Phospholipid Turnover Research Articles

Disruption of phospholipase B‐mediated turnover upon loss of Ypk1 in S. cerevisiae

Ypk1, the yeast homolog of the human serum‐ and glucocorticoid‐induced kinase (Sgk1), is a serine/threonine protein kinase known to affect various downstream processes, including plasma membrane flippase activity and sphingolipid homeostasis. The results described here indicate that Ypk1 also impinges upon the turnover of phospholipids. Pulse‐chase experiments reveal that a ypk1 mutant exhibits alterations in phospholipid deacylation and resultant glycerophosphodiester production as compared to a wild type strain. Furthermore, loss of Plb1, a plasma membrane (PM) associated phospholipase, in a ypk1 background results in further alterations in phospholipid deacylation and a growth defect. We are in the process of examining potential explanations for the altered Plb1 activity in the ypk1 mutant. Strains in which other phospholipase B‐encoding genes are deleted in combination with YPK1 do not display a growth defect, suggesting a specific functional interaction between Plb1 and Ypk1. Preliminary studies have identified model PM proteins, including the Git1 permease, whose activity is affected by loss of Ypk1 and/or Plb1. Our data is consistent with the idea that Ypk1 and Plb1 alter plasma membrane phospholipid composition, which in turn impinges upon the activity of integral plasma membrane proteins.

Serine 32 targeted mutation abolishes Prdx6 trafficking to lamellar bodies in vivo

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid (PL) turnover and protects lung epithelium from oxidative stress. Prdx6 null mice accumulate PL in lung tissue, compatible with the role of PLA2 activity in lung phospholipid turnover. Prdx6 has been localized to cytosolic and acidic compartments (lamellar bodies (LB) and lysosomes) in lung epithelium. Our previous studies indicated that serine at position 32 (S32) is necessary for proper Prdx6 targeting to LB. We have generated a mouse model with an S32T mutation in the Prdx6 LB targeting sequence. Homozygous mice carrying the S32T mutation exhibited phospholipid (PL) accumulation in LB (PL/protein ratio 14.36) vs. WT (PL/protein ratio 9.51). Western blot analysis demonstrated absence of Prdx6 in LBs isolated from S32T mice despite equal amounts of Prdx6 in lung homogenates. Immunofluorescence analysis of mouse lung tissue sections showed co‐localization of Prdx6 with LAMP1, a marker of lysosome‐related organelles, in wild type but not in S32T mouse alveolar type II cells. These findings indicate that S32 is required for Prdx6 targeting to LB in mouse lung epithelium. [HL102016 and HL19737]

Acyl-CoA Synthetase 1 Is Induced by Gram-negative Bacteria and Lipopolysaccharide and Is Required for Phospholipid Turnover in Stimulated Macrophages

The enzyme acyl-CoA synthetase 1 (ACSL1) is induced by peroxisome proliferator-activated receptor α (PPARα) and PPARγ in insulin target tissues, such as skeletal muscle and adipose tissue, and plays an important role in β-oxidation in these tissues. In macrophages, however, ACSL1 mediates inflammatory effects without significant effects on β-oxidation. Thus, the function of ACSL1 varies in different tissues. We therefore investigated the signals and signal transduction pathways resulting in ACSL1 induction in macrophages as well as the consequences of ACSL1 deficiency for phospholipid turnover in LPS-activated macrophages. LPS, Gram-negative bacteria, IFN-γ, and TNFα all induce ACSL1 expression in macrophages, whereas PPAR agonists do not. LPS-induced ACSL1 expression is dependent on Toll-like receptor 4 (TLR4) and its adaptor protein TRIF (Toll-like receptor adaptor molecule 1) but does not require the MyD88 (myeloid differentiation primary response gene 88) arm of TLR4 signaling; nor does it require STAT1 (signal transducer and activator of transcription 1) for maximal induction. Furthermore, ACSL1 deletion attenuates phospholipid turnover in LPS-stimulated macrophages. Thus, the regulation and biological function of ACSL1 in macrophages differ markedly from that in insulin target tissues. These results suggest that ACSL1 may have an important role in the innate immune response. Further, these findings illustrate an interesting paradigm in which the same enzyme, ACSL1, confers distinct biological effects in different cell types, and these disparate functions are paralleled by differences in the pathways that regulate its expression.

Transport and Metabolism of glycerophosphocholine by Candida albicans: Role of CaGIT3, CaGIT4, and CaGDE1

Phospholipase B‐mediated turnover of phospholipids results in the production of glycerophosphodiesters. Candida albicans contains 4 ORFs (CaGIT1–4) predicted to be glycerophosphodiester transporters. Here we have identified CaGit3 and CaGit4 as glyerophosphocholione (GroPCho) transporters, with CaGit3 having the greatest transport activity. Deletion of CaGIT2–4 abolishes the cells ability to utilize GroPCho as a sole phosphate source during early growth (<12 hours after inoculation) and abolishes short term (2 min) transport of [3H]GroPCho. Upon reintegration of CaGIT3 (orf19.1979) and CaGIT4 (orf19.1980), both growth and transport of GroPCho is rescued, albeit minimally for CaGIT4. In addition, we have identified CaGDE1 (orf19.3936), as affecting the catabolism of internalized GroPCho, presumably by acting as a glycerophosphodiesterase. As is the case for CaGIT1, the transcriptional regulator, CaPHO4, regulates CaGIT3–4 expression. To our knowledge, this is the first evidence of a eukaryotic permease specific for GroPCho.

Patatin-Related Phospholipase pPLAIIIδ Increases Seed Oil Content with Long-Chain Fatty Acids in Arabidopsis

The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, β, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.

항산화반응을 유발하는 물질의 검색에 적용할 수 있는 형질전환 마우스 생산을 위한 새로운 Prx 6/Luc 벡터시스템의 제조 및 폐암세포주에서 반응성 확인

Peroxiredoxin 6 (Prx 6) is a member of the thiol-specific antioxidant protein family, which may play a role in protection against oxidative stress and in regulating phospholipid turnover. The aim of this study was to determine whether a human Prx 6/Luc vector was stably expressed and responded to antioxidants in a lung cell line (NCI-H460). To achieve this, the luciferase signal, hPrx 6 mRNA expression, and superoxide dismutase (SOD) activity were measured in transfectants with a hPrx 6/Luc plasmid after treatment with four antioxidant extracts, including Korea white ginseng (KWG), Korea red ginseng (KRG), Liriope platyphylla (LP), and red Liriope platyphylla (RLP). First, the hPrx 6/Luc plasmid was successfully constructed with DNA fragments of human Prx 6 promoter, amplified by PCR using genomic DNA isolated from NCI-H460 cells, and cloned into the pTransLucent reporter vector. The orientation and sequencing of the hPrx 6/Luc plasmid were identified with restriction enzyme and automatic sequencing. A luciferase assay revealed significant enhancement of luciferase activity in the four treatment groups compared with a vehicle-treated group, although the ratio of the increase was different within each group. The KRG- and LP-treated groups showed higher activity than the KWG- and RLP-treated groups. Furthermore, the luciferase activity against RLP occurred roughly in a dose-dependent manner. However, the level of endogenous hPrx 6 mRNA did not change in any group treated with the four extracts. The SOD activity was in agreement with the luciferase activity. Therefore, these results indicate that the hPrx 6/Luc vector system may successfully express and respond to antioxidant compounds in NCI-H460 cells. The data also suggest that the Prx 6/Luc vector system may be effectively applied in screening the response of hPrx 6 to antioxidant compounds in transgenic mice.

Synthetic phosphoethanolamine induces cell cycle arrest and apoptosis in human breast cancer MCF-7 cells through the mitochondrial pathway

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes. In a recent study, we showed that Pho-s has antitumor effect in the several tumor cells. In this study we evaluated the antitumor activity of synthetic Pho-s on MCF-7 breast cancer cells. Here we demonstrate that Pho-s is cytotoxic to MCF-7 cells in a dose-dependent manner, while it is cytotoxic to MCF10 only at higher concentrations. In addition, Pho-s induces a disruption in mitochondrial membrane potential (Δψm). Furthermore, Pho-s induces mitochondria aggregates in the cytoplasm and DNA fragmentation of MCF-7 cells visualized by confocal microscopy. In agreement with the reduction on Δψm, we showed that Pho-s induces apoptosis followed by an increase in cytochrome c expression and capase-3-like activity in MCF-7 cells. Our results demonstrate that Pho-s induces a cell cycle arrest in the G1 phase through an inhibition of cyclin D1 and stimulates p53. An additional highlight of this study is the finding that Pho-s inhibits Bcl-2, inducing apoptosis through the mitochondrial pathway. Taken together, these results show that Pho-s is a promising compound in the fight against cancer.

Cholesterol and phospholipids in frontal cortex and synaptosomes of suicide completers: Relationship with endosomal lipid trafficking genes

Cholesterol (CHL) and phospholipid (PL) levels in synaptosomal membranes in particular can have an impact on cell signalling. Alterations in peripheral CHL measures have been consistently reported in suicidal behaviour. As CHL and PL turnover in the brain are important in synapse maintenance and function, the objective of this study was to determine if differences exist in synaptosomal cholesterol and phospholipid levels between suicide completers and controls. Expression measures of genes involved in lipid trafficking suggest an association between Lysosomal acid lipase A, cholesteryl ester hydrolase (LIPA) and brain PL levels, with LIPA being significantly increased in violent suicides and associated with alterations in brain PL. The results of this study suggest an altered PL content mediated by LIPA expression in violent suicides in the prefrontal cortex, which would have important consequences for inhibitory neurotransmission.

Neutral Lipid Metabolism Influences Phospholipid Synthesis and Deacylation in Saccharomyces cerevisiae

Establishment and maintenance of equilibrium in the fatty acid (FA) composition of phospholipids (PL) requires both regulation of the substrate available for PL synthesis (the acyl-CoA pool) and extensive PL turnover and acyl editing. In the present study, we utilize acyl-CoA synthetase (ACS) deficient cells, unable to recycle FA derived from lipid deacylation, to evaluate the role of several enzymatic activities in FA trafficking and PL homeostasis in Saccharomyces cerevisiae. The data presented show that phospholipases B are not contributing to constitutive PL deacylation and are therefore unlikely to be involved in PL remodeling. In contrast, the enzymes of neutral lipid (NL) synthesis and mobilization are central mediators of FA trafficking. The phospholipid:DAG acyltransferase (PDAT) Lro1p has a substantial effect on FA release and on PL equilibrium, emerging as an important mediator in PL remodeling. The acyl-CoA dependent biosynthetic activities of NL metabolism are also involved in PL homeostasis through active modulation of the substrate available for PL synthesis. In addition TAG mobilization makes an important contribution, especially in cells from stationary phase, to FA availability. Beyond its well-established role in the formation of a storage pool, NL metabolism could play a crucial role as a mechanism to uncouple the pools of PL and acyl-CoAs from each other and thereby to allow independent regulation of each one.

Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence

Leishmania parasites are intracellular protozoans capable of salvaging and remodeling lipids from the host. To understand the role of lipid metabolism in Leishmania virulence, it is necessary to characterize the enzymes involved in the uptake and turnover of phospholipids. This study focuses on a putative phospholipase A2 (PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) in Leishmania major. In mammals, PAF-AH is a subgroup of PLA2 catalyzing the hydrolysis/inactivation of platelet-activating factor (PAF), a potent mediator of many leukocyte functions. By immunofluorescence microscopy, L. major PLA2/PAF-AH is predominantly localized in the ER. While wild type L. major parasites are able to hydrolyze PAF, this activity is completely absent in the PLA2/PAF-AH-null mutants. Meanwhile, deletion of PLA2/PAF-AH had no significant effect on the turnover of common glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. PLA2/PAF-AH is not required for the growth of L. major parasites in culture, or the production of GPI-anchored virulence factors. Nonetheless, it does play a key role in the mammalian host as the PLA2/PAF-AH null mutants exhibit attenuated virulence in BALB/c mice. In conclusion, these data suggest that Leishmania parasites possess a functional PAF-AH and the degradation of PAF or PAF-like lipids is an important step in infection.

Acute depletion of plasma membrane phospholipids—dissecting the roles of PtdIns(4)P and PtdIns(4,5)P2

Acute depletion of plasma membrane phospholipids—dissecting the roles of PtdIns(4)P and PtdIns(4,5)P2

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Pho-s has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma.

