Apoptosis-inducing membrane vesicles. A novel agent with unique properties

The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novo protein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL.     

Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.     

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.     

Docosahexaenoic acid: membrane properties of a unique fatty acid

Docosahexaenoic acid (DHA) with 22-carbons and 6 double bonds is the extreme example of an omega-3 polyunsaturated fatty acid (PUFA). DHA has strong medical implications since its dietary presence has been positively linked to the prevention of numerous human afflictions including cancer and heart disease. The PUFA, moreover, is essential to neurological function. It is remarkable that one simple molecule has been reported to affect so many seemingly unrelated biological processes. Although details of a molecular mode of action remain elusive, DHA must be acting at a fundamental level common to many tissues that is related to the high degree of conformational flexibility that the multiple double bonds have been identified to confer. One likely target for DHA action is at the cell membrane where the fatty acid is known to readily incorporate into membrane phospholipids. Once esterified into phospholipids DHA has been demonstrated to significantly alter many basic properties of membranes including acyl chain order and "fluidity", phase behavior, elastic compressibility, permeability, fusion, flip-flop and protein activity. It is concluded that DHA's interaction with other membrane lipids, particularly cholesterol, may play a prominent role in modulating the local structure and function of cell membranes.     

Ceramide phosphoethanolamine,an enigmatic cellular membrane sphingolipid

Ceramide phosphoethanolamine (CPE) is the major sphingolipid in invertebrates and in some bacterial species. It has been also detected in mammalian cells, although only in trace amounts. Complete understanding of the biophysical and physiological relevance of CPE is still lacking, and its biological role is still an open question. CPE differs in its biosynthetic mechanisms from sphingomyelin, due to the specific CPE synthase in invertebrates. In contrast to well-established sphingomyelin/cholesterol interactions that result in the formation of ordered membrane domains, the formation of ordered CPE/cholesterol domains is not favored. CPE might be crucial for the early development of Drosophila melanogaster, and it might be involved in the developmental stages of Trypanosoma brucei. As a Bacteroidetes-associated sphingolipid, CPE might also be involved in maintenance of these bacteria in their ecological niches. Therefore, efficient detection of CPE in biological systems is needed to better define its distribution and biological role(s).     

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.     

Neuronal membrane dynamics as fine regulator of sphingolipid composition

Sphingolipid metabolism is an intricate network of several interdependent and co-regulated pathways. In addition to the mainstream biosynthetic and catabolic pathways, several processes, even if less important in contributing to the final tissue sphingolipid composition from the quantitative point of view, might become relevant when sphingolipid metabolism is for any reason dysregulated and concur to the onset of neuronal pathologies. The main subcellular sites involved in the mainstream metabolic pathway are represented by the Golgi apparatus (for the biosynthesis) and by the lysosomes (for catabolism). On the other hand, the minor collateral pathways are associated with the plasma membrane and membranes of other organelles, and likely play important roles in the local regulation of membrane dynamics and contribute to maintain a perfect membrane organization functional to the physiology of the cell. In this review, we will consider few aspects of the sphingolipid metabolic pathway depending by the dynamic of the membranes that seems to become relevant in neurodegenerative diseases.     

Evaluation of synthetic sphingolipid analogs as ligands for peroxisome proliferator-activated receptors

In this Letter, we assessed newly synthesized sphingolipid analogs as ligands for peroxisome proliferator-activated receptor (PPAR)α, PPARβ or PPARγ, using a dual-luciferase reporter system. We tested 640 sphingolipid analogs for ligand activity. As a result, seven types: A9, B9, C9, C50, F66, G66 and H66, were found to show agonistic activities for PPARs.     

Cancer and sphingolipid storage disease therapy using novel synthetic analogs of sphingolipids

Sphingolipid metabolites have become recognized for their participation in cell functions and signaling events that control a wide array of cellular activities. Two main sphingolipids, ceramide and sphingosine-1-phosphate, are involved in signaling pathways that regulate cell proliferation, apoptosis, motility, differentiation, angiogenesis, stress responses, protein synthesis, carbohydrate metabolism, and intracellular trafficking. Ceramide and S1P often exert opposing effects on cell survival, ceramide being pro-apoptotic and S1P generally promoting cell survival. Therefore, the conversion of one of these metabolites to the other by sphingolipid enzymes provides a vast network of regulation and provides a useful therapeutic target. Here we provide a survey of the current knowledge of the roles of sphingolipid metabolites in cancer and in lipid storage disease. We review our attempts to interfere with this network of regulation and so provide new treatments for a range of diseases. We synthesized novel analogs of sphingolipids which inhibit the hydrolysis of ceramide or its conversion to more complex sphingolipids. These analogs caused elevation of ceramide levels, leading to apoptosis of a variety of cancer cells. Administration of a synthetic analog to tumor-bearing mice resulted in reduction and even disappearance of the tumors. Therapies for sphingolipid storage diseases, such as Niemann-Pick and Gaucher diseases were achieved by two different strategies: inhibition of the biosynthesis of the substrate (substrate reduction therapy) and protection of the mutated enzyme (chaperone therapy). Sphingolipid metabolism was monitored by the use of novel fluorescent sphingolipid analogs. The results described in this review indicate that our synthetic analogs could be developed both as anticancer drugs and for the treatment of sphingolipid storage diseases.     

5-Iminodaunomycin. An anthracycline with unique properties

5-Iminodaunomycin forms a 3:1 complex with Fe(III) at pH 7.4. Drug-metal complex formation is associated with a marked decline in absorbance at 548 and 593 nm and the appearance of a broad band above 625 nm. The 5-iminodaunomycin-Fe(III) complex reacts with hydrogen peroxide to yield .OH radicals. This reaction is at a maximum at a drug/iron ratio of 2:1, and the yield is far less than that obtained with the doxorubicin-iron complex. In contrast to the results with doxorubicin, the production of .OH declines markedly at high 5-iminodaunomycin/iron ratios. There is a close parallel between the formation of hydroxyl radicals and the ability of the 5-iminodaunomycin complex to nick supercoiled SV40 DNA. The suppression of both .OH and DNA damage at high 5-iminodaunomycin:iron ratios is the result of several factors. 1) The presence of DNA stimulates .OH production from the doxorubicin complex, but not 5-iminodaunomycin; 2) doxorubicin reduces its chelated Fe(III) to Fe(II), but 5-iminodaunomycin does not; 3) 5-iminodaunomycin forms such a stable drug-metal complex that solvent water and, therefore, presumably H2O2, has diminished access to the chelated iron. The affinity of 5-iminodaunomycin is such that it can quantitatively abstract iron from doxorubicin. As a result, 5-iminodaunomycin is an effective competitive inhibitor of .OH radical formation by the doxorubicin-iron complex.     

Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots

The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization (+ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.     

The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition

The influence of agents, known to affect the membrane dipole potential, phloretin and RH 421, on the multi channel activity of amphotericin B in lipid bilayers of various compositions, was studied. It was shown that the effects were dependent on the membrane’s phospholipid and sphingolipid type. Phloretin enhanced amphotericin B induced steady-state transmembrane current through bilayers made from binary mixtures of POPC (DOPC) and ergosterol and ternary mixture of DPhPC, ergosterol and stearoylphytosphingosine. RH 421 increased steady-state polyene induced transmembrane current through membranes made from binary mixtures of DPhPC (DPhPS) and ergosterol and ternary mixture of DPhPS, ergosterol and stearoylphytosphingosine. It was proposed that the observed effects reflect the fine balance of the interactions between the various components present: amphotericin B, ergosterol, phospholipid, sphingolipid and dipole modifier. The shape of lipid molecules seems to be an important factor impacting the responses of amphotericin B modified bilayers to dipole modifiers. The influence of different phospholipids and sphingolipids on the physical and structural properties of ordered lipid microdomains, enriched in AmB, was also discussed. It was also shown that RH 421 enhanced the antifungal activity of amphotericin B in vitro.     

Angioarrestin: a unique angiopoietin-related protein with anti-angiogenic properties

The process of angiogenesis plays a pivotal role in embryogenesis, wound healing, and tumorigenesis through the growth of new blood vessels from pre-existing vasculature. Among the angiogenic factors recently identified as specific for vascular endothelium are the angiopoietins. In depth characterization of the angiopoietins has allowed investigators to better understand the molecular basis of blood vessel formation and vascular endothelial cell function. In this review, we describe angiopoietins and related family members, with particular emphasis on a recently identified protein known as angioarrestin. Our investigations clearly demonstrate that angioarrestin is an anti-angiogenic molecule. The effects of angioarrestin on tumor cell progression and specific aspects of the angiogenic cascade in in vitro models are further discussed.     

Methylation of glycosylated sphingolipid modulates membrane lipid topography and pathogenicity of Cryptococcus neoformans

In previous studies we showed that the replication of Cryptococcus neoformans in the lung environment is controlled by the glucosylceramide (GlcCer) synthase gene (GCS1), which synthesizes the membrane sphingolipid GlcCer from the C9-methyl ceramide. Here, we studied the effect of the mutation of the sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position 9 of the sphingosine backbone of ceramide. The C. neoformans Δsmt1 mutant does not make C9-methyl ceramide and, thus, any methylated GlcCer. However, it accumulates demethylated ceramide and demethylated GlcCer. The Δsmt1 mutant loses more than 80% of its virulence compared with the wild type and the reconstituted strain. Interestingly, growth of C. neoformans Δsmt1 in the lung was decreased and C. neoformans cells were contained in lung granulomas, which significantly reduced the rate of their dissemination to the brain reducing the onset of meningoencephalitis. Thus, using fluorescent spectroscopy and atomic force microscopy we compared the wild type and Δsmt1 mutant and found that the altered membrane composition and GlcCer structure affects fungal membrane rigidity, suggesting that specific sphingolipid structures are required for proper fungal membrane organization and integrity. Therefore, we propose that the physical structure of the plasma membrane imparted by specific classes of sphingolipids represents a critical factor for the ability of the fungus to establish virulence.     

Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells

Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple "microdomains" of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-D-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.     

Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.     

Annexin V staining during reperfusion detects cardiomyocytes with unique properties

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and further elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in approximately 3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.