Cooperative induction of CXCL chemokines by inflammatory cytokines and oxidized phospholipids.

Inflammation is initiated and driven by a mixture of mediators, which modify effects of each other. This study analysed in vitro pro-inflammatory activity of inflammatory cytokines (TNFα and IL-1ß) in a combination with a lipid DAMP molecule, oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC). The study was performed on endothelial and monocytic cell lines. The cells were treated with different concentrations of TNFα or IL-1ß, OxPAPC and their combinations, either in the presence or absence of drugs regulating inflammation. Pro-inflammatory effects of TNFα/IL-1ß and OxPAPC were estimated by analysis of chemokines CXCL8, CXCL2 and CXCL3 by ELISA and RT-PCR. Toxicity was determined by analysis of metabolic activity. Statistical significance was estimated by ANOVA and Dunnett's test. OxPAPC was a much weaker chemokine inducer as compared to TNFα or IL-1ß. However, OxPAPC and TNFα/IL-1ß together induced effects that were significantly stronger than the arithmetical sum of individual effects. This cooperative action of OxPAPC and TNFα was reversed by inhibitors of p38 MAPK. We hypothesise that the boosting of TNFα and IL-1ß effects by OxPAPC may be more pathologically important than the action of the lipid alone. Inhibitors of p38 MAPK may become a tool for analysis of pathological role of oxidized phospholipids.

Pharmacological heat-shock protein inducers and chemical chaperones inhibit upregulation of interleukin-8 by oxidized phospholipids.

Oxidised phospholipids such as oxidised palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) are increasingly recognised as danger-associated molecular patterns (DAMPs) inducing cyto- and chemokines. The pathological impact of oxidised phosphatidylcholine in vivo has been demonstrated in several animal models, as well as in human association studies. In this work, we have tested a number of small molecules with known or potential anti-inflammatory properties for their ability to inhibit secretion of interleukin-8 by OxPAPC-treated endothelial cells. Six compounds capable of inhibiting the induction of IL-8 were selected. Analysis of gene expression has shown that all these substances reduced the OxPAPC-induced elevation of IL-8 mRNA but potentiated induction of heat-shock proteins (HSPs). We further found that drug-like HSP inducers also prevented the induction of IL-8 by OxPAPC. Similar inhibitory action was demonstrated by two chemical chaperones, which stabilise proteins through physicochemical mechanisms thus mimicking effects of HSPs. Our data suggest that proteostatic stress plays an important mechanistic role in the pro-inflammatory effects of OxPAPC and that stabilisation of proteome by overexpression of HSPs or by chemical chaperones can reduce the pro-inflammatory effects of OxPLs.

12/15-lipoxygenase orchestrates the clearance of apoptotic cells and maintains immunologic tolerance.

Noninflammatory clearance of apoptotic cells (ACs) is crucial to maintain self-tolerance. Here, we have reported a role for the enzyme 12/15-lipoxygenase (12/15-LO) as a central factor governing the sorting of ACs into differentially activated monocyte subpopulations. During inflammation, uptake of ACs was confined to a population of 12/15-LO-expressing, alternatively activated resident macrophages (resMΦ), which blocked uptake of ACs into freshly recruited inflammatory Ly6C(hi) monocytes in a 12/15-LO-dependent manner. ResMΦ exposed 12/15-LO-derived oxidation products of phosphatidylethanolamine (oxPE) on their plasma membranes and thereby generated a sink for distinct soluble receptors for ACs such as milk fat globule-EGF factor 8, which were essential for the uptake of ACs into inflammatory monocytes. Loss of 12/15-LO activity, in turn, resulted in an aberrant phagocytosis of ACs by inflammatory monocytes, subsequent antigen presentation of AC-derived antigens, and a lupus-like autoimmune disease. Our data reveal an unexpected key role for enzymatic lipid oxidation during the maintenance of self-tolerance.

Oxidized phospholipids stimulate production of stem cell factor via NRF2-dependent mechanisms.

