Засоби масової комунікації в культурі суспільства постмодерну

Проаналізовано місце, роль та вплив засобів масової комунікації на суспільство й індивіда в умовах інформаційного суспільства.Проанализировано место, роль и влияние средств массовой коммуникации на общество и индивида в условиях информационного общества.A place, role and influencing of facilities of mass communication, is analized on society and individual in the conditions of informative societ

Bugging the cell wall of bacteria : novel insights into the biosynthesis of peptidoglycan and its inhibition

The last few decades saw an alarming rise of resistance against antibiotics, including the infamous methicillin-resistant Staphylococcus aureus (MRSA) that is resistant to the large group of antibiotics. This has turned the development of new antimicrobial compounds into a crucial necessity. The bacterial cell wall forms an invaluable target for antibiotics as it is essential for the viability of the cell and its location at the cell’s exterior gives it a relatively high accessibility. Many aspects concerning the biochemical processes involving the cell wall remain unclear, which likely conceals potential targets for new antibiotics. It is therefore important to deepen our understanding of cell wall physiology, which requires the development of additional strategies for cell wall analysis. This thesis describes the development and application of novel techniques to gain more insight into peptidoglycan metabolism and the enzymes involved herein with an emphasis on the role of the cell wall precursor Lipid II in the targeting of transglycosylases. The high tolerance of the bacterial cell wall synthesis machinery is fully exploited by the use of a variety of labeled peptidoglycan derivatives. A fluorescent cell wall labeling approach is set up, which is used to study peptidoglycan metabolism in vivo. It is shown that externally supplied NBD-labeled murein tripeptide is taken up by E. coli cells and metabolically incorporated into the cell wall via the peptidoglycan recycling pathway. By analyzing the cell wall labeling patterns in wild-type cells and several division and amidase mutants, FtsZ-dependent hydrolase activity during preseptal elongation was discovered. Additionally, we could visualize the major peptidoglycan hydrolase activity of AmiC during septation. The above developed method of labeling the cell wall of E. coli with reporter groups in vivo is adapted into a proteomics format. Using a tripeptide derivative containing a photoactivatable crosslinker and an alkyne moiety, proteins interacting with the cell wall and/or its precursors were crosslinked. Using the alkyne as bait, azide derivatized tags or beads could be covalently attached to the crosslink products via click chemistry, enabling their selective detection and purification. A number of interesting proteins were identified. The focus turned to the transglycosylation process, which for long was a black box in peptidoglycan biosynthesis and a potentially interesting target for novel antibiotics. In vitro photo-crosslinking in combination with mass spectrometry techniques provided information on the substrate (Lipid II) binding site in the transglycosylase penicillin-binding protein 1b (PBP1b) of E. coli. By reacting a photoactivatable analogue of Lipid II with the enzyme in the presence of moenomycin, it is shown that the substrate is covalently captured in one specific region, possibly encompassing the binding site of Lipid II. The mechanism of action of the potential transglycosylase inhibitor compound 5b is investigated. 5b is shown to disturb the functional integrity of negatively charged membranes. Moreover, nisin-Lipid II pore formation in model membranes was inhibited by 5b, which was reduced by an excess presence of undecaprenylpyrophosphate. This points to a specific interaction of 5b with Lipid I

Novel insights into the function of the conserved domain of the CAP superfamily of proteins

Members of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) superfamily are found in a remarkable variety of biological species. The presence of a highly conserved CAP domain defines the CAP family members, which in many cases is linked to other functional protein domains. As a result, this superfamily of proteins is involved in a large variety of biological processes such as reproduction, tumor suppression, and immune regulation. The role of the CAP domain and its conserved structure throughout evolution in relation to the diverse functions of CAP proteins is, however, poorly understood. Recent studies on the mammalian Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), which consists almost exclusively of a CAP domain, may shed new light on the function of the CAP domain. GAPR-1 was shown to form amyloid fibrils but also to possess anti-amyloidogenic properties against other amyloid forming peptides. Amyloid prediction analysis reveals the presence of potentially amyloidogenic sequences within the highly conserved sequence motifs of the CAP domain. This review will address the structural properties of GAPR-1 in combination with existing knowledge on CAP protein structure-function relationships. We propose that the CAP domain is a structural domain, which can regulate protein-protein interactions of CAP family members using its amyloidogenic properties

Metal ions and redox balance regulate distinct amyloid-like aggregation pathways of GAPR-1

Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-Related 1 proteins) are characterized by the presence of a structurally conserved CAP domain. The common structure-function relationship of this domain is still poorly understood. In this study, we unravel specific molecular mechanisms modulating the quaternary structure of the mammalian CAP protein GAPR-1 (Golgi-Associated plant Pathogenesis-Related protein 1). Copper ions are shown to induce a distinct amyloid-like aggregation pathway of GAPR-1 in the presence of heparin. This involves an immediate shift from native multimers to monomers which are prone to form amyloid-like fibrils. The Cu2+-induced aggregation pathway is independent of a conserved metal-binding site and involves the formation of disulfide bonds during the nucleation process. The elongation process occurs independently of the presence of Cu2+ ions, and amyloid-like aggregation can proceed under oxidative conditions. In contrast, the Zn2+-dependent aggregation pathway was found to be independent of cysteines and was reversible upon removal of Zn2+ ions. Together, our results provide insight into the regulation of the quaternary structure of GAPR-1 by metal ions and redox homeostasis with potential implications for regulatory mechanisms of other CAP proteins

Regulation of Functional Protein Aggregation by Multiple Factors: Implications for the Amyloidogenic Behavior of the CAP Superfamily Proteins

The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases

The less conserved metal-binding site in human CRISP1 remains sensitive to zinc ions to permit protein oligomerization

Cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and PR-1 (CAP) superfamily that is characterized by the presence of a conserved CAP domain. Two conserved histidines in the CAP domain are proposed to function as a Zn2+-binding site with unknown function. Human CRISP1 is, however, one of the few family members that lack one of these characteristic histidine residues. The Zn2+-dependent oligomerization properties of human CRISP1 were investigated using a maltose-binding protein (MBP)-tagging approach in combination with low expression levels in XL-1 Blue bacteria. Moderate yields of soluble recombinant MBP-tagged human CRISP1 (MBP-CRISP1) and the MBP-tagged CAP domain of CRISP1 (MBP-CRISP1ΔC) were obtained. Zn2+ specifically induced oligomerization of both MBP-CRISP1 and MBP-CRISP1ΔC in vitro. The conserved His142 in the CAP domain was essential for this Zn2+ dependent oligomerization process, confirming a role of the CAP metal-binding site in the interaction with Zn2+. Furthermore, MBP-CRISP1 and MBP-CRISP1ΔC oligomers dissociated into monomers upon Zn2+ removal by EDTA. Condensation of proteins is characteristic for maturing sperm in the epididymis and this process was previously found to be Zn2+-dependent. The Zn2+-induced oligomerization of human recombinant CRISP1 may shed novel insights into the formation of functional protein complexes involved in mammalian fertilization

Regulation of Functional Protein Aggregation by Multiple Factors: Implications for the Amyloidogenic Behavior of the CAP Superfamily Proteins

The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases