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Studies in the last decade have established the roles of oxidized phospholipids as modulators of various cellular processes, from inflammation and immunity to cell death. Oxidized lysophospholipids, formed through the activity of phospholipases and oxidative enzymes and lacking an acyl chain in comparison with parent phospholipids, are now emerging as novel bioactive lipid mediators. Their detection and structural characterization have been limited in the past due to low amounts and the complexity of their biosynthetic and removal pathways, but recent studies have unequivocally demonstrated their formation under inflammatory conditions. The involvement of oxidized lysophospholipids in immune regulation classifies them as damage-associated molecular patterns (DAMPs), which can promote sterile inflammation and contribute to autoimmune and chronic diseases as well as aging-related diseases. Their signaling pathways are just beginning to be revealed. As the first publications indicate that oxidized lysophospholipids use the same receptors as pathogen-associated molecular patterns (PAMPs), it is likely that the inhibition of signaling pathways activated by oxidized lysophospholipids would affect innate immunity per se. On the other hand, inhibition or modulation of their enzymatic formation, which would not interfere with the response to pathogens, might be beneficial and is potentially a promising new field of research.

When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia‐like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca2+, likely to enhance the LD–organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress‐induced lipotoxicity., Main points Astroglial lipid droplets are dynamic organelles with confined mobility.Lipid droplet turnover is important for astrocyte survival/proliferation.Stressed astrocytes accumulate lipid droplets likely to provide energy and reduce lipotoxicity.

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A(2) (sPLA(2)) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA(2) activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA(2) promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA(2) enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

Superparamagnetic iron oxide nanoparticles (SPIONs) have great potential for use in medicine, but they may cause side effects due to oxidative stress. In our study, we investigated the effects of silica-coated SPIONs on endothelial cells and whether oleic acid (OA) can protect the cells from their harmful effects. We used viability assays, flow cytometry, infrared spectroscopy, fluorescence microscopy, and transmission electron microscopy. Our results show that silica-coated SPIONs are internalized by endothelial cells, where they increase the amount of reactive oxygen species (ROS) and cause cell death. Exposure to silica-coated SPIONs induced accumulation of lipid droplets (LD) that was not dependent on diacylglycerol acyltransferase (DGAT)-mediated LD biogenesis, suggesting that silica-coated SPIONs suppress LD degradation. Addition of exogenous OA promoted LD biogenesis and reduced SPION-dependent increases in oxidative stress and cell death. However, exogenous OA protected cells from SPION-induced cell damage even in the presence of DGAT inhibitors, implying that LDs are not required for the protective effect of exogenous OA. The molecular phenotype of the cells determined by Fourier transform infrared spectroscopy confirmed the destructive effect of silica-coated SPIONs and the ameliorative role of OA in the case of oxidative stress. Thus, exogenous OA protects endothelial cells from SPION-induced oxidative stress and cell death independent of its incorporation into triglycerides.

Lipid droplets are dynamic organelles with a central role in fatty acid metabolism. They protect cells from lipotoxicity by sequestering excess fatty acids but also provide fatty acids for metabolic reactions and signalling events. Here we show that lipid droplet turnover in cancer cells is required for production of {omega}-3 and {omega}-6 polyunsaturated fatty acid (PUFA)-derived inflammatory lipid mediators, including eicosanoids and specialised pro-resolving mediators. We show that incorporation of PUFAs into triglycerides mediated by diacylglycerol acyltransferase 1 (DGAT1), and their release by adipose triglyceride lipase (ATGL), are required for cyclooxygenase- and lipoxygenase-dependent lipid mediator production and cancer cell proliferation. The human group X secreted phospholipase A2 (hGX sPLA2) drives the delivery of membrane-derived PUFAs into lipid droplets, while ATGL promotes the incorporation of lipid droplet-derived PUFAs into phospholipids. The group IVA cytosolic PLA2 (cPLA2) acts on membrane phospholipids and complements ATGL in the regulation of PUFA trafficking between phospholipids and triglycerides. This study identifies lipid droplets as essential cellular hubs that control PUFA availability for production of lipid mediators involved in inflammation and tumorigenesis. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/470010v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@13df1c6org.highwire.dtl.DTLVardef@1cc25c8org.highwire.dtl.DTLVardef@1c47cc0org.highwire.dtl.DTLVardef@da4508_HPS_FORMAT_FIGEXP M_FIG C_FIG This study shows that lipid droplets in cancer cells control the supply of {omega}-3 and {omega}-6 polyunsaturated fatty acids (PUFAs) for the production of lipid mediators, which in turn drive cancer cell proliferation. The esterification of PUFAs into triacylglycerols (TAGs) and their release from lipid droplets are necessary for PUFA entry into lipid mediator production pathways. O_LILipid mediator production induced by the human group X secreted phospholipase A2 (hGX sPLA2), which releases PUFAs from the plasma membrane and serum lipoproteins, depends on diacylglycerol acyltransferase 1 (DGAT1)-mediated TAG synthesis. C_LIO_LIAdipose triglyceride lipase (ATGL) liberates {omega}-3 and {omega}-6 PUFAs from TAGs and drives lipid mediator production via cyclooxygenase (COX) and lipoxygenase (LOX) pathways. C_LIO_LIATGL promotes the incorporation of lipid droplet-derived PUFAs into phospholipids, which are targeted by the group IVA cytosolic PLA2 (cPLA2), thereby selectively supplying arachidonic acid for lipid mediator production. C_LIO_LILipid droplets are required for cPLA2-induced lipid mediator production also in cells that do not depend on ATGL for the supply PUFAs into lipid mediator pathways. C_LI

