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Targeting of viral nanoparticles to cancer specific receptors
Žáčková Suchanová, Jiřina ; Španielová, Hana (advisor) ; Němečková, Šárka (referee) ; Ulbrich, Pavel (referee)
The aim of this thesis is to reveal the potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as possible nanocarriers for directed delivery of therapeutic or diagnostic compounds to specific cells or tissues. We have chosen mouse polyomavirus VLPs because they do not contain viral DNA and are considered safe for utilization in bio-applications. In our research, we used a chemical approach for retargeting of MPyV based VLPs from their natural receptor to cancer cells. The chemical modification of the capsid surface exposed lysines by an aldehyde-containing reagent enabled conjugation of VLPs to selected molecules: transferrin and inhibitor of glutamate carboxypeptidase II (GCPII). Transferrin, as a transporter of iron to metabolically active cells, targeted VLPs to numerous types of cancer cells overexpressing the transferrin receptor. On the other hand, GCPII serves as a transmembrane marker specific for prostate cancer cells and conjugation of its inhibitor to VLPs resulted in successful recognition of these cells. Electron microscopy was used for visualization of modified VLPs and flow cytometry together with confocal microscopy for investigation of cell specific interactions and VLP uptake. Furthermore, we explored the influence of serum proteins on VLPs. The abundance of...
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Structural study of S6A mutant of matrix protein from Mason-Pfizer monkey virus
Kamal, Rašíd ; Hrabal, Richard (advisor) ; Ulbrich, Pavel (referee)
Mason-Pfizer monkey virus (M-PMV) belongs to the morphogenetic type D retroviruses which produce viral particles in the cytoplasm. This process is temporally and spatially separated from the cytoplasmic membrane budding and therefore M-PMV is a suitable model organism for studying the life cycle of retroviruses. Matrix protein, which is N-terminal portion of Gag polyprotein precursor, plays a central role in this life cycle. One of the key areas of the protein is its myristoylated N-terminus which is particularly important for binding to the cytoplasmic membrane during budding from host cells. A hydrogen bond exists between serine 6 and glutamate 9 of the wild-type protein. We have studied its influence on the structure and molecular mechanics of the corresponding area of the protein by means of disruption of this interaction by replacing serine 6 with alanine. This thesis describes the preparation of recombinant mutant of the M-PMV matrix protein, resonance assignment of its backbone atoms with nuclear magnetic resonance spectroscopy and compares observed secondary structure with that of other mutants and the wild type.
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NMR structure of an assembly defective mutant of the N-terminal domain of the capsid protein from Mason-Pfizer monkey virus.
Obr, Martin ; Rumlová, Michaela (advisor) ; Ulbrich, Pavel (referee)
Retroviral polyprotein precursor Gag is cleaved by viral protease during the course of virus particle maturation to yield individual structural components - matrix protein (MA), capsid protein (CA) and nucleocapsid protein (NC). CA consists of two domains: N-terminal (NTD) and C-terminal (CTD) and forms a hexameric lattice. Conformational switch upon proteolytic release of CA mediates disassembly of an immature and assembly of a mature capsid. 3D structure of M-PMV CANTD consists of six -helices and N-terminal -hairpin. The -hairpin is stabilized by a salt bridge between Pro1 and Asp57 and another, yet uncharacterized interface between Arg14 and helix 5. Proper -hairpin formation is critical for mature particle assembly. This thesis analyses the impact of the helix 5 and -hairpin point mutations (D111N and R14K, respectively) on the CANTD structure by NMR spectroscopy. None of the mutations affect overall fold of the protein. Comparison of NOE contacts of WT and D111N indicates structural identity of these proteins. R14K mutation shows significant changes in the helix 5 and the -hairpin regions in comparison with WT. These results confirm presence of the interface stabilized by -hairpin and helix 5 interactions, with Arg14 being the critical part. Role of Asp111 in this interface was not confirmed and other...
