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Protein phosphorylation is the most common post-translational modification of proteins and functions as a molecular switch for their regulation. This modification is reversibly regulated by protein kinases and phosphatases. In most cases, the phosphorylation of enzymes positively or negatively regulates enzyme activity. However, we found that the phosphorylation of DDHD1 phospholipase A1 (PLA1) did not affect PLA1 activity. Integrated analyses, including phospho-proteomics, Phos-tag SDS-PAGE, PLA1 enzyme assays, and immunofluorescent microscopy, revealed the subcellular localization of DDHD1 without greatly affecting its PLA1 activity. Our findings may contribute to understanding rare clinical cases that concern the implications of protein phosphorylation.

BRAF and NRAS are the most reported mutations associated to melanomagenesis. The lack of accurate diagnostic markers in response to therapeutic treatment in BRAF/NRAS-driven melanomagenesis is one of the main challenges in melanoma personalized therapy. In order to assess the diagnostic value of phosphatidylserine-specific phospholipase A1-alpha (PLA1A), a potent lysophospholipid mediating the production of lysophosphatidylserine, PLA1A mRNA and serum levels were compared in subjects with malignant melanoma (n = 18), primary melanoma (n = 13), and healthy subjects (n = 10). Additionally, the correlation between histopathological subtypes of BRAF/NRAS-mutated melanoma and PLA1A was analyzed. PLA1A expression was significantly increased during melanogenesis and positively correlated to disease severity and histopathological markers of metastatic melanoma. PLA1A mRNA and serum levels were significantly higher in patients with BRAF-mutated melanoma compared to the patients with NRAS-mutated melanoma. Notably, PLA1A can be used as a diagnostic marker for an efficient discrimination between naïve melanoma samples and advanced melanoma samples (sensitivity 91%, specificity 57%, and AUC 0.99), as well as BRAF-mutated melanoma samples (sensitivity 62%, specificity 61%, and AUC 0.75). Our findings suggest that PLA1A can be considered as a potential diagnostic marker for advanced and BRAF-mutated melanoma.

Through the insertion of nonpolar side chains into the bilayer, the hydrophobic effect has long been accepted as a driving force for membrane protein folding. However, how the changing chemical composition of the bilayer affects the magnitude side chain transfer free energieshas historically not been well understood. A particularly challenging region for experimental interrogation is the bilayer interfacial region that is characterized by a steep polarity gradient. In this study we have determined thefor nonpolar side chains as a function of bilayer position using a combination of experiment and simulation. We discovered an empirical correlation between the surface area of nonpolar side chain, the transfer free energies, and the local water concentration in the membrane that allows forto be accurately estimated at any location in the bilayer. Using these water-to-bilayervalues, we calculated the interface-to-bilayer transfer free energy. We find that theare similar to the “biological”, translocon-based transfer free energies, indicating that the translocon energetically mimics the bilayer interface. Together these findings can be applied to increase the accuracy of computational workflows used to identify and design membrane proteins, as well as bring greater insight into our understanding of how disease-causing mutations affect membrane protein folding and function.

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Phospholipase C (PLC) represents an important type of enzymes with the feature of hydrolyzing phospholipids at the position of the glycerophosphate bond, among which PLC extracted from

Synovial fluids from rheumatoid and psoriatic arthritis patients have high levels of PLA1A. The current study was to understand PLA1A functions in the pathophysiology of rheumatic diseases. We generated Pla1a−/− mice to assess their phenotype and the impact of PLA1A deficiency on the development of mannan-induced psoriatic arthritis (MIP). Mice were evaluated routinely for the induced symptoms. On the day of sacrifice, blood samples were collected for hematology analysis and prepared for plasma. Livers were collected. Lymph node immune cells were analyzed using flow cytometry. We performed μCT scans of hind paws from naïve and mannan-induced female mice. Cytokines/chemokines were quantified using Luminex in hind paw tissues and plasma of female mice. Pla1a−/− mice showed a slight increase in circulating and lymph node lymphocytes. CD4+ T cells contributed most to this increase in lymph nodes (p = 0.023). In the MIP model, the lymph node ratios of CD3+ to CD19+ and CD4+ to CD8+ were higher in Pla1a−/− mice. Pla1a−/− mice were less susceptible to MIP (p < 0.001) and showed reduced bone erosions. Pla1a−/− mice also showed reduced IL-17, KC, IP-10, MIP-1β, LIF, and VEGF in hind paw tissues as compared to WT mice (p < 0.05). These findings indicated that PLA1A deficiency protected from the development of the MIP disease. The data suggested that PLA1A could contribute to MIP through increased activation of lymphocytes, possibly those producing IL-17.

Macrophages are a key in innate immune responses and play vital roles in homeostasis and inflammatory diseases. Phosphatidylserine-specific phospholipase A1 (PS-PLA1) is a specific phospholipase which hydrolyzes fatty acid from the sn-1 position of phosphatidylserine (PS) to produce lysophosphatidylserine (lysoPS). Both PS and lysoPS are associated with activation of immune cells including macrophages. However, the effect of PS-PLA1 on macrophage inflammation remains unclear. The purpose of this study is to evaluate the role of PS-PLA1 in lipopolysaccharide (LPS)-induced macrophage inflammation. Alterations of PS-PLA1 expression in LPS-stimulated RAW264.7 macrophages were investigated via Western blot. PS-PLA1 stable knockdown and overexpression RAW264.7 cell lines were generated by infecting cells with appropriate lentiviral vectors, respectively. PS-PLA1 expression was found to be dramatically upregulated in RAW264.7 macrophages after LPS stimulation. PS-PLA1 knockdown promotes while PS-PLA1 overexpression ameliorates the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and nitric oxide from RAW264.7 cells and M1 macrophage polarization. Additionally, PS-PLA1 knockdown facilitates phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PS-PLA1 overexpression attenuates their phosphorylation. Moreover, mitogen-activated protein kinase (MAPK) inhibitors block the release of TNF-α and IL-1β in PS-PLA1 knockdown RAW264.7 cells after LPS stimulation. These findings suggest PS-PLA1 ameliorates LPS-induced macrophage inflammation by inhibiting MAPKs activation, and PS-PLA1 might be considered as a target for modulating macrophage inflammation.

Six kinds of Mo-basing nanomaterials (MoO

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2(±), lpar1(−/−) or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2(±) and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion. Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2(±): Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1(−/−): lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Lipid metabolic abnormalities in cancer cells are increasingly being studied. Several studies have reported that phosphatidylserine-specific phospholipase A1 (PLA1A) might be involved in the pathogenesis of cancers. Nevertheless, the function and mechanistic details of PLA1A in lung adenocarcinoma (LUAD) progression remain largely undefined. In the present study, low PLA1A expression was correlated with poor prognosis in patients with LUAD. Results from in vitro and in vivo animal studies showed that overexpressed PLA1A suppressed the proliferation of LUAD cells in vitro and tumor growth in vivo through regulation of cyclin abundance, thereby inducing S-phase arrest. Meanwhile, PLA1A overexpression attenuated migration and invasion of LUAD cells, including by inhibiting the epithelial-mesenchymal transition. Mechanistically, PLA1A overexpression inhibited aggressiveness of LUAD cells through elevated lysophosphatidylserine, which acts via G-protein-coupled receptor 174, further activating cAMP/protein kinase A pathway. Activating G-protein-coupled receptor 174/protein kinase A pathway may involve effects on cell cycle regulators and transcription factors-regulated epithelial-mesenchymal transition. The study uncovered the mechanism through which PLA1A regulates LUAD proliferation, invasion, and migration. These results demonstrate the potential use of PLA1A as a biomarker for diagnosing LUAD, which may therefore potentially serve as a therapeutic target for LUAD.

Phospholipase A

Since the clinical outcome of patients with sarcoidosis is still unpredictable, a good prognostic biomarker is necessary. Autotaxin (ATX) and phosphatidylserine-specific phospholipase A1 (PS-PLA1) function as main enzymes to produce lysophospholipids (LPLs), and these enzymes are attracting attention as useful biomarkers for several chronic inflammatory diseases. Here, we investigated the relationships between LPLs-producing enzymes and the disease activity of sarcoidosis. In total, 157 patients with sarcoidosis (active state, 51%) were consecutively enrolled. Using plasma or urine specimens, we measured the values of LPLs-producing enzymes. Urine ATX (U-ATX) levels were significantly lower in the active state compared to those in the inactive state, while the plasma ATX (P-ATX) and PS-PLA1 levels showed no significant difference between these two states. Concerning the comparison with existing clinical biomarkers for sarcoidosis, U-ATX showed a weak negative correlation to ACE, P-ATX a weak positive correlation to both ACE and sIL-2R, and PS-PLA1 a weak positive one to sIL-2R. Notably, only the U-ATX levels inversely fluctuated depending on the status of disease activity whether OCS had been used or not. These findings suggest that U-ATX is likely to be a novel and useful molecule for assessing the disease activity of sarcoidosis.

Cotton is the most important crop for the production of natural fibres used in the textile industry. High salinity, drought, cold and high temperature represent serious abiotic stresses, which seriously threaten cotton production. Phospholipase AS has an irreplaceable role in lipid signal transmission, growth and development and stress events. Phospholipase A can be divided into three families: PLA1, PLA2 and pPLA. Among them, the PLA1 family is rarely studied in plants. In order to study the potential functions of the PLA1 family in cotton, the bioinformatics analysis of the PLA1 family was correlated with cotton adversity, and tissue-specific analysis was performed. Explore the structure-function relationship of PLA1 members. It is found that the expression of GbPLA1-32 gene is affected by a variety of environmental stimuli, indicating that it plays a very important role in stress and hormone response, and closely associates the cotton adversity with this family. Through further functional verification, we found that virus-induced GbPLA1-32 gene silencing (VIGS) caused Gossypium barbadense to be sensitive to salt stress. This research provides an important basis for further research on the molecular mechanism of cotton resistance to abiotic stress.

Giant lipid vesicles are used to study artificial cell models, as well as the encapsulation of biomolecules, and the reconstitution of membrane proteins on these vesicles. Recently, complex reactions in giant vesicles have been controlled by reconstituting numerous kinds of biomolecules. However, it is challenging to generate giant lipid vesicles containing a diverse set of proteins at concentrations sufficient to ensure proper functioning. Here, we describe an artificial cell model showing dual functions of small molecule transportation and small vesicle budding, using a dual functional membrane protein (transportation and phosphatase activity) called the outer membrane phospholipase (OmpLA). To the best of our knowledge, we have revealed for the first time the transportation of ions or small molecules through OmpLA on the charged lipid bilayer. The lipid composition controlled the orientation of OmpLA through proteinase K digestion. Finally, OmpLA enzyme activity of phospholipid hydrolysis caused the budding of small vesicles.

Phospholipids (PL) rich in conjugated linolenic acid (CLA) have important health effects. Yields of phosphatidylcholine (PC) acidolysis with CLA use to be limited to 30%, due to competitive side-hydrolysis. Duolite A658-Lecitase is a very suitable biocatalyst for this reaction. In this study, PC hydrolysis has been practically eliminated using extremely dried lyophilized PC (279 ± 4 mg water/Kg PC), obtaining close to 100% molar yield of modified PC (72.3% CLA) with Duolite-Lecitase in 24 h, the highest yield reported in the literature for this reaction. It has been better improved by changing the immobilization support, using three food grade hydrophobic supports (Styrene, and two Octadecyl methacrylates (OM and OMC)). In only 2 h, with a 1/12 PC/CLA molar ratio at 50 °C, similar almost quantitative yields of PC with 74.4% CLA content has been obtained using OM-Lecitase. The fatty acid composition of modified PCs is not affected by the enzyme immobilization protocol.

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

A two-stage enzymatic hydrolysis method, in which phospholipase A1 (PLA1) was added after phospholipase A2 (PLA2) was added for a certain time, was successfully carried out to prepare glycerylphosphorylcholine (GPC) from soybean concentrated phospholipid. Effects of reaction variables on hydrolysis reaction were optimized using response surface methodology, and the optimal conditions were as follows: PLA2 load of 1.25%, PLA1 load of 0.70%, substrate concentration of 13%, reaction temperature of 41°C, and stirring rate of 680 rpm. Under the optimal conditions, the GPC yield reached 83.07%, which is close to the predicted value by the fitted model. This paper not only provides an efficient and low-cost method to prepare GPC, but also improves the high-value utilization of soybean concentrated phospholipid.

DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1), a phospholipase A1, utilizes galactolipids (18:3) to generate α-linolenic acid (ALA) in the initial step of jasmonic acid (JA) biosynthesis in Arabidopsis thaliana. In this study, we isolated the JcDAD1 gene, an ortholog of Arabidopsis DAD1 in Jatropha curcas, and found that it is mainly expressed in the stems, roots, and male flowers of Jatropha. JcDAD1-RNAi transgenic plants with low endogenous jasmonate levels in inflorescences exhibited more and larger flowers, as well as a few abortive female flowers, although anther and pollen development were normal. In addition, fruit number was increased and the seed size, weight, and oil contents were reduced in the transgenic Jatropha plants. These results indicate that JcDAD1 regulates the development of flowers and fruits through the JA biosynthesis pathway, but does not alter androecium development in Jatropha. These findings strengthen our understanding of the roles of JA and DAD1 in the regulation of floral development in woody perennial plants.

This study sought to prepare a cognitive enhancer l-α-glycerylphosphorylcholine (l-α-GPC) using an immobilized Lecitase Ultra (LU, phospholipase A

Enzymatic treatment of egg yolk with phospholipases can enhance its emulsifying properties and thermal stability. Additionally, a two-step process (primary and secondary homogenization) could form emulsions with better stability. Thus, in this study we used a split-split-plot in time design to assess the effect of enzymatic treatment, processing, and storage conditions on the encapsulation efficiency, stability, toxicity, and permeability of egg yolk/fish oil emulsions stored up to 10 days at 45 °C. Egg yolk solutions before and after treatment with phospholipase A

An affinity ligand was designed from 1-aminocyclohexane based on the crystal structure of Streptomyces albidoflavus phospholipase A

Recent studies revealed that lysophospholipids (LPLs) and related molecules, such as autotaxin (ATX) and phosphatidylserine-specific phospholipase AIn this retrospective cohort study, serum samples were collected from 39 patients with LN and 37 patients with other glomerular diseases. The serum levels of ATX and PS-PLAThe serum levels of ATX and PS-PLAAlthough the serum levels of ATX and PS-PLA

Introduction and objective. Non-alcoholic fatty liver disease remains as one of the main liver disorders worldwide. It is widely accepted that is the kind of lipid, rather than the amount deposited in the cells that determines cell damage. Cholesterol and saturated free fatty acids are deleterious lipids when accumulated but, in contrast, there are some valuable lipids that could counteract those with harmful properties. Much of this knowledge arises from studies using a single fatty acid, but the effects of a combination of fatty acids, as obtained by diet has been poorly addressed. In the present work, we were focused to figure out the cellular effect of two different mixes of fatty acids, one with high proportion of saturated fatty acids, and another one with high proportion of unsaturated fatty acids (Mediterranean-like) in a cellular model of steatosis. Material and methods. Primary mouse hepatocytes from animals fed with a western diet (high fat and carbohydrates diet), were treated with both mixes of fatty acids for 24 h. Results. Our data clearly show that only the high unsaturated fatty acid mix induced a decrease in triglycerides (47.5%) and cholesterol (59%) content in steatotic hepatocytes mediating cellular protection associated to the decrement of ROS and oxidative damage. The mixture of high saturated fatty acids exhibited no effects, preserving high levels of cholesterol and triglycerides and oxidative damage. In conclusion, our results show that Mediterranean-like mix of fatty acids exerts cellular protection in steatosis by decreasing triglycerides, cholesterol, ROS content and oxidative damage.

Insect venom can cause systemic allergic reactions, including anaphylaxis. Improvements in diagnosis and venom immunotherapy (VIT) are based on a better understanding of an immunological response triggered by venom allergens. Previously, we demonstrated that the recombinant phospholipase A1 (rPoly p 1) from Polybia paulista wasp venom induces specific IgE and IgG antibodies in sensitized mice, which recognized the native allergen. Here, we addressed the T cell immune response of rPoly p 1-sensitized BALB/c mice. Cultures of splenocytes were stimulated with Polybia paulista venom extract and the proliferation of CD8+ and CD4+ T cells and the frequency of T regulatory cells (Tregs) populations were assessed by flow cytometry. Cytokines were quantified in cell culture supernatants in ELISA assays. The in vitro stimulation of T cells from sensitized mice induces a significant proliferation of CD4+ T cells, but not of CD8+ T cells. The cytokine pattern showed a high concentration of IFN-γ and IL-6, and no significant differences to IL-4, IL-1β and TGF-β1 production. In addition, the rPoly p 1 group showed a pronounced expansion of CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ Tregs. rPoly p 1 sensitization induces a Th1/Treg profile in CD4+ T cell subset, suggesting its potential use in wasp venom immunotherapy.

Deleterious mutations in the serine hydrolase DDHD1 cause the SPG28 subtype of the neurological disease Hereditary Spastic Paraplegia (HSP), which is characterized by axonal neuropathy and gait impairments. DDHD1 has been shown to display PLA1-type phospholipase activity with a preference for phosphatidic acid. However, the endogenous lipid pathways regulated by DDHD1 in vivo remain poorly understood. Here we use a combination of untargeted and targeted metabolomics to compare the lipid content of brain tissue from DDHD1(+/+) and DDHD1(–/–) mice, revealing that DDHD1 inactivation causes a substantial decrease in polyunsaturated lysophosphatidylinositol (lyso-PI) lipids and an corresponding increase in phosphatidylinositol (PI) lipids. Other phospholipids were mostly unchanged with the exception of decreases in select polyunsaturated lyso-phosphatidylserine (lyso-PS) and lyso-phosphatidylcholine (lyso-PC) lipids and a striking remodeling of PI phosphates (e.g., PIP, PIP2) in DDHD1(–/–) brain tissue. Biochemical assays confirmed that DDHD1 hydrolyzes PI/PS to lyso-PI/PS with sn-1-selectivity and accounts for a substantial fraction of the PI/PS lipase activity in mouse brain tissue. These data indicate that DDHD1 is a principal regulator of bioactive lyso-PI and other lysophospholipids, as well as PI phosphates, in the mammalian nervous system, pointing to a potential role for these lipid pathways in HSP.

There is no information on the chemical composition of camelina seed lecithin; therefore, the objective of this study was to investigate the chemical composition and emulsifying properties of lecithin recovered from camelina seed oil by water (WDCL) and enzymatic degumming (EDCL) using phospholipase A1 (PLA1). The lecithin obtained by both WDLC and EDLC was rich in phosphatidylinositol (PI), and contents were 37.8 and 25.2wt%, respectively. Lecithin recovered by enzymatic degumming contained more lysophospholipids compared to water degumming. The saturated fatty acid content of the EDCL was significantly higher than that of the WDCL. Emulsions stabilized using EDCL resulted in the highest stability when deionized water was used as the aqueous phase (original pH); however, at pH=7.5, emulsions stabilized using EDCL and WDCL were less stable compared to the emulsion stabilized with soy lecithin. Results showed that camelina seed lecithin is a promising alternative PI-rich emulsifier for various food applications.

The aim of the study was to determine the prevalence of virulence genes responsible for the adhesion (flaA, cadF and racR) and invasion (virB11, iam and pldA) in Campylobacter isolates from cattle and swine and determine their adherence and invasion abilities. The studies conducted revealed high prevalence rate of adherence and invasion associated genes irrespective of the isolates origin. All Campylobacter strains of swine and cattle origin adhered to HeLa cells at mean level 0.1099% ± SD 0.1341% and 0.0845% ± SD 0.1304% of starting viable inoculum, respectively. However swine isolates exhibited higher invasion abilities (0.0012% ± SD 0.0011%) compared to bovine isolates (0.00038% ± SD 0.00055%). The results obtained revealed significantly positive correlation between invasion and adherence abilities of swine origin isolates (R = 0.4867 in regard to C. jejuni and R = 0.4507 in regard to C. coli) and bovine origin isolates (R = 0.726 in regard to C. jejuni). Bacterial virulence is multifactorial and it is affected by the expression of virulence genes. Moreover the presence of virulence genes determines the ability of Campylobacter isolates to adhere and invade the cells.

Intrahepatic cholestasis of pregnancy (ICP) is frequently accompanied by pruritus, whose etiology has been associated with an enhanced production of lysophosphatidic acid (LPA) by the combined action of phospholipase A1/A2 (PLA1/PLA2) and autotaxin (ATX). Here, we have investigated whether the placenta is involved in LPA release to maternal circulation during ICP. Serum levels of ATX and LPA (determined by ELISA) were elevated in women with ICP, and a correlation between both parameters was found. No relationship between serum levels of ATX or LPA and bile acids was found. Expression levels of ATX and PLA2 were determined by RT-qPCR and Western blot. Placenta ATX but not PLA2 was significantly upregulated in ICP, and a tendency to increase was found at the protein level. A correlation between serum ATX and placental ATX mRNA levels was found. In human placenta at term, ATX was clearly detected (by immunofluorescence) in Hofbauer cells, but only faintly in trophoblast cells. In pregnant rats, the expression of Atx and Pla2 in placenta was lower than in liver. When obstructive cholestasis was imposed by bile duct ligation from day 14 of gestation until term, placenta Atx and Pla2 expression was markedly enhanced, and overexpression was confirmed at the protein level for Pla2, whereas Atx protein was not detected. In conclusion, the placenta substantially participates in LPA production during gestation. This contribution is markedly higher during maternal cholestasis and hence, may be involved in ICP-associated pruritus. NEW & NOTEWORTHY Fetal placental macrophages and, to a lesser extent, trophoblast cells express high levels of autotaxin at term. An increased expression of mRNA and protein autotaxin, the key secretory enzyme responsible for the production of lysophosphatidic acid in serum, has been observed in placentas of women with cholestasis of pregnancy, which supports that the placenta can contribute to an increased production of this pruritogenic compound in women suffering from this liver disease.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Various human tissues and cells express phospholipase A1 member A (PLA1A), including the liver, lung, prostate gland, and immune cells. The enzyme belongs to the pancreatic lipase family. PLA1A specifically hydrolyzes sn-1 fatty acid of phosphatidylserine (PS) or 1-acyl-lysophosphatidylserine (1-acyl-lysoPS). PS externalized by activated cells or apoptotic cells or extracellular vesicles is a potential source of substrate for the production of unsaturated lysoPS species by PLA1A. Maturation and functions of many immune cells, such as T cells, dendritic cells, macrophages, and mast cells, can be regulated by PLA1A and lysoPS. Several lysoPS receptors, including GPR34, GPR174 and P2Y10, have been identified. High serum levels and high PLA1A expression are associated with autoimmune disorders such as Graves' disease and systemic lupus erythematosus. Increased expression of PLA1A is associated with metastatic melanomas. PLA1A may contribute to cardiometabolic disorders through mediating cholesterol transportation and producing lysoPS. Furthermore, PLA1A is necessary for hepatitis C virus assembly and can play a role in the antivirus innate immune response. This review summarizes recent findings on PLA1A expression, lysoPS and lysoPS receptors in autoimmune disorders, cancers, cardiometabolic disorders, antivirus immune responses, as well as regulations of immune cells.

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.

Deleterious mutations in the serine lipase DDHD2 are a causative basis of complex hereditary spastic paraplegia (HSP, subtype SPG54) in humans. We recently found that DDHD2 is a principal triglyceride hydrolase in the central nervous system (CNS) and that genetic deletion of this enzyme in mice leads to ectopic lipid droplet (LD) accumulation in neurons throughout the brain. Nonetheless, how HSP-related mutations in DDHD2 relate to triglyceride metabolism and LD formation remains poorly understood. Here, we have characterized a set of HSP-related mutations in DDHD2 and found that they disrupt triglyceride hydrolase activity in vitro and impair the capacity of DDHD2 to protect cells from LD accumulation following exposure to free fatty acid, an outcome that was also observed with a DDHD2-selective inhibitor. We furthermore isolated and characterized LDs from brain tissue of DDHD2−/− mice, revealing that they contain both established LD-associated proteins identified previously in other organs and CNS-enriched proteins, including several proteins with genetic links to human neurological disease. These data, taken together, indicate that the genetic inactivation of DDHD2, as caused by HSP-associated mutations, substantially perturbs lipid homeostasis and the formation and content of LDs, underscoring the importance of triglyceride metabolism for normal CNS function and the key role that DDHD2 plays in this process.

The H-Ras-like suppressor (HRASLS) is a protein family consisting of five members in humans. Despite their discovery as tumor suppressors, we demonstrated that all these proteins are phospholipid-metabolizing enzymes, such as phospholipase (PL) A1 /A2 and acyltransferase. We thus proposed to rename HRASLS1-5 as PLA/acyltransferase (PLAAT)-1-5. Notably, PLAATs exhibit N-acyltransferase activity to biosynthesize N-acylated ethanolamine phospholipids, including N-acyl-plasmalogen, which serve as precursors of bioactive N-acylethanolamines. Furthermore, the overexpression of PLAAT-3 in animal cells causes disappearance of peroxisomes and a remarkable reduction in plasmalogen levels. This finding might be related to the inhibitory effect of PLAAT-3 on the chaperone activity of the peroxin PEX19. In this article, we will review our recent findings about PLAAT proteins, with special reference to their roles in peroxisome biogenesis and plasmalogen metabolism.

Objective: We present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 21q11.2-q21.1, and we review the literature of an sSMC(21) with a duplication of 21q11.2-q21.1. Case report: A 40-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XX,+mar [18]/46,XX [4]. The parental karyotypes were normal. Prenatal ultrasound findings were unremarkable. aCGH analysis of cultured amniocytes revealed a 2.855-Mb duplication of 21q11.2-q21.1 encompassing the genes of LIPI, ABCC13 and NRIP1. Metaphase fluorescence in situ hybridization analysis on cultured amniocytes revealed a result of 47,XX,+mar .ish der(13/21) (D13/21Z1+) [10]. Spectral karyotyping analysis determined the origin of chromosome 21 in the sSMC. A female fetus was delivered with no phenotypic features of Down syndrome and no structural abnormalities. We discuss the genotype–phenotype correlation of LIPI, ABCC13 and NRIP1, and review the literature of an sSMC(21) associated with dup(21)(q11.2q21.1). Conclusion: aCGH is useful for identification of the nature and genetic component of a prenatally detected sSMC.

The lipid composition of thylakoid membranes inside chloroplasts is conserved from leaves to developing embryos. A finely tuned lipid assembly machinery is required to build these membranes during Arabidopsis thaliana development. Contrary to thylakoid lipid biosynthetic enzymes, the functions of most predicted chloroplast lipid-degrading enzymes remain to be elucidated. Here, we explore the biochemistry and physiological function of an Arabidopsis thylakoid membrane-associated lipase, PLASTID LIPASE1 (PLIP1). PLIP1 is a phospholipase A1 In vivo, PLIP1 hydrolyzes polyunsaturated acyl groups from a unique chloroplast-specific phosphatidylglycerol that contains 16:1 Δ3trans as its second acyl group. Thus far, a specific function of this 16:1 Δ3trans -containing phosphatidylglycerol in chloroplasts has remained elusive. The PLIP1 gene is highly expressed in seeds, and plip1 mutant seeds contain less oil and exhibit delayed germination compared with the wild type. Acyl groups released by PLIP1 are exported from the chloroplast, reincorporated into phosphatidylcholine, and ultimately enter seed triacylglycerol. Thus, 16:1 Δ3trans uniquely labels a small but biochemically active plastid phosphatidylglycerol pool in developing Arabidopsis embryos, which is subject to PLIP1 activity, thereby contributing a small fraction of the polyunsaturated fatty acids present in seed oil. We propose that acyl exchange involving thylakoid lipids functions in acyl export from plastids and seed oil biosynthesis.

Lysophosphatidic acid (LysoPA) has been proposed to be involved in the pathogenesis of various cancers. Moreover, glycero-lysophospholipids (glycero-LysoPLs) other than LysoPA are now emerging as novel lipid mediators. Therefore, we aimed to elucidate the possible involvement of glycero-LysoPLs in the pathogenesis of gastric cancer by measuring glycero-LysoPLs, autotaxin (ATX), and phosphatidylserine-specific phospholipase A1 (PS-PLA1) in ascites obtained from patients with gastric cancer and those with cirrhosis (as a control). We observed that after adjustments according to the albumin levels, the lysophosphatidylserine (LysoPS) and lysophosphatidylglycerol (LysoPG) levels were significantly higher, while the LysoPA and ATX levels were lower, in the ascites from patients with gastric cancer. We also found that multiple regression analyses revealed that ATX was selected as a significant explanatory factor for all the detectable LysoPA species only in the cirrhosis group and that a significant positive correlation was observed between LysoPS and PS-PLA1 only in the gastric cancer group. In conclusion, the LysoPA levels might be determined largely by LysoPC and LysoPI (possible precursors) and the PS-PLA1-mediated pathway might be involved in the production of LysoPS in gastric cancer. Glycero-LysoPLs other than LysoPA might also be involved in the pathogenesis of cancer directly or through being converted into LysoPA.

Lysophosphatidic acids (LysoPAs) and lysophosphatidylserine (LysoPS) are emerging lipid mediators proposed to be involved in the pathogenesis of acute coronary syndrome (ACS). In this study, we attempted to elucidate how LysoPA and LysoPS become elevated in ACS using human blood samples collected simultaneously from culprit coronary arteries and peripheral arteries in ACS subjects. We found that: 1) the plasma LysoPA, LysoPS, and lysophosphatidylglycerol levels were not different, while the lysophosphatidylcholine (LysoPC), lysophosphatidylinositol, and lysophosphatidylethanolamine (LysoPE) levels were significantly lower in the culprit coronary arteries; 2) the serum autotaxin (ATX) level was lower and the serum phosphatidylserine-specific phospholipase A1 (PS-PLA1) level was higher in the culprit coronary arteries; 3) the LysoPE and ATX levels were significant explanatory factors for the mainly elevated species of LysoPA, except for 22:6 LysoPA, in the peripheral arteries, while the LysoPC and LysoPE levels, but not the ATX level, were explanatory factors in the culprit coronary arteries; and 4) 18:0 and 18:1 LysoPS were significantly correlated with PS-PLA1 only in the culprit coronary arteries. In conclusion, the origins of LysoPA and LysoPS might differ between culprit coronary arteries and peripheral arteries, and substrates for ATX, such as LysoPC and LysoPE, might be important for the generation of LysoPA in ACS.

Structure-based coarse-grained Gō-like models have been used extensively in deciphering protein folding mechanisms because of their simplicity and tractability. Meanwhile, explicit-solvent molecular dynamics (MD) simulations with physics-based all-atom force fields have been applied successfully to simulate folding/unfolding transitions for several small, fast-folding proteins. To explore the degree to which coarse-grained Gō-like models and their extensions to incorporate nonnative interactions are capable of producing folding processes similar to those in all-atom MD simulations, here we systematically compare the computed unfolded states, transition states, and transition paths obtained using coarse-grained models and all-atom explicit-solvent MD simulations. The conformations in the unfolded state in common Gō models are more extended, and are thus more in line with experiment, than those from all-atom MD simulations. Nevertheless, the structural features of transition states obtained by the two types of models are largely similar. In contrast, the folding transition paths are significantly more sensitive to modeling details. In particular, when common Gō-like models are augmented with nonnative interactions, the predicted dimensions of the unfolded conformations become similar to those computed using all-atom MD. With this connection, the large deviations of all-atom MD from simple diffusion theory are likely caused in part by the presence of significant nonnative effects in folding processes modelled by current atomic force fields. The ramifications of our findings to the application of coarse-grained modeling to more complex biomolecular systems are discussed.

Leishmaniasis is caused by several species of protozoan parasites of the genus Leishmania and represents an important global health problem. Leishmania braziliensis in particular is responsible of cutaneous and mucocutaneous forms of this parasitosis, with prevalence in Latin America. In the present work, we describe in L. braziliensis promastigotes and amastigotes the presence of a Phospholipase A

The interfacial hydrolysis of phospholipids catalyzed by phospholipase A

Phospholipases A

Objectives Although a single nucleotide polymorphism in a specific receptor for lysophosphatidylserine, a lysophospholipid mediator involved in the immune system, is reportedly associated with Graves’ disease, the association between lysophosphatidylserine and thyroid disorders remains to be elucidated. Therefore, we aimed to investigate the association between the level of phosphatidylserine-specific phospholipase A1 (PS-PLA1), which produces lysophosphatidylserine, and thyroid disorders. Methods We measured serum PS-PLA1 levels in the patients with various thyroid disorders (n = 120) and normal subjects (n = 58). Results We observed that the serum PS-PLA1 levels were higher in the subjects with Graves’ disease, subacute thyroiditis, or silent thyroiditis, while they were not modulated in the patients with hypothyroidism. The serum PS-PLA1 levels were strongly correlated with the levels of thyroid hormones, especially in the subjects with Graves’ disease. Moreover, we found that the serum PS-PLA1 levels were lowered by treatment with anti-thyroid reagents in subjects with Graves’ disease and that the changes in PS-PLA1 were strongly correlated with those in thyroid hormones. Conclusion These results suggest that PS-PLA1 might be a novel target in the treatment of hyperthyroidism, especially Graves’ disease, and that its measurement might be useful as a supplementary diagnostic test for thyroid function.

International audience; Singlet oxygen produced from triplet excited chlorophylls in photosynthesis is a signal molecule that can induce programmed cell death (PCD) through the action of the OXIDATIVE STRESS INDUCIBLE 1 (OXI1) kinase. Here, we identify two negative regulators of light-induced PCD that modulate OXI1 expression: DAD1 and DAD2, homologs of the human antiapoptotic protein DEFENDER AGAINST CELL DEATH. Overexpressing OXI1 in Arabidopsis ($Arabidopsis\ thaliana$) increased plant sensitivity to high light and induced early senescence of mature leaves. Both phenomena rely on a marked accumulation of jasmonate and salicylate. DAD1 or DAD2 overexpression decreased OXI1 expression, jasmonate levels, and sensitivity to photooxidative stress. Knock-out mutants of DAD1 or DAD2 exhibited the opposite responses. Exogenous applications of jasmonate upregulated salicylate biosynthesis genes and caused leaf damage in wild-type plants but not in the salicylate biosynthesis mutant Salicylic acid induction-deficient2, indicating that salicylate plays a crucial role in PCD downstream of jasmonate. Treating plants with salicylate upregulated the DAD genes and downregulated OXI1. We conclude that OXI1 and DAD are antagonistic regulators of cell death through modulating jasmonate and salicylate levels. High light-induced PCD thus results from a tight control of the relative activities of these regulating proteins, with DAD exerting a negative feedback control on OXI1 expression.

To assess the utility of serum levels of phosphatidylserine-specific phospholipase ASerum PS-PLASerum PS-PLASerum PS-PLA