共查询到20条相似文献,搜索用时 31 毫秒
1.
Katherine E. Ward James P. Ropa Emmanuel Adu-Gyamfi Robert V. Stahelin 《Journal of lipid research》2012,53(12):2656-2666
Group IVA cytosolic phospholipase A2 (cPLA2α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA2α binds zwitterionic lipids such as phosphatidylcholine in a Ca2+-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA2α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA2α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA2α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA2α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA2α. 相似文献
2.
Katherine E. Ward Nitin Bhardwaj Mohsin Vora Charles E. Chalfant Hui Lu Robert V. Stahelin 《Journal of lipid research》2013,54(3):636-648
Group IVA cytosolic phospholipase A2 (cPLA2α), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affinity for a cationic patch in the C2 domain β-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA2α-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specificity is mediated by Arg59, Arg61, and His62 (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confirm the origin of C1P specificity, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the first study demonstrating the molecular origin of C1P recognition. 相似文献
3.
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. 相似文献
4.
Song Xue Diane E. Brockman Donna M. Slater Leslie Myatt 《Prostaglandins & other lipid mediators》1995,49(6)
The objective of this study was to examine the expression and activity of cytosolic phospholipase A2 (cPLA2) in relation to prostaglandin E2 (PGE2) synthesis in human amnion-derived WISH cells in response to stimulation by interleukin-1β (IL-1β). cPLA2 activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3). Treatment of WISH cells with IL-1β (0.01–1 ng/mL) for up to 24 h resulted in a significant increase in PGE2 release in a concentration- and time-dependent manner accompanied by increases both in total cellular cPLA2 activity and in cPLA2 protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA2 activity and cPLA2 protein level indicates that IL-1β may induce the synthesis of CPLA2. Incubation of the cells with 10 μM AACOCF3 for 24 h significantly inhibited IL-1β-induced PGE2 production strongly suggesting that cPLA2 mediates IL-1β-induced PGE2 formation. In unstimulated cells, there is appreciable total cellular cPLA2 activity and protein, but these cells produce low amounts of PGE2 until stimulated by IL-1β, suggesting that cPLA2 translocation from cytosol to the membrane is necessary for its bioactivity. In contrast to IL-1β, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10−10−10−6 M) for 24 h significantly inhibited total cellular cPLA2 activity in a concentration-dependent manner. The amount of total cellular cPLA2 protein seen on Western blot remained unchanged following TPA treatment. These data suggest that in WISH cells, IL-1β induces both translocation to the membrane and de novo synthesis of cPLA2 protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only cause cPLA2 translocation but no increase in cPLA2 protein synthesis, resulting in limited PFG synthesis. Our results provide a mechanism for the effect of IL-1β on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA2 in the mechanism initiating human parturition. 相似文献
5.
H. P. Kim H. T. Pham V. A. Ziboh 《Prostaglandins, leukotrienes, and essential fatty acids》2001,65(5-6)
Cytosolic phospholipase A2 (cPLA2) is believed to involve the regulation of essential cellular processes. Like other cell types, epidermal cPLA2 may participate in various metabolic processes including eicosanoid generation. In this investigation, we demonstrated the presence of cPLA2 in guinea pig epidermis. The epidermal cPLA2 is Ca2+-dependent, active at micromolar concentration of Ca2+ and resistant to disulfide-reducing agents. Furthermore, it is inhibited by methyl arachidonyl fluorophosphonate (MAFP), a selective inhibitor of cPLA2, while 12-epi-scalardial (a sPLA2 inhibitor) did not cause inhibition. A test of several flavonoids revealed that quercetin (flavonol) weakly inhibited cPLA2, while flavanone had negligible inhibitory activity. In contrast, amentoflavone and ginkgetin (biflavones) markedly inhibited cPLA2 activity in the epidermis. These results underscore that different flavonoids do vary in their capability to exert differential effects on arachidonate metabolism in the skin via modulation of epidermal cPLA2 activity. 相似文献
6.
Farideh Ghomashchi Gajendra S. Naika James G. Bollinger Ahmed Aloulou Matthias Lehr Christina C. Leslie Michael H. Gelb 《The Journal of biological chemistry》2010,285(46):36100-36111
The cytosolic (group IV) phospholipase A2 (cPLA2s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA2s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA2β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca2+ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca2+ on the interfacial binding of mouse cPLA2β and its C2 domain to vesicles. Ca2+-dependent binding of mouse cPLA2β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca2+-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA2δ also displays Ca2+- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A1, and phospholipase A2 activities of the full set of mammalian cPLA2s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA2δ stands out as having relatively high phospholipase A1 activity. We also tested the susceptibility of all cPLA2 family members to a panel of previously reported inhibitors of human cPLA2α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA2β. These in vitro studies help determine the regulation and function of the cPLA2 family members. 相似文献
7.
《Prostaglandins & other lipid mediators》2007,82(3-4):113-125
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. 相似文献
8.
Cytosolic calcium-dependent phospholipase A2 (cPLA2) has multiple roles including production of arachidonic acid (a key player in cellular signaling pathways) and membrane remodeling.
Additionally, since catabolism of arachidonic acid generates free radicals, the enzyme is also implicated in ischemic injury
to mammalian organs. Regulation of cPLA2 could be important in the suppression and prioritization of cellular pathways in animals that undergo reversible transitions
into hypometabolic states. The present study examines the responses and regulation of cPLA2 in skeletal muscle and liver of hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. cPLA2 activity decreased significantly by 43% in liver during hibernation, compared with euthermic controls, and Km values for arachidonoyl thio-PC substrate fell in both organs during hibernation to 61% in liver and 28% in muscle of the
corresponding euthermic value. To determine whether these responses were due to a change in the phosphorylation state of the
enzyme, Western blotting was employed using antibodies recognizing phospho-Ser505 on α-cPLA2. The amount of phosphorylated α-cPLA2 in hibernator liver was just 38% of the value in euthermic liver. Furthermore, incubation of liver extracts under conditions
that enhanced protein phosphatase action caused a greater reduction in the detectable amount of phospho-Ser505 enzyme content in euthermic, versus hibernator, extracts. The data are consistent with a suppression of cPLA2 function during torpor via enzyme dephosphorylation, an action that may contribute to the well-developed ischemia tolerance
and lack of oxidative damage found in hibernating species over cycles of torpor and arousal. 相似文献
9.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(2):223-234
Neutrophils (PMN) contain two types of phospholipase A2 (PLA2), a 14 kDa ‘secretory’ Type II PLA2 (sPLA2) and an 85 kDa ‘cytosolic’ PLA2 (cPLA2), that differ in a number of key characteristics: (1) cPLA2 prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA2 is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA2 is active at nM calcium (Ca2+) concentrations; sPLA2 requires μM Ca2+ levels. (3) cPLA2 activity is regulated by phosphorylation; sPLA2 lacks phosphorylation sites. (4) cPLA2 is insensitive to reduction; sPLA2 is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA2 were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [3H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca2+, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [3H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA2 activation. Dithiothreitol treatment had little affect on [3H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA2 activity and the cytosolic buffer utilized did not support activity of recombinant sPLA2. These results strongly suggested that cPLA2 was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA2 in PMN. 相似文献
10.
11.
Efrosini Barbayianni Daren Stephens Andrej Grkovich Victoria Magrioti Yuan-Hao Hsu Panagiotis Dolatzas Dimitrios Kalogiannidis Edward A. Dennis George Kokotos 《Bioorganic & medicinal chemistry》2009,17(13):4833-4843
A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A2 (GIVA cPLA2 and GVIA iPLA2) and one human secretory phospholipase A2 (GV sPLA2) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA2 inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA2. The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA2 inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC50 value of 2 μM. 相似文献
12.
Hiroyuki Nakamura Yuta Moriyama Tomohiko Makiyama Shunsuke Emori Hisahiro Yamashita Risa Yamazaki Toshihiko Murayama 《The Journal of biological chemistry》2013,288(32):23264-23272
Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A2α (cPLA2α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA2α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA2α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA2α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA2α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA2α. Phosphatidylcholine vesicles containing LacCer increased cPLA2α activity. LacCer bound to cPLA2α and its C2 domain in a Ca2+-independent manner. Thus, we propose that LacCer is a direct activator of cPLA2α. A23187相似文献
13.
Matthieu Rousseau Gajendra S. Naika Jean Perron Frederic Jacques Michael H. Gelb Eric Boilard 《PloS one》2015,10(5)
The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α. 相似文献
14.
Mark Hess Alwine Schulze Elfringhoff 《Journal of enzyme inhibition and medicinal chemistry》2013,28(6):946-957
A series of 3-pyrrol-3-yl-3H-isobenzofuran-1-ones was synthesized and assessed for the ability to inhibit cytosolic phospholipase A2α (cPLA2α). Several of these compounds were found to be active in both a cell based assay and an isolated enzyme assay. The most potent inhibitor was the thiazolidine-2,4-dione substituted derivative 35. With IC50-values of 0.7 μM and 7.3 μM in the cellular and isolated enzyme assay, respectively, it possesses similar inhibitory potency as the known cPLA2α inhibitor arachidonyltrifluoromethyl ketone (AACOCF3). Structure–activity relationship studies revealed that the evaluated isobenzofuran-1-ones seem to exert their cellular activities not only by a direct interaction with the enzyme but also by other as yet unknown mechanisms. 相似文献
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16.
The association of cytosolic phospholipase A2-α (cPLA2α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A2 in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA2α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA2α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA2α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA2α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA2α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA2α with its membrane substrate via interaction with actin. 相似文献
17.
18.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(1):23-33
UVB irradiation has previously been shown to significantly increase phospholipase activity and prostaglandin synthesis. Because UVB irradiation is a potent oxidative stress, the role of active oxygen species in regulating UV-induced cPLA2 synthesis and phosphorylation was examined. In the present study, irradiation produced a 3-fold increase in synthesis within 6 h following irradiation. Phosphorylation of cPLA2 was also increased to a similar extent. UVB-induced synthesis and phosphorylation of cPLA2 could be inhibited by pretreatment with the antioxidants 2,2,5,7,8-pentamethyl-6-hydroxychromane (50 μM) or N-acetylcysteine (10 mM). Treatment of unirradiated cultures with the potent oxidant tert-butyl hydroperoxide (500 μM) also increased cPLA2 synthesis and phosphorylation, suggesting that oxidative injury is an important regulator of cPLA2 synthesis. Increased synthesis of cPLA2 correlated well with increased [3H]arachidonic acid release, PGE2 synthesis and lipid peroxidation in epidermis after oxidant or UVB treatment. The results indicate that UVB-induced upregulation of cPLA2 synthesis is mediated by UVB-induced formation of free radicals. 相似文献
19.
Sheng Hua Mu Yao Soma Vignarajan Paul Witting Leila Hejazi Zhen Gong Ying Teng Marzieh Niknami Stephen Assinder Des Richardson Qihan Dong 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(6):1146-1157
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. 相似文献
20.
Afshin Ghasemi Alwine Schulze Elfringhoff Matthias Lehr 《Journal of enzyme inhibition and medicinal chemistry》2013,28(5):429-437
Recently we found that 1-methyldodecanoylindole-2-carboxylic acid (1) and 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) were inhibitors of the cytosolic phospholipase A2α (cPLA2α)-mediated arachidonic acid release in calcium ionophore A23187-stimulated human platelets with IC50-values of 4.8 μM (1) and 0.86 μM (4). We have now replaced the 3-acyl residue of these compounds by alkylated sulfinyl-, sulfony-, sulfinamoyl-, sulfamoyl-, carbonylamino-, or carbonylaminomethyl-substituents. Structure–activity relationship studies revealed that the pronounced cellular activity of 4 strongly depends on the presence of the 3-acyl moiety. Surprisingly, when testing 4 and its derivatives in an assay with the isolated cPLA2, none of these compounds showed an inhibitory potency at 10 μM indicating that they do not inhibit cPLA2α in the cells by a direct interaction with the active site of the enzyme. 相似文献