BACKGROUND: The formation of apoptosomes is well-established in the mechanism of programmed cell death. The interaction of APAF-1, caspase-3 and -9, which by adding of cytochrome C only in the presence of macroergs (dATP or ATP) generates apoptosome. Besides the cytoplasmic protein concentration is critical for the assembly of apoptosomes since in vitro it can induced only at values starting from ~2 mg/ml. The study of this feature led us to the discovery of at least one more protein that is critically involved in their formation.

METHODS: Cytoplasmic fraction from brain homogenates of newborn rats was obtained by centrifugation. Nucleoside di- and triphosphates, Na⁺, K⁺, RNAse A, DNAse1, phalloidin etc., as well as non-muscle F (filamentous) and G (monomeric) actin were added to study their effect on formation of active apoptosomes in the cytoplasm. Preliminary analysis showed that the proteasome activity of the "hydrolyzing peptidyl-glutamyl peptide" constitutes a significant part of all activities in the cleavage of caspase substrates. In this regard, experiments to study the activity of caspases were carried out in the presence of proteasome inhibitors (bortezomib or AdaAhx3L3VS), which do not affect the assembly of apoptosomes. Caspase activity was confirmed by the use of a selective caspase inhibitor emricasane.

RESULTS: It was found that manipulations with the cytoplasm (its concentration variation, the presence of monovalent cations, membrane fragments) in a dose-dependent manner nonlinearly led to an increasing of formed apoptosomes and the activity of caspase-3. These modifications changed the protease activity: maximal velocity from 0.15 to 2.4 mkmol/mg/min and K_m from 4.2 to 0.13 mkM. A more detailed analysis showed that substances influencing on the assembly and disassembly of actin filaments directly affect both the formation of apoptosomes and the caspase-3 activity. This influence is critically significant, changing the activity by at least an order of magnitude. In this case, the effect of proteins that directly inhibit the activities of caspases (for example, XIAP) did not change.

CONCLUSIONS: Thus the actin G/F ratio (balance assembly/disruption of actin filaments) is a key regulator of the assembly of apoptosomes and it depends on the presence of nucleoside triphosphates. It explains the critical dependence of apoptosis on the production of macroergs.

Background: Recently, the list of intercellular communicators has been extended by extracellular vesicles, whose contents and membrane proteins are involved in signaling or in its modulation. In particular, it is known that exosomes, the most studied form of extracellular vesicles, can contain 20S proteasome subunits. It is also known that extracellular proteasomes exist in a vesicle-free form, and their activity has a physiological significance. In this work we investigated proteasomes and already activated apoptosomes that are secreted by adult human hematopoietic stem cells (aHSCs).

Methods: aHSCs were isolated from human bone marrow, cultivated in Serum-Free Expansion Medium (SFEM), supplemented with Flt3L, SCF, IL-3, IL-6 and TPO. The cultured cells were concentrated by positive selection for CD34+ antigen. The cells were permeabilized and apoptosis was induced with cytochrome C and dATP. Exosomes were purified from culture medium by centrifugation or using a special kit. The concentrated culture medium was fractionated on a gel filtration column. Caspase-3 activity was measured with (Z-DEVD)2-R110 substrate in the presence of bortezomib. Chymotrypsin-like proteasome activities were determined with Suc-LLYV-AMC and different proteasome inhibitors were used for the specific identification.

Results: Attempts to induce apoptosis in aHSCs were unsuccessful. Minor specific caspase activity was found in cell membranes and in exosomes but it was constitutive one and activation of apoptosome assembly by cytochrome C/dATP does not affect this in any way. At the same time, a notable caspase activity was found in SFEM freed from exosomes. Gel filtration of SFEM showed that this activity associated with components of a high molecular weight and is separated by gel filtration together with the proteasomes. It turned out that exosomes also contain proteosomal activity. This suggests that exosomes contain proteasomes and activated caspases. The presence of these active enzymes in the culture medium could be explained by the action of sphingomyelinase or phospholipases, which release them from exosomes.

