Lysosomal phospholipase A2

Lysosomal phospholipase A2 (PLA2G15) is a ubiquitous enzyme uniquely characterized by a subcellular localization to the lysosome and late endosome. PLA2G15 has an acidic pH optimum, is calcium independent, and acts as a transacylase in the presence of N-acetyl-sphingosine as an acceptor. Recent studies aided by the delineation of the crystal structure of PLA2G15 have clarified further the catalytic mechanism, sn-1 versus sn-2 specificity, and the basis whereby cationic amphiphilic drugs inhibit its activity. PLA2G15 has recently been shown to hydrolyze short chain oxidized phospholipids which access the catalytic site directly based on their aqueous solubility. Studies on the PLA2G15 null mouse suggest a role for the enzyme in the catabolism of pulmonary surfactant. PLA2G15 may also have a role in host defense and in the processing of lipid antigens for presentation by CD1 proteins. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.     

Nonbactericidal secreted phospholipase A2s are potential anti-inflammatory factors in the mammary gland

The recent burst of duplication and divergence of the bovine PLA2G2D genes is considered typical of immune response genes, and it was recently shown that PLA2G2D is abundantly expressed in mouse leukocytes and acts as an immunosuppressive phospholipase. Analysis of 1,143 Holstein bulls indicated that the four common haplotypes spanning PLA2G2D display copy number variation ranging from 1 to 4 per haploid genome. Association of the fourth haplotype with negative total merit remained significant (P?<?0.002) when corrected for population relatedness. We compared the lipase and bactericidal activities of bovine pancreatic PLA2G1B with human PLA2G2A and G2D and bovine PLA2G2D1 and G2D4 proteins, which had been subcloned, expressed, and refolded by us, and the impact of point mutations in the calcium binding site was investigated. All tested phospholipases were ineffective bactericides of Escherichia coli isolated from bovine mastitis. However, in lactating mice treated with E. coli or lipopolysaccharide (LPS), intramammary injection of bovine PLA2G1B relieved visual and histological inflammation and reduced blood levels of infiltrating lactose. Further studies are warranted to determine whether the observed anti-inflammatory effect involves competitive binding of the receptor Pla2r1 which may mimic the LPS resistance effect in Pla2r1-deficient mice.     

Catalytic function of PLA2G6 is impaired by mutations associated with infantile neuroaxonal dystrophy but not dystonia-parkinsonism

Background

Mutations in the PLA2G6 gene have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystonia-parkinsonism. These clinical syndromes display two significantly different disease phenotypes. NBIA and INAD are very similar, involving widespread neurodegeneration that begins within the first 1–2 years of life. In contrast, patients with dystonia-parkinsonism present with a parkinsonian movement disorder beginning at 15 to 30 years of age. The PLA2G6 gene encodes the PLA2G6 enzyme, also known as group VIA calcium-independent phospholipase A₂, which has previously been shown to hydrolyze the sn-2 acyl chain of phospholipids, generating free fatty acids and lysophospholipids.

Methodology/Principal Findings

We produced purified recombinant wildtype (WT) and mutant human PLA2G6 proteins and examined their catalytic function using in vitro assays with radiolabeled lipid substrates. We find that human PLA2G6 enzyme hydrolyzes both phospholipids and lysophospholipids, releasing free fatty acids. Mutations associated with different disease phenotypes have different effects on catalytic activity. Mutations associated with INAD/NBIA cause loss of enzyme activity, with mutant proteins exhibiting less than 20% of the specific activity of WT protein in both lysophospholipase and phospholipase assays. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity, and two mutations produce a significant increase in specific activity for phospholipid but not lysophospholipid substrates.

Conclusions/Significance

These results indicate that different alterations in PLA2G6 function produce the different disease phenotypes of NBIA/INAD and dystonia-parkinsonism. INAD/NBIA is caused by loss of the ability of PLA2G6 to catalyze fatty acid release from phospholipids, which predicts accumulation of PLA2G6 phospholipid substrates and provides a mechanistic explanation for the accumulation of membranes in neuroaxonal spheroids previously observed in histopathological studies of INAD/NBIA. In contrast, dystonia-parkinsonism mutations do not appear to directly impair catalytic function, but may modify substrate preferences or regulatory mechanisms for PLA2G6.     

Low-Molecular-Weight, Calcium-Dependent Phospholipase A2Genes Are Linked and Map to Homologous Chromosome Regions in Mouse and Human

The Group IIA phospholipase gene (PLA2G2A) protein coding regions exhibit significant homology with recently described Group IIC (PLA2G2C) and Group V (PLA2GV) genes. All three genes are present in many mammalian species and are expressed in a tissue-specific pattern. Here, we demonstrate in human that they are tightly linked and map to chromosome 1p34–p36.1. We also show that the homologous mouse loci are tightly linked (no observed recombination) on the distal part of chromosome 4, a region exhibiting synteny with human 1p34–p36. Unlike its rodent counterpart, humanPLA2G2Cappears to be a nonfunctional pseudogene.     

Structural Insight into the Activation Mechanism of Human Pancreatic Prophospholipase A2

Pancreatic phospholipase A2 (phospholipase A2 group 1B, G1B) belongs to the superfamily of secreted phospholipase A2 (PLA2) enzymes. G1B has been proposed to be a potential target for diseases such as hypertension, obesity, and diabetes. Human pancreatic prophospholipase A2 (pro-hG1B) is activated by cleavage of the first seven-residue propeptide (phospholipase A2 propeptide, PROP). However, questions still remain on the mode of action for pro-hG1B. In this work, we expressed pro-hG1B in Pichia pastoris and determined the crystal structure at 1.55-Å resolution. The x-ray structure demonstrates that pro-hG1B forms a trimer. In addition, PROP occupies the catalytic cavity and can be self-cleaved at 37 °C. A new membrane-bound surface and activation mechanism are proposed based on the trimeric model of pro-hG1B. We also propose a new autoproteolytic mechanism for pro-hG1B by the reaction triad Asp49-Arg0-Ser(-2) that is similar to the serine protease catalytic triad.Phospholipase A2 (PLA2,⁵ EC 3.1.1.4) hydrolyzes glycerophospholipids at the sn-2 acyl bond to produce free fatty acids. The PLA2 family currently comprises five categories: the secreted PLA2s, the cytosolic PLA2s, the Ca²⁺-independent PLA2s, the platelet-activating factor acetyl hydrolases, and the lysosomal PLA2s. To date, based on the catalytic mechanism as well as functional and structural information, 15 different groups of PLA2 have been reported and named (1).G1B is a member of the secreted PLA2 enzymes. This lipolytic enzyme releases glycerophospholipids and arachidonic acid that serve as the precursors of signal molecules that mediate a multitude of biological functions, such as inflammation. The G1B gene has been reported to be linked to hypertension in three sample populations (2). The concentration of G1B protein in serum is a potential marker for pancreatic acinar cell carcinoma (3). Knock-out mice experiments showed that G1B knockdown can prevent diet-induced obesity and obesity-related insulin resistance (4). After being fed with glucose-rich meals, knock-out mice showed lower postprandial glycemia than wild-type mice (5). A recent report also pointed to the linkage between hG1B and ophthalmic diseases (6). Therefore, hG1B is considered to be a potential target for treatment of obesity, diabetes (4,5), or ophthalmic diseases (6).Pro-hG1B is a digestive zymogen secreted from pancreatic acinar cells in its inactive form (7). It is activated by trypsin in the duodenum. The activation of pro-hG1B by cleavage of the PROP, a heptapeptide of the sequence DSGISPR, is linked to diseases like pancreatitis (8) and acute lung injury (9). Circulating hG1B, mostly in the form of pro-hG1B, indicates pancreatic injury in acute pancreatitis (10). PROP can be used in assays to characterize the severity of acute pancreatitis (11). The C-terminal pentapeptide of PROP (GISPR) is essential for the inhibition of enzyme activity (12). Besides trypsin, pro-hG1B can also be activated by thrombin (13) and 29-kDa trypsin-like endogenous type 1-proPLA2 activator (11). PROP may also play an important role in the folding process as suggested by the refolding experiments of pro-hG1B and hG1B (14).Helix 1 at the N-terminal of hG1B is known to play an important role in enzyme function. An engineered hG1B lacking the N-terminal helix 1 bound to membranes with weaker affinity and exhibited ∼100-fold lower enzymatic activity compared with that of the full-length hG1B. It is inferred that this helix 1 facilitates the membrane binding, thus enhances the enzymatic activity based on polarized infrared spectroscopic experiments (15). Experiments using semi-synthetic hG1B demonstrated that helix 1 residues act as a regulatory domain and mediate interfacial activation (16).It has been generally believed that PLA2 possesses an interfacial binding surface (i-face), which orientates PLA2 for binding to membrane so that PLA2 subsequently hydrolyzes the sn-2 acyl bond of glycerophospholipids (17). A model for this i-face was postulated based on the crystal structure of dimeric porcine G1B (18). Questions still remain on the details of this i-face and the mechanism of its binding to membrane.To date, numerous structures of G1B proteins from various species, including bovine (17, 19–21) and porcine (22,23), have been published. However, there is no report of structure for hG1B or pro-hG1B. The crystal structure of pro-hG1B presented here will illustrate the structural difference of G1B between the human and other species. It will provide structural insight into the activation mechanism of pro-hG1B and potentially facilitate the structure-based design of inhibitor for the treatment of a number of diseases including hypertension, obesity, and diabetes.     

PLA2R1: Expression and function in cancer

The phospholipase A2 receptor 1 (PLA2R1 or PLA2R) was isolated twenty years ago for its ability to bind several secretory phospholipase A2 proteins (sPLA2). Since its identification, it has attracted only a limited interest, mainly in the sPLA2 biology field, as it is viewed uniquely as a regulator of sPLA2 activities. Recent discoveries outline novel important functions of this gene in cancer biology. Indeed, PLA2R1 gain or loss of function experiments in vitro and in vivo shows that this receptor promotes several tumor suppressive responses including senescence, apoptosis and inhibition of transformation. Supporting a tumor suppressive role of PLA2R1, its expression decreases in numerous cancers, and known oncogenes such as HIF2α and c-MYC repress its expression. PLA2R1 promoter methylation, a classical way to repress tumor suppressive gene expression in cancer cells, is observed in leukemia, in kidney and in breast cancer cells. Mechanistically, PLA2R1 activates the kinase JAK2 and orients its activity towards a tumor suppressive one. PLA2R1 also promotes accumulation of reactive oxygen species which induce cell death and senescence. This review compiles recent data demonstrating an unexpected tumor suppressive role of PLA2R1 and outlines the future work needed to improve our knowledge of the functions of this gene in cancer.     

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: Presence of an amino-terminal helix in solution

Summary ¹H, ¹⁵N and ¹³C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007–1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G.H., Kaptein, R. and Verheij, H.M. (1995) Nature Struct. Biol., 2, 402–406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned aminoterminal helix in the absence of substrate. Therefore, there is a clear difference between the conformations of group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.Abbreviations PLA2 phospholipase A2 - App-D49 phospholipase from Agkistrodon piscivorus piscivorus - NOE nuclear Overhauser effect     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Selection of reference genes for real-time polymerase chain reaction analysis in tissues from Bombus terrestris and Bombus lucorum of different ages

Quantitative real-time polymerase chain reaction (PCR) is an accurate and sensitive technique for gene expression analysis. However, it requires data normalization using reference genes. Here we assessed the stability of eight reference genes in the labial gland and fat body of the bumblebees Bombus terrestris and Bombus lucorum of different ages. To date, no reference genes have been identified for these species. Our data show that arginine kinase (AK) and phospholipase A2 (PLA2) are the most stable genes in both tissues of B. terrestris. The most stable genes for the labial gland and fat body of B. lucorum were found to be elongation factor 1α (EEF1A) and PLA2.     

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.     

Elevated PLA2G7 Gene Promoter Methylation as a Gender-Specific Marker of Aging Increases the Risk of Coronary Heart Disease in Females

PLA2G7 gene product is a secreted enzyme whose activity is associated with coronary heart disease (CHD). The goal of our study is to investigate the contribution of PLA2G7 promoter DNA methylation to the risk of CHD. Using the bisulphite pyrosequencing technology, PLA2G7 methylation was measured among 36 CHD cases and 36 well-matched controls. Our results indicated that there was a significant association between PLA2G7 methylation and CHD (adjusted P = 0.025). Significant gender-specific correlation was observed between age and PLA2G7 methylation (males: adjusted r = −0.365, adjusted P = 0.037; females: adjusted r = 0.373, adjusted P = 0.035). A breakdown analysis by gender showed that PLA2G7 methylation was significantly associated with CHD in females (adjusted P = 0.003) but not in males. A further two-way ANOVA analysis showed there was a significant interaction between gender and status of CHD for PLA2G7 methylation (gender*CHD: P = 6.04E−7). Moreover, PLA2G7 methylation is associated with the levels of total cholesterols (TC, r = 0.462, P = 0.009), triglyceride (TG, r = 0.414, P = 0.02) and Apolipoprotein B (ApoB, r = 0.396, P = 0.028) in females but not in males (adjusted P>0.4). Receiver operating characteristic (ROC) curves showed that PLA2G7 methylation could predict the risk of CHD in females (area under curve (AUC) = 0.912, P = 2.40E−5). Our results suggest that PLA2G7 methylation changes with aging in a gender-specific pattern. The correlation between PLA2G7 methylation and CHD risk in females is independent of other parameters including age, smoking, diabetes and hypertension. PLA2G7 methylation might exert its effects on the risk of CHD by regulating the levels of TC, TG, and ApoB in females. The gender disparities in the PLA2G7 methylation may play a role in the molecular mechanisms underlying the pathophysiology of CHD.     

Studies on the hemolytic and hydrolytic actions of phospholipases against mammalian erythrocyte membranes.

The hemolytic actions of three kinds of phospholipase C on horse and sheep erythrocytes were studied in relation to their hydrolytic activities on the phospholipid components of these red cells. Clostridium novyi (oedematiens) type A phospholipase C hemolyzed horse red cells by hydrolyzing phosphatidylcholine. However, the enzyme did not lyse sheep cells nor did it hydrolyze any phospholipid under the same conditions, although this enzyme hydrolyzed both sphingomyelin and phosphatidylethanolamine in the phospholipid mixture extracted from sheep red cells. Clostridium perfringens phospholipase C hemolyzed not only horse red cells by hydrolyzing phosphatidylcholine but also sheep red cells by hydrolyzing sphingomyelin. Sphingomyelin on sheep red cell membrane was hydrolyzed 10 times faster by this enzyme than that on horse red cell membrane. Pseudomonas aureofaciens phospholipase C hemolyzed horse red cells by attacking phosphatidylcholine and phosphatidylethanolamine. The enzyme did not attack sheep red cells but it did hydrolyze phosphatidylethanolamine in the extracted phospholipid mixture from sheep cells. The hemolytic activity of phospholipase C depends not only on the enzyme and the asymmetric distribution of phospholipids in the erythrocyte membrane but also on the accessibility of the enzymes to the phospholipids in the surface of the membranes. Hemolysis by phospholipase C belongs to a hot-cold type of lysis.     

Expression,purification and biophysical characterization of recombinant Streptomyces violaceoruber phospholipase PLA2 overproduced in Pichia pastoris

Abstract

Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties.

Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism.

Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris. Functional active phospholipase A2 with specific activity 73?U/mg was purified with a concentration of at least 3?mg/mL. The role of different divalent ions in PLA2 thermostability were evaluated. Ca²⁺ and Ba²⁺ ions significantly increased thermostability of the enzyme.     

Myeloid- and hepatocyte-specific deletion of group VIA calcium-independent phospholipase A2 leads to dichotomous opposing phenotypes during MCD diet-induced NASH

Polymorphisms of phospholipase A2VIA (iPLA2β or PLA2G6) are associated with body weights and blood C-reactive protein. The role of iPLA2β/PLA2G6 in non-alcoholic steatohepatitis (NASH) is still elusive because female iPla2β-null mice showed attenuated hepatic steatosis but exacerbated hepatic fibrosis after feeding with methionine- and choline-deficient diet (MCDD). Herein, female mice with myeloid- (MPla2g6^?/?) and hepatocyte- (LPla2g6^?/?) specific PLA2G6 deletion were generated and phenotyped after MCDD feeding. Without any effects on hepatic steatosis, MCDD-fed MPla2g6^?/? mice showed further exaggeration of liver inflammation and fibrosis as well as elevation of plasma TNFα, CCL2, and circulating monocytes. Bone-marrow-derived macrophages (BMDMs) from MPla2g6^?/? mice displayed upregulation of PPARγ and CEBPα proteins, and elevated release of IL6 and CXCL1 under LPS stimulation. LPS-stimulated BMDMs from MCDD-fed MPla2g6^?/? mice showed suppressed expression of M1 Tnfa and Il6, but marked upregulation of M2 Arg1, Chil3, IL10, and IL13 as well as chemokine receptors Ccr2 and Ccr5. This in vitro shift was associated with exaggeration of hepatic M1/M2 cytokines, chemokines/chemokine receptors, and fibrosis genes. Contrarily, MCDD-fed LPla2g6^?/? mice showed a complete protection which was associated with upregulation of Ppara/PPARα and attenuated expression of Pparg/PPARγ, fatty-acid uptake, triglyceride synthesis, and de novo lipogenesis genes. Interestingly, LPla2g6^?/? mice fed with chow or MCDD displayed an attenuation of blood monocytes and elevation of anti-inflammatory lipoxin A4 in plasma and liver. Thus, PLA2G6 inactivation specifically in myeloid cells and hepatocytes led to opposing phenotypes in female mice undergoing NASH. Hepatocyte-specific PLA2G6 inhibitors may be further developed for treatment of this disease.     

Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation

Anandamide and other bioactive N-acylethanolamines (NAEs) are a class of lipid mediators and are produced from glycerophospholipids via N-acylphosphatidylethanolamines (NAPEs). Although the generation of NAPE by N-acylation of phosphatidylethanolamine is thought to be the rate-limiting step of NAE biosynthesis, the enzyme responsible, N-acyltransferase, remains poorly characterized. Recently, we found that five members of the HRAS-like suppressor (HRASLS) family, which were originally discovered as tumor suppressors, possess phospholipid-metabolizing activities including NAPE-forming N-acyltransferase activity, and proposed to call HRASLS1–5 phospholipase A/acyltransferase (PLA/AT)-1–5, respectively. Among the five members, PLA/AT-1 attracts attention because of its relatively high N-acyltransferase activity and predominant expression in testis, skeletal muscle, brain and heart of human, mouse and rat. Here, we examined the formation of NAPE by PLA/AT-1 in living cells. As analyzed by metabolic labeling with [¹⁴C]ethanolamine or [¹⁴C]palmitic acid, the transient expression of human, mouse and rat PLA/AT-1s in COS-7 cells as well as the stable expression of human PLA/AT-1 in HEK293 cells significantly increased the generation of NAPE and NAE. Liquid chromatography–tandem mass spectrometry also exhibited that the stable expression of PLA/AT-1 enhanced endogenous levels of NAPE, N-acylplasmenylethanolamine, NAE and glycerophospho-NAE. Furthermore, the knockdown of endogenous PLA/AT-1 in mouse ATDC5 cells lowered NAPE levels. Interestingly, the dysfunction of peroxisomes, which was caused by PLA/AT-2 and -3, was not observed in the PLA/AT-1-expressing HEK293 cells. Altogether, these results suggest that PLA/AT-1 is at least partly responsible for the generation of NAPE in mammalian cells.     

ADP ribosylation factors 1 and 4 and group VIA phospholipase A₂ regulate morphology and intraorganellar traffic in the endoplasmic reticulum-Golgi intermediate compartment

Organelle morphology of the endomembrane system is critical for optimal organelle function. ADP ribosylation factors (Arfs), a family of small GTPases, are required for maintaining the structure of the Golgi and endosomes. What determines the discontinuous nature of the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) as tubulovesicular clusters is unknown. In search of morphological determinants for the ERGIC, we found that a double knockdown of Arf1+Arf4 induced dynamic ERGIC tubules that connect ERGIC clusters, indicating that the tubules mediated lateral intraERGIC traffic. Tubule formation was inhibited by an antagonist of group VI calcium-independent phospholipase A₂ (PLA2G6) and by silencing the A isoform of PLA2G6 (PLA2G6-A). Arf1+Arf4 depletion altered the expression of PLA2G6-A splice variants and relocalized PLA2G6-A from the cytosol to ERGIC clusters and tubules, suggesting that the enzyme became locally active. We show that changes in Arf1 can modulate the activity of PLA2G6-A. We propose that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC in a way that is predicted to impact the rate and possibly the destination of cargos. Our findings have identified key components in the molecular mechanism underlying the regulation of tubules in the ERGIC and uncover tubular carriers as tightly controlled machinery.     

The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A1 (PLA1), PLA2, and Lyso-PLA2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA₂ and lyso-PLA₂ activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA₁ activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.