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Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.
Sphingolipids serve important structural and functional roles in cellular membranes and myelin sheaths. Plasma sphingolipids have been shown to predict cognitive decline and Alzheimer's disease. However, the association between plasma sphingolipid levels and brain white matter (WM) microstructure has not been examined. We investigated whether plasma sphingolipids (ceramides and sphingomyelins) were associated with magnetic resonance imaging-based diffusion measures, fractional anisotropy (FA), and mean diffusivity, 10.5 years later in 17 WM regions of 150 cognitively normal adults (mean age 67.2). Elevated ceramide species (C20:0, C22:0, C22:1, and C24:1) were associated with lower FA in multiple WM regions, including total cerebral WM, anterior corona radiata, and the cingulum of the cingulate gyrus. Higher sphingomyelins (C18:1 and C20:1) were associated with lower FA in regions such as the anterior corona radiata and body of the corpus callosum. Furthermore, lower sphingomyelin to ceramide ratios (C22:0, C24:0, and C24:1) were associated with lower FA or higher mean diffusivity in regions including the superior and posterior corona radiata. However, although these associations were significant at the a priori p < 0.05, only associations with some regional diffusion measures for ceramide C22:0 and sphingomyelin C18:1 survived correction for multiple comparisons. These findings suggest plasma sphingolipids are associated with variation in WM microstructure in cognitively normal aging.
Copyright © 2016 Elsevier Inc. All rights reserved.
The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death globally. To develop better diagnostics and more effective treatments, research in the past decades has focused on identification of molecular changes in the genome, transcriptome, proteome, and more recently also the metabolome. Phospholipids, which nevertheless play a central role in cell functioning, remain poorly explored. Here, using a mass spectrometry (MS)-based phospholipidomics approach, we profiled 179 phospholipid species in malignant and matched non-malignant lung tissue of 162 NSCLC patients (73 in a discovery cohort and 89 in a validation cohort). We identified 91 phospholipid species that were differentially expressed in cancer versus non-malignant tissues. Most prominent changes included a decrease in sphingomyelins (SMs) and an increase in specific phosphatidylinositols (PIs). Also a decrease in multiple phosphatidylserines (PSs) was observed, along with an increase in several phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species, particularly those with 40 or 42 carbon atoms in both fatty acyl chains together. 2D-imaging MS of the most differentially expressed phospholipids confirmed their differential abundance in cancer cells. We identified lipid markers that can discriminate tumor versus normal tissue and different NSCLC subtypes with an AUC (area under the ROC curve) of 0.999 and 0.885, respectively. In conclusion, using both shotgun and 2D-imaging lipidomics analysis, we uncovered a hitherto unrecognized alteration in phospholipid profiles in NSCLC. These changes may have important biological implications and may have significant potential for biomarker development.
© 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.
BACKGROUND - Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease and a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. Previously, we reported that dietary docosahexaenoic acid (DHA, 22:6,n-3) was more effective than eicosapentaenoic acid (EPA, 20:5,n-3) at reversing western diet (WD) induced NASH in LDLR(-/-) mice.
METHODS - Using livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism.
RESULTS - Livers from WD + olive oil (WD + O)-fed mice displayed histological and gene expression features consistent with NASH. The metabolomic analysis of 320 metabolites established that the WD and n-3 polyunsaturated fatty acid (PUFA) supplementation had broad effects on all major metabolic pathways. Livers from WD + O-fed mice were enriched in saturated (SFA) and monounsaturated fatty acids (MUFA), palmitoyl-sphingomyelin, cholesterol, n-6 PUFA, n-6 PUFA-containing phosphoglycerolipids, n-6 PUFA-derived oxidized lipids (12-HETE) and depleted of C20-22 n-3 PUFA-containing phosphoglycerolipids, C20-22 n-3 PUFA-derived oxidized lipids (18-HEPE, 17,18-DiHETE) and S-lactoylglutathione, a methylglyoxal detoxification product. WD + DHA was more effective than WD + EPA at attenuating WD + O-induced changes in NASH gene expression markers, n-6 PUFA and oxidized lipids, citrate and S-lactosyl glutathione. Diet-induced changes in hepatic MUFA and sphingolipid content were associated with changes in expression of enzymes involved in MUFA and sphingolipid synthesis. Changes in hepatic oxidized fatty acids and S-lactoylglutathione, however, correlated with hepatic n-3 and n-6 C20-22 PUFA content. Hepatic C20-22 n-3 PUFA content was inversely associated with hepatic α-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress.
CONCLUSION - DHA regulation of hepatic SFA, MUFA, PUFA, sphingomyelin, PUFA-derived oxidized lipids and S-lactoylglutathione may explain the protective effects of DHA against WD-induced NASH in LDLR(-/-) mice.
This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼ 12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ∼ 12 nm and 10 nm diameter, contained ∼ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ∼ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.
To devise successful imaging and therapeutic strategies, the identification of β-cell surface markers is one of the challenges in diabetes research that has to be resolved. We previously showed that IC2, a rat monoclonal IgM antibody, can be used for ex vivo determination of β-cell mass by imaging. Further progress toward the development of an antibody-based imaging agent was hampered by the lack of knowledge regarding the nature and composition of the IC2 antigen. Here, we show a series of systematic experiments involving classical lipid extraction and chromatography techniques combined with immunochemistry, which led to the identification of sphingomyelin as the target antigen assembled in the form of patches on the β-cell surface. Our findings were verified by modulating SM by enzymatic cleavage, downregulation, upregulation, and perturbation of membrane SM and observation of corresponding changes in IC2 binding. Cholesterol participates in stabilization of these patches, as its removal results in loss of IC2 binding. We believe that these findings have implications for identifying future ligands for the proposed antigen for imaging purposes as well as for potential therapy, as sphingomyelin has been shown to play a role in the apoptotic cascade in pancreatic β cells.
Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.
Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCT alpha-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCT alpha is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCT alpha-deficient cells. Pro-tumor necrosis factor alpha and interleukin-6 remain lodged in the secretory compartment of CCT alpha-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1 beta secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCT alpha-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor alpha or interleukin-6. These results identify CCT alpha as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.