|
|
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...
|
|
|
Impact of membrane properties on clustering of transmembrane peptides
Sabó, Ján ; Cebecauer, Marek (advisor) ; Vít, Ondřej (referee)
Unfolded protein response (UPR) is a complex cellular mechanism induced upon ER stress caused by various environmental factors. Single spanning signal transducers of UPR were reported to recognise also lipid-induced ER stress. Studies of these transducers, namely PERK and IRE1 uncovered that they can sense change in membrane properties and activate themselves by clustering. Moreover, signal transducer IRE1 retained ability to sense changes in the membrane properties with TMD exchanged for a polyLeu α-helix. It was thus unclear what mechanism drives lipid-induced UPR via IRE1. We employed model membrane system in form of LUVs, where properties of membranes can be readily altered by specific lipid composition. As a simplified model of the UPR signal transducers in the ER, synthetic transmembrane peptides with polyLeu core were used. Dynamic light scattering (DLS) has been used for qualitative and semi-quantitative analysis of LUVs. Clustering of synthetic peptides was determined by time resolved anisotropy of fluorescence. DLS results demonstrate successful formation of vesicles with a desired size in all planned composition. On the contrary to the studies in living cells, the presence of cholesterol or palmitic acid in model membranes did not induce the aggregation of transmembrane peptides....
|
|
|
Mass spectrometry analysis of integral membrane peptides
Sabó, Ján ; Cebecauer, Marek (advisor) ; Pompach, Petr (referee)
Biological membranes ensure a large scale of vital processes in the living organisms. Lipid and protein composition is very complex in the membranes. Therefore, simplified model systems were developed to study basic mechanisms regulating the function of membranes. To simulate transmembrane proteins of the type I and II, the short, α-helix-forming, synthetic peptides are employed. The hydrophobic character of the peptides and their transbilayer positioning in membranes well represents transmembrane domains of proteins in the living organisms. One of the simplest and most widely used model membrane systems are liposomes. Methods for their formation has been known for a long time. Quantification of their components after the process of liposome preparation is challenging, but for the maximal control over given model system very desirable. In this work, we adapted a formerly described protocol for delipidisation of the transmembrane peptides for their consequent characterisation by LC/MS. A relative amount of peptide successfully incorporated into vesicles was acquired by the analysis of extracted chromatograms of peptide ions. We demonstrate that analysis of vesicles with peptide content of 5-10 mol% is feasible and the loss of the peptide is below 50 %. Such vesicles can be used for further...
|
|
|
Enhanced biomechanical characteristics of connective tissues and development of artificial implants
Varga, Ferdinand ; Amler, Evžen (advisor) ; Rosina, Jozef (referee) ; Daniel, Matej (referee) ; Sabo, Ján (referee)
(english) Hyaline cartilages are designed to enable smooth life-long articular move- ment. They serve also substantial role in load bearing, being loaded especially in normal compression. Eventual deterioration of articular cartilage tissue therefore has crucial consequences for affected individ- ual. Characterization of native articular cartilage and application of gained knowledge in reconstructive cartilage treatment is therefore de- sired. Mechanical characteristics are of prime importance. Blunt impact testing method in pendulum setup was developed to characterize cartilage tissue under physiological values of dynamic com- pressive loading. Information on impact process was simultaneously read by piezoelectric accelerometer and laser doppler vibrometer. Acquired data were processed to form loading diagrams and to evaluate standard mechanical quantities. The method was found to be consistent, reliable and effective way of mechanical characterization. Introduced technique was used for qualitative and quantitative de- scription of native articular cartilage, correlation of its condition with mechanical properties, assessment of tissue engineered materials quality and approval of newly designed artificial materials suitability. Consider- ing native cartilage status, dissipated energy was found to be...
|
guest ::
RSS feed.