Extracellular vesicles that are shed from the plasma membrane contain a wide range of molecules, among which are proteins, lipids, nucleic acids, and sugars. The cytotoxic proteins of natural killer cells play a key role in the implementation of their cytolytic functions. One of the important steps in understanding the distant communication of cells is the determination of the proteome of microvesicles. This study was aimed at the protein profiling of the microvesicles produced by the NK-92 natural killer cell line. 986 proteins with a variety of functions were identified in the lysate of microvesicles using the MALDI-TOF mass spectrometric analysis. With automated methods of functional analysis applied, it has been shown that the largest protein groups are hypothetical proteins, proteins with unknown functions, and domains. The most representative groups are also comprised by transcription regulators; intracellular signaling proteins; RNA translation, transcription, processing, and utilization regulators; receptors; protein processing and proteolysis regulators; amino acid metabolism enzymes, as well as transport proteins and transport regulators. Minor functional groups are represented by vitamins and mineral metabolism enzymes, membrane and microdomain-forming proteins, hormones, hemostatic regulators, regulators of sensory systems, specific mitochondrial and Golgi apparatus proteins, and extracellular signaling proteins. An intermediate position is occupied by various functional groups, including cytoskeleton and motor proteins; proteins of centrioles; ion channels and their regulators; proteins of the ubiquitin-proteasome pathway of protein degradation; lipid, steroid, and fatty acid metabolism enzymes; nucleic acid base and carbohydrate metabolism enzymes, as well as energy metabolism enzymes and other proteins involved in intermediate metabolism; proteins of the immune response and inflammation; antigens and histocompatibility proteins; cytokines and growth factors; regulators of apoptosis, autophagy, endocytosis, and exocytosis; regulators of the cell cycle and division; regulators of proliferation, cell differentiation, and morphogenesis; regulators of cell adhesion and matrix metabolism; nuclear transport proteins; transposition proteins; DNA replication and repair proteins, as well as inactive proteins. The data obtained expand the existing knowledge of the distant communication of cells and indicate new mechanisms of interaction between natural killer and target cells.
Extracellular vesicles that are shed from the plasma membranes take an active part in intercellular communication, transporting a wide range of molecules, including proteins, lipids, nucleic acids and carbohydrates, being of great functional importance. One of the steps to better understanding of distant communications of cells and their regulatory mechanisms is a proteomic study of various extracellular vesicles, including microvesicles and exosomes. Pro-inflammatory cytokines produced by monocytes and individual complement system components play a key role in their specific functioning. The aim of this work was to study proteomic composition of THP-1 monocyte-like cells and their microvesicles. The MALDI-mass spectrometric analysis of electrophoretic protein fractions of cell lysates and microvesicles allowed for identifying 107 proteins that perform various functions. Among 19 determined functional groups, the largest ones comprise transcription regulators and proteins with unknown functions. The smallest functional groups include regulators of cell differentiation and development, proteins participating in immune response and inflammation, cellular receptors and their regulators, transporter and transport regulatory proteins, as well as cell proteins mediating adhesion and matrix structures, processing regulators, proteins of ubiquitin-proteasome system, intracellular signaling, autophagy and exocytosis regulators, chromatin structural proteins, hemostatic regulators, and peptide hormones. An intermediate position is occupied by cytokines and growth factors, enzymes, cytoskeleton and motor proteins, as well as RNA processing and translation regulators. The subsequent DAVID Functional Annotation Clustering analysis allowed for identifying the most common groups distributed by their molecular function, biological processes, and cellular component. Separately, in the microvesicles derived from THP-1 monocyte-like cells, proteins of the immune response and inflammation, cytokines and growth factors, intracellular signaling proteins, cell differentiation regulators and developmental proteins, as well as cell adhesion and matrix proteins were identified among other protein molecules. The data obtained on the partial proteome of THP-1 monocyte-like cells and their microvesicles extend the existing knowledge on distant communications between the cells and suggest new mechanisms of interaction between monocytes/macrophages and their microenvironment.
Microvesicles are membrane-derived formations ranging in size from 100 to 1000 nm, being produced by a variety of resting and activated cells. They can transfer their cargo to target cells, regulate physiological processes, and participate in the development of clinical disorders. Among the microvesicles of different origin, natural killers are of special interest. They represent a subpopulation of lymphocytes that eliminate aberrant cells, including virally infected and malignant cells, and participate in regulation of angiogenesis. By producing various stimuli and inhibitors of the latter process, natural killers are able to change functional activity of endothelial cells by means of microvesicle-mediated contacts. There are only scarce literature data on ability of the extracellular vesicles to influence endothelial functions, depending on the intrinsic balance of pro- and anti-angiogenic factors. Therefore, the aim of our study was to evaluate the effect of protein fractions derived from microvesicle lysate of the NK-92 natural killer cell line upon phenotype and functional characteristics of EA.hy926 endothelial cell line under in vitro experimental conditions. Using chromatographic micro-preparatory separation, twelve protein fractions (inducers) were obtained from the lysate. It was found that proliferation and migration of EA.hy926 cells after their cultivation with 10 of 12 protein fractions, were changed in different directions. These effects were dose-dependent, or remained unchanged, at distinct concentrations of active components in the fractions. The inducing factors from these fractions exerted predominantly stimulating effects on proliferation of the target cells, thus suggesting presence of proteins which are able of regulating endothelial functions. However, the size of residual area free of migrating endothelial cells treated by the inducers did not always correlate with the migration intensity and did not inversely correlate with the number of migrating cells. Moreover, it was found that the obtained protein fractions had no effect upon expression of CD54 (ICAM-1), CD34, CD31 (PECAM-1) and CD119 (IFNγR1) receptors by EA.hy926 cells. The data obtained confirm an involvement of microvesicles in communications between natural killer cells and endothelial cells, and presume different participation modes of microvesicle-derived effector proteins in the angiogenesis machinery.
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