PLD2 deficiency alleviates endothelial glycocalyx degradation in LPS-induced ARDS/ALI.

- Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a life-threatening condition marked by severe lung inflammation and increased lung endothelial barrier permeability. Endothelial glycocalyx deterioration is the primary factor of vascular permeability changes in ARDS/ALI. Although previous studies have shown that phospholipase D2 (PLD2) is closely related to the onset and progression of ARDS/ALI, its role and mechanism in the damage of endothelial cell glycocalyx remains unclear. We used LPS-induced ARDS/ALI mice (in vivo) and LPS-stimulated injury models of EA.hy926 endothelial cells (in vitro). We employed C57BL/6 mice, including wild-type and PLD2 knockout (PLD2-/-) mice, to establish the ARDS/ALI model. We applied immunofluorescence and ELISA to examine changes in syndecan-1 (SDC-1), matrix metalloproteinase-9 (MMP9), inflammatory cytokines (TNF-α, IL-6, and IL-1ß) levels and the effect of external factors, such as phosphatidic acid (PA), 1-butanol (a PLD inhibitor), on SDC-1 and MMP9 expression levels. We found that PLD2 deficiency inhibits SDC-1 degradation and MMP9 expression in LPS-induced ARDS/ALI. Externally added PA decreases SDC-1 levels and increases MMP9 in endothelial cells, hence underlining PA's role in SDC-1 degradation. Additionally, PLD2 deficiency decreases the production of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in LPS-induced ARDS/ALI. In summary, these findings suggest that PLD2 deficiency plays a role in inhibiting the inflammatory process and protecting against endothelial glycocalyx injury in LPS-induced ARDS/ALI.

H2S is involved in drought-mediated stomatal closure through PLDα1 in Arabidopsis.

MAIN CONCLUSION: PLDα1 promoted H2S production by positively regulating the expression of LCD. Stomatal closure promoted by PLDα1 required the accumulation of H2S under drought stress. Phospholipase Dα1 (PLDα1) acting as one of the signal enzymes can respond to drought stress. It is well known that hydrogen sulfide (H2S) plays an important role in plant responding to biotic or abiotic stress. In this study, the functions and relationship between PLDα1 and H2S in drought stress resistance in Arabidopsis were explored. Our results indicated that drought stress promotes PLDα1 and H2S production by inducing the expression of PLDα1 and LCD genes. PLDα1 and LCD enhanced plant tolerance to drought by regulating membrane lipid peroxidation, proline accumulation, H2O2 content and stomatal closure. Under drought stress, the H2O2 content of PLDα1-deficient mutant (pldα1), L-cysteine desulfhydrase (LCD)-deficient mutant (lcd) was higher than that of ecotype (WT), the stomatal aperture of pldα1 and lcd was larger than that of WT. The transcriptional and translational levels of LCD were lower in pldα1 than that in WT. Exogenous application of the H2S donor NaHS or GYY reduced the stomatal aperture of WT, pldα1, PLDα1-CO, and PLDα1-OE lines, while exogenous application of the H2S scavenger hypotaurine (HT) increased the stomatal aperture. qRT-PCR analysis of stomatal movement-related genes showed that the expression of CAX1, ABCG5, SCAB1, and SLAC1 genes in pldα1 and lcd were down-regulated, while ACA1 and OST1 gene expression was significantly up-regulated. Thus, PLDα1 and LCD are required for stomatal closure to improve drought stress tolerance.

ß-Hydroxy-ß-methylbutyrate (HMB) leads to phospholipase D2 (PLD2) activation and alters circadian rhythms in myotubes.

ß-Hydroxy-ß-methylbutyrate (HMB) is a breakdown product of leucine, which promotes muscle growth. Although some studies indicate that HMB activates AKT and mTOR, others show activation of the downstream effectors, P70S6K and S6, independent of mTOR. Our aim was to study the metabolic effect of HMB around the circadian clock in order to determine more accurately the signaling pathway involved. C2C12 myotubes were treated with HMB and clock, metabolic and myogenic markers were measured around the clock. HMB-treated C2C12 myotubes showed no activation of AKT and mTOR, but did show activation of P70S6K and S6. Activation of P70S6K and S6 was also found when myotubes were treated with HMB combined with metformin, an indirect mTOR inhibitor, or rapamycin, a direct mTOR inhibitor. The activation of the P70S6K and S6 independent of AKT and mTOR, was accompanied by increased activation of phospholipase D2 (PLD). In addition, HMB led to high amplitude and advanced circadian rhythms. In conclusion, HMB induces myogenesis in C2C12 by activating P70S6K and S6 via PLD2, rather than AKT and mTOR, leading to high amplitude advanced rhythms.

Populus euphratica PeNADP-ME interacts with PePLDδ to mediate sodium and ROS homeostasis under salinity stress.

Populus euphratica phospholipase Dδ (PePLDδ) is transcriptionally regulated and mediates reactive oxygen species (ROS) and ion homeostasis under saline conditions. The purpose of this study is to explore the post-transcriptional regulation of PePLDδ in response to salt environment. P. euphratica PePLDδ was shown to interact with the NADP-dependent malic enzyme (NADP-ME) by screening the yeast two-hybrid libraries. The transcription level of PeNADP-ME increased upon salt exposure to NaCl (200 mM) in leaves and roots of P. euphratica. PeNADP-ME had a similar subcellular location with PePLDδ in the cytoplasm, and the interaction between PeNADP-ME and PePLDδ was further verified by GST pull-down and yeast two-hybrid. To clarify whether PeNADP-ME interacts with PePLDδ to enhance salt tolerance, PePLDδ and PeNADP-ME were overexpressed singly or doubly in Arabidopsis thaliana. Dual overexpression of PeNADP-ME and PePLDδ resulted in an even more pronounced improvement in salt tolerance compared with single transformants overexpressing PeNADP-ME or PePLDδ alone. Greater Na+ limitation and Na+ efflux in roots were observed in doubly overexpressed plants compared with singly overexpressed plants with PeNADP-ME or PePLDδ. Furthermore, NaCl stimulation of SOD, APX, and POD activity and transcription were more remarkable in the doubly overexpressed plants. It is noteworthy that the enzymic activity of NADP-ME and PLD, and total phosphatidic acid (PA) concentrations were significantly higher in the double-overexpressed plants than in the single transformants. We conclude that PeNADP-ME interacts with PePLDδ in Arabidopsis to promote PLD-derived PA signaling, conferring Na+ extrusion and ROS scavenging under salt stress.

PLD2 deletion ameliorates sepsis-induced cardiomyopathy by suppressing cardiomyocyte pyroptosis via the NLRP3/caspase 1/GSDMD pathway.

OBJECTIVE: Sepsis-induced cardiomyopathy (SICM) is a life-threatening complication. Phospholipase D2 (PLD2) is crucial in mediating inflammatory reactions and is associated with the prognosis of patients with sepsis. Whether PLD2 is involved in the pathophysiology of SICM remains unknown. This study aimed to investigate the effect of PLD2 knockout on SICM and to explore potential mechanisms. METHODS: The SICM model was established using cecal ligation and puncture in wild-type and PLD2-knockout mice and lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Transfection with PLD2-shRNA lentivirus and a PLD2 overexpression plasmid were used to interfere with PLD2 expression in H9C2 cells. Cardiac pathological alterations, cardiac function, markers of myocardial injury, and inflammatory factors were used to evaluate the SICM model. The expression of pyroptosis-related proteins (NLRP3, cleaved caspase 1, and GSDMD-N) was assessed using western blotting, immunofluorescence, and immunohistochemistry. RESULTS: SICM mice had myocardial tissue damage, increased inflammatory response, and impaired heart function, accompanied by elevated PLD2 expression. PLD2 deletion improved cardiac histological changes, mitigated cTNI production, and enhanced the survival of the SICM mice. Compared with controls, PLD2-knockdown H9C2 exhibits a decrease in inflammatory markers and lactate dehydrogenase production, and scanning electron microscopy results suggest that pyroptosis may be involved. The overexpression of PLD2 increased the expression of NLRP3 in cardiomyocytes. In addition, PLD2 deletion decreased the expression of pyroptosis-related proteins in SICM mice and LPS-induced H9C2 cells. CONCLUSION: PLD2 deletion is involved in SICM pathogenesis and is associated with the inhibition of the myocardial inflammatory response and pyroptosis through the NLRP3/caspase 1/GSDMD pathway.

Whole-Cell Display of Phospholipase D in Escherichia coli for High-Efficiency Extracellular Phosphatidylserine Production.

Phospholipids are widely utilized in various industries, including food, medicine, and cosmetics, due to their unique chemical properties and healthcare benefits. Phospholipase D (PLD) plays a crucial role in the biotransformation of phospholipids. Here, we have constructed a super-folder green fluorescent protein (sfGFP)-based phospholipase D (PLD) expression and surface-display system in Escherichia coli, enabling the surface display of sfGFP-PLDr34 on the bacteria. The displayed sfGFP-PLDr34 showed maximum enzymatic activity at pH 5.0 and 45 °C. The optimum Ca2+ concentrations for the transphosphatidylation activity and hydrolysis activity are 100 mM and 10 mM, respectively. The use of displayed sfGFP-PLDr34 for the conversion of phosphatidylcholine (PC) and L-serine to phosphatidylserine (PS) showed that nearly all the PC was converted into PS at the optimum conditions. The displayed enzyme can be reused for up to three rounds while still producing detectable levels of PS. Thus, Escherichia coli/sfGFP-PLD shows potential for the feasible industrial-scale production of PS. Moreover, this system is particularly valuable for quickly screening higher-activity PLDs. The fluorescence of sfGFP can indicate the expression level of the fused PLD and changes that occur during reuse.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Phospholipase D, a Novel Therapeutic Target Contributes to the Pathogenesis of Neurodegenerative and Neuroimmune Diseases.

Phospholipase D (PLD) is an enzyme that consists of six isoforms (PLD1-PLD6) and has been discovered in different organisms including bacteria, viruses, plants, and mammals. PLD is involved in regulating a wide range of nerve cells' physiological processes, such as cytoskeleton modulation, proliferation/growth, vesicle trafficking, morphogenesis, and development. Simultaneously, PLD, which also plays an essential role in the pathogenesis of neurodegenerative and neuroimmune diseases. In this review, family members, characterizations, structure, functions and related signaling pathways, and therapeutic values of PLD was summarized, then five representative diseases including Alzheimer disease (AD), Parkinson's disease (PD), etc. were selected as examples to tell the involvement of PLD in these neurological diseases. Notably, recent advances in the development of tools for studying PLD therapy envisaged novel therapeutic interventions. Furthermore, the limitations of PLD based therapy were also analyzed and discussed. The content of this review provided a thorough and reasonable basis for further studies to exploit the potential of PLD in the treatment of neurodegenerative and neuroimmune diseases.

Phospholipase D Mediates Glutamine-Induced mTORC1 Activation to Promote Porcine Intestinal Epithelial Cell Proliferation.

BACKGROUND: The intestinal epithelium is one of the fastest self-renewal tissues in the body, and glutamine plays a crucial role in providing carbon and nitrogen for biosynthesis. In intestinal homeostasis, phosphorylation-mediated signaling networks that cause altered cell proliferation, differentiation, and metabolic regulation have been observed. However, our understanding of how glutamine affects protein phosphorylation in the intestinal epithelium is limited, and identifying the essential signaling pathways involved in regulating intestinal epithelial cell growth is particularly challenging. OