Intracellular cathepsin C levels determine sensitivity of cells to leucyl-leucine methyl ester-triggered apoptosis.

L-leucyl-leucine methyl ester (LLOMe) is a lysosomotropic detergent, which was evaluated in clinical trials in graft-vs-host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in cancer cells, suggesting that the drug might have potential in anticancer therapy. Using U-937 and THP-1 promonocytes as models for monocytic cells, U-87-MG and HeLa cells as models for cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid cathepsin C-dependent lysosomal membrane permeabilization, followed by the release of other cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of cathepsin C-the most upstream molecule in the pathway-in monocytic cell lines as compared to cancer cells. Overexpression of cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of cathepsin C. Major involvement of cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of cysteine cathepsins, stefin B, in primary mouse cancer cells, and simultaneous ablation of two major cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of stefin B resulted in sensitizing primary murine breast cancer cells to cell death without affecting the release of cathepsins, whereas simultaneous ablation of cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer therapy due to risk of systemic toxicity.

Salivary Tick Cystatin OmC2 Targets Lysosomal Cathepsins S and C in Human Dendritic Cells.

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells.

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.

Is Nano-Silver Safe within Bioactive Hydroxyapatite Composites?

Because of the abounded presence of the silver-containing products in the market and intensively studied silver-containing biomaterials in the literature, we investigated a hypothesis that stabilizing silver within bioactive hydroxyapatite composite is capable to provide safe and effective antibacterial action. For that purpose nanocomposite made of bioactive, mineral hydroxyapatite (HAp) and antibacterial silver (Ag) is studied for interactions with both, bacterial and human cells. The nanocomposite provides controlled release of Ag ions; induces severe damages in bacterial cells and is capable for strong bacteriostatic and bactericidal action. On the other hand, investigations of the material's interactions with human cells confirm that lower concentrations are highly compatible with osteosarcoma and fetal lung fibroblasts, but at higher concentrations (that provide bacteriostatic and bactericidal influences in bacteria) the material is toxic and capable of inducing morphological changes similar to those observed in bacterial cells.

Human stefin B role in cell's response to misfolded proteins and autophagy.

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

Gain in toxic function of stefin B EPM1 mutants aggregates: correlation between cell death, aggregate number/size and oxidative stress.

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.

N-terminally truncated forms of human cathepsin F accumulate in aggresome-like inclusions.

The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala(20)-Asp(484) (Δ(19)CatF), Pro(126)-Asp(484) (Δ(125)CatF), and Met(147)-Asp(484) (Δ(146)CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ(19)CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms.

Poly(D,L-lactide-co-glycolide)/hydroxyapatite core-shell nanospheres. Part 3: properties of hydroxyapatite nano-rods and investigation of a distribution of the drug within the composite.

A step-by-step analysis of the formation and the drug loading of the poly(D,L-lactide-co-glycolide)/hydroxyapatite (PLGA/HAp) composite was carried out in a perspective of the following parameters: the structure, the morphology and the adsorption/desorption properties of the composite's bioceramic part. The authors demonstrated the importance of the material's capacity to form a fine dispersion of solid HAp particles, as an initial step, for the further loading of the drug and for the formation of the core-shell structures. The nanometer-sized rods of HAp have the capacity of ensuring a rapid adsorption and a controlled desorption of the drug from their surface, and they can act as a nucleating site for the formation of polymeric cores. Each component of this material was labeled with fluorescence dye, which enabled an insight into the distribution of the components in the core-shells that were obtained as the final outcome. Such an analysis showed a high level of uniformity among the cores enclosed within polymeric shells. From a practical perspective, the labeling of each component of the composite can be regarded as an additional functionality of the material: labeling can enable us to monitor its action during the healing process. This ability to be easily detected is expected to enhance the procedure for the controlled delivery of antibiotics after their local implantation of carriers loaded with the antibiotic and to provide more careful control over this process.

Intracellular aggregation of human stefin B: confocal and electron microscopy study.

BACKGROUND: Protein aggregation is a major contributor to the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the CSTB (cystatin B) gene [StB (stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with features of neurodegeneration and increased oxidative stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has also been observed that StB is overexpressed after seizures and in certain neurodegenerative conditions, which could potentially lead to its aggregation. Human StB proved to be a good model system to study amyloid fibril formation in vitro and, as we show here, to study protein aggregation in cells. RESULTS: Endogenous human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin-proteasome system) led to an increase in the amount of the endogenous protein and also increased its aggregation. Further, we characterized both the untagged and T-Sapphire-tagged StB on overexpression in mammalian cells. Compared with wild-type StB, the EPM1 missense mutant (G4R), the aggregate-prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear aggregates accompanied by cytoskeletal reorganization. Non-homogeneous morphology of these large aggregates was revealed using TEM (transmission electron microscopy) with StB detected by immunogold labelling. StB-positive cytoplasmic aggregates were partially co-localized with ubiquitin, proteasome subunits S20 and S26 and components of microfilament and microtubular cytoskeleton using confocal microscopy. StB aggregates also co-localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed that protein aggregation was associated with reduced cell viability. CONCLUSIONS: We have shown that endogenous StB aggregates within cells, and that aggregation is increased upon protein overexpression or proteasome inhibition. From confocal and TEM analyses, we conclude that aggregates of StB show some of the molecular characteristics of aggresomes and may be eliminated from the cell by autophagy. Intracellular StB aggregation shows a negative correlation with cell survival.

Cystatin protease inhibitors and immune functions.

Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. They are wide spread in all living organisms (mammals, nematodes, arthropods etc.) and are involved in various biological processes where they regulate normal proteolysis and also take part in disease pathology. Many cystatins show changes in expression and/or localization, as well as changes in secretion, following certain stimuli acting on immune cells. In immune cells, cystatins interfere with antigen processing and presentation, phagocytosis, expression of cytokines and nitric oxide and these ways modify the immune response. Further, it has been suggested that cystatin-type molecules secreted from parasites down-modulate the host immune response. Precise understanding of the regulatory roles on proteolytic enzymes of endogenous and exogenous cystatins, such as those from parasites, will provide us with valuable insight into how immune response could be modulated to treat a specific disease. This review covers some specific functions of individual cystatins, with a particular focus on the relevance of cystatins to the immune response.

Cysteine proteases: destruction ability versus immunomodulation capacity in immune cells.

Cysteine proteases (cathepsins) play a pivotal role in various physiological processes, as well as in several diseases. In the immune response, maturation of major histocompatibility class II (MHC II) molecules and processing of antigens for further presentation by MHC II is tightly linked to the enzymes of the endosomal/lysosomal system, of which cysteine proteases constitute a major proportion. Furthermore, the process of autophagy provides access for cytosolic antigens to proteolysis by lysosomal cathepsins and subsequent MHC II presentation. Other specific functions of proteolytic enzymes associated with the immune response, such as activation of granzymes by cathepsin C in T-lymphocytes, are introduced and covered in this review.

Cystatin C as a potential marker for relapse in patients with non-Hodgkin B-cell lymphoma.

The concentration of cysteine protease inhibitor cystatin C was determined in sera from 59 patients with non-Hodgkin B-cell lymphoma using ELISA. The sera from 43 age and sex matched healthy blood donors served as controls. Cystatin C was significantly increased in sera of patients without therapy (mean 1136+/-SE 105.7ng/ml, p=0.00001) and with therapy (mean 1073+/-52ng/ml, p=0.001) compared to controls (mean 819+/-28ng/ml). The highest levels were determined in sera of patients with a relapse (mean 1680+/-196ng/ml). By using immunofluorescence staining and confocal microscopy we determined immature dendritic cells as a major population of cystatin C positive cells in affected lymph nodes. Our study reports for the first time that cystatin C is a potential marker for relapse in patients with non-Hodgkin B-cell lymphoma.

Carboxypeptidase cathepsin X mediates beta2-integrin-dependent adhesion of differentiated U-937 cells.

Cathepsin X is a lysosomal carboxypeptidase with a potential role in processes of inflammation and immune response. The integrin-binding motifs RGD and ECD, present in the pro- and in mature forms of cathepsin X, respectively, suggest that this enzyme might have a function in cell signaling and adhesion. In this study, we report that cysteine protease inhibitors E-64 and CA-074 and 2F12 monoclonal antibody, all of which inhibit cathepsin X activity, significantly reduced adhesion of differentiated U-937 cells to polystyrene- and fibrinogen-coated surfaces via Mac-1 integrin receptor, whereas their binding to vitronectin, fibronectin or Matrigel was not affected. On the other hand, cathepsin X, added to differentiating U-937 cells, stimulated their adhesion. Using confocal microscopy, we demonstrated that the pro-form of cathepsin X was co-localized with beta(2) and beta(3) integrin subunits and its mature form solely with the beta(2) integrin subunit with the most intense signal in cell-cell junctions in differentiated U-937 cells and in co-cultures with endothelial cells. Our results indicate that active cathepsin X mediates the function of beta(2) integrin receptors during cell adhesion and that it could also be involved in other processes associated with beta(2) integrin receptors such as phagocytosis and T cell activation.

Actinonin induces apoptosis in U937 leukemia cells.

We have previously shown that actinonin causes inhibition of cellular proliferation in U937 leukemia cells. In this report we demonstrate that the inhibition of cell growth by actinonin occurs through the induction of apoptosis. Signs of apoptosis at high actinonin concentration included DNA fragmentation, exposure of phosphatidylserine and condensation of cell nuclei. Apoptosis caused by actinonin was inhibited by Z-VAD-FMK, a broad specificity inhibitor of caspases, implicating the caspase pathway of apoptosis. Further, apoptosis was associated with a large increase in intracellular caspase-3 and -7 activities.

Differentiation- and maturation-dependent content, localization, and secretion of cystatin C in human dendritic cells.

Antigen-presenting cells (APC) play a pivotal role in the initiation of the T cell-mediated and antigen-specific immune response. The suggested role of endogenous inhibitor cystatin C (CyC) is to modulate cysteine proteases (cathepsins) present in human APC. To test this hypothesis, dendritic cells (DC) were generated in vitro from isolated monocytes, and changes in content, localization, and secretion of CyC and cathepsins S, L, and H (CatS, -L, and -H, repsectively) were followed in response to interleukin-4, enabling monocyte differentiation, and to tumor necrosis factor alpha (TNF-alpha), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF-alpha, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, -L, and -H in immature and mature DC shows that the regulatory potential of CyC toward CatS, -L, and -H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, -L, and -H, and they may facilitate DC migration to lymph nodes.