Cytosolic phospholipase A2α blockade abrogates disease during the tissue-damage effector phase of experimental autoimmune encephalomyelitis by its action on APCs

Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.     

Pharmacophore-based discovery of a novel cytosolic phospholipase A(2)α inhibitor

The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)α (cPLA(2)α). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA(2)α inhibitor in cell-free and cell-based in vitro assays.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

A new assay for rhamnolipid detection—important virulence factors of Pseudomonas aeruginosa

Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two l-rhamnose sugars and one or two β-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC₅₀) of 40 μM (23.2 μg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p?=?0.0008), highlighting the role of RLs in certain infections.     

Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Background  

Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.     

Localization and function of cytosolic phospholipase A2α at the Golgi

Cytosolic phospholipase A₂α (cPLA₂α, Group IVA phospholipase A₂) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA₂α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA₂α translocation to the Golgi. However, other features of cPLA₂α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA₂α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA₂α is targeted. Increasing evidence strongly suggests that cPLA₂α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA₂α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell–cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA₂α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA₂α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA₂α. Thus, there is continued need for studies employing highly specific cPLA₂α antagonists in addition to genetic deletion of cPLA₂α in mice.     

Benzhydrylquinazolinediones: Novel cytosolic phospholipase A2α inhibitors with improved physicochemical properties

The synthesis and optimization of a class of trisubstituted quinazoline-2,4(1H,3H)-dione cPLA₂α inhibitors are described. Utilizing pharmacophores that were found to be important in our indole series, we discovered inhibitors with reduced lipophilicity and improved aqueous solubility. These compounds are active in whole blood assays, and cell-based assay results indicate that prevention of arachidonic acid release arises from selective cPLA₂α inhibition.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α

Cytosolic phospholipase A₂-α (cPLA₂) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA₂ is dependent on a rise in cytosolic Ca²⁺ concentration, membrane association via the Ca²⁺-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA₂ via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), including the role of a “pleckstrin homology (PH)-like” region of cPLA₂ (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca²⁺ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP₂) attenuated cPLA₂ activity. Stimulated cPLA₂ activity was also attenuated by the expression of cPLA₂ 135-366, or cPLA₂ 2-366, and expression of a PIP₂-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP₂, with high affinity, only cPLA₂ 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA₂s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA₂ activity can be modulated by sequestration or depletion of cellular PIP₂, although the interaction of cPLA₂ with membrane PIP₂ appears to be indirect, or of weak affinity.     

A VE-cadherin-PAR3-α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A(2)α in endothelial cells

Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not β-catenin, regulates cPLA(2)α Golgi localization linked to cell confluence. Furthermore, cPLA(2)α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA(2)α-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-α-catenin adhesion complex regulates cPLA(2)α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.     

Human cancer-associated mutations in the Aα subunit of protein phosphatase 2A increase lung cancer incidence in Aα knock-in and knockout mice

Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding Aα subunit, one of several regulatory B subunits, and the catalytic Cα subunit. Aα mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant Aα subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated Aα point mutants defective in binding B' subunits, one knockout mouse expressing truncated Aα defective in B and C subunit binding, and a floxed mouse for generating conditional Aα knockouts. We found that the cancer-associated Aα mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of Aα mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the Aα mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.     

Virtual screening of AmpC/β-lactamase as target for antimicrobial resistance in Pseudomonas aeruginosa

AmpC is a group I, class C -lactamase present in most Enterobacteriaceae and in Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli. The β-lactam class of antibiotics is one of the most important structural classes of antibacterial compounds and act by inhibiting the bacterial D ,D - transpeptidases that are responsible for the final step of peptidoglycan cross-linking. Our main aim in the study is to screen possible inhibitors against AmpC / β - lactamase (an enzyme responsible for antimicrobial activity in Pseudomonas aeruginosa), through virtual screening of 1364 NCI (National Cancer Institute) diversity set II compounds. Homology Model of AmpC / β - lactamase was constructed using MODELLER and the Model was validated using PROCHECK and Verify 3D programs to obtain a stable structure, which was further used for virtual screening of NCI (National Cancer Institute) diversity set II compounds through molecular Docking studies using Autodock. The amino acid sequence of the β - lactamase was also subjected to ScanProsite web server to find any pattern present in the sequence. After the prediction of 3-dimensional model of AmpC/ β-lactamase, the possible Active sites ofβ - lactamase were determined using LIGSITE(csc) and CastP web servers simultaneously. The Docked complexes were validated and Enumerated based on the Autodock Scoring function to pick out the best inhibitor based on Autodock energy score. Thus from the entire 1364 NCI diversity set II compounds which were Docked, the best four docking solutions were selected (ZINC12670903, ZINC17465965, ZINC11681166 and ZINC13099024). Further the Complexes were analyzed through LIGPLOT for their interaction for the 4 best docked NCI diversity set II compounds. Thus from the Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds could be promising inhibitors for Pseudomonas aeruginosa using AmpC /β - lactamase as Drug target yet pharmacological studies have to confirm it.     

LPS-induced activation of phospholipase A_2 phospholipase Cand protein kinase C of murine macrophage-like cell lines （J774 and P388D1）

A murine macrophage-like cell line,J774,acquried,in response to LPS,an ability to kill tumor necrosisfactor(TNF)-insensitive target P815 mastocytoma cells,whereas another cell line,P388D1,did not.LPS-triggered signaling mechanisms between the two celllines were compared with an aim to inquire about thepossible nature of the above-mentioned difference.Theresults showed that two cell lines respond to LPS-treatment by parallel activation of both phospholipasesC and A_2(PLC and PLA_2)to approximately the sameextent.The maximum response of both enzymes of J774cells was noted within 10 min of the treatment,whereas that of P388D1 cells required more than 20min.The other properties of LPS-responsive enzymesstudied were similar between two cell lines,ineludingActivation of PLC and PLA_2 and PKC in macrophages by LPSCa~(2 )augmentation of enzyme activation,participationof guanine nucleotide binding (G) proteins in theinitial activation processes,and inhibition of enzymeactivation by the prior treatment of cells with choleraorpartussis toxins etc.Moreover,LPS-triggered activationof PLC and PLA2 was found to be followed by theincrease of PKC activities in both cell lines.In spite ofthese similarities,J774 cells possessed both basic andacidic forms of PKC activities,while P 388D1 cells ownedonly PKC of basic form.Nevertheless,the question whyJ774 cells,but not P388D1 cells,can acquire thetumoricidal actiyity,aganist P815 cells following LPS-treatment remains to be answered.     

Major urinary metabolites of 6-keto-prostaglandin F2α in mice

Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF_2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF_2α urinary metabolites included dinor-6-keto-PGF_2α (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF_1α (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI₃ by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF_2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases.     

A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase

A carbapenem-resistant strain of Pseudomonas aeruginosa, NCGM1984, was isolated in 2012 from a hospitalized patient in Japan. Immunochromatographic assay showed that the isolate was positive for IMP-type metallo-β-lactamase. Complete genome sequencing revealed that NCGM1984 harbored two copies of bla_IMP-34, located at different sites on the chromosome. Each bla_IMP-34 was present in the same structures of the class 1 integrons, tnpA(ISPa7)-intI1-qacG-bla_IMP-34-aac(6'')-Ib-qacEdelta1-sul1-orf5-tniBdelta-tniA. The isolate belonged to multilocus sequence typing ST235, one of the international high-risk clones. IMP-34, with an amino acid substitution (Glu126Gly) compared with IMP-1, hydrolyzed all β-lactamases tested except aztreonam, and its catalytic activities were similar to IMP-1. This is the first report of a clinical isolate of an IMP-34-producing P. aeruginosa harboring two copies of bla_IMP-34 on its chromosome.     

Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.