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Targeting of viral nanoparticles to cancer specific receptors
Žáčková Suchanová, Jiřina ; Španielová, Hana (advisor) ; Němečková, Šárka (referee) ; Ulbrich, Pavel (referee)
The aim of this thesis is to reveal the potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as possible nanocarriers for directed delivery of therapeutic or diagnostic compounds to specific cells or tissues. We have chosen mouse polyomavirus VLPs because they do not contain viral DNA and are considered safe for utilization in bio-applications. In our research, we used a chemical approach for retargeting of MPyV based VLPs from their natural receptor to cancer cells. The chemical modification of the capsid surface exposed lysines by an aldehyde-containing reagent enabled conjugation of VLPs to selected molecules: transferrin and inhibitor of glutamate carboxypeptidase II (GCPII). Transferrin, as a transporter of iron to metabolically active cells, targeted VLPs to numerous types of cancer cells overexpressing the transferrin receptor. On the other hand, GCPII serves as a transmembrane marker specific for prostate cancer cells and conjugation of its inhibitor to VLPs resulted in successful recognition of these cells. Electron microscopy was used for visualization of modified VLPs and flow cytometry together with confocal microscopy for investigation of cell specific interactions and VLP uptake. Furthermore, we explored the influence of serum proteins on VLPs. The abundance of...
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NMR structure of an assembly defective mutant of the N-terminal domain of the capsid protein from Mason-Pfizer monkey virus.
Obr, Martin ; Rumlová, Michaela (advisor) ; Ulbrich, Pavel (referee)
Retroviral polyprotein precursor Gag is cleaved by viral protease during the course of virus particle maturation to yield individual structural components - matrix protein (MA), capsid protein (CA) and nucleocapsid protein (NC). CA consists of two domains: N-terminal (NTD) and C-terminal (CTD) and forms a hexameric lattice. Conformational switch upon proteolytic release of CA mediates disassembly of an immature and assembly of a mature capsid. 3D structure of M-PMV CANTD consists of six -helices and N-terminal -hairpin. The -hairpin is stabilized by a salt bridge between Pro1 and Asp57 and another, yet uncharacterized interface between Arg14 and helix 5. Proper -hairpin formation is critical for mature particle assembly. This thesis analyses the impact of the helix 5 and -hairpin point mutations (D111N and R14K, respectively) on the CANTD structure by NMR spectroscopy. None of the mutations affect overall fold of the protein. Comparison of NOE contacts of WT and D111N indicates structural identity of these proteins. R14K mutation shows significant changes in the helix 5 and the -hairpin regions in comparison with WT. These results confirm presence of the interface stabilized by -hairpin and helix 5 interactions, with Arg14 being the critical part. Role of Asp111 in this interface was not confirmed and other...
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Structural study of S6A mutant of matrix protein from Mason-Pfizer monkey virus
Kamal, Rašíd ; Hrabal, Richard (advisor) ; Ulbrich, Pavel (referee)
Mason-Pfizer monkey virus (M-PMV) belongs to the morphogenetic type D retroviruses which produce viral particles in the cytoplasm. This process is temporally and spatially separated from the cytoplasmic membrane budding and therefore M-PMV is a suitable model organism for studying the life cycle of retroviruses. Matrix protein, which is N-terminal portion of Gag polyprotein precursor, plays a central role in this life cycle. One of the key areas of the protein is its myristoylated N-terminus which is particularly important for binding to the cytoplasmic membrane during budding from host cells. A hydrogen bond exists between serine 6 and glutamate 9 of the wild-type protein. We have studied its influence on the structure and molecular mechanics of the corresponding area of the protein by means of disruption of this interaction by replacing serine 6 with alanine. This thesis describes the preparation of recombinant mutant of the M-PMV matrix protein, resonance assignment of its backbone atoms with nuclear magnetic resonance spectroscopy and compares observed secondary structure with that of other mutants and the wild type.
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