Conclusion: Content of exosomes from aHSCs includes proteases, such as proteasomes and caspases, which could perform signaling and other functions.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBE) in human hematopoietic stem and progenitor cells (HSPC) potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBE were analyzed in human CD34+ cells cultivated in fractions of filtered medium conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double strand-breaks) and chromosomal instability (CIN) were evaluated in CD34+ cells grown for 3 days in (a) < 10 kDa, (b) 10 – 100 kDa and (c) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, substantial bystander effects were detected predominantly in CD34+ cells grown in 10 – 100 kDa fractions of MSC conditioned medium when compared to < 10 kDa or > 100 kDa fractions of MSC conditioned medium or fractionated control medium. Taken together, our data suggest that RIBE are predominantly mediated by the 10 – 100 kDa fractions of conditioned medium. This finding might be important for the identification of key bystander signals by in depth proteome analysis, which might contribute to the development of next-generation anti-leukemic drugs.

Cardiolipin (CL) is a mitochondrial phospholipid, which has a well-defined role in regulating metabolic processes and maintaining homeostasis. While CL is normally confined to the inner mitochondrial membrane, it may be released extracellularly from damaged or dying cells during physiological and pathological conditions characterized by cellular stress, tissue damage, and cell death. Once released, CL can act as a signalling molecule by interacting with cells in an autocrine and paracrine manner. Previous research demonstrates that extracellular CL regulates select immune functions of microglia, which are the innate immune cells of the brain. However, the effects of CL on astrocyte functions are unknown. Astrocytes support metabolic homeostasis of neurons, facilitate neurotransmission, and are also active participants in neuroimmune responses of the brain. During chronic neuroinflammatory states, astrocytes become over-activated and may cause damage to the surrounding tissues and cells. We hypothesized that extracellular CL modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity. Therefore, we studied the effects of CL added to the culture media of primary murine astrocytes and human U118 MG astrocytic cells. The phagocytic activity of primary murine astrocytes was measured using fluorescent latex microspheres. The secretion of inflammatory cytokines was monitored by the enzyme-linked immunosorbent assay or immunoblotting. The expression of glial fibrillary acidic protein (GFAP) was measured using immunoblotting. Extracellular CL alone upregulated the phagocytic activity of primary murine astrocytes, as well as the release of monocyte chemoattractant protein (MCP)-1 and interferon (IFN)-β by human U118 MG astrocytic cells. CL inhibited the lipopolysaccharide (LPS)-induced increase in GFAP expression by U118 MG astrocytic cells, as well as the secretion of cytotoxins by the same cell type. Our study demonstrates that extracellular CL regulates select functions of astrocytes, which become dysregulated in chronic neuroinflammatory states, such as those observed in neurodegenerative diseases.

Microglia are the resident immune cells of the brain, which become activated in response to diverse immune stimuli. Following activation, microglia signal to other central nervous system (CNS) cells by secreting a variety of cytokines. Under specific stimulatory conditions, microglia can also release a spectrum of cytotoxins, including reactive oxygen species (ROS), reactive nitrogen species (RNS), proteases, and other enzymes. Molecules secreted by microglia have specific functions in the neuroimmune response to pathogens and endogenous stimuli. They also contribute to the pathogenesis of neurodegenerative diseases. Neurotrophins (NTs) are proteins that support development, maturation and normal metabolic functions of neurons by interacting with neurotrophin receptors (NTRs) on these cells. Microglia also express several NTRs that signal in response to binding of NTs. These include tropomyosin receptor kinase (Trk)A, TrkB and p75NTR. Binding and activation of these receptors can alter the phenotype, metabolism, and other functions of microglia. We hypothesized that NTR ligands, such as the small molecule ligand LM11A-31 and the NT precursor pro-brain derived neurotrophic factor (proBDNF), bind p75NTR and modulate the immune functions of microglia. We studied the effects of NTR ligands on the following immune functions by using in vitro models of microglia: 1) secretion of cytotoxins and cytokines by lipopolysaccharide (LPS) plus interferon-γ stimulated human THP-1 monocytic cells; 2) release of ROS through the LPS-primed respiratory burst response of dimethyl sulfoxide-differentiated human HL-60 myelomonocytic cells; 3) secretion of nitric oxide by LPS-stimulated murine BV-2 microglia; and 4) phagocytic activity of BV-2 cells. LM11A-31 and proBDNF inhibited the respiratory burst response of differentiated HL-60 microglia-like cells but did not have a significant effect in most other cellular assays. Our data indicate that NTs could serve as intercellular signaling molecules of the CNS by regulating select immune functions of microglia, such as their production of ROS.