Receptor tyrosine kinase c-Kit and its ligand stem cell factor (SCF) regulate resident vascular wall cells and recruit circulating progenitors. We tested whether SCF may be induced by oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) known to accumulate in atherosclerotic vessels. Gene expression analysis demonstrated OxPAPC-induced upregulation of SCF mRNA and protein in different types of endothelial cells (ECs). Elevated levels of SCF mRNA were observed in aortas of ApoE-/- knockout mice. ECs produced biologically active SCF because conditioned medium from OxPAPC-treated cells stimulated activation (phosphorylation) of c-Kit in naïve ECs. Induction of SCF by OxPAPC was inhibited by knocking down transcription factor NRF2. Inhibition or stimulation of NRF2 by pharmacological or molecular tools induced corresponding changes in SCF expression. Finally, we observed decreased levels of SCF mRNA in aortas of NRF2 knockout mice. We characterize OxPLs as a novel pathology-associated stimulus inducing expression of SCF in endothelial cells. Furthermore, our data point to transcription factor NRF2 as a major mediator of OxPL-induced upregulation of SCF. This mechanism may represent one of the facets of pleiotropic action of NRF2 in vascular wall.

Effects of prostaglandin lipid mediators on agonist-induced lung endothelial permeability and inflammation.

Prostaglandins (PG), the products of cyclooxygenase-mediated conversion of arachidonic acid, become upregulated in many situations including allergic response, inflammation, and injury, and exhibit a variety of biological activities. Previous studies described barrier-enhancing and anti-inflammatory effects of PGE2 and PGI2 on vascular endothelial cells (EC). Yet, the effects of other PG members on EC barrier and inflammatory activation have not been systematically analyzed. This study compared effects of PGE2, PGI2, PGF2α, PGA2, PGJ2, and PGD2 on human pulmonary EC. EC permeability was assessed by measurements of transendothelial electrical resistance and cell monolayer permeability for FITC-labeled tracer. Anti-inflammatory effects of PGs were evaluated by analysis of expression of adhesion molecule ICAM1 and secretion of soluble ICAM1 and cytokines by EC. PGE2, PGI2, and PGA2 exhibited the most potent barrier-enhancing effects and most efficient attenuation of thrombin-induced EC permeability and contractile response, whereas PGI2 effectively suppressed thrombin-induced permeability but was less efficient in the attenuation of prolonged EC hyperpermeability caused by interleukin-6 or bacterial wall lipopolysaccharide, LPS. PGD2 showed a modest protective effect on the EC inflammatory response, whereas PGF2α and PGJ2 were without effect on agonist-induced EC barrier dysfunction. In vivo, PGE2, PGI2, and PGA2 attenuated LPS-induced lung inflammation, whereas PGF2α and PGJ2 were without effect. Interestingly, PGD2 exhibited a protective effect in the in vivo model of LPS-induced lung injury. This study provides a comprehensive analysis of barrier-protective and anti-inflammatory effects of different prostaglandins on lung EC in vitro and in vivo and identifies PGE2, PGI2, and PGA2 as prostaglandins with the most potent protective properties.

Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2.

Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.

A simplified procedure for semi-targeted lipidomic analysis of oxidized phosphatidylcholines induced by UVA irradiation.

Oxidized phospholipids (OxPLs) are increasingly recognized as signaling mediators that are not only markers of oxidative stress but are also "makers" of pathology relevant to disease pathogenesis. Understanding the biological role of individual molecular species of OxPLs requires the knowledge of their concentration kinetics in cells and tissues. In this work, we describe a straightforward "fingerprinting" procedure for analysis of a broad spectrum of molecular species generated by oxidation of the four most abundant species of polyunsaturated phosphatidylcholines (OxPCs). The approach is based on liquid-liquid extraction followed by reversed-phase HPLC coupled to electrospray ionization MS/MS. More than 500 peaks corresponding in retention properties to polar and oxidized PCs were detected within 8 min at 99 m/z precursor values using a single diagnostic product ion in extracts from human dermal fibroblasts. Two hundred seventeen of these peaks were fluence-dependently and statistically significantly increased upon exposure of cells to UVA irradiation, suggesting that these are genuine oxidized or oxidatively fragmented species. This method of semitargeted lipidomic analysis may serve as a simple first step for characterization of specific "signatures" of OxPCs produced by different types of oxidative stress in order to select the most informative peaks for identification of their molecular structure and biological role.

Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of inflammation.

Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4. In this study, we show that half-maximal inhibition of LPS-induced elevation of E-selectin mRNA in endothelial cells developed at concentrations of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) 10-fold lower than those required to induce proinflammatory response. Similar concentration difference was observed for other classes and molecular species of OxPLs. Upon injection into mice, OxPAPC did not elevate plasma levels of IL-6 and keratinocyte chemoattractant but strongly inhibited LPS-induced upregulation of these inflammatory cytokines. Thus, both in vitro and in vivo, anti-LPS effects of OxPLs are observed at lower concentrations than those required for their proinflammatory action. Quantification of the most abundant oxidized phosphatidylcholines by HPLC/tandem mass spectrometry showed that circulating concentrations of total oxidized phosphatidylcholine species are close to the range where they demonstrate anti-LPS activity but significantly lower than that required for induction of inflammation. We hypothesize that low levels of OxPLs in circulation serve mostly anti-LPS function and protect from excessive systemic response to TLR4 ligands, whereas proinflammatory effects of OxPLs are more likely to develop locally at sites of tissue deposition of OxPLs (e.g., in atherosclerotic vessels).

Low Concentrations of Oxidized Phospholipids Increase Stress Tolerance of Endothelial Cells.

Oxidized phospholipids (OxPLs) are generated by enzymatic or autooxidation of esterified polyunsaturated fatty acids (PUFAs) residues. OxPLs are present in circulation and atherosclerotic plaques where they are thought to induce predominantly proinflammatory and toxic changes in endothelial (ECs) and other cell types. Unexpectedly, we found that low concentrations of OxPLs were not toxic but protected ECs from stress induced by serum deprivation or cytostatic drugs. The protective effect was observed in ECs obtained from different vessels and was monitored using a variety of readouts based on different biological and chemical principles. Analysis of the structure−activity relationship identified oxidized or missing fatty acid residue (OxPLs or Lyso-PLs, respectively) as a prerequisite for the protective action of a PL. Protective OxPLs or Lyso-PLs acquired detergent-like properties and formed in solution aggregates <10 nm in diameter (likely micelles), which were in striking contrast with large aggregates (>1000 nm, likely multilayer liposomes) produced by nonoxidized precursor PLs. Because surfactants, OxPLs, and Lyso-PLs are known to extract membrane cholesterol, we tested if this effect might trigger the protection of endothelial cells. The protective action of OxPLs and Lyso-PLs was inhibited by cotreatment with cholesterol and mimicked by cholesterol-binding beta-cyclodextrin but not inactive α-cyclodextrin. Wide-scale mRNA expression analysis in four types of ECs showed the induction of genes encoding for heat shock proteins (HSPs) and secreted prosurvival peptides and proteins. Inducers of HSPs, chemical chaperones, and pure prosurvival factors mimicked the protective action of OxPLs/Lyso-PLs. We hypothesize that oxidation changes the physicochemical properties of PLs, thus promoting membrane cholesterol redistribution or extraction leading to the expression of intra- and extracellular prosurvival factors.

Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids.

Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.

Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.

OBJECTIVE: The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. METHODS AND RESULTS: Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. CONCLUSION: Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.

Oxidized phospholipids on alkyl-amide scaffold demonstrate anti-endotoxin and endothelial barrier-protective properties.

Oxidized phospholipids (OxPLs) containing enzymatically or non-enzymatically oxidized fatty acids (oxylipins) are increasingly recognized as lipid mediators involved in pathogenesis of diseases. Further understanding of structure-activity relationship and molecular mechanisms activated by OxPLs is hampered by the complexity of synthesis of individual molecular species. Although dozens of individual free oxylipins are commercially available, their attachment to the phospholipid scaffold requires relatively harsh conditions during activation of carboxy-group, which may lead to decomposition of unstable oxylipins. Furthermore, additional protection-deprotection steps are required for oxylipins containing hydroxy-groups. In this work we describe synthesis of OxPLs containing oxylipins bound at the sn-2-position via an amide-bond that is characteristic of sphingophospholipids. Activation of oxylipins and attachment to the phospholipid scaffold are performed under mild conditions and characterized by high yield. Hydroxy-groups of oxylipins do not interfere with reactions and therefore no protection/deprotection steps are needed. In order to prevent oxylipin migration, a fatty acid residue at the sn-1 was bound through an alkyl bond, which is a common bond present in a large proportion of naturally occurring phospholipids. An additional advantage of combining alkyl and amide bonds in a single phospholipid molecule is that both types of bonds are phospholipase A1/A2-resistant, which may be expected to improve biological stability of OxPLs and thus simplify analysis of their effects. As proof of principle, several alkyl-amide oxidized phosphatidylcholines (OxPCs) containing either linear or prostane ring oxylipins have been synthesized. Importantly, we show here that alkyl-amide-OxPCs demonstrated biological activities similar to those of di-acyl-OxPCs. Alkyl-amide-OxPCs inhibited pro-inflammatory action of LPS and increased endothelial cellular barrier in vitro and in mouse models. The effects of alkyl-amide and di-acyl-OxPCs developed in a similar range of concentrations. We hypothesize that alkyl-amide-OxPLs may become a useful tool for deeper analysis of the structure-activity relationship of OxPLs.

ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response.

We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques.

Multi-hit inhibition of circulating and cell-associated components of the toll-like receptor 4 pathway by oxidized phospholipids.

OBJECTIVE: Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro- and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS). METHODS AND RESULTS: We show, using several cell types and various inflammatory genes as readouts, that different classes and molecular species of OxPLs do not stimulate TLR4 but exert prominent inhibitory effects on LPS-induced reactions. Our data demonstrate that binding of OxPLs to the LPS-binding protein (LBP) and CD14 prevents recognition of LPS by these proteins, thus impairing activation of TLR4. In addition, OxPLs inhibited LBP- and CD14-independent activation of TLR4 by the synthetic TLR4 agonist E6020 indicating that in parallel with LBP and CD14, OxPLs target cell-associated steps in TLR4 cascade. CONCLUSIONS: Our data suggest that OxPLs inhibit action of LPS via a multi-hit mechanism. These results support the notion that OxPLs are endogenous inhibitors of TLR4 produced in response to oxidative stress.

Analysis of fragmented oxidized phosphatidylcholines in human plasma using mass spectrometry: Comparison with immune assays.

Circulating oxidized phospholipids are increasingly recognized as biomarkers of atherosclerosis. Clinical association studies have been mainly performed using an immune assay based on monoclonal antibody E06, which recognizes a variety of molecular species of oxidized phosphatidylcholine (OxPC) in lipoproteins, cell membranes or covalently bound to plasma proteins. Accumulating evidence shows that individual molecular species of OxPC demonstrate different biological activities and have different half-life times. Therefore, it is likely that certain molecular species can be associated with pathology more strongly than others. This hypothesis can only be tested using LC-MS/MS allowing quantification of individual molecular species of OxPCs. In order to ensure that laborious LC-MS/MS methods do not simply replicate the results of a technically simpler E06-OxPCs assay, we have performed relative quantification of 8 truncated molecular species of OxPCs in plasma of 132 probands and compared the data with the results of the E06-OxPCs and OxLDL assays. We have found a strong correlation between individual molecular species of OxPCs but only a weak correlation of LC-MS/MS-OxPCs data with the E06-OxPCs assay and no correlation with the OxLDL assay. Furthermore, in contrast to the results of E06-OxPCs or OxLDL assays, 7 out of 8 OxPC species were associated with hypertension. The data suggest that the results of the LC-MS/MS-OxPCs assay do not replicate the results of two ELISA-based lipid oxidation tests and therefore may produce additional diagnostic information. These findings necessitate development of simplified mass spectrometric procedures for high-throughput and affordable analysis of selected molecular species of OxPCs.

Off-Target Anti-Inflammatory Activity of the P2X7 Receptor Antagonist AZ11645373.