Lipid droplets have a unique structure among organelles consisting of a dense hydrophobic core of neutral lipids surrounded by a single layer of phospholipids decorated with various proteins. Often labeled merely as passive fat storage repositories, they in fact have a remarkably dynamic life cycle. Being formed within the endoplasmic reticulum membrane, lipid droplets rapidly grow, shrink, traverse the cytosol, and engage in contacts with other organelles to exchange proteins and lipids. Their lipid and protein composition changes dynamically in response to cellular states and nutrient availability. Remarkably, their biogenesis is induced when cells experience various forms of nutrient, energy, and redox imbalances, including lipid excess and complete nutrient deprivation. Cancer cells are continuously exposed to nutrient and oxygen fluctuations and have the capacity to switch between alternative nutrient acquisition and metabolic pathways in order to strive even during severe stress. Their supply of lipids is ensured by a series of nutrient uptake and scavenging mechanisms, upregulation of de novo lipid synthesis, repurposing of their structural lipids via enzymatic remodeling, or lipid recycling through autophagy. Importantly, most of these pathways of lipid acquisition converge at lipid droplets, which combine different lipid fluxes and control their usage based on specific cellular needs. It is thus not surprising that lipid droplet breakdown is an elaborately regulated process that occurs via a complex interplay of neutral lipases and autophagic degradation. Cancer cells employ lipid droplets to ensure energy production and redox balance, modulate autophagy, drive membrane synthesis, and control its composition, thereby minimizing stress and fostering tumor progression. As regulators of (poly)unsaturated fatty acid trafficking, lipid droplets are also emerging as modulators of lipid peroxidation and sensitivity to ferroptosis. Clearly, dysregulated lipid droplet turnover may also be detrimental to cancer cells, which should provide potential therapeutic opportunities in the future. In this review, we explore how lipid droplets consolidate lipid acquisition and trafficking pathways in order to match lipid supply with the requirements for cancer cell survival, growth, and metastasis.

Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14- fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.

Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A2 (hGX sPLA2) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA2 and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA2 releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA2 and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA2 and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA2-induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions.

Integrins are plasma membrane proteins, whose dysfunction frequently results in cancer pathology, and therefore they represent important targets of anti-tumour therapy. Snake venoms are a rich source of disintegrins (Dis), proteins that specifically bind integrins and thus interfere with their functions. In an attempt to discover new molecules for treatment of breast cancer, the major type of cancer in women, we isolated a dimeric Dis (Vaa-Dis) from the venom of the nosehorned viper. By cell viability testing we demonstrated that 50 nM and higher concentrations of Vaa-Dis were toxic to highly invasive human breast adenocarcinoma cell line MDA-MB-231. Wound-healing assay revealed that already at one order of magnitude lower concentrations Vaa-Dis efficiently inhibited MDA-MB-231 cell migration. This exposed a promising anti-metastatic potential of Vaa-Dis and a good perspective of these natural snake venom proteins for further research and development towards the application in breast cancer treatment.

