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Analysis of intermediate filament structure by chemical cross-linking
Dlabolová, Lada ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
Intermediate filament proteins create a dynamic cytoskeletal filamentous network, which due to its elastic properties, significantly contributes to the resistance of cells and tissues to mechanical stress. An important protein from the family of intermediate filaments, vimentin, is expressed mainly in cells of mesenchymal origin. Vimentin has been associated with a large number of pathophysiological conditions, and current studies consider vimentin as clinically promising target for the diagnosis, prognosis and treatment of a wide range of diseases from cancer to infectious and inflammatory diseases. Although in terms of structural characterization, vimentin belongs to one of the most studied proteins from the family of intermediate filaments, our knowledge is currently limited to the structure of the vimentin tetramer. Vimentin is capable of self-assembly into filaments formed by homo-oligomeric ULF subunits and the assembly process involves several steps of the organization of subunits. Structural characterization of the oligomeric subunits involved in the assembly of vimentin filaments is a prerequisite for elucidating the architecture of mature filaments, which can significantly contribute to understanding and connecting the mechanisms of many diseases associated with changes in vimentin...
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Utilization of chemical cross-linking for studying intermediate filaments organization
Dlabolová, Lada ; Novák, Petr (advisor) ; Sabó, Ján (referee)
Intermediate filaments are cytoskeleton components formed by a large family of fibrous proteins specifically expressed in nearly all differentiated cells. Under physiological conditions, they spontaneously assemble into fibers in a process that involves several stages in the organization of subunits. These fibers provide elastic properties to the cells, allowing them to maintain their structural and mechanical integrity. While the structure of other cytoskeletal components is now well researched, detailed information on the structure of intermediate filaments at various stages of assembly is still not available. Thus, new insights into the structure of these proteins could be of great benefit in understanding of various pathological mechanisms associated with changes in their expression in cells. This thesis studies interactions of dimeric subunits in the tetrameric assembly of vimentin, class III protein of intermediate filaments. By chemical cross-linking of isotopically labeled and unlabeled tetrameric vimentin mixture, followed by proteolytic cleavage and mass spectrometry analysis, interdimeric, intradimeric and intrapeptide cross-linking products were identified. Quantification yielded information on interdimeric and intradimeric distance constraints, which allow the characterization of a...
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