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Biochemical and Biophysical Research... Oct 2022Activation of Gq protein-coupled receptors triggers the phospholipase C (PLC) pathway, which yields a pair of second messengers: diacylglycerol (DG) and inositol...
Activation of Gq protein-coupled receptors triggers the phospholipase C (PLC) pathway, which yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP). DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA), which serves as another second messenger. Along with PLC-DGK pathway, PA is produced directly by the action of phospholipase D (PLD), which hydrolyzes the major membrane phospholipid: phosphatidylcholine (PC). PA is converted to DG by phosphatidic acid phosphatase, suggesting that PLD, together with DGK, is a key enzyme regulating DG and PA. PLD has been implicated in a broad range of cellular processes. However, cellular expression and subcellular localization of PLD remain elusive because of a lack of specific antibodies against PLDs. For this study, we raised specific antibodies against major mammalian PLD isoforms: PLD1 and PLD2. Immunocytochemical analysis using specific antibodies showed clearly that native PLD1 and PLD2 localize to distinct subcellular regions as dot-like structures in cultured cells. PLD1 predominantly localizes to the plasma membrane, whereas PLD2 mostly localizes within the cytoplasm. These findings suggest that PLD1 and PLD2 have different roles in the phosphoinositide signaling pathway in distinct subcellular regions.
Topics: Animals; Cells, Cultured; Immunohistochemistry; Mammals; Phosphatidic Acids; Phospholipase D; Type C Phospholipases
PubMed: 35963162
DOI: 10.1016/j.bbrc.2022.07.118 -
Scientific Reports Mar 2022Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and...
Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and pathogenicity. A few phospholipases (PL) have been characterised so far at the gene and protein level in unicellular parasites including African trypanosomes (AT). They could play a role in different processes such as host-pathogen interaction, antigenic variation, intermediary metabolism. By mining the genome database of AT we found putative new phospholipase candidate genes and here we provided biochemical evidence that one of these has lipolytic activity. This protein has a unique non-canonical glycosome targeting signal responsible for its dual localisation in the cytosol and the peroxisomes-related organelles named glycosomes. We also show that this new phospholipase is excreted by these pathogens and that antibodies directed against this protein are generated during an experimental infection with T. brucei gambiense, a subspecies responsible for infection in humans. This feature makes this protein a possible tool for diagnosis.
Topics: Humans; Lipase; Microbodies; Phospholipases; Trypanosoma; Trypanosoma brucei brucei
PubMed: 35306507
DOI: 10.1038/s41598-022-08546-w -
Accounts of Chemical Research Dec 2022Water-soluble proteins as well as membrane-bound proteins associate with membrane surfaces and bind specific lipid molecules in specific sites on the protein. Membrane...
Water-soluble proteins as well as membrane-bound proteins associate with membrane surfaces and bind specific lipid molecules in specific sites on the protein. Membrane surfaces include the traditional bilayer membranes of cells and subcellular organelles formed by phospholipids. Monolayer membranes include the outer monolayer phospholipid surface of intracellular lipid droplets of triglycerides and various lipoproteins including HDL, LDL, VLDL, and chylomicrons. These lipoproteins circulate in our blood and lymph systems and contain triglycerides, cholesterol, cholesterol esters, and proteins in their interior, and these are sometimes interspersed on their surfaces. Similar lipid-water interfaces also occur in mixed micelles of phospholipids and bile acids in our digestive system, which may also include internalized triglycerides and cholesterol esters. Diacyl phospholipids constitute the defining molecules of biological membranes. Phospholipase A (PLA) hydrolyzes phospholipid acyl chains at the -1 position of membrane phospholipids, phospholipase A (PLA) hydrolyzes acyl chains at the -2 position, phospholipase C (PLC) hydrolyzes the glycerol-phosphodiester bond, and phospholipase D (PLD) hydrolyzes the polar group-phosphodiester bond. Of the phospholipases, the PLAs have been the most well studied at the mechanistic level. The PLA superfamily consists of 16 groups and numerous subgroups, and each is generally described as one of 6 types. The most well studied of the PLAs include extensive genetic and mutational studies, complete lipidomics specificity characterization, and crystallographic structures. This Account will focus principally on results from deuterium exchange mass spectrometric (DXMS) studies of PLA interactions with membranes and extensive molecular dynamics (MD) simulations of their interactions with membranes and specific phospholipids bound in their catalytic and allosteric sites. These enzymes either are membrane-bound or are water-soluble and associate with membranes before extracting their phospholipid substrate molecule into their active site to carry out their enzymatic hydrolytic reaction. We present evidence that when a PLA associates with a membrane, the membrane association can result in a conformational change in the enzyme whereby the membrane association with an allosteric site on the enzyme stabilizes the enzyme in an active conformation on the membrane. We sometimes refer to this transition from a "closed" conformation in aqueous solution to an "open" conformation when associated with a membrane. The enzyme can then extract a single phospholipid substrate into its active site, and catalysis occurs. We have also employed DXMS and MD simulations to characterize how PLAs interact with specific inhibitors that could lead to potential therapeutics. The PLAs constitute a paradigm for how membranes interact allosterically with proteins, causing conformational changes and activation of the proteins to enable them to extract and bind a specific phospholipid from a membrane for catalysis, which is probably generalizable to intracellular and extracellular transport and phospholipid exchange processes as well as other specific biological functions. We will focus on the four main types of PLA, namely, the secreted (sPLA), cytosolic (cPLA), calcium-independent (iPLA), and lipoprotein-associated PLA (Lp-PLA) also known as platelet-activating factor acetyl hydrolase (PAF-AH). Studies on a well-studied specific example of each of the four major types of the PLA superfamily demonstrate clearly that protein subsites can show precise specificity for one of the phospholipid hydrophobic acyl chains, often the one at the -2 position, including exquisite sensitivity to the number and position of double bonds.
Topics: Cholesterol Esters; Phospholipases A2; Phospholipids; Phospholipases; Lipoproteins; Triglycerides; Water; Polyesters; Substrate Specificity
PubMed: 36315840
DOI: 10.1021/acs.accounts.2c00497 -
Circulation Sep 2022Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol...
BACKGROUND
Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, , has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family.
METHODS
Using next-generation sequencing, we identified a novel dominant rare variant in the gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this -E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after -E97G overexpression.
RESULTS
Family members carrying the -E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of -E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the -E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human -E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver.
CONCLUSIONS
We identified and characterized a novel rare variant in the gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes the second identified gene, after , causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.
Topics: Angiopoietin-Like Protein 3; Angiopoietin-like Proteins; Animals; Cholesterol, HDL; Cholesterol, LDL; Gain of Function Mutation; Humans; Lipase; Lipoproteins; Mice; Phospholipases
PubMed: 35899625
DOI: 10.1161/CIRCULATIONAHA.121.057978 -
Free Radical Biology & Medicine Jun 2024Peroxiredoxin 6 (Prdx6) repairs peroxidized membranes by reducing oxidized phospholipids, and by replacing oxidized sn-2 fatty acyl groups through hydrolysis/reacylation...
Peroxiredoxin 6 (Prdx6) repairs peroxidized membranes by reducing oxidized phospholipids, and by replacing oxidized sn-2 fatty acyl groups through hydrolysis/reacylation by its phospholipase A (aiPLA) and lysophosphatidylcholine acyltransferase activities. Prdx6 is highly expressed in the lung, and intact lungs and cells null for Prdx6 or with single-point mutations that inactivate either Prdx6-peroxidase or aiPLA activity alone exhibit decreased viability, increased lipid peroxidation, and incomplete repair when exposed to paraquat, hyperoxia, or organic peroxides. Ferroptosis is form of cell death driven by the accumulation of phospholipid hydroperoxides. We studied the role of Prdx6 as a ferroptosis suppressor in the lung. We first compared the expression Prdx6 and glutathione peroxidase 4 (GPx4) and visualized Prdx6 and GPx4 within the lung. Lung Prdx6 mRNA levels were five times higher than GPx4 levels. Both Prdx6 and GPx4 localized to epithelial and endothelial cells. Prdx6 knockout or knockdown sensitized lung endothelial cells to erastin-induced ferroptosis. Cells with genetic inactivation of either aiPLA or Prdx6-peroxidase were more sensitive to ferroptosis than WT cells, but less sensitive than KO cells. We then conducted RNA-seq analyses in Prdx6-depleted cells to further explore how the loss of Prdx6 sensitizes lung endothelial cells to ferroptosis. Prdx6 KD upregulated transcriptional signatures associated with selenoamino acid metabolism and mitochondrial function. Accordingly, Prdx6 deficiency blunted mitochondrial function and increased GPx4 abundance whereas GPx4 KD had the opposite effect on Prdx6. Moreover, we detected Prdx6 and GPx4 interactions in intact cells, suggesting that both enzymes cooperate to suppress lipid peroxidation. Notably, Prdx6-depleted cells remained sensitive to erastin-induced ferroptosis despite the compensatory increase in GPx4. These results show that Prdx6 suppresses ferroptosis in lung endothelial cells and that both aiPLA and Prdx6-peroxidase contribute to this effect. These results also show that Prdx6 supports mitochondrial function and modulates several coordinated cytoprotective pathways in the pulmonary endothelium.
Topics: Ferroptosis; Peroxiredoxin VI; Phospholipid Hydroperoxide Glutathione Peroxidase; Lung; Animals; Endothelial Cells; Mice; Lipid Peroxidation; Humans; Phospholipases A2; Mice, Knockout; Piperazines; Group VI Phospholipases A2
PubMed: 38579937
DOI: 10.1016/j.freeradbiomed.2024.04.208 -
Toxins Oct 2022Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms.... (Review)
Review
Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation calls for critical review and analysis. The epithelium is a primary target for injected viper venom's toxic substances, and therefore, is a focus in modern toxinology. We used the Vero epithelial cell line as a model to compare the actions of a crude (Levantine viper) venom with the actions of the same venom with two key enzymatic components inhibited (specifically, phospholipase A2 (PLA2) and metalloproteinases) in the bioenergetic cellular response, i.e., oxygen uptake and reactive oxygen species generation. In addition to the rate of free-radical oxidation and lipid peroxidation, we measured real-time mitochondrial respiration (based on the oxygen consumption rate) and glycolysis (based on the extracellular acidification rate) using a Seahorse analyzer. Our data show that viper venom drives an increase in both glycolysis and respiration in Vero cells, while the blockage of PLA2 or/and metalloproteinases affects only the rates of the oxidative phosphorylation. PLA2-blocking in venom also increases cytotoxic activity and the overproduction of reactive oxygen species. These data show that certain components of the venom may have a different effect within the venom cocktail other than the purified enzymes due to the synergy of the venom components.
Topics: Animals; Chlorocebus aethiops; Viper Venoms; Vero Cells; Reactive Oxygen Species; Viperidae; Phospholipases A2; Metalloproteases; Lipid Peroxidation
PubMed: 36355974
DOI: 10.3390/toxins14110724 -
Journal of Cellular and Molecular... Aug 2022This study aimed to further investigate the effect of PLD1 on the biological characteristics of human cervical cancer (CC) cell line, CASKI and the potential related...
This study aimed to further investigate the effect of PLD1 on the biological characteristics of human cervical cancer (CC) cell line, CASKI and the potential related molecular mechanism. CRISPR/Cas9 genome editing technology was used to knock out the PLD1 gene in CASKI cells. Cell function assays were performed to evaluate the effect of PLD1 on the biological function of CASKI cells in vivo and in vitro. A PLD1-overexpression rescue experiment in these knockout cells was performed to further confirm its function. Two PLD1-knockout CASKI cell lines (named PC-11 and PC-40, which carried the ins1/del4 mutation and del1/del2/ins1 mutation, respectively), were constructed by CRISPR/Cas9. PLD1 was overexpressed in these knockout cells (named PC11-PLD1 and PC40-PLD1 cells), which rescued the expression of PLD1 by approximately 71.33% and 74.54%, respectively. In vivo, the cell function assay results revealed that compared with wild-type (WT)-CASKI cells, the ability of PC-11 and PC-40 cells to proliferate, invade and migrate was significantly inhibited. The expression of H-Ras and phosphorylation of Erk1/2 (p-Erk1/2) was decreased in PC-11 and PC-40 cells compared with WT-CASKI cells. PC-11 and PC-40 cells could sensitize CASKI cells to cisplatin. More importantly, the proliferation, migration and invasion of PC11-PLD1 and PC40-PLD1 cells with PLD1 overexpression were significantly improved compared with those of the two types of PLD1 knockout cells. The sensitivity to cisplatin was decreased in PC11-PLD1 and PC40-PLD1 cells compared with PC-11 and PC-40 cells. In vivo, in the PC-11 and PC-40 tumour groups, tumour growth was significantly inhibited and tumour weight (0.95 ± 0.27 g and 0.66 ± 0.43 g vs. 1.59 ± 0.67 g, p = 0.0313 and 0.0108) and volume (1069.41 ± 393.84 and 1077.72 mm ± 815.07 vs. 2142.94 ± 577.37 mm , p = 0.0153 and 0.0128) were significantly reduced compared to those in the WT-CASKI group. Tumour differentiation of the PC-11 and PC40 cells was significantly better than that of the WT-CASKI cells. The immunohistochemistry results confirmed that the expression of H-Ras and p-Erk1/2 was decreased in PC-11 and PC-40 tumour tissues compared with WT-CASKI tumour tissues. PLD1 promotes CC progression by activating the RAS pathway. Inhibition of PLD1 may serve as an attractive therapeutic modality for CC.
Topics: Cell Line, Tumor; Cell Proliferation; Cisplatin; Female; Humans; Immunohistochemistry; Phospholipase D; Uterine Cervical Neoplasms
PubMed: 35775110
DOI: 10.1111/jcmm.17439 -
Developmental Cell Jun 2021The development of the lens in the vertebrate eye requires the degradation of all organelles. In a recent issue of Nature, Morishita et al. (2021) identify a conserved...
The development of the lens in the vertebrate eye requires the degradation of all organelles. In a recent issue of Nature, Morishita et al. (2021) identify a conserved phospholipase that appears to achieve this by simply digesting organelle membranes away.
Topics: Animals; Lens, Crystalline; Organelles; Phospholipases; Vertebrates
PubMed: 34102103
DOI: 10.1016/j.devcel.2021.05.009 -
Journal of Plant Research Jul 2020Non-specific phospholipase C (NPC) is a novel class of phospholipase C found only in bacteria and higher plants. NPC hydrolyzes major phospholipid classes such as... (Review)
Review
Non-specific phospholipase C (NPC) is a novel class of phospholipase C found only in bacteria and higher plants. NPC hydrolyzes major phospholipid classes such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to produce diacylglycerol (DAG) and a corresponding phosphate-containing polar head group. Originally known as a toxin in certain bacteria to invade the host cell, this class of phospholipase has been well-investigated in bacteriology. Since the first discovery of eukaryotic NPC in Arabidopsis in 2005, this emerging class of phospholipase has received greater attention in plant biology in elucidating the biochemical characteristics and physiological function in the context of plant growth regulation and stress response. Particularly in the last few years, there has been significant progress made in understanding the fundamental character of 6 NPC isoforms in Arabidopsis, as well as novel function in other plant models. Now that research with plant NPC is entering into a new phase, this review aims to summarize recent progress in plant NPC along with some future perspectives.
Topics: Arabidopsis; Arabidopsis Proteins; Growth and Development; Phosphatidylcholines; Phospholipase D; Type C Phospholipases
PubMed: 32372398
DOI: 10.1007/s10265-020-01199-8 -
Toxins Mar 2020Spiders of the genus , popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts... (Review)
Review
Spiders of the genus , popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.
Topics: Animals; Catalysis; History, 20th Century; History, 21st Century; Humans; Phospholipase D; Phosphoric Diester Hydrolases; Proteomics; Recombinant Proteins; Spider Bites; Spider Venoms
PubMed: 32155765
DOI: 10.3390/toxins12030164