Cytosolic lipid droplets (LDs) store excess fatty acids (FAs) in the form of neutral lipids and prevent starvation-induced cancer cell death. Here we studied the ability of mono- and polyunsaturated FAs to affect LD formation and survival in HeLa cervical cancer cells. We found that the LD content in HeLa cells increases with cell density, but it decreases in MDA-MB-231 breast cancer cells. Exogenously-added unsaturated FAs, including oleic (OA), linoleic (LA), arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) displayed a similar ability to alter LD formation in HeLa cells. There was a dual, concentration-dependent effect on neutral lipid accumulation: low micromolar concentrations of LA, AA and DHA reduced, while all FAs induced LD formation at higher concentrations. In serum starved He-La cells, OA stimulated LD formation, but, contrary to expectations, it promoted cell death. Our results reveal a link between cell population density and LD formation in HeLa cells and show that unsaturated FAs may both suppress or stimulate LD formation. This dynamic regulation of LD content must be accounted for when studying the effects of lipids and lipid metabolism-targeting drugs on LD metabolism in HeLa cells.

Lipid droplets have a unique structure among organelles consisting of a dense hydrophobic core of neutral lipids surrounded by a single layer of phospholipids decorated with various proteins. Often labeled merely as passive fat storage repositories, they in fact have a remarkably dynamic life cycle. Being formed within the endoplasmic reticulum membrane, lipid droplets rapidly grow, shrink, traverse the cytosol, and engage in contacts with other organelles to exchange proteins and lipids. Their lipid and protein composition changes dynamically in response to cellular states and nutrient availability. Remarkably, their biogenesis is induced when cells experience various forms of nutrient, energy, and redox imbalances, including lipid excess and complete nutrient deprivation. Cancer cells are continuously exposed to nutrient and oxygen fluctuations and have the capacity to switch between alternative nutrient acquisition and metabolic pathways in order to strive even during severe stress. Their supply of lipids is ensured by a series of nutrient uptake and scavenging mechanisms, upregulation of de novo lipid synthesis, repurposing of their structural lipids via enzymatic remodeling, or lipid recycling through autophagy. Importantly, most of these pathways of lipid acquisition converge at lipid droplets, which combine different lipid fluxes and control their usage based on specific cellular needs. It is thus not surprising that lipid droplet breakdown is an elaborately regulated process that occurs via a complex interplay of neutral lipases and autophagic degradation. Cancer cells employ lipid droplets to ensure energy production and redox balance, modulate autophagy, drive membrane synthesis, and control its composition, thereby minimizing stress and fostering tumor progression. As regulators of (poly)unsaturated fatty acid trafficking, lipid droplets are also emerging as modulators of lipid peroxidation and sensitivity to ferroptosis. Clearly, dysregulated lipid droplet turnover may also be detrimental to cancer cells, which should provide potential therapeutic opportunities in the future. In this review, we explore how lipid droplets consolidate lipid acquisition and trafficking pathways in order to match lipid supply with the requirements for cancer cell survival, growth, and metastasis.

The β-neurotoxic secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals. Damage inflicted by such sPLA2s (β-ntx) on neuronal mitochondria is characteristic, very similar to that induced by structurally homologous endogenous group IIA sPLA2 when its activity is elevated, as, for example, in the early phase of Alzheimer’s disease. Using ammodytoxin (Atx), the β-ntx from the venom of the nose-horned viper (Vipera a. ammodytes), the sPLA2 receptor R25 has been detected in neuronal mitochondria. This receptor has been purified from porcine cerebral cortex mitochondria by a new Atx-affinity-based chromatographic procedure. Mass spectrometry analysis revealed R25 to be the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. CCOX was confirmed as being the first intracellular membrane receptor for sPLA2 by alternative Atx-affinity-labellings of purified CCOX, supported also by the encounter of Atx and CCOX in PC12 cells. This discovery suggests the explanation of the mechanism by which β-ntx hinders production of ATP in poisoned nerve endings. It also provides a new insight into the potential function and dysfunction of endogenous GIIA sPLA2 in mitochondria.

