Cellular Phospholipid Metabolism Research Articles

Molecular mechanisms of ferroptosis and its effects on bladder cancer.

Bladder cancer (BC) is one of the 3 common malignant tumors in the urinary system, with high incidence, easy metastasis, poor therapeutic efficacy, and poor prognosis, which seriously threatens the health of human. Tumor cells exhibit a strong demand for iron, and iron overload can induce ferroptosis, which is an iron dependent cell death caused by lipid peroxidation and cell membrane damage. Therefore, ferroptosis has strong anti-tumor potential. The molecular mechanisms of ferroptosis is associated with abnormalities in cellular phospholipid metabolism and iron metabolism, and dysregulation of antioxidant and non-antioxidant systems Xc-/glutathione (GSH)/glutathione peroxidase 4 (GPX4). Ferroptosis relevant molecules play important roles in the occurrence and development, metastasis, drug resistance, and immune response of BC, and are expected to become targets for the treatment of BC.

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

ABSTRACTGammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.

Quantitative Analysis of Cellular Phospholipid Metabolism by Enzymatic Fluorometric Assays

Quantitative Analysis of Cellular Phospholipid Metabolism by Enzymatic Fluorometric Assays

Therapeutic Target Metabolism Observed Using Hyperpolarized 15N Choline

Choline is a precursor of cellular phospholipid metabolism that provides Magnetic Resonance (MR) and Positron Emission Tomography (PET) biomarkers for cancer detection and response assessment. Employing Dynamic Nuclear Polarization we show that the MR signal of 15N in choline can be enhanced by at least 4 orders of magnitude with a relaxation time of ca. 4 min, providing a method to observe the action of choline kinase, an important target for novel cancer therapeutics.

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Dynamic lipidomics of the nucleus

Once nuclear envelope membranes have been removed from isolated nuclei, around 6% of mammalian cell phospholipid is retained within the nuclear matrix, which calculations suggest may occupy 10% of the volume of this subcellular compartment. It is now acknowledged that endonuclear phospholipid, largely ignored for the past 40 years, provides substrate for lipid-mediated signaling events. However, given its abundance, it likely also has other as yet incompletely defined roles. Endonuclear phosphatidylcholine is the predominant phospholipid comprising distinct and highly saturated molecular species compared with that of the whole cell. Moreover, this unusual composition is subject to tight homeostatic maintenance even under conditions of extreme dietary manipulation, presumably reflecting a functional requirement for highly saturated endonuclear phosphatidylcholine. Recent application of new lipidomic technologies exploiting tandem electrospray ionization mass spectrometry in conjunction with deuterium stable isotope labeling have permitted us to probe not just molecular species compositions but endonuclear phospholipid acquisition and turnover with unparalleled sensitivity and specificity. What emerges is a picture of a dynamic pool of endonuclear phospholipid subject to autonomous regulation with respect to bulk cellular phospholipid metabolism. Compartmental biosynthesis de novo of endonuclear phosphatidylcholine contrasts with import of phosphatidylinositol synthesized elsewhere. However, irrespective of the precise temporal-spatial-dynamic relationships underpinning phospholipid acquisition, derangement of endonuclear lipid-mediated signaling from these parental phospholipids halts cell growth and division indicating a pivotal control point. This in turn defines the manipulation of compartmentalized endonuclear phospholipid acquisition and metabolism as intriguing potential targets for the development of future antiproliferative strategies.

Bromoenol Lactone Promotes Cell Death by a Mechanism Involving Phosphatidate Phosphohydrolase-1 Rather than Calcium-independent Phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

ATP-binding Cassette Transporter A7 (ABCA7) Binds Apolipoprotein A-I and Mediates Cellular Phospholipid but Not Cholesterol Efflux

ATP-binding cassette transporter 1 (ABCA1), the defective transporter in Tangier disease, binds and promotes cellular cholesterol and phospholipid efflux to apolipoprotein I (apoA-I). Based on a high degree of sequence homology between ABCA1 and ABCA7, a transporter of unknown function, we investigated the possibility that ABCA7 might be involved in apolipoprotein binding and lipid efflux. Similarly to cells expressing ABCA1, HEK293 cells overexpressing ABCA7 showed specific binding and cross-linking of lipid-poor apoA-I. ABCA7 expression increased cellular phosphatidylcholine and sphingomyelin efflux to apoA-I in a manner similar to ABCA1 but had no effect on cholesterol efflux. Western analysis showed a high protein level of ABCA7 in mouse spleen, lung, adrenal, and brain but low expression in liver. In contrast to ABCA1, ABCA7 showed moderate basal mRNA and protein levels in macrophages and lymphocytes but no induction by liver X receptor activation. These studies show that ABCA7 has the ability to bind apolipoproteins and promote efflux of cellular phospholipids without cholesterol, and they suggest a possible role of ABCA7 in cellular phospholipid metabolism in peripheral tissues.

Structure of apo-phosphatidylinositol transfer protein alpha provides insight into membrane association.

Phosphatidylinositol transfer protein alpha (PITP alpha) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three-dimensional crystal structure of a phospholipid-free mouse PITP alpha at 2.0 A resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid-binding cavity on one side by displacement of the C-terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central beta-sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane-bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).

Paper Alert

Paper Alert

Structure of a Multifunctional Protein: MAMMALIAN PHOSPHATIDYLINOSITOL TRANSFER PROTEIN COMPLEXED WITH PHOSPHATIDYLCHOLINE

Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The three-dimensional structure of the alpha-isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 A resolution by x-ray diffraction techniques. A single beta-sheet and several long alpha-helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.

Lithium and protein kinase C modulators regulate swelling-activated K-Cl cotransport and reveal a complete phosphatidylinositol cycle in low K sheep erythrocytes.

K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li(i)) but not external (Li(o)) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K(i) of approximately 7 mm. In Cl, Li(i) decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li(i) reduced the V(max) and increased the K(m) for K efflux in Cl. Furthermore, Li(i) increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li(i) appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume.

Hereditary Ataxias

Hereditary Ataxias

Brain phospholipids and fatty acids in Friedreich's ataxia and spinocerebellar atrophy type-1.

Previous studies of patients with spinocerebellar atrophy type 1 (SCA-1) and Friedreich's ataxia (FA) have suggested the occurrence of membrane disturbances in both disorders. We measured concentrations of phosphatidylcholine (PC), diacyl and plasmalogen phosphatidylethanolamine (PE), and phosphatidylserine (PS), along with their fatty acid profiles, in the brains of eight patients with Friedreich's ataxia (FA) and nine patients with dominantly inherited spinocerebellar atrophy type 1 (SCA-1). Compared with the controls, levels of all phospholipid types (PE, PS, and PC) were reduced in the cerebellar but not occipital cortex of SCA-1 patients. In contrast, in the FA group, levels of PS and PE, but not PC, were reduced in both cerebellar and occipital cortices. The fatty acid composition of individual brain phospholipids was altered in both FA and SCA-1 patients, most markedly in the plasmalogen PE and PS classes of cerebellar phospholipids. Given the neuropathologic characteristics of each disorder, it is likely that altered fatty acid composition and phospholipid levels in SCA-1 cerebellar cortex occur as a consequence of pronounced cerebellar degeneration. In contrast, reduced phospholipid levels in FA cerebellar and occipital cortex, areas characterized by, at most, minimal neuronal loss in FA, may represent a widespread alteration in cellular phospholipid metabolism occurring in response to the specific gene defect in the disorder.

31P NMR measurements of the effects of unsaturated fatty acids on cellular phospholipid metabolism.

31P NMR measurements on extracts prepared from a variety of cultured mammalian cell lines and primary rat hepatocytes have shown changes in the levels of several phospholipid metabolites after incubation of cells with unsaturated fatty acids. These data suggest a possible link between the accumulation of neutral lipid and the changes in phospholipid metabolite concentrations that have been observed in some tumor cells and other rapidly growing tissues such as the regenerating liver and mitogen-stimulated lymphocytes.

The antitumor phospholipid analog, hexadecylphosphocholine, activates cellular phospholipase D.

Hexadecylphosphocholine (HePC), a glycerol-free phospholipid analog, belongs to a new class of drugs that demonstrate selective anticancer activity. The mechanisms underlying the anticancer activity are unclear. To investigate possible signal transduction relationships we examined the influence of HePC on cellular phospholipid metabolism. When HePC was added to cultured human breast fibroblasts (CCD-986-SK cells) that had been radiolabeled with fatty acid, phosphatidylethanol (PEt, the transphosphatidylation product of phospholipase D (PLD)) formation was stimulated as early as 5 min after addition. In cells labeled with [3H]choline, HePC treatment caused release of choline-containing metabolites to the culture medium, concurrent with PEt formation. HePC also elicited formation of diacylglycerol (DG) which, after 30 min increased 3.5-fold over control. As little is known regarding HePC and PLD, attention was directed towards studies on PC metabolism by PLD. PEt formation was shown to be optimal at 20-50 microM HePC, and structure-activity studies showed HePC to be more potent than either lyso-phosphatidylcholine or 1-hexadecyl-2-O-methyl-rac-glycero-3-phosphocholine for PLD activation. PLD activity induced by HePC was totally inhibited by cellular pretreatment with phorbol dibutyrate, and 59% diminished by pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor. Our results demonstrate for the first time that HePC activates PLD, and suggest that PKC participates in this response. The relationship of PLD to the anticancer properties of HePC may be clinically relevant to drug actions.

An original method for rapid serial determination of phospholipid biosynthesis. Applications to mammalian lymphocytic cells and a lower eucaryote, Plasmodium falciparum

A rapid, convenient, and efficient method is presented to measure phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol biosynthesis from [ 3H]choline, [ 3H]ethanolamine, and [ 3H]inositol, respectively. After incubation of the cells in 96-multiwell dishes with the appropriate radioactive precursor, cells were lysed with water and the water-insoluble materials, particularly cellular membranes which contain the bulk of phospholipids, were serially collected on glass-fiber papers using a cell harvester. The method was first applied to human lymphocytic cell lines then adapted to Plasmodium falciparum-infected human erythrocytes which in both cases allowed recovery of more than 90% of the newly biosynthesized phospholipids. With this quick method, adapted to short incubation periods (less than 5 h), we were able to determine optimal conditions such as the best medium (RPMI devoid of serum, thus avoiding interference from endogenous precursors, notably choline present in significant quantities in serum) and the lowest specific activity to be used for each radioactive precursor and the minimum quantity of cells. This method could be adapted to other cell systems, provided that the precursors are specific to phospholipids and that the bulk of biosynthesized phospholipids are present as membrane components. Finally, by this method the activity of effectors of phospholipid metabolism can be tested on a large scale, thus allowing rapid screening of original molecules specifically affecting cellular phospholipid metabolism.

Cytidine diphosphate diglyceride analogs of antiretroviral dideoxynucleosides: evidence for release of dideoxynucleoside-monophosphates by phospholipid biosynthetic enzymes in rat liver subcellular fractions

We recently synthesized phospholipid analogs with antiviral nucleosides in the polar headgroup and demonstrated their antiretroviral activity in vitro in human immunodeficiency virus-infected cells (Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van den Bosch, H. and Richman, D.D. (1990) J. Biol. Chem. 265, 6112-6117). Dideoxynucleoside analogs of cytidine diphosphate diglyceride (CDP-DG) represent one class of such phospholipid prodrugs from which the antiviral active principle may be released through established pathways of cellular phospholipid metabolism. We now demonstrate that the liponucleotides of dideoxycytidine, 3'-deoxythymidine and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) can substitute to varying extents for CDP-DG in the biosynthesis of phosphatidylinositol, phosphatidylglycerol or diphosphatidylglycerol by rat liver subcellular fractions. In all three biosynthetic pathways dideoxycytidine diphosphate diglyceride was the most active donor of the phosphatidyl unit. The nearly stoichiometric formation of dideoxycytidine-5'-monophosphate during phosphatidylinositol biosynthesis supports the rationale that the antiretroviral liponucleotides may provide cells with a depot form from which the antiviral drug can be released in 5'-monophosphorylated form, thus bypassing the initial phosphorylation of free dideoxynucleosides.

Erythrocytes attached to a wheat germ agglutinin coated surface display an altered phospholipid metabolism.

Erythrocytes were bound to a lectin-coated surface; the multivalent attachment to this surface resulted in a severe deformation of the cells and an alteration in the cellular phospholipid metabolism. Human erythrocytes were allowed to bind for 20 min at 20 degrees C to polystyrene beads coated with wheat germ agglutinin (WGA beads). The bound erythrocytes were then lysed to produce stroma bound to WGA beads. Control stroma and stroma-WGA beads were incubated at 37 degrees C with gamma-32P-ATP to examine the phospholipid labeling patterns. The control stroma incorporated 32P-label into phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, in agreement with earlier studies. However, the stroma-WGA beads showed incorporation of 32P-label into phosphatidic acid in addition to that in the phosphoinositides. The quantity of 32P-phosphatidic acid produced during the 20-min assay was 3.23 +/- 0.84 (n = 7) picomoles/micrograms stromal cholesterol; the amount synthesized, however, was dependent on the procedure used to prepare the stroma-WGA beads. If the erythrocytes were bound to the WGA beads at 0 degrees C instead of 20 degrees C, the quantity of 32P-phosphatidic acid produced during the subsequent 37 degrees C assay with gamma-32P-ATP was decreased 4.2 fold; the phosphoinositide labeling pattern was unchanged. In addition, when the time for binding of intact erythrocytes to the WGA beads was varied from 1 to 20 minutes, there was a time-dependent increase in the amount of 32P-phosphatidic acid produced. This induction of phosphatidic acid synthesis could not be duplicated with fluid phase WGA. Therefore, the multivalent binding of intact erythrocytes to WGA beads causes an alteration in phospholipid metabolism.

A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2.

Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism.