18 research outputs found
The Ether Lipid Precursor Hexadecylglycerol Causes Major Changes in the Lipidome of HEp-2 Cells
<div><p>The ether-lipid precursor <i>sn-1</i>-O-hexadecylglycerol (HG) can be used to compensate for early metabolic defects in ether-lipid biosynthesis. To investigate a possible metabolic link between ether-linked phospholipids and the rest of the cellular lipidome, we incubated HEp-2 cells with HG. Mass spectrometry analysis revealed major changes in the lipidome of HG-treated cells compared to that of untreated cells or cells treated with palmitin, a control substance for HG containing an acyl group instead of the ether group. We present quantitative data for a total of 154 species from 17 lipid classes. These species are those constituting more than 2% of their lipid class for most lipid classes, but more than 1% for the ether lipids and glycosphingolipids. In addition to the expected ability of HG to increase the levels of ether-linked glycerophospholipids with 16 carbon atoms in the <i>sn-1</i> position, this precursor also decreased the amounts of glycosphingolipids and increased the amounts of ceramide, phosphatidylinositol and lysophosphatidylinositol. However, incubation with palmitin, the fatty acyl analogue of HG, also increased the amounts of ceramide and phosphatidylinositols. Thus, changes in these lipid classes were not ether lipid-dependent. No major effects were observed for the other lipid classes, and cellular functions such as growth and endocytosis were unaffected. The data presented clearly demonstrate the importance of performing detailed quantitative lipidomic studies to reveal how the metabolism of ether-linked glycerophospholipids is coupled to that of glycosphingolipids and ester-linked glycerophospholipids, especially phosphatidylinositols.</p></div
Quantitative analysis of ceramide and glycosphingolipids after HG or palmitin treatment.
<p>The major species of (<b>A</b>) Cer, (<b>B</b>) GlcCer, (<b>C</b>) LacCer, and (<b>D</b>) Gb3 in HEp-2 cells treated with HG (20 µM), palmitin (20 µM) or ethanol (0.1%, as control) for 24 hours. The species shown here are species comprising more than 1% of the total mass of any of the classes.</p
Overall changes in the lipidome of HEp-2 cells after treatment with HG or palmitin.
<p>HEp-2 cells were treated for 24 hours with HG (20 µM), palmitin (20 µM) or ethanol (0.1%, as control) before analyzing the lipidome by MS. (<b>A</b>) The total amount of the different lipid classes are shown as absolute values (note the logarithmic scale) and (<b>B</b>) the difference between treated cells and control cells are expressed as relative values.</p
Effect of low temperature and DMSO on PC biosynthesis.
<p>CHO-K1 cells stably expressing PC-wt or PC-A267T were incubated at 26°C (A) or in culture medium containing 2% DMSO (B). PC concentrations were quantified in cell lysates (▪) and culture medium (□), and normalized to total protein (TP) concentrations of the corresponding lysate samples. Histograms and the bars represent mean + SEM values, n = 9. * (p≤0.05, Mann-Whithey test), PC-wt/PC-A267T treated versus PC-wt/PC-A267T untreated.</p
Quantitative analysis of glycerophospholipids after HG or palmitin treatment.
<p>The major species of (<b>A</b>) PC O, (<b>B</b>) PC P, (<b>C</b>) PE O, (<b>D</b>) PE P, (<b>E</b>) PC and (<b>F</b>) PE, in HEp-2 cells treated with HG (20 µM), palmitin (20 µM) or ethanol (0.1%, as control) for 24 hours. The species shown here are species comprising more than 1% of the total mass of the ether lipids and more than 2% of PC and PE for at least one of the samples.</p
DNA fragmentation analysis in CHO-K1 cells stably expressing PC-wt or PC-A267T.
<p>The levels of cytoplasmic DNA fragments (mono- and oligonucleosomes) in cells stably expressing PC variants was measured by the Cell Death Detection ELISA<sup>PLUS</sup> kit. The results were normalized to total protein (TP) concentrations and presented as mean + SEM values of four independent experiments (each experiment performed in triplicates). * (p≤0.05, Mann-Whithey test).</p
Biosynthesis of ether and ester glycerophospholipids and the chemical structures of the precursors used.
<p>(<b>A</b>) Schematic overview of the biosynthesis of ether and ester glycerophospholipids. Note that 1-alkylglycerols such as HG (red box) may enter the pathway through phosphorylation to 1-alkylglycerol 3-phosphate by an alkylglycerol kinase. For abbreviations of the compounds and enzymes, see below. (<b>B</b>) The chemical structure of the compounds used in this study, HG and palmitin. DHAP; dihydroxyacetone phosphate, G3P; glycerol 3-phoshpate, DHAPAT; dihydroxyacetone phosphate acyltransferase, GPAT; glycerol phosphate acyltransferase, ADHAPS; alkyldihydroxyacetone phosphate synthase, LPA; lysophosphatidic acid, LPAAT; lysophosphatidic acid acyltransferase, PAP; phosphatidic acid phosphatase, EPT; ethanolamine phosphotransferase, CEPT; choline/ethanolamine phosphotransferase.</p
Identification of PC-chaperone complexes in CHO-K1 cells stably expressing PC-wt or PC-A267T.
<p>The cells were pretreated with dithiobis[succinimydylpropionate] (DSP) before cell lysis. Cell lysates with equivalent amounts of PC were separated on SDS-PAGE under reducing conditions and immunoblotted with anti-PC to detect PC-chaperone complexes. Similar results were obtained in three independent experiments. The molecular weight standards are shown on the right (kDa) and the positions of the PC-chaperone complexes are marked with the arrows on the left (A). Identification of chaperones associated with the PC mutant. The cell lysates pretreated with DSP were subjected to SDS-PAGE and Western blot analysis with antibodies against indicated chaperones. Two independent experiments with similar results were performed. (B).</p
Expression levels of ER stress and UPR activation markers.
<p>Immunoglobulin-binding protein (BiP) (A) and phosphorylated eukaryotic initiation factor 2α (P-eIF2α) (B) levels were analyzed by Western blotting in lysates from CHO-K1 cells stably expressing PC-wt (lane 1) or PC-A267T (lane 2). Fold increase of BiP and P-eIF2α levels in CHO-K1 cells expressing PC-A267T are shown as histograms and bars, which represent mean + SEM values of expression levels detected in three independent experiments. BiP bands were quantified and normalized to corresponding amounts of α-tubulin. P-eIF2α bands were quantified and normalized to corresponding amounts of eIF2α.</p
Mixed-valent EPR spectra of crucian carp R2ii and p53R2i compared to mammalian homologs.
<p>Mixed-valent EPR spectra of crucian carp R2ii and p53R2i compared to mammalian homologs.</p