Cancer cells possess remarkable abilities to adapt to adverse environmental conditions. Their survival during severe nutrient and oxidative stress depends on their capacity to acquire extracellular lipids and the plasticity of their mechanisms for intracellular lipid synthesis, mobilisation, and recycling. Lipid droplets, cytosolic fat storage organelles present in most cells from yeast to men, are emerging as major regulators of lipid metabolism, trafficking, and signalling in various cells and tissues exposed to stress. Their biogenesis is induced by nutrient and oxidative stress and they accumulate in various cancers. Lipid droplets act as switches that coordinate lipid trafficking and consumption for different purposes in the cell, such as energy production, protection against oxidative stress or membrane biogenesis during rapid cell growth. They sequester toxic lipids, such as fatty acids, cholesterol and ceramides, thereby preventing lipotoxic cell damage and engage in a complex relationship with autophagy. Here, we focus on the emerging mechanisms of stress-induced lipid droplet biogenesis; their roles during nutrient, lipotoxic, and oxidative stress; and the relationship between lipid droplets and autophagy. The recently discovered principles of lipid droplet biology can improve our understanding of the mechanisms that govern cancer cell adaptability and resilience to stress.

Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A

The pulmonary delivery of nanoparticles (NPs) is a promising approach in nanomedicine. For the efficient and safe use of inhalable NPs, understanding of NP interference with lung surfactant metabolism is needed. Lung surfactant is predominantly a phospholipid substance, synthesized in alveolar type II cells (ATII), where it is packed in special organelles, lamellar bodies (LBs). In vitro and in vivo studies have reported NPs impact on surfactant homeostasis, but this phenomenon has not yet been sufficiently examined. We showed that in ATII-like A549 human lung cancer cells, silica-coated superparamagnetic iron oxide NPs (SiO

Secreted phospholipases A2 (sPLA2s) hydrolyse cell and lipoprotein phospholipid membranes to release free fatty acids and lysophospholipids, and can also bind to specific proteins. Several sPLA2s have been associated with various cancers, including prostate, colon, gastric, lung and breast cancers, yet, their role is controversial and seems to be dependent on the cancer type, the local microenvironment and the enzyme studied. There is strong evidence that the expression of some sPLA2s, most notably the group IIA, III and X enzymes, is dysregulated in various malignant tissues, where, as described in a number of in vitro and in vivo studies using mouse models and according to correlations between sPLA2 expression and patient survival, a particular enzyme may exert either a pro- or an anti-tumourigenic role. It is becoming clear that there are multiple, context-dependent mechanisms of action of sPLA2s in different cancers. First, the role of sPLA2s in cancer has traditionally been associated with their enzymatic activity and ability to participate in the release of potent biologically active lipid mediators, in particular arachidonic acid-derived eicosanoids, which promote tumourigenesis by stimulating cell proliferation and cell survival, by abrogating apoptosis and by increasing local inflammation and angiogenesis. Second, several biological effects of sPLA2s were found to be independent of sPLA2 enzymatic activity, arguing for a receptor-mediated mechanism of action. Finally, recent studies have implicated sPLA2s in the regulation of basal lipid metabolism, opening a new window to the understanding of the diverse roles of sPLA2s in cancer. In this short review, we highlight the newest findings on the biological roles of sPLA2s in cancer, with emphasis on their diverse mechanisms of action.

Background. Functional erythropoietin (EPO) signaling is not specific only to erythroid lineages and has been confirmed in several solid tumors, including breast. Three different isoforms of erythropoietin receptor (EPOR) have been reported, the soluble (EPOR-S) and truncated (EPOR-T) forms acting antagonistically to the functional EPOR. In this study, we investigated the effect of human recombinant erythropoietin (rHuEPO) on cell proliferation, early gene response and the expression of EPOR isoforms in the MCF-7 breast cancer cell line. Materials and Methods. The MCF-7 cells were cultured with or without rHuEPO for 72 h or 10 weeks and assessed for their growth characteristics, expression of early response genes and different EPOR isoforms. The expression profile of EPOR and EPOR-T was determined in a range of breast cancer cell lines and compared with their invasive properties. Results. MCF-7 cell proliferation after rHuEPO treatment was dependent on the time of treatment andthe concentration used. High rHuEPO concentrations (40 U/ml) stimulated cell proliferation in dependently of a preceding long-term exposure of MCF-7 cells to rHuEPO, while lower concentrations increased MCF-7 proliferation only after 10 weeks of treatment. Gene expression analysis showed activation of EGR1 and FOS, confirming the functionality of EPOR. rHuEPO treatment also slightly increased the expression of the functional EPOR isoform,which, however, persisted throughout the 10 weeks of treatment. The expression levels of EPOR-T were not influenced.There were no correlations between EPOR expression and the invasiveness of MCF-7, MDA-MB-231, Hs578T, Hs578Bst, SKBR3,T-47D and MCF-10A cell lines. Conclusions. rHuEPO modulates MCF-7 cell proliferation in time- and concentration-dependent manner. We confirmed EGR1, FOS and EPOR as transcription targets of the EPO-EPOR signaling loop but could not correlate the expression of different EPOR isoforms with the invasiveness of breast cancer cell lines. Keywords: Breast cancer; erythropoietin; erythropoietin receptor isoforms; proliferation; gene expression

Ammodytoxin (Atx), a neurotoxic secreted phospholipase A(2) (sPLA(2)), forms a high-affinity complex with calmodulin (CaM). The latter substantially increases the enzymatic activity of Atx under both non-reducing and reducing conditions, and the activity enhancement was accompanied, but not caused, by conformational stabilization of the enzyme. In this work, the energetically most favorable model of the complex was generated, making use of interaction site mapping, mutagenesis data and protein-docking algorithms. The model explains, in structural terms, the observed effects of stabilization and activity enhancement of the neurotoxic sPLA(2) by CaM. The structures of four mammalian sPLA(2) isoforms, groups IB, IIA, V and X, having the same fold as Atx, were superimposed on the structure of Atx in the complex with CaM. According to the generated models, the group V and X sPLA(2)s, but not the group IB and IIA enzymes, form stable complexes with CaM, which should also result in the augmentation of their enzymatic activity. By confirming the latter, the presented model is validated as a valuable tool to investigate the as yet unexplained role of CaM in the pathophysiology of snake venom and mammalian sPLA(2)s.

Ammodytoxins are presynaptically neurotoxic snake venom group IIA secreted phospholipase A(2) enzymes that interact specifically with calmodulin in the cytosol of nerve cells. We show that calmodulin behaves as an activator of ammodytoxin under both nonreducing and reducing (cytosol-like) conditions by stimulating its enzymatic activity up to 21-fold. Kinetic analysis, using a general modifier mechanism, and surface plasmon resonance measurements reveal that calmodulin influences both the catalytic and the vesicle binding properties of the enzyme without affecting its calcium binding properties. The equilibrium dissociation constant of the ammodytoxin-calmodulin complex under cytosol-like conditions is in the low nanomolar range (3 nM), while under nonreducing conditions, the binding affinity is in the subnanomolar range (0.07-0.18 nM). Upon exposure to cytosol-like conditions, ammodytoxin undergoes a slow hysteretic transition to a less active state. Calmodulin stabilizes the conformation of ammodytoxin and thereby restores its activity. These results provide insights into the neurotoxic action of ammodytoxins and the mechanisms involved in the regulation of secreted phospholipase A(2) activity within the cytosol.

Phospholipases A2 (PLA2s), lipolytic enzymes that release fatty acids (FAs) and lysophospholipids from the hydrolysis of the sn-2 ester bond of membrane glycerophospholipids, modulate (phospho)lipid-related homeostatic and stimulus-induced intracellular processes, linking lipid metabolism with cell signaling [1, 2]. Although the diverse products released by their activity on cell membranes have signaling and metabolic activities in and of themselves, the products may be further converted into hundreds of lipid mediator molecules, including arachidonic acid (AA)derived eicosanoids and x-3 polyunsaturated FA-derived pro-resolving mediators [3]. Unsurprisingly, PLA2s are involved in diverse and fundamental biological processes, including inflammation, immunity, reproduction, atherosclerosis, cancer, and neurodegeneration [2]. Among the human PLA2s, eleven members belong to the secreted PLA2 (sPLA2) family of low-molecular-mass Ca -dependent enzymes that function in the extracellular space on cell membranes, lipoproteins, bacteria, microparticles, and viruses [3, 4]. Most PLA2s are, however, intracellular enzymes, including the Ca-dependent cytosolic PLA2s (cPLA2s or group IV PLA2s) and the Ca -independent PLA2s (iPLA2s or group VI PLA2s). Among the nine enzymes within the iPLA2 family, also named the patatinlike phospholipase domain-containing lipase (PNPLA) family, iPLA2b (also PNPLA9 or group VIA PLA2) is far and away the most studied enzyme [5]. The human iPLA2b gene (PLA2G6) may undergo several alternative splicing events that generate isoforms with varying enzymatic activity and function [6]. The ‘‘classic’’ isoform of iPLA2b (group VIA-1 PLA2) is an 85-kDa enzyme with a Ser-Asp catalytic dyad, a conserved glycine-rich, nucleotide-binding motif (GXGXXG) and eight N-terminal ankyrin repeats [5]. The cellular activity of iPLA2b is regulated by ATP binding, oligomerization, binding to calmodulin, caspase-3-mediated proteolytic cleavage, and alternative splicing [5, 6]. In contrast to cPLA2, iPLA2b is not specific for AA, hydrolyzing phospholipids with no preference for their FA substituents or head groups. The biological activity of iPLA2b has been traditionally described as homeostatic membrane remodeling, which includes deacylation/reacylation cycles important for AA incorporation into phospholipids, for phosphatidylcholine (PC) turnover and cell growth, but also for excision-repair mechanisms that maintain mitochondrial membrane integrity [5]. However, recent studies have revealed a number of cell signaling-based roles for the enzyme, such as the regulation of endoplasmic reticulum (ER) stress, apoptosis, cell proliferation, differentiation, and immune cell function [5]. Analyses of mutant mouse models and clinical phenotypes have provided important insights into its (patho)physiological effects, which include osteoblastic differentiation, male fertility, b-cell function, cancer, and neurodegeneration [5]. In this issue of Digestive Diseases and Sciences, Jiao et al. [7] report a novel function for iPLA2b in the maintenance of intestinal homeostasis. The authors reported that iPLA2b deficiency significantly exacerbates pathological features in an experimental mouse model of dextran & Toni Petan toni.petan@ijs.si

The enzymatic activity of ammodytoxins (Atxs), secreted phospholipases A(2) (sPLA(2)s) in snake venom, is essential for expression of their presynaptic neurotoxicity, but its exact role in the process is unknown. We have analyzed in detail the enzymatic properties of Atxs, their mutants, and homologues. The apparent rates of phospholipid hydrolysis by the sPLA(2)s tested vary by up to 4 orders of magnitude, and all enzymes display a strong preference for vesicles containing anionic phospholipids, phosphatidylglycerol or phosphatidylserine (PS), over those containing zwitterionic phosphatidylcholine (PC). Nevertheless, Atxs are quite efficient in hydrolyzing pure PC vesicles as well as PC-rich plasma membranes of intact HEK293 cells. The presence of anionic phospholipids in PC vesicles dramatically increases the interfacial binding affinity and catalytic activity of Atxs, but not of their nontoxic homologue ammodytin I(2), that displays unusually low binding affinity and enzymatic activity on PS-containing vesicles and HEK293 plasma membranes. Aromatic and hydrophobic residues on the interfacial binding surface of Atxs are important for productive binding to both zwitterionic and anionic vesicles, while basic and polar residues have a negative impact on binding to zwitterionic vesicles. When tightly bound to the membrane interface, Atxs can reach full enzymatic activity at low micromolar concentrations of Ca(2+). Although Atxs have evolved to function as potent neurotoxins that specifically target presynaptic nerve terminals, they display a high degree of phospholipolytic efficiency on various phospholipid membranes.

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the h-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the h-region of AtxA contribute to, but are not necessary for, its neurotoxic effect. D 2004 Elsevier B.V. All rights reserved.

Ammodytoxins (Atxs) are group II phospholipases A(2) (PLA(2)s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA(2) toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe(24), in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp(24) mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40-80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA(2) activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe(24) also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn(24) mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe(24) in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.

Our previous gene expression analysis identified phospholipase A2 group IIA (PLA2G2A) as a potential biomarker of ovarian endometriosis. The aim of this study was to evaluate PLA2G2A mRNA and protein levels in tissue samples (endometriomas and normal endometrium) and in serum and peritoneal fluid of ovarian endometriosis patients and control women. One-hundred and sixteen women were included in this study: the case group included 70 ovarian endometriosis patients, and the control group included 38 healthy women and 8 patients with benign ovarian cysts. We observed 41.6-fold greater PLA2G2A mRNA levels in endometrioma tissue, compared to normal endometrium tissue. Using Western blotting, PLA2G2A was detected in all samples of endometriomas, but not in normal endometrium, and immunohistochemistry showed PLA2G2A-specific staining in epithelial cells of endometrioma paraffin sections. However, there were no significant differences in PLA2G2A levels between cases and controls according to ELISA of peritoneal fluid (6.0 ± 4.4 ng/ml, 6.6 ± 4.3 ng/ml; p = 0.5240) and serum (2.9 ± 2.1 ng/ml, 3.1 ± 2.2 ng/ml; p = 0.7989). Our data indicate that PLA2G2A is implicated in the pathophysiology of ovarian endometriosis, but that it cannot be used as a diagnostic biomarker.

Secreted phospholipases A2 (sPLA2s) have recently been associated with several cancers, but their role in breast cancer is unknown. Here we demonstrate that mRNA expression of group IIA, III and X sPLA2s differs both in vivo in tumour biopsies and in breast cancer cells in vitro. Their expression is differentially regulated by DNA methylation and histone acetylation and, significantly, all three genes are silenced in aggressive triple negative cells due to both mechanisms. The transcription start site promoter region and the upstream CpG islands, exclusive to the group X sPLA2 gene, have variable roles in the regulation of sPLA2 expression. Our results suggest that the differential expression of hGIIA, hGIII and hGX sPLA2s in breast cancer cells is a consequence of various degrees of epigenetic silencing due to DNA hypermethylation and histone deacetylation.

Background The role of AID/APOBEC proteins in the mammalian immune response against retroviruses and retrotransposons is well established. G to A hypermutations, the hallmark of their cytidine deaminase activity, are present in several mammalian retrotransposons. However, the role of AID/APOBEC proteins in non-mammalian retroelement restriction is not completely understood. Results Here we provide the first evidence of anti-retroelement activity of a reptilian APOBEC protein. The green anole lizard A1 protein displayed potent DNA mutator activity and inhibited ex vivo retrotransposition of LINE1 and LINE2 ORF1 protein encoding elements, displaying a mechanism of action similar to that of the human A1 protein. In contrast, the human A3 proteins did not require ORF1 protein to inhibit LINE retrotransposition, suggesting a differential mechanism of anti-LINE action of A1 proteins, which emerged in amniotes, and A3 proteins, exclusive to placental mammals. In accordance, genomic analyses demonstrate differential G to A DNA editing of LINE retrotransposons in the lizard genome, which is also the first evidence for G to A DNA editing in non-mammalian genomes. Conclusion Our data suggest that vertebrate APOBEC proteins differentially inhibit the retrotransposition of LINE elements and that the anti-retroelement activity of APOBEC proteins predates mammals.

Ammodytoxins (Atxs) and ammodytins (Atns) are group IIA phospholipases A2 (sPLA2s) and their homologues, secreted by venom glands of the nose-horned viper (Vipera a. ammodytes). The molecular mechanisms underlying their various pharmacological effects, including neurotoxicity, myotoxicity and anticoagulant activity, are still not completely understood. The structure-function relationships of Atxs and Atns have been studied by site-directed and cassette mutagenesis. We cloned their complementary DNA reversely transcribed from the mRNA isolated from the venom glands, and expressed the mature protein regions in Escherichia coli. The recombinant proteins were isolated in their inactive forms and renatured in vitro to the properly folded and biologically active forms. More than fifty site-directed mutants and chimeric sPLA2 proteins of Atxs and Atns were produced and their properties analysed. In the course of these studies, the three-dimensional crystal structure was determined of the most neurotoxic venom sPLA2, AtxA, that induces complete failure of vertebrate neuromuscular transmission, using the recombinant protein. The results have contributed significantly to a better understanding of the molecular mechanism of presynaptic toxicity of sPLA2 neurotoxins. In addition, the activity of enzymatically inactive sPLA2 homologues and their evolution are now better understood.

Background Alterations in lipid metabolism are inherent to the metabolic transformations that support tumorigenesis. The relationship between the synthesis, storage and use of lipids and their importance in cancer is poorly understood. The human group X secreted phospholipase A2 (hGX sPLA2) releases fatty acids (FAs) from cell membranes and lipoproteins, but its involvement in the regulation of cellular FA metabolism and cancer is not known. Results Here we demonstrate that hGX sPLA2 induces lipid droplet (LD) formation in invasive breast cancer cells, stimulates their proliferation and prevents their death on serum deprivation. The effects of hGX sPLA2 are shown to be dependent on its enzymatic activity, are mimicked by oleic acid and include activation of protein kinase B/Akt, a cell survival signaling kinase. The hGX sPLA2-stimulated LD biogenesis is accompanied by AMP-activated protein kinase (AMPK) activation, up-regulation of FA oxidation enzymes and the LD-coating protein perilipin 2, and suppression of lipogenic gene expression. Prolonged activation of AMPK inhibited hGX sPLA2-induced LD formation, while etomoxir, an inhibitor of FA oxidation, abrogated both LD formation and cell survival. The hGX sPLA2-induced changes in lipid metabolism provide a minimal immediate proliferative advantage during growth under optimal conditions, but they confer to the breast cancer cells a sustained ability to resist apoptosis during nutrient and growth factor limitation. Conclusion Our results identify hGX sPLA2 as a novel modulator of lipid metabolism that promotes breast cancer cell growth and survival by stimulating LD formation and FA oxidation.

The molecular mechanism of action of presynaptically neurotoxic secreted phospholipases A2 (sPLA2s) has not been fully elucidated. We have recently proposed a model to explain one of the hallmarks of their action - the reduction in endocytosis leading to synaptic vesicle depletion in nerve terminals. Our results speak strongly in favor of a mechanism in which both specific protein-protein interactions and enzymatic activity of the neurotoxic sPLA2 ammodytoxin A (AtxA) are necessary for impairment of clathrin-dependent endocytosis in yeast cells. The reduction of endocytosis was strictly dependent on the enzymatic activity of sPLA2s expressed ectopically in our yeast model cells and was not observed with the catalytically inactive, non-neurotoxic AtxA-homolog, ammodytin L (AtnL). Here we confirm the validity of the model in mammalian cells also, by demonstrating that the enzymatically active mutant of AtnL, shown to inhibit endocytosis in yeast, acts as a presynaptically neurotoxic sPLA2 at the mammalian neuromuscular junction.

Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

BACKGROUND: Presynaptically neurotoxic phospholipases A(2) inhibit synaptic vesicle recycling through endocytosis. PRINCIPAL FINDINGS: Here we provide insight into the action of a presynaptically neurotoxic phospholipase A(2) ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin. CONCLUSIONS: We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein-protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A(2) work can open new ways to regulate endocytosis.

Ammodytoxin A (AtxA) is a presynaptically neurotoxic secretory phospholipase A(2) from snake venom. The aim of this study was to investigate the mechanism of its cytotoxicity expressed against mouse motoneuronal NSC34 cells. AtxA displayed a potent dose- and time-dependent cytotoxicity that was associated with apoptosis and not necrosis, as revealed by a reduction of mitochondrial membrane potential, activation of caspase-3, and by the absence of propidium iodide staining. The cytotoxic- and apoptosis-inducing effects of AtxA were specific for the motoneuronal cells; human embryonic kidney (HEK293) and mouse myoblast (C2C12) cells were shown to be resistant to the toxin.

The structural features of presynaptically neurotoxic secretory phospholipases A 2 (sPLA 2 s) that are responsible for their potent and specific action are still a matter of debate. To identify the residues that distinguish a highly neurotoxic sPLA 2 , ammodytoxin A (AtxA), from a structurally similar but more than two orders of magnitude less toxic Russell's viper sPLA 2 , VIIIa, we prepared a range of mutants and compared their properties. The results show that the structural features that confer high neurotoxicity to AtxA extend from its C-terminal part, with a central role of the residues Y115, I116, R118, N119 (the YIRN cluster) and F124, across the interfacial binding surface (IBS) in the vicinity of F24, to the N-terminal helix whose residues M7 and G11 are located on the edges of the IBS. Competition binding studies indicate that the surface of interaction with the neuronal M-type sPLA 2 receptor R180 extends over a similar region of the molecule. In addition, the YIRN cluster of AtxA is crucial for the high-affinity interaction with two intracellular binding proteins, calmodulin and R25. The concept of a single “presynaptic neurotoxic site” on the surface of snake venom sPLA 2 s is not consistent with these results which suggest that different parts of the toxin molecule are involved in distinct steps of presynaptic neurotoxicity.