522). In the second publication we investigated another predicted transmembrane αhelix(565591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...
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Protein translocation into hydrogenosomes of "Trichomonas vaginalis"
Radhakrishna Makki, Abhijith ; Tachezy, Jan (advisor) ; Hashimi, Hassan (referee) ; JACKSON, Catherine Lynn (referee)
Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...
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Protein translocation into hydrogenosomes of "Trichomonas vaginalis"
Radhakrishna Makki, Abhijith ; Tachezy, Jan (advisor) ; Hashimi, Hassan (referee) ; JACKSON, Catherine Lynn (referee)
Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...
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Fluorescence studies of bacterial membrane proteins and cell signalling.
Fišer, Radovan ; Konopásek, Ivo (advisor) ; Hof, Martin (referee) ; Forstová, Jitka (referee)
(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cationselective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; αhemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic αhelix(502522). In the second publication we investigated another predicted transmembrane αhelix(565591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...
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