Specific Phospholipase Research Articles

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission.

ATP, being a well-known universal high-energy compound, plays an important role as a signaling molecule and together with its metabolite adenosine they both attenuate the release of acetylcholine in the neuro-muscular synapse acting through membrane P2 and P1 receptors, respectively. In this work, using a mechanomyographic method, we analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat m. soleus and m. extensor digitorum longus. N-ethylmaleimide, a G-protein antagonist, prevents the modulating effects of both ATP and adenosine. The action of ATP is abolished by chelerythrin, a specific phospholipase C inhibitor, while the inhibitory effect of adenosine is slightly increased by Rp-cAMPS, an inhibitor of protein kinase A, and by nitrendipine, a blocker of L-type Ca2+ channels. The addition of DPCPX, an A1 receptor antagonist, fully prevents the inhibitory action of adenosine in both muscles. Our data indicate that the inhibitory action of ATP involves metabotropic P2Y receptors and is mediated by phospholipase C dependent processes in rat motor neuron terminals. We suggest that the presynaptic effect of adenosine consists of negative and positive actions. The negative action occurs by stimulation of adenosine A1 receptors while the positive action is associated with the stimulation of adenosine A2A receptors, activation of protein kinase A and opening of L-type calcium channels. The combined mechanism of the modulating action of ATP and adenosine provides fine tuning of the synapse to fast changing conditions in the skeletal muscles.

Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation.

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1-/-) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1-/--sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1-/- sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1-/- sperm and Plcz1-/- and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1-/-eCS-/-), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1-/- sperm + PLCζ mRNA, Plcz1-/- sperm + SrCl2 and Plcz1-/-eCS-/- sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1-/- sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1-/- sperm + SrCl2:4.0 ± 4.3% and Plcz1-/-eCS-/- sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1-/- sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1-/- sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.

Endoplasmic reticulum localization of phosphoinositide specific phospholipase C enzymes in U73122 cultured human osteoblasts

<abstract> <p>Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzyme family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. In addition to the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected <italic>PLC</italic> genes and different localization of PLC enzymes, depending on the cell line, in different cell lines.</p> <p>In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of <italic>PLC</italic> genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122.</p> <p>Our results confirm that all <italic>PLC</italic> genes are transcribed in hOBs, that probably splicing variants of selected <italic>PLC</italic> genes are expressed and that all PLC enzymes are present in hOBs, except for PLC δ3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 on cell survival. Our results indicate that U-73122 did not significantly affect the transcription of <italic>PLC</italic> genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles and/or in the endoplasmic reticulum.</p> </abstract>

Two Different Phospholipases C, Isc1 and Pgc1, Cooperate To Regulate Mitochondrial Function.

The absence of Isc1, the yeast homologue of mammalian neutral sphingomyelinase type 2, leads to severe mitochondrial dysfunction. We show that the deletion of another type C phospholipase, the phosphatidylglycerol (PG)-specific phospholipase Pgc1, rescues this defect. Phosphatidylethanolamine (PE) levels and cytochrome c oxidase activity, which were reduced in isc1Δ cells, were restored to wild-type levels in the pgc1Δ isc1Δ mutant. The Pgc1 substrate PG inhibited the in vitro activities of Isc1 and the phosphatidylserine decarboxylase Psd1, an enzyme crucial for PE biosynthesis. We also identify a mechanism by which the balance between the current demand for PG and its consumption is controlled. We document that the product of PG hydrolysis, diacylglycerol, competes with the substrate of PG-phosphate synthase, Pgs1, and thereby inhibits the biosynthesis of excess PG. This feedback loop does not work in the absence of Pgc1, which catalyzes PG degradation. Finally, Pgc1 activity is partially inhibited by products of Isc1-mediated hydrolysis. The described functional interconnection of the two phospholipases contributes significantly to lipid homeostasis throughout the cellular architecture. IMPORTANCE In eukaryotic cells, mitochondria are constantly adapting to changes in the biological activity of the cell, i.e., changes in nutrient availability and environmental stresses. We propose a model in which this adaptation is mediated by lipids. Specifically, we show that mitochondrial phospholipids regulate the biosynthesis of cellular sphingolipids and vice versa. To do this, lipids move by free diffusion, which does not require energy and works under any condition. This model represents a simple way for the cell to coordinate mitochondrial structure and performance with the actual needs of overall cellular metabolism. Its simplicity makes it a universally applicable principle of cellular regulation.

Phosphatidylserine-Specific Phospholipase A1 Alleviates Lipopolysaccharide-Induced Macrophage Inflammation by Inhibiting MAPKs Activation

Macrophages are a key in innate immune responses and play vital roles in homeostasis and inflammatory diseases. Phosphatidylserine-specific phospholipase A1 (PS-PLA1) is a specific phospholipase which hydrolyzes fatty acid from the sn-1 position of phosphatidylserine (PS) to produce lysophosphatidylserine (lysoPS). Both PS and lysoPS are associated with activation of immune cells including macrophages. However, the effect of PS-PLA1 on macrophage inflammation remains unclear. The purpose of this study is to evaluate the role of PS-PLA1 in lipopolysaccharide (LPS)-induced macrophage inflammation. Alterations of PS-PLA1 expression in LPS-stimulated RAW264.7 macrophages were investigated via Western blot. PS-PLA1 stable knockdown and overexpression RAW264.7 cell lines were generated by infecting cells with appropriate lentiviral vectors, respectively. PS-PLA1 expression was found to be dramatically upregulated in RAW264.7 macrophages after LPS stimulation. PS-PLA1 knockdown promotes while PS-PLA1 overexpression ameliorates the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and nitric oxide from RAW264.7 cells and M1 macrophage polarization. Additionally, PS-PLA1 knockdown facilitates phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PS-PLA1 overexpression attenuates their phosphorylation. Moreover, mitogen-activated protein kinase (MAPK) inhibitors block the release of TNF-α and IL-1β in PS-PLA1 knockdown RAW264.7 cells after LPS stimulation. These findings suggest PS-PLA1 ameliorates LPS-induced macrophage inflammation by inhibiting MAPKs activation, and PS-PLA1 might be considered as a target for modulating macrophage inflammation.

Decreasing GPLD 1-levels in patients after ST-elevated myocardial infarction

Background and Aims : Recently, higher levels of phosphatidyl-inositol-glycan specific phospholipase D (GPLD1) were associated with an impaired glucose tolerance and diabetes mellitus, factors of the metabolic syndrome, being a risk factor for myocardial infarctions. A previous study already showed higher serum GPLD1-levels in stroke patients compared with a healthy control group. Our pilot study analyzed the serum concentration of GPLD1 in patients with ST-elevated myocardial infarction (STEMI) at two different time points.

Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming.

The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80°C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80°C.

Transient early life growth hormone exposure permanently alters brain, muscle, liver, macrophage, and adipocyte statusin long-lived Ames dwarf mice.

The exceptional longevity of Ames dwarf (DF) mice can be abrogated by a brief course of growth hormone (GH) injections started at 2 weeks of age. This transient GH exposure also prevents the increase in cellular stress resistance and decline in hypothalamic inflammation characteristic of DF mice. Here, we show that transient early‐life GH treatment leads to permanent alteration of pertinent changes in adipocytes, fat‐associated macrophages, liver, muscle, and brain that are seen in DF mice. Ames DF mice, like Snell dwarf and GHRKO mice, show elevation of glycosylphosphatidylinositol specific phospholipase D1 in liver, neurogenesis in brain as indicated by BDNF and DCX proteins, muscle production of fibronectin type III domain‐containing protein 5 (a precursor of irisin), uncoupling protein 1 as an index of thermogenic capacity in brown and white fat, and increase in fat‐associated anti‐inflammatory macrophages. In each case, transient exposure to GH early in life reverts the DF mice to the levels of each protein seen in littermate control animals, in animals evaluated at 15–18 months of age. Thus, many of the traits seen in long‐lived mutant mice, pertinent to age‐related changes in inflammation, neurogenesis, and metabolic control, are permanently set by early‐life GH levels.

The 5-HT and PLC Signaling Pathways Regulate the Secretion of IL-1β, TNF-α and BDNF from NG2 Cells

The present study was clarified the relationship between NG2 glial cells and 5-hydroxytryptamine (5-HT) to further revealed a role in the regulation of cortical excitability. The co-localization of NG2 cells and 5-HT in rat prefrontal cortex was determined using immunofluorescence. Different concentrations of 5-HT were applied to cultured NG2 cells. Real-time PCR measured the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and brain-derived neurotrophic factor (BDNF). Changes in the expression of IL-1β, TNF-α, and BDNF in NG2 cells were detected after the addition of 5-HT receptor specific blockers and phospholipase C (PLC) specific activators and inhibitors. The results confirmed that the NG2 protein and 5-HT co-localized in the prefrontal cortex. 5-HT treatment of NG2 cells significantly reduced the expression of IL-1β and BDNF mRNA and increased the expression of TNF-α. The 5-HT receptor specific inhibitors alverine citrate, ketanserin, ondansetron and SB-399885 blocked the regulatory effects of 5-HT on NG2 cells. The PLC signal was linked to the secretion of IL-1β, TNF-α and BDNF in NG2 cells. These results indicated that 5-HT affected IL-1β, TNF-α, and BDNF secretion from NG2 cells via the 5-HT1A, 5-HT2A, 5-HT3, 5-HT6 receptors and the PLC signaling pathway.

Tricyclodecan-9-yl-Xanthogenate (D609): Mechanism of Action and Pharmacological Applications.

Tricyclodecan-9-yl xanthogenate (D609) is a synthetic tricyclic compound possessing a xanthate group. This xanthogenate compound is known for its diverse pharmacological properties. Over the last three decades, many studies have reported the biological activities of D609, including antioxidant, antiapoptotic, anticholinergic, anti-tumor, anti-inflammatory, anti-viral, anti-proliferative, and neuroprotective activities. Its mechanism of action is extensively attributed to its ability to cause the competitive inhibition of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS). The inhibition of PCPLC or SMS affects secondary messengers with a lipidic nature, i.e., 1,2-diacylglycerol (DAG) and ceramide. Various in vitro/in vivo studies suggest that PCPLC and SMS inhibition regulate the cell cycle, block cellular proliferation, and induce differentiation. D609 acts as a pro-inflammatory cytokine antagonist and diminishes Aβ-stimulated toxicity. PCPLC enzymatic activity essentially requires Zn2+, and D609 might act as a potential chelator of Zn2+, thereby blocking PCPLC enzymatic activity. D609 also demonstrates promising results in reducing atherosclerotic plaque formation, post-stroke cerebral infarction, and cancer progression. The present compilation provides a comprehensive mechanistic insight into D609, including its chemistry, mechanism of action, and regulation of various pharmacological activities.

High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

LncRNA ACTA2-AS1 inhibits malignant phenotypes of gastric cancer cells.

Gastric cancer (GC) is one of the most common malignancies in digestive system. Accumulating evidence reveals the critical role of long noncoding RNAs (lncRNAs) in GC development. The study aimed to explore the functions and mechanism of lncRNA actin alpha 2, smooth muscle antisense RNA 1 (ACTA2-AS1) in GC. Reverse transcription-quantitative polymerase chain reaction analyses and subcellular fractionation assays showed that ACTA2-AS1 was lowly expressed in GC cells and was mainly distributed in the cytoplasm. Overexpressed ACTA2-AS1 inhibited GC cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition process, as suggested by cell counting kit-8 assays, colony formation assays, wound healing assays, Transwell assays and Western blot analyses. Mechanistically, ACTA2-AS1 served as a competing endogenous RNA (ceRNA) to bind with miR-378a-3p and thereby, antagonized the inhibitory effect of miR-378a-3p on the expression of messenger RNA phosphatidylinositol specific phospholipase C X domain containing 2 (PLCXD2). The binding capacity between miR-378a-3p and ACTA2-AS1 (or PLCXD2) was detected by RNA pulldown assays, luciferase reporter assays and RNA immunoprecipitation assays. Moreover, PLCXD2 knockdown rescued the inhibitory effect of ACTA2-AS1 overexpression on malignant behaviors of GC cells. Overall, ACTA2-AS1 inhibits malignant phenotypes of GC cells by acting as a ceRNA to target miR-378a-3p/PLCXD2 axis.

Synthesis and antiviral effect of phosphamide modified vidarabine for treating HSV 1 infections

Vidarabine (ARA) was one of the earliest marine-related compounds to be used clinically for antiviral therapy, however, its fast metabolism is the main defect of this drug. To overcome this, we designed and synthesized a group of phosphamide-modified ARA compounds using ProTide technology. With a phosphamide modification, these compounds could become the substrate of specific phospholipase enzymes expressed in the liver. Among all 16 synthesized compounds, most showed stronger activity against herpes simplex virus type 1 (HSV-1) than ARA (EC50 of approximately 10 μM). The top three compounds were compound 2 (EC50 = 0.52 ± 0.04 μM), compound 6 (EC50 = 1.05 ± 0.09 μM) and compound 15 (EC50 = 1.18 ± 0.08 μM) (about 2 times higher than Sp type compound 2). This study provides evidence for use of the phosphamide modification, which could give ARA higher activity and liver cell targeting.

Nitric oxide positively affects endometrial receptivity via FAAH and NAPE-PLD in vitro.

SummaryObjectiveTo determine if models of human 'receptive' and 'non-receptive endometrium' differ in their responses to nitric oxide (NO) supplementation by measuring the levels of the enzymes of the endocannabinoid system (ECS) (fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD)), which control the 'anandamide tone' essential for successful pregnancy.DesignA study of FAAH and NAPE-PLD expression (using human endometrium) through the menstrual cycle and an in vitro using a model of 'receptive' (Ishikawa) and 'non-receptive' (HEC-1A) human endometrial cell lines treated with the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP).ResultsImmunoreactivity measured by optimised H-score for both FAAH and NAPE-PLD was reduced in secretory (receptive) endometrium compared to proliferative (non-receptive) endometrium (P = 0.0009 and <0.0001, respectively). FAAH and NAPE transcript levels were significantly higher in untreated Ishikawa cells than in HEC-1A cells (P = 0.0228 and 0.0001, respectively). Treatment of cultures with SNAP resulted in an increase in the amount of FAAH mRNA produced by Ishikawa cells and a decrease in NAPE-PLD mRNA. No effect of SNAP was observed in HEC-1A cells. Similarly, FAAH protein was significantly decreased in endometria representative of the receptive endometrium.ConclusionThese data suggest that NO most likely affects the expression of ECS enzymes in the implantation site of a receptive endometrium; a phenomenon not seen in a non-receptive endometrium. These effects are most marked with FAAH expression, suggesting that FAAH may play the more critical role in ensuring the correct 'anandamide tone' for successful embryo implantation than NAPE-PLD.Lay summaryEmbryo implantation into the wall of the uterus is only successful when the inner wall of the uterus (the endometrium) is ‘receptive’, because if it is ‘non-receptive’, implantation will fail. Previous work showed that enzymes of the 'endocannabinoid system' are critical for implantation by maintaining the correct level of a fat called anandamide. This is by balancing its synthesis (by N-acylphosphatidylethanolamine specific phospholipase D, NAPE-PLD) and degradation (by fatty acid amide hydrolase, FAAH). Using immortalised cell lines as models of ‘receptive’ and ‘non-receptive’ human endometrium, we demonstrate a key stimulator of implantation, nitric oxide, has a positive effect on implantation by both increasing the mRNA levels of the degrading enzyme (FAAH) and decreasing the expression of the synthesising enzyme (NAPE-PLD). These effects are most marked with the degrading enzyme, suggesting that FAAH plays a more critical role than NAPE-PLD in ensuring the correct 'anandamide tone' for successful embryo implantation.

Supernatants From Human Osteosarcoma Cultured Cell Lines Induce Modifications in Growth and Differentiation of THP-1 Cells and Phosphoinositide- Specific Phospholipase C Enzymes

Supernatants From Human Osteosarcoma Cultured Cell Lines Induce Modifications in Growth and Differentiation of THP-1 Cells and Phosphoinositide- Specific Phospholipase C Enzymes

High-Density Lipoprotein Carries Markers That Track With Recovery From Stroke

Prospective cohort studies question the value of HDL-C (high-density lipoprotein cholesterol) for stroke risk prediction. Investigate the relationship between long-term functional recovery and HDL proteome and function. Changes in HDL protein composition and function (cholesterol efflux capacity) in patients after acute ischemic stroke at 2 time points (24 hours, 35 patients; 96 hours, 20 patients) and in 35 control subjects were measured. The recovery from stroke was assessed by 3 months, the National Institutes of Health Stroke Scale and modified Rankin scale scores. When compared with control subject after adjustments for sex and HDL-C levels, 12 proteins some of which participate in acute phase response and platelet activation (APMAP [adipocyte plasma membrane-associated protein], GPLD1 [phosphate inositol-glycan specific phospholipase D], APOE [apolipoprotein E], IHH [Indian hedgehog protein], ITIH4 [inter-alpha-trypsin inhibitor chain H4], SAA2 [serum amyloid A2], APOA4 [apolipoprotein A-IV], CLU [clusterin], ANTRX2 [anthrax toxin receptor 2], PON1 [serum paraoxonase/arylesterase], SERPINA1 [alpha-1-antitrypsin], and APOF [apolipoprotein F]) were significantly (adjusted P<0.05) altered in stroke HDL at 96 hours. The first 8 of these proteins were also significantly altered at 24 hours. Consistent with inflammatory remodeling, cholesterol efflux capacity was reduced by 32% (P<0.001) at both time points. Baseline stroke severity adjusted regression model showed that changes within 96-hour poststroke in APOF, APOL1, APMAP, APOC4 (apolipoprotein C4), APOM (apolipoprotein M), PCYOX1 (prenylcysteine oxidase 1), PON1, and APOE correlate with stroke recovery scores (R2=0.38-0.73, adjusted P<0.05). APOF (R2=0.73) and APOL1 (R2=0.60) continued to significantly correlate with recovery scores after accounting for tPA (tissue-type plasminogen activator) treatment. Changes in HDL proteins during early acute phase of stroke associate with recovery. Monitoring HDL proteins may provide clinical biomarkers that inform on stroke recuperation.

BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.

Inhibition of LIPG phospholipase activity suppresses tumor formation of human basal-like triple-negative breast cancer

The endothelial lipase LIPG possesses serine phospholipase activity and is involved in lipoprotein metabolism. Our previous studies have revealed that LIPG overexpression is required for tumor formation and metastasis of human basal-like triple-negative breast cancer (TNBC). We also demonstrated that LIPG differentially regulates TNBC malignancy through its enzymatic and non-enzymatic functions. The present studies were aimed at determining how XEN445, a specific inhibitor targeting LIPG phospholipase activity, impacts on TNBC tumor formation and malignant features. We established a cell-based LIPG enzymatic assay system to measure the inhibitory effect of XEN445 on LIPG phospholipase activity and determine its IC50. We found that XEN445 preferentially inhibited the proliferation of LIPG-expressing TNBC cells but not LIPG-negative luminal breast cancer cells. XEN445 inhibited the self-renewal of cancer stem cells (CSCs) in vitro and TNBC tumor formation in vivo. However, XEN445 had no inhibitory effect on the invasiveness and CSC stemness of TNBC cells. Our studies suggest that targeting both LIPG enzymatic and non-enzymatic functions is an important strategy for the treatment of TNBC.

Phospholipases in neuronal function: A role in learning and memory?

Despite the human brain being made of nearly 60% fat, the vast majority of studies on the mechanisms of neuronal communication which underpin cognition, memory and learning, primarily focus on proteins and/or (epi)genetic mechanisms. Phospholipids are the main component of all cellular membranes and function as substrates for numerous phospholipid-modifying enzymes, including phospholipases, which release free fatty acids (FFAs) and other lipid metabolites that can alter the intrinsic properties of the membranes, recruit and activate critical proteins, and act as lipid signalling molecules. Here, we will review brain specific phospholipases, their roles in membrane remodelling, neuronal function, learning and memory, as well as their disease implications. In particular, we will highlight key roles of unsaturated FFAs, particularly arachidonic acid, in neurotransmitter release, neuroinflammation and memory. In light of recent findings, we will also discuss the emerging role of phospholipase A1 and the creation of saturated FFAs in the brain.