Inhibition of human cytosolic phospholipase A2 by human annexin V

The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. However, there are only a very limited number of reported studies involving annexins and the high-molecular-mass cytosolic PLA2 (cPLA2). In this study we have examined the effect of human recombinant annexin V, a potentially abundant cytosolic protein, on the ability of human recombinant cPLA2 to hydrolyse a variety of phospholipid substrates. The results show clearly that, under the conditions of our study, annexin V can inhibit cPLA2 activity by a mechanism of substrate depletion and that this inhibition is dependent on the nature of the phospholipids and the concentration of Ca2+ ions in the assay. The hydrolysis of 1-stearoyl 2-arachidonyl phosphatidylcholine by cPLA2 was not significantly affected by annexin V over a range of Ca2+ concentrations (1 μM-2.5 mM), a result that presumably reflects the zwitterionic nature of the phospholipid and the known inability of annexins to bind to such interfaces. In contrast, the hydrolysis of dioleoyl phosphatidylglycerol, which is an effective anionic phospholipid substrate for this enzyme, and more significantly that of 1-stearoyl 2-arachidonyl phosphatidic acid, were readily inhibited by annexin V, although these effects were Ca2+-dependent. The Ca2+ concentrations required for inhibition in the assay system in vitro are greater than those associated with Ca2+-stimulated events within the cell, suggesting that a role for annexin V in regulating cPLA2 activity might not involve a substrate depletion mechanism in vivo unless factors in addition to Ca2+ and phospholipids contribute to the binding of annexin V to cell membranes.

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The role of cytosolic phospholipase A2 in gastric damage induced by helicobacter pylori infection: in vivo and in vitro studies

Gastroenterology ◽

10.1016/s0016-5085(00)85063-5 ◽

2000 ◽

Vol 118 (4) ◽

pp. A732-A733

Author(s):

Gerardo Nardone ◽

Eileen Holicky ◽

Jim R. Uhl ◽

Vittorio Colantuoni ◽

Lina Sabatino ◽

...

Keyword(s):

Helicobacter Pylori ◽

Phospholipase A2 ◽

Cytosolic Phospholipase A2 ◽

In Vitro Studies ◽

Helicobacter Pylori Infection ◽

Gastric Damage ◽

Cytosolic Phospholipase

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Discrete Role for Cytosolic Phospholipase A2α in Platelets

Journal of Experimental Medicine ◽

10.1084/jem.20011443 ◽

2002 ◽

Vol 196 (3) ◽

pp. 349-357 ◽

Cited By ~ 40

Author(s):

Dennis A. Wong ◽

Yoshihiro Kita ◽

Naonori Uozumi ◽

Takao Shimizu

Keyword(s):

Therapeutic Potential ◽

Genetically Engineered ◽

Collagen Receptors ◽

Cytosolic Phospholipase ◽

Genetically Engineered Mice ◽

Cytosolic Pla2 ◽

In Vivo Effects ◽

Deficient Mice

Among several different types of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2)α and group IIA (IIA) secretory PLA2 (sPLA2) have been studied intensively. To determine the discrete roles of cPLA2α in platelets, we generated two sets of genetically engineered mice (cPLA2α−/−/sPLA2-IIA−/− and cPLA2α−/−/sPLA2-IIA+/+) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA2α+/+/sPLA2-IIA−/−) and C3H/HeN (cPLA2α+/+/sPLA2-IIA+/+). We found that cPLA2α is needed for the production of the vast majority of thromboxane (TX)A2 with collagen stimulation of platelets. In cPLA2α-deficient mice, however, platelet aggregation in vitro is only fractionally decreased because small amounts of TX produced by redundant phospholipase enzymes sufficiently preserve aggregation. In comparison, adenosine triphosphate activation of platelets appears wholly independent of cPLA2α and sPLA2-IIA for aggregation or the production of TX, indicating that these phospholipases are specifically linked to collagen receptors. However, the lack of high levels of TX limiting vasoconstriction explains the in vivo effects seen: increased bleeding times and protection from thromboembolism. Thus, cPLA2α plays a discrete role in the collagen-stimulated production of TX and its inhibition has a therapeutic potential against thromboembolism, with potentially limited bleeding expected.

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Cytosolic phospholipase A2 expression during Helicobacter pylori infection: In vivo and in vitro studies

Digestive and Liver Disease ◽

10.1016/s1590-8658(00)80443-0 ◽

2000 ◽

Vol 32 ◽

pp. A66

Author(s):

G. Nardone ◽

E. Holicky ◽

J.R. Uhl ◽

V. Colantuoni ◽

L. Sabatino ◽

...

Keyword(s):

Helicobacter Pylori ◽

Phospholipase A2 ◽

Cytosolic Phospholipase A2 ◽

In Vitro Studies ◽

Helicobacter Pylori Infection ◽

Cytosolic Phospholipase

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Role of cytosolic phospholipase A2 in vivo and in vitro

Prostaglandins & Other Lipid Mediators ◽

10.1016/s0090-6980(99)90272-1 ◽

1999 ◽

Vol 59 (1-6) ◽

pp. 37

Author(s):

Takao Shimizu ◽

Naonori Uozumi ◽

Noriaki Nakatani ◽

Tetsuya Hirabayashi ◽

Kazuhiko Kume

Keyword(s):

Phospholipase A2 ◽

Cytosolic Phospholipase A2 ◽

Cytosolic Phospholipase

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Enhanced hydrolysis of phosphatidylcholine by human group II non-pancreatic secreted phospholipase A2 as a result of interfacial activation by specific anions. Potential role of cholesterol sulphate

Biochemical Journal ◽

10.1042/bj3080507 ◽

1995 ◽

Vol 308 (2) ◽

pp. 507-512 ◽

Cited By ~ 21

Author(s):

A R Kinkaid ◽

D C Wilton

Keyword(s):

Phospholipase A2 ◽

Anionic Phospholipid ◽

Interfacial Activation ◽

Secreted Phospholipase A2 ◽

Cell Plasma ◽

Membrane Hydrolysis ◽

Membrane Bound ◽

Group Ii ◽

Hydrolysis Of

The extracellular concentration of the Group II human non-pancreatic secreted phospholipase A2 (hnpsPLA2) is elevated in a variety of inflammatory disorders. This enzyme is remarkable because it demonstrates almost zero activity with egg phosphatidylcholine (PC) or synthetic dioleoyl-phosphatidylcholine (DOPC) as substrate, but expresses high activity with the anionic phospholipid dioleoyl-phosphatidylglycerol (DOPG), a feature shared with the Group II enzyme from rat liver. The presence of certain membrane-bound anions can enhance hydrolysis of PC by the mammalian secreted PLA2S. In this study the ability of various non-polar anions to stimulate DOPC hydrolysis by secreted PLA2S has been investigated. The naturally occurring membrane anion, cholesterol sulphate, was particularly effective in stimulating the hydrolysis of both DOPC and also 1-stearoyl-2-arachidonyl phosphatidylcholine by hnpsPLA2. Activation of DOPC hydrolysis was also achieved with dioleoyl-phosphatidylserine (DOPS); however, DOPS was less effective than cholesterol sulphate. In contrast, the dianion dioleoyl-phosphatidic acid, a known activator of pig pancreatic PLA2, failed to activate the human enzyme. It remains to be established whether cell plasma-membrane hydrolysis by extracellular hnpsPLA2 can be activated in vivo by the presence of suitable membrane anions such as cholesterol sulphate and thus promote an inflammatory response within the cell.

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Fexofenadine inhibits TNF signaling through targeting to cytosolic phospholipase A2 and is therapeutic against inflammatory arthritis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2019-215543 ◽

2019 ◽

Vol 78 (11) ◽

pp. 1524-1535 ◽

Cited By ~ 4

Author(s):

Ronghan Liu ◽

Yuehong Chen ◽

Wenyu Fu ◽

Shuya Wang ◽

Yazhou Cui ◽

...

Keyword(s):

Phospholipase A2 ◽

Inflammatory Arthritis ◽

Catalytic Domain ◽

Cytosolic Phospholipase A2 ◽

Inflammatory Rheumatic Diseases ◽

Cytosolic Phospholipase ◽

Tnf Α ◽

Approved Drugs

ObjectiveTumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis.MethodsIn vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine’s anti-TNF activity on cPLA2.ResultsSerial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine’s anti-TNF activity.ConclusionCollectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.

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cPLA2α and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e11-10-0881 ◽

2012 ◽

Vol 23 (10) ◽

pp. 1874-1888 ◽

Cited By ~ 26

Author(s):

Bishuang Cai ◽

Steve Caplan ◽

Naava Naslavsky

Keyword(s):

Phospholipase A2 ◽

Membrane Curvature ◽

Cytosolic Phospholipase ◽

Early Endosomes ◽

Lysophospholipid Acyltransferase ◽

Curved Membranes ◽

Hydrolysis Of

The lipid modifier phospholipase A2 catalyzes the hydrolysis of phospholipids to inverted-cone–shaped lysophospholipids that contribute to membrane curvature and/or tubulation. Conflicting findings exist regarding the function of cytosolic phospholipase A2 (cPLA2) and its role in membrane regulation at the Golgi and early endosomes. However, no studies addressed the role of cPLA2 in the regulation of cholesterol-rich membranes that contain glycosylphosphatidylinositol-anchored proteins (GPI-APs). Our studies support a role for cPLA2α in the vesiculation of GPI-AP–containing membranes, using endogenous CD59 as a model for GPI-APs. On cPLA2α depletion, CD59-containing endosomes became hypertubular. Moreover, accumulation of lysophospholipids induced by a lysophospholipid acyltransferase inhibitor extensively vesiculated CD59-containing endosomes. However, overexpression of cPLA2α did not increase the endosomal vesiculation, implying a requirement for additional factors. Indeed, depletion of the “pinchase” EHD1, a C-terminal Eps15 homology domain (EHD) ATPase, also induced hypertubulation of CD59-containing endosomes. Furthermore, EHD1 and cPLA2α demonstrated in situ proximity (<40 nm) and interacted in vivo. The results presented here provide evidence that the lipid modifier cPLA2α and EHD1 are involved in the vesiculation of CD59-containing endosomes. We speculate that cPLA2α induces membrane curvature and allows EHD1, possibly in the context of a complex, to sever the curved membranes into vesicles.

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Fexofenadine inhibits TNF signaling through targeting to cytosolic phospholipase A2 and is therapeutic against autoimmune diseases

10.1101/584540 ◽

2019 ◽

Author(s):

Ronghan Liu ◽

Yuehong Chen ◽

Shuya Wang ◽

Yazhou Cui ◽

Xiangli Zhang ◽

...

Keyword(s):

Autoimmune Diseases ◽

Phospholipase A2 ◽

Catalytic Domain ◽

Underlying Mechanism ◽

Cytosolic Phospholipase A2 ◽

Therapeutic Interventions ◽

Cytosolic Phospholipase ◽

Tnf Α

SUMMARYTNF-α signaling plays a central role in the pathogenesis of various diseases, particularly autoimmune diseases. Screening of a library composed of FDA approved drugs led to the identification of Terfenadine and its active metabolite Fexofenadine as inhibitors of TNF-α signaling. Both Fexofenadine and Terfenadine inhibited TNF/NF-κB signaling in vitro and in vivo, and ameliorated disease symptoms in various autoimmune disease models, including TNF-α transgenic mice, collagen-induced arthritis, and inflammatory bowel disease. Subsequent studies identified cytosolic phospholipase A2 (cPLA2) as a novel target of Fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished Fexofenadine’s anti-TNF activity. Collectively, these findings not only provide new insights into the understanding of Fexofenadine action and underlying mechanism, but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly autoimmune diseases.

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