We have found that a well-characterized P2X7 receptor antagonist AZ11645373 blocked production of pro-inflammatory chemokine IL-8 in endothelial cells treated with OxPAPC. The effect was not due to toxicity of AZ11645373 as documented by cellular metabolic activity assay. The mechanism of inhibition by AZ11645373 was apparently independent of the P2X7 receptor because this receptor was not involved in induction of IL-8 under our experimental conditions. In support of this notion, two P2X7 agonists ATP and BzATP did not upregulate IL-8. On the other hand, a chemically different P2X7 receptor antagonist A740003 did not inhibit OxPAPC-induced production of IL-8. The inhibitory action of AZ11645373 was observed at the level of IL-8 protein and messenger RNA (mRNA) induction. Furthermore, AZ11645373 inhibited induction of mRNA encoding for COX-2 (PTGS2) suggesting that its anti-inflammatory potential is not limited to suppression of IL-8 production. In addition to inhibiting stimulation by OxPAPC, AZ11645373 suppressed induction of IL-8 by TNFα and LPS. To summarize, AZ11645373 inhibits in a P2X7-independent manner action of chemically different inflammatory agonists such as OxPLs, LPS, and TNFα. Thus, AZ11645373 may be especially effective for treatment of inflammatory disorders due to a beneficial combination of P2X7 receptor-dependent effects (inhibition of inflammasome activation, antinociceptive effects) with P2X7-independent general anti-inflammatory action described in this paper.

Pleiotropic effects of oxidized phospholipids.

Oxidized phospholipids (OxPLs) are increasingly recognized to play a role in a variety of normal and pathological states. OxPLs were implicated in regulation of inflammation, thrombosis, angiogenesis, endothelial barrier function, immune tolerance and other important processes. Rapidly accumulating evidence suggests that OxPLs are biomarkers of atherosclerosis and other pathologies. In addition, successful application of experimental drugs based on structural scaffold of OxPLs in animal models of inflammation was recently reported. This review briefly summarizes current knowledge on generation, methods of quantification and biological activities of OxPLs. Furthermore, receptor and cellular mechanisms of these effects are discussed. The goal of the review is to give a broad overview of this class of lipid mediators inducing pleiotropic biological effects.

Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae.

The genome of the yeast, Saccharomyces cerevisiae, contains three highly similar genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preferences in vitro and relative contributions to phospholipid catabolism in vivo [Merkel, Fido, Mayr, Pruger, Raab, Zandonella, Kohlwein and Paltauf (1999) J. Biol. Chem. 274, 28121-28127]. It is shown in the present study that, in vitro, pH markedly affects the substrate preference of Plb1p and Plb2p, but not of Plb3p. At the pH optimum of 2.5-3.5, the order of substrate preference of Plb1p and Plb2p is PtdSer (phosphatidylserine)>PtdIns>PtdCho (phosphatidylcholine>PtdEtn (phosphatidylethanolamine). At pH values of 5 and above, the substrate preferences change to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns/PtdCho breakdown, as reflected in the ratio of GroPIns (glycerophosphoinositol)/GroPCho (glycerophosphocholine) released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p, because Plb2p does not contribute to the degradation of PtdIns and PtdCho in vivo. Bivalent and tervalent cations activate phospholipases B at pH 5.5, but are inhibitory at pH 2.5. Al3+ at a concentration of 20 mM increases Plb1p activity in vitro by 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo, cycloheximide strongly inhibits the breakdown of PtdIns, and to a lesser extent PtdCho. However, Al3+-stimulated GroPCho release is almost completely inhibited by cycloheximide. Deletion of PLB3 leads to increased sensitivity to toxic Al3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by cycloheximide. Deletion mutants defective in the PLB1 gene are significantly more resistant to SDS than are wild-type cells.

Hormetic and anti-inflammatory properties of oxidized phospholipids.

Oxidized phospholipids are generally recognized as deleterious factors involved in disease pathogenesis. This review summarizes the data suggesting that under certain biological conditions the opposite is correct, namely that OxPLs can also induce protective effects. Examples that are discussed in the review include upregulation of antioxidant genes, inhibition of inflammatory signaling pathways through Nrf2-dependent and -independent mechanisms, antagonism of Toll-like receptors, immuno-modulating and immuno-suppressive action of OxPLs in adaptive immunity and autoimmune disease, activation of PPARs known for their anti-inflammatory action, as well as protective action against lung edema in acute lung inflammation. The data support the notion that oxidation of phospholipids provides a negative feedback preventing damage to host tissues due to uncontrolled inflammation and oxidative stress.

Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases.

Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis. Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents. The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates. The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL). The resulting lipid-protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes. We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water. It was much higher when the protein was dissolved in aqueous ethanol. These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents.