Effects of glycosaminoglycans and glycosphingolipids on cytosolic phospholipases A2 from bovine brain.
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ABSTRACT:
SUBMITTER: Yang HC
PROVIDER: S-EPMC1138025 | biostudies-other | 1994 Apr
REPOSITORIES: biostudies-other
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Project description:1. [(32)P]Lecithin and [(32)P]phosphatidylethanolamine were prepared by incubating rat liver mince with [(32)P]phosphate. With these (32)P-labelled phospholipids conditions for the quantitative assay of phospholipase A activity were established. 2. The distribution of phospholipase A activity between subcellular fractions of the bovine adrenal medulla was determined. Phospholipases A(1) and A(2), with pH optima at 4.2 and 6.5 respectively, were found in the large-granule fraction. By means of sucrose-density-gradient centrifugation it was shown that both these phospholipases were localized in lysosomes. 3. Lysosomal phospholipase A(1) catalysed the hydrolysis of [(32)P]lecithin and [(32)P]phosphatidylethanolamine at the same rate. The enzymic activity was inhibited by 70% in the presence of 10mm-calcium chloride. 4. Lysosomal phospholipase A(2) catalysed the hydrolysis of [(32)P]phosphatidylethanolamine more rapidly than it hydrolysed [(32)P]lecithin. The hydrolysis of [(32)P]phosphatidylethanolamine, but not that of [(32)P]lecithin, by phospholipase A(2) was activated by 0.8mm-calcium chloride. However, the hydrolysis of both substrates was inhibited by 8mm-calcium chloride. 5. The significance of the presence of phospholipase activity in lysosomes is discussed in relation to the functions of lysosomes in general and in the adrenal medulla.
| S-EPMC1198894 | biostudies-other
Project description:Studying phospholipases A2 (PLA2s) is a challenging task since they act on membrane-like aggregated substrates and not on monomeric phospholipids. Multidisciplinary approaches that include hydrogen/deuterium exchange mass spectrometry (DXMS) and computational techniques have been employed with great success in order to address important questions about the mode of interactions of PLA2 enzymes with membranes, phospholipid substrates and inhibitors. Understanding the interactions of PLA2s is crucial since these enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid (AA) and other polyunsaturated fatty acids (PUFA). The liberation of AA by PLA2 enzymes sets off a cascade of molecular events that involves downstream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX) metabolites leading to inflammation. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting COX, while Zileuton inhibits LOX and both rely on PLA2 enzymes to provide them with AA. That means PLA2 enzymes can potentially also be targeted to diminish inflammation at an earlier point in the process. In this review we describe extensive efforts reported in the past to define the interactions of PLA2 enzymes with membranes, substrate phospholipids and inhibitors using DXMS, molecular docking, and molecular dynamics (MD) simulations.
| S-EPMC4884535 | biostudies-literature
Project description:Phospholipases type A2 (PLA2s) are the most abundant proteins found in Viperidae snake venom. They are quite fascinating from both a biological and structural point of view. Despite similarity in their structures and common catalytic properties, they exhibit a wide spectrum of pharmacological activities. Besides being hydrolases, secreted phospholipases A2 (sPLA2) are an important group of toxins, whose action at the molecular level is still a matter of debate. These proteins can display toxic effects by different mechanisms. In addition to neurotoxicity, myotoxicity, hemolytic activity, antibacterial, anticoagulant, and antiplatelet effects, some venom PLA2s show antitumor and antiangiogenic activities by mechanisms independent of their enzymatic activity. This paper aims to discuss original finding against anti-tumor and anti-angiogenic activities of sPLA2 isolated from Tunisian vipers: Cerastes cerastes and Macrovipera lebetina, representing new tools to target specific integrins, mainly, ?5?1 and ?v integrins.
| S-EPMC3581298 | biostudies-literature
Project description:Mitochondrial dysfunction has been implicated in the pathophysiology of Alzheimer's disease (AD) brains. To unravel the mechanism(s) underlying this dysfunction, we demonstrate that phospholipases A2 (PLA2s), namely the cytosolic and the calcium-independent PLA2s (cPLA2 and iPLA2), are key enzymes mediating oligomeric amyloid-beta peptide (Abeta(1-42))-induced loss of mitochondrial membrane potential and increase in production of reactive oxygen species from mitochondria in astrocytes. Whereas the action of iPLA2 is immediate, the action of cPLA2 requires a lag time of approximately 12-15 min, probably the time needed for initiating signaling pathways for the phosphorylation and translocation of cPLA2 to mitochondria. Western blot analysis indicated the ability of oligomeric Abeta(1-42) to increase phosphorylation of cPLA2 in astrocytes through the NADPH oxidase and mitogen-activated protein kinase pathways. The involvement of PLA2 in Abeta(1-42)-mediated perturbations of mitochondrial function provides new insights to the decline in mitochondrial function, leading to impairment in ATP production and increase in oxidative stress in AD brains.
| S-EPMC6674660 | biostudies-literature
Project description:BackgroundSecreted phospholipases A(2) (sPLA(2)s) are released in plasma and other biologic fluids of patients with inflammatory, autoimmune, and allergic diseases.ObjectiveWe sought to evaluate sPLA(2) activity in the bronchoalveolar lavage fluid (BALF) of asthmatic patients and to examine the expression and release of sPLA(2)s from primary human lung mast cells (HLMCs).MethodssPLA(2) activity was measured in BALF and supernatants of either unstimulated or anti-IgE-activated HLMCs as hydrolysis of oleic acid from radiolabeled Escherichia coli membranes. Expression of sPLA(2)s was examined by using RT-PCR. The release of cysteinyl leukotriene (LT) C(4) was measured by means of enzyme immunoassay.ResultsPhospholipase A(2) (PLA(2)) activity was higher in the BALF of asthmatic patients than in the control group. BALF PLA(2) activity was blocked by the sPLA(2) inhibitors dithiothreitol and Me-Indoxam but not by the cytosolic PLA(2) inhibitor AZ-1. HLMCs spontaneously released a PLA(2) activity that was increased on stimulation with anti-IgE. This PLA(2) activity was blocked by dithiothreitol and Me-Indoxam but not by AZ-1. HLMCs constitutively express mRNA for group IB, IIA, IID, IIE, IIF, III, V, X, XIIA, and XIIB sPLA(2)s. Anti-IgE did not modify the expression of sPLA(2)s. The cell-impermeable inhibitor Me-Indoxam significantly reduced (up to 40%) the production of LTC(4) from anti-IgE-stimulated HLMCs.ConclusionssPLA(2) activity is increased in the airways of asthmatic patients. HLMCs express multiple sPLA(2)s and release 1 or more of them when activated by anti-IgE. The sPLA(2)s released by mast cells contribute to LTC(4) production by acting in an autocrine fashion. Mast cells can be a source of sPLA(2)s in the airways of asthmatic patients.
| S-EPMC2788562 | biostudies-literature
Project description:Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein-protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein-protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.
| S-EPMC8073766 | biostudies-literature
Project description:We report a series of inhibitors of secreted phospholipases A2 (sPLA2s) based on substituted indoles, 6,7-benzoindoles, and indolizines derived from LY315920, a well-known indole-based sPLA2 inhibitor. Using the human group X sPLA2 crystal structure, we prepared a highly potent and selective indole-based inhibitor of this enzyme. Also, we report human and mouse group IIA and IIE specific inhibitors and a substituted 6,7-benzoindole that inhibits nearly all human and mouse sPLA2s in the low nanomolar range.
| S-EPMC2965735 | biostudies-literature
Project description:PurposeTo achieve a better understanding of the involvement of phospholipases in the inflammation and wound-healing processes in human corneal epithelial cells (HCECs), expression of phospholipase A2s (PLA2s) and phospholipase Cs (PLCs) was examined in the human corneal epithelium.MethodsSpecific primers were designed for RT-PCR amplification of the known secreted (s)PLA2, cytosolic (c)PLA2, and PLC mRNAs. Corresponding PCR products were cloned and the DNA sequenced. Immunofluorescence of flatmounted corneal sections and Western blot analyses were used to detect the PLA2s and PLCs expressed by HCECs.ResultsThe mRNAs for the following phospholipases were detected by RT-PCR in the HCECs: sPLA2GIII, -GX, and -GXIIA; cPLA2alpha and -gamma; PLCbeta1, -beta2, -beta3, -beta4, -gamma1, -gamma2, -delta1, -delta3, -delta4, and -epsilon. Immunofluorescence analyses conducted on corneal epithelium cryosections and Western blot on freshly isolated HCECs demonstrated the presence of sPLA2GIII, -GX, and -GXIIA; cPLA2alpha and -gamma; and PLCbeta2, -beta3, -gamma1, -gamma2, and -delta3.ConclusionsMany phospholipase isoforms are expressed by HCECs and may play a major role in signal transduction (PLCs) as well as in the release of precursors of potent mediators of inflammation, such as leukotrienes and prostaglandins (PLA2s). Moreover, the sPLA2s expressed by the corneal epithelium could be involved in the normal antibacterial activity in the tears and in wound healing.
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Project description:Relationships between phospholipases are poorly understood, but phosphatidic acid (PA) and diglycerides (DGs), produced by phospholipase D (PLD) and phosphatidate phosphohydrolase actions, might function as second messengers coupling cell stimulation to cellular responses. This study investigates the role of PLD-mediated PA and DG formation in inducing phospholipase A2 (PLA2) activity in intact human neutrophils (PMNs) and in PMNs permeabilized with Staphylococcus aureus alpha-toxin. PMNs were labelled with [3H]arachidonic acid (AA) to assess AA release and metabolism and diacylglycerol formation, or with [3H]1-O-hexadecyl-2-lyso-glycerophosphatidylcholine for the determination of platelet-activating factor (PAF), PA and alkylacylglycerol production. In intact PMNs primed with tumour necrosis factor alpha before stimulation with N-formyl-Met-Leu-Phe, AA release and metabolism and PAF formation increased in parallel with enhanced PA and DG formation, and inhibition of PA and DG production led to a decrease in both AA release and PAF accumulation. In alpha-toxin-permeabilized PMNs, AA release and PAF production result from the specific activation of cytosolic PLA2 (cPLA2). In this system, PA and DG formation were always present when cPLA2 activation occurred; blocking PA and DG production inhibited AA release and PAF accumulation. Adding either PA or DG back to permeabilized cells (with endogenous PA and DG formation blocked) led to a partial restoration of AA release and PAF formation; a combination of PA and DGs reconstituted full cPLA2 activity. These results strongly suggest that products of PLD participate in activating cPLA2 in PMNs.
| S-EPMC1218199 | biostudies-other
Project description:Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly (ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω-6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1 ± 0.3 g/kg/day), or control (dextrose) ± veliparib (25 mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.
| S-EPMC6760246 | biostudies-literature