Abstract 5165: Fatty acid diversification in prostate cancer progression

Abstract Two clinically significant indices of prostate cancer progression are the dedifferentiation of the gland (as measured by histopathology and graded by Gleason scoring, GS) and extension beyond the prostatic capsule (extracapsular extension, ECE). In order to better understand the underlying biology of prostate cancer progression, we undertook the metabolomic characterization of prostatectomy tissues and correlated our metabolite findings with these post-surgical endpoints. Briefly, frozen tissues from 298 prostatectomy cases were characterized by frozen sections and then disaggregated and the biochemical constituents were extracted into cold methanol as previously reported. These extracts were analyzed in three parallel workflows: gas chromatography-mass spectrometry and ultrahigh performance liquid chromatography and tandem mass spectrometry. The resulting data sets were compared against an in-house chemical reference library of over 2500 authentic standards. Three hundred and fifty-six metabolites were identified across all biochemical classes (amino acids, carbohydrates, lipids, nucleotides, cofactors and xenobiotics (primarily dietary and pharmaceutical compounds)). The steady state abundance profiles of numerous long chain fatty acids increased with Gleason pattern progression. The products of α-oxidation of long chain fatty acids (long chain α-hydroxy fatty acids (2-hydroxypalmitate and 2-hydroxystearate) and odd chain length fatty acids (such as C19:0) increased in tissue samples in conjunction with the amount of Gleason pattern 4 tissue that was present in the tumor. Branched chain fatty acids such as isopalmitate and polyunsaturated C22 species (22:2n6 and 22:3n3) increased in abundance from Gleason 7 to 8. In addition to these changes with Gleason pattern progression, extracapsular extension was found to have biochemical correlates in fatty acid metabolism. Specifically, the monounsaturated fatty acids (14:1n5, 16:1n7, 17:1n7, 18:1n9 and 19:1n9) were all found to increase in abundance in prostate tissue samples where capsular integrity was compromised compared to those tissues where the tumor was confined to the gland. These data demonstrate that during the progression of human prostate cancer, there is tremendous fatty acid diversification. Taken together with other data demonstrating phospholipid turnover and membrane remodeling in prostate cancer, these data suggest that prostate cancer cells can dramatically alter membrane composition. The reliance of many prostate cancers on these fatty acid diversification pathways can be appreciated by the demonstration of chemotherapeutic activities of inhibitors of both fatty acid synthase and stearoyl-coA dehydrogenase. In addition to target validation, metabolomic profiling may also provide insight into therapeutic selection for and monitoring of the prostate cancer patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5165. doi:1538-7445.AM2012-5165

Coordination of stress response signaling and phospholipid metabolism in yeast

Previously we showed that the availability of the lipid precursor inositol influences the levels and rates of synthesis and turnover of inositol phospholipids, phosphoinositides and complex sphingolipids. To discover the inositol‐dependent mechanisms that lead to activation of lipid‐mediated signaling pathways, we compared the simultaneous changes in lipid metabolism with those occurring in gene expression in yeast cells during a 3‐hour period of inositol starvation followed by a 30‐min period of inositol add‐back. We found that inositol starvation and add‐back led to dramatic changes in the abundance of inositol‐containing phospholipids, including phosphatidylinositol, the phosphoinositides, and complex sphingolipids. These changes were accompanied by the altered expression of hundreds of genes involved in diverse stress response pathways. Significantly we found that many mutants deleted for nonessential genes in the same stress response pathways exhibit inositol auxotrophy. These results suggest that activation of stress response signaling pathways is coordinated with inositol‐containing phospholipid metabolism.

Lipid composition of Silybum marianum cell cultures treated with methyl jasmonate

Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated acyl species, 36:5 and 36:6 and an increase in 36:2 species. A drop in the ratio of compounds with 18:3 in PI and PE was also observed. The presence of the lysophospholipids (LP) LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and the high contents of PA, represented by the molecular species 34:3, 34:2 and 36:5 and 36:4, indicates high basal level of phospholipase activity in cultures and a high phospholipid turnover. MeJA treatment did not quantitatively alter these lipid classes.

Preclinical Activity of the Rational Combination of Selumetinib (AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models

Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, (18)F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G(1), and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups. These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects.

Lipid composition of Silybum marianum cell cultures treated with methyl jasmonate

Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated acyl species, 36:5 and 36:6 and an increase in 36:2 species. A drop in the ratio of compounds with 18:3 in PI and PE was also observed. The presence of the lysophospholipids (LP) LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and the high contents of PA, represented by the molecular species 34:3, 34:2 and 36:5 and 36:4, indicates high basal level of phospholipase activity in cultures and a high phospholipid turnover. MeJA treatment did not quantitatively alter these lipid classes.

Phospholipid turnover and phospholipase D activity in tobacco hairy roots exposed to phenol

Plants are exposed to a great variety of environmental factors, which can affect their survival. In addition, the rapid urbanization and the increased release of different pollutants, such as phenol, to the environment, produce another stressful condition to the development and growth of the plants. In this work, we studied the effects on the [ 32P]P iphospholipid turnover and phospholipase D (PLD) activity after phenol treatment, using tobacco hairy roots (HRs), double transgenic (DT) for two peroxidase genes (tpx1 and tpx2) and wild type (WT) ones. In both HRs, the [ 32P]phospholipid turnover of the most abundant phospholipids (PLs), such as phosphatidylcholine (PC), phosphatydilethanolamine (PE), phosphatydilglycerol (PG) and cardiolipin (CL) did not show any changes after phenol treatment. However, modifications in the minor PLs of both HRs were observed. Phenol treatment significantly increases the turnover of phosphatidic acid (PA) and phosphatydilinositol (PI), in WT HRs. In DT HRs, phenol produced significant increase in the turnover of PI, lisophosphatidic acid (LPA), diacylglycerolpyrophosphate (DGPP) and PA with a concomitant decrease in the phosphatidylinositol monophosphate (PIP). Moreover, phosphatidylinositol bisphosphate (PIP 2) was detected, but its level did not change in presence of the pollutant. Phenol treatment significantly increased the PLD activity of both HRs. In WT HRs the increase was 100% higher than the control, whereas in DT HRs it was about to 50%. These results suggest the participation of minor PLs, mainly PA, and the PLD pathway as one source of PA production in the activation of intracellular mechanisms that might be important in the response of these plant tissues to phenol treatment.

Lipid Markers of “Geometrical” Radical Stress: Synthesis of Monotrans Cholesteryl Ester Isomers and Detection in Human Plasma

Heteroatom-centered free radicals are able to transform cis unsaturated fatty acids to the thermodynamically more stable, but unnatural, trans configuration. The "geometrical" radical stress can be estimated in biological samples using trans fatty acid isomers as lipid markers. Regioselectivity is an important feature of the "geometrical" radical stress, because the supramolecular organization of the polyunsaturated fatty acid moieties of phospholipids can lead to preferential monotrans isomer formation. The regioisomer recognition is a crucial step, which is helped by appropriate molecular libraries available through radical-based synthetic methodologies. Cholesteryl linoleate and arachidonate esters are interesting targets for their biochemical connection with membrane phospholipid turnover and their well-known roles in cardiovascular health. The synthesis of monotrans isomers of PUFA cholesteryl esters was achieved by a thiyl radical-catalyzed cis-trans isomerization. Valuable NMR, IR, and Raman spectroscopic data have been collected for promising application in metabolomics and lipidomics. The identification of monotrans cholesteryl ester isomers was carried out in human plasma by GC, Raman, and IR analyses, demonstrating for the first time the presence of specific regiosiomers connected to free radical stress. This work helps to highlight the chemical biology approach for the access to molecular libraries applicable to biomarker development, and the cis-trans isomerization as a relevant process to be integrated in the puzzling scenario of free radical-mediated lipid modifications.

Deficiency of Prdx6 in lens epithelial cells induces ER stress response-mediated impaired homeostasis and apoptosis

The multifunctional cytoprotective protein peroxiredoxin 6 (Prdx6) maintains cellular homeostasis and membrane integrity by regulating expression of intracellular reactive oxygen species (ROS) and phospholipid turnover. Using cells derived from targeted inactivation of Prdx6 gene or its depletion by RNA interference or aging, we showed that Prdx6 deficiency in cells evoked unfolded protein response (UPR), evidenced by increased expression or activation of proapoptotic factors, CHOP, ATF4, PERK, IRE-α and eIF2-α and by increased caspases 3 and 12 processing. Those cells displayed enhanced and sustained expression of endoplasmic reticulum (ER) stress-related chaperon proteins, Bip/glucose-regulated protein 78, calnexin, and calreticulin. Under cellular stress induced by hypoxia (1% O(2) or CoCl(2) treatment) or tunicamycin, Prdx6-deficient cells exhibited aberrant activation of ER stress-responsive genes/protein with higher expression of ROS, and died with apoptosis. Wild-type cells exposed to tunicamycin or hypoxia remained relatively insensitive with lower expression of ROS and ER-responsive genes than did Prdx6-deficient cells, but upregulation of ER stress responsive proteins or chaperones mimicked the UPR response of Prdx6-deficient or aging cells. Expression of Prdx6 blocked ER stress-induced deleterious signaling by optimizing physiologically aberrant expression of ER stress responsive genes/proteins in Prdx6-deficient cells or cells facing stressors, and rescued the cells from apoptosis. These findings demonstrate that impaired homeostasis and progression of pathogenesis in Prdx6-deficient lens epithelial cells or in aging cells should be blocked by a supply of Prdx6. The results provide a new molecular basis for understanding the etiology of several age-associated degenerative disorders, and potentially for developing antioxidant Prdx6-based therapeutics.