Inhibitors of sphingolipid metabolism enzymes

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P₁ and S1P₃, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.     

Release, purification, and characterization of platelet-activating factor (PAF)

Platelet-activating factor (PAF) is a phospholipid mediator, released by basophils, macrophages and neutrophils under immunological and non immunological stimuli. It aggregates platelets and liberates their vasoactive contents. We studied the "spontaneous" release of PAF from hog blood leukocytes : optimal conditions were 22 degrees C, pH 9.5 in BSA and Ca2+-containing Tyrode's. This release was inhibited by the Ca2+-chelating agent, EDTA, and by the phospholipase A2 inhibitor, bromophenacyl bromide. Disruption of the cells did not yield PAF, indicating that it is not a "preformed" mediator. A preparative procedure for the extraction and purification of bulk quantities of PAF was developed. Purification was performed by silicic acid columns followed by high pressure liquid chromatography. The active fraction was eluted between sphingomyelin and lysophosphatidylcholine. The PAF purest fractions were still contaminated with these phospholipids as shown by thin layer chromatography and chemical ionization mass spectrometry. PAF activity was not affected by treatment with diazomethane, acetylation or hydrogenation. Our results combined with those obtained from our previous studies of the PAF structure using specific phospholipases indicate that PAF is a glycero-phospholipid devoid of ester function at position 1. This allowed us to establish precise criteria to distinguish PAF from other aggregating agents.     

The leucine 10 residue in the pleckstrin homology domain of ceramide kinase is crucial for its catalytic activity

Ceramide kinase (CERK) converts ceramide (Cer) to ceramide-1-phosphate (C1P), a newly recognized bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. However, little is known regarding the functional roles of this domain in CERK. In this study, we have demonstrated that the PH domain of CERK is essential for its enzyme activity. Using site-directed mutagenesis, we have further determined that Leu10 in the PH domain has an important role in CERK activity. Replacing this residue with a neutral alanine or isoleucine, caused a dramatic decrease in CERK activity to 1% and 29%, respectively, compared to CERK, but had no effect on substrate affinity. The study presented here suggests that the PH domain of CERK is not only indispensable for its activity but also act as a regulator of CERK activity.     

Ceramide kinase: the first decade

It has been some 20 years since the initial discovery of ceramide 1-phosphate (C1P) and nearly a decade since ceramide kinase (CERK) was cloned. Many studies have shown that C1P is important for membrane biology and for the regulation of membrane-bound proteins, and the CERK enzyme has appeared to be tightly regulated in order to control both ceramide levels and production of C1P. Furthermore, C1P made by CERK has emerged as a genuine signalling entity. However, it represents only part of the C1P pool that is available in the cell, therefore suggesting that alternative unknown C1P-producing mechanisms may also play a role. Recent technological developments for measuring complex sphingolipids in biological samples, together with the availability of Cerk-deficient animals as well as potent CERK inhibitors, have now provided new grounds for investigating C1P biology further. Here, we will review the current understanding of CERK and C1P in terms of biochemistry and functional implications, with particular attention to C1P produced by CERK.     

FTY720-phosphate is dephosphorylated by lipid phosphate phosphatase 3

FTY720 is a novel immunomodulatory drug efficacious in the treatment of multiple sclerosis. The drug is converted in vivo to the monophosphate, FTY720-P, by sphingosine kinase 2. This conversion is incomplete, suggesting opposing actions of kinase and phosphatase activities. To address which of the known lipid phosphatases might dephosphorylate FTY720-P, we overexpressed the broad specificity lipid phosphatases LPP1-3, and the specific S1P phosphatases (SPP1 and 2) in HEK293 cells, and performed in vitro assays using lysates of transfected cells. Among LPPs, only LPP3 was able to dephosphorylate FTY720-P; among SPPs, only SPP1 showed activity against FTY720-P. On intact cells, LPP3 acted as an ecto-phosphatase or FTY720-P, thus representing the major phosphatase involved in the equilibrium between FTY720 and FTY720-P observed in vivo.     

Dose dependent actions of LCL521 on acid ceramidase and key sphingolipid metabolites

The function of acid ceramidase (ACDase), whose congenital deficiency leads to Farber disease, has been recognized to be vital to tumor cell biology, and inhibition of its activity may be beneficial in cancer therapy. Therefore, manipulation of the activity of this enzyme may have significant effect, especially on cancer cells. LCL521, Di-DMG-B13, is a lysosomotropic inhibitor of ACDase. Here we define complexities in the actions of LCL521 on ACDase. Systematic studies in MCF7 cells showed dose and time divergent action of LCL521 on ACDase protein expression and sphingolipid levels. Low dose of LCL521 (1?µM) effectively inhibited ACDase in cells, but the effects were transient. A higher dose of LCL521 (10?µM) caused a profound decrease of sphingosine and increase of ceramide, but additionally affected the processing and regeneration of the ACDase protein, with biphasic and reversible effects on the expression of ACDase, which paralleled the long term changes of cellular sphingosine and ceramide. Finally, the higher concentrations of LCL521 also inhibited Dihydroceramide desaturase (DES-1). In summary, LCL521 exhibits significant effects on ACDase in a dose and time dependent manner, but dose range and treatment time need to be paid attention to specify its future exploration on ACDase targeted cancer treatment.     

Proximate composition, fatty acids, cholesterol, minerals in frozen red porgy

The proximate composition of frozen red porgy (Pagrus pagrus) was determined. The moisture, ash, protein and total lipids (45.5+/-1.4% PL of which 90.4+/-2.0% PhL) were found to be 71.7+/-1.0%, 1.73+/-0.12%, 21.5+/-0.8% and 0.81+/-0.09% of the wet muscle tissue, respectively. 16:0 and 18:0 were the main SFA, 18:1 (omega-9 and omega-7) the main MUFA while DHA, EPA and arachidonic acid were the main polyunsaturated fatty acids (PUFA). The SFA/PUFA ratio was 1.5 and the omega-3/omega-6 ratio was 3.02. The cholesterol content was found to be 8.18+/-0.34 mg/100 g of the wet muscle tissue. Ni, Cr, Mn, Cu, Zn, Fe and Mg were determined in the muscles, skin, hepatopancreas and head of the fish. The covering percentage of the recommended daily allowance/intake (RDA/RDI) for each mineral, in the muscle tissue, has been calculated to 14.2% (males) and 7.89% (females) for Fe, 2.87% for Cu, 4.07% for Zn 0.4% for Mn, 13.9% for Ni, 20.2% for Cr and 10.4% for Mg.