Simona Kavaliauskiene

Lipid requirements for entry of protein toxins into cells

The plant toxin ricin and the bacterial toxin Shiga toxin both belong to a group of protein toxins having one moiety that binds to the cell surface, and another, enzymatically active moiety, that enters the cytosol and inhibits protein synthesis by inactivating ribosomes. Both toxins travel all the way from the cell surface to endosomes, the Golgi apparatus and the ER before the ribosome-inactivating moiety enters the cytosol. Shiga toxin binds to the neutral glycosphingolipid Gb3 at the cell surface and is therefore dependent on this lipid for transport into the cells, whereas ricin binds both glycoproteins and glycolipids with terminal galactose. The different steps of transport used by these toxins have specific requirements for lipid species, and with the recent developments in mass spectrometry analysis of lipids and microscopical and biochemical dissection of transport in cells, we are starting to see the complexity of endocytosis and intracellular transport. In this article we describe lipid requirements and the consequences of lipid changes for the entry and intoxication with ricin and Shiga toxin. These toxins can be a threat to human health, but can also be exploited for diagnosis and therapy, and have proven valuable as tools to study intracellular transport.

Entrapment in phospholipid vesicles quenches photoactivity of quantum dots

Quantum dots have emerged with great promise for biological applications as fluorescent markers for immunostaining, labels for intracellular trafficking, and photosensitizers for photodynamic therapy. However, upon entry into a cell, quantum dots are trapped and their fluorescence is quenched in endocytic vesicles such as endosomes and lysosomes. In this study, the photophysical properties of quantum dots were investigated in liposomes as an in vitro vesicle model. Entrapment of quantum dots in liposomes decreases their fluorescence lifetime and intensity. Generation of free radicals by liposomal quantum dots is inhibited compared to that of free quantum dots. Nevertheless, quantum dot fluorescence lifetime and intensity increases due to photolysis of liposomes during irradiation. In addition, protein adsorption on the quantum dot surface and the acidic environment of vesicles also lead to quenching of quantum dot fluorescence, which reappears during irradiation. In conclusion, the in vitro model of phospholipid vesicles has demonstrated that those quantum dots that are fated to be entrapped in endocytic vesicles lose their fluorescence and ability to act as photosensitizers.

Blebbistatin, a myosin inhibitor, is phototoxic to human cancer cells under exposure to blue light

BACKGROUND Blebbistatin is a new inhibitor of cell motility. It is used to study dynamics of cytokinesis machinery in cells. However, the potential of this inhibitor as an anticancer agent has not been studied so far. METHODS Cytotoxicity of blebbistatin was evaluated in five human cell lines, FEMX-I melanoma, U87 glioma, androgen independent Du145 and androgen sensitive LNCaP prostate adenocarcinoma, and F11-hTERT immortalized fibroblasts. Phototoxicity of blebbistatin was assessed in these cell lines after their exposure to a blue light (390-470 nm). Photostability of blebbistatin and its reactive oxygen species (ROS) generating properties were measured during irradiation with the blue light. RESULTS Blebbistatin at a concentration range of 10-200 μmol/L was toxic to all studied cells. Toxic concentrations (TC) were about 10-25 μmol/L corresponding to TC10, 50-100 μmol/L to TC50 and 140-190 μmol/L to TC90. Only for the U87 glioma cells TC90 could not be measured as the highest studied concentration of 200 μmol/L gave around 70% toxicity. However, after exposure to the blue light blebbistatin exhibited phototoxicity on the cells, with a cytotoxicity enhancement ratio that was greatest for the FEMX-I cells (about 9) followed by LNCaP (5), Du145 (3), U87 (2) and F11-hTERT (1.7) cells. CONCLUSIONS Blebbistatin inhibits cell motility and viability. Under exposure to the blue light blebbistatin exhibits photodynamic action on human cancer cells. During the irradiation blebbistatin oxidizes dihydrorhodamine 123 but not Singlet Oxygen Sensor Green. GENERAL SIGNIFICANCE Our findings offer new possibilities for blebbistatin as a potential anticancer and photodynamic agent.

Protection against Shiga Toxins

Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans.

Cell density-induced changes in lipid composition and intracellular trafficking.

Abstract Cell density is one of the extrinsic factors to which cells adapt their physiology when grown in culture. However, little is known about the molecular changes which occur during cell growth and how cellular responses are then modulated. In many cases, inhibitors, drugs or growth factors used for in vitro studies change the rate of cell proliferation, resulting in different cell densities in control and treated samples. Therefore, for a comprehensive data analysis, it is essential to understand the implications of cell density on the molecular level. In this study, we have investigated how lipid composition changes during cell growth, and the consequences it has for transport of Shiga toxin. By quantifying 308 individual lipid species from 17 different lipid classes, we have found that the levels and species distribution of several lipids change during cell growth, with the major changes observed for diacylglycerols, phosphatidic acids, cholesterol esters, and lysophosphatidylethanolamines. In addition, there is a reduced binding and retrograde transport of Shiga toxin in high density cells which lead to reduced intoxication by the toxin. In conclusion, our data provide novel information on how lipid composition changes during cell growth in culture, and how these changes can modulate intracellular trafficking.

Determining the Turnover of Glycosphingolipid Species by Stable-Isotope Tracer Lipidomics ☆

In this study, we used water-soluble stable mass isotope precursors to measure the turnover of endogenous ceramide (Cer) and glycosphingolipids (GSLs) in HEp-2 cells. Cells incubated in the presence of [13C-U]glucose showed rapid incorporation of hexose residues with an increased mass of 6Da into GSLs. Different turnover rates of GSL classes and their molecular species were observed. Approximately 30% of the glucosylceramide, 50% of the lactosylceramide, and 50% of the globotriaosylceramide species showed a much slower turnover than the rest. This demonstrates the existence of different lipid pools, where a certain fraction of species survived for a long time in the cells. The species with the shortest N-amidated fatty acyl groups (C16:0 and C18:0) showed a more rapid turnover than those with the longest N-amidated fatty acids (C24:0 and C24:1). Experiments with addition of [13C-U]serine were performed to study de novo synthesis of Cer from serine and palmitoyl-CoA. These experiments revealed that de novo synthesis contributes to a minor extent to the total synthesis of new sphingolipids and showed that there is a more rapid formation of the longest Cer species (C24:0 and C24:1) than of the shortest species (C16:0), that is, the opposite as observed for the GSLs in the experiments with [13C-U]glucose. In conclusion, this FLUX lipidomics experimental approach with the addition of [13C-U]glucose to cells allows us to not only study the total turnover but also permit observations of lipid intermediates and metabolic flow of endogenous GSL species at the molecular lipid level.

Quantum dots affect expression of CD133 surface antigen in melanoma cells.

Background In novel treatment approaches, therapeutics should be designed to target cancer stem cells (CSCs). Quantum dots (QDs) are a promising new tool in fighting against cancer. However, little is known about accumulation and cytotoxicity of QDs in CSCs. Methods Accumulation and cytotoxicity of CdTe-MPA (mercaptopropionic acid) QDs in CSCs were assessed using flow cytometry and fluorescence-activated cell sorting techniques as well as a colorimetric cell viability assay. Results We investigated the expression of two cell surface-associated glycoproteins, CD44 and CD133, in four different cancer cell lines (glioblastoma, melanoma, pancreatic, and prostate adenocarcinoma). Only the melanoma cells were positive to both markers of CD44 and CD133, whereas the other cells were only CD44-positive. The QDs accumulated to a similar extent in all subpopulations of the melanoma cells. The phenotypical response after QD treatment was compared with the response after ionizing radiation treatment. The percentage of the CD44high−CD133high subpopulation decreased from 72% to 55%–58% for both treatments. The stem-like subpopulation CD44highCD133low/− increased from 26%–28% in the untreated melanoma cells to 36%–40% for both treatments. Conclusion Treatment of melanoma cells with QDs results in an increase of stem-like cell subpopulations. The changes in phenotype distribution of the melanoma cells after the treatment with QDs are comparable with the changes after ionizing radiation.

Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic

Glycosphingolipids (GSLs) are predominantly found in the outer leaflet of the plasma membrane, where they play a role in important processes such as cell adhesion, migration and signaling. However, by which mechanisms GSLs regulate these processes remains elusive. In this study, we therefore took advantage of the fact that some GSLs also serve as receptors for certain protein toxins, which rely on receptor binding for internalization and intoxication. Here, we demonstrate that Shiga and cholera toxins, which both possess multivalent GSL-binding capacity, induce dissociation of the cytosolic cPLA2α–AnxA1 complex in HeLa and HMEC-1 cells. The dissociation is mediated through an increase in cytosolic calcium levels and activation of the tyrosine kinase Syk. Ricin, a protein toxin that does not cross-link surface molecules, has no effect on the same complex. Importantly, we find that antibody-mediated cross-linking of Gb3 and GM1, the GSL receptors for Shiga and cholera toxin, respectively, also induces dissociation. These data demonstrate that cross-linking of GSLs at the plasma membrane mediates the intracellular signaling events resulting in dissociation of the complex. After dissociation, cPLA2α and AnxA1 are translocated to intracellular membranes where they are known to function in regulating membrane transport processes. In conclusion, we have characterized a novel mechanism for cell surface-induced initiation of intracellular signaling and transport events.

The anti-tumor drug 2-hydroxyoleic acid (Minerval) stimulates signaling and retrograde transport

2-hydroxyoleic acid (OHOA, Minerval®) is an example of a substance used for membrane lipid therapy, where the cellular membranes rather than specific proteins constitute the therapeutical target. OHOA is thought to mediate its anti-tumor effect by affecting the biophysical properties of membranes, which leads to altered recruitment and activation of amphitropic proteins, altered cellular signaling, and eventual cell death. Little is known about the initial signaling events upon treatment with OHOA, and whether the altered membrane properties would have any impact on the dynamic intracellular transport system. In the present study we demonstrate that treatment with OHOA led to a rapid release of intracellular calcium and activation of multiple signaling pathways in HeLa cells, including the PI3K-AKT1-MTOR pathway and several MAP kinases, in a process independent of the EGFR. By lipidomics we confirmed that OHOA was incorporated into several lipid classes. Concomitantly, OHOA potently increased retrograde transport of the plant toxin ricin from endosomes to the Golgi and further to the endoplasmic reticulum. The OHOA-stimulated ricin transport seemed to require several amphitropic proteins, including Src, phospholipase C, protein kinase C, and also Ca2+/calmodulin. Interestingly, OHOA induced a slight increase in endosomal localization of the retromer component VPS35. Thus, our data show that addition of a lipid known to alter membrane properties not only affects signaling, but also intracellular transport.

Novel actions of 2-deoxy-D-glucose: protection against Shiga toxins and changes in cellular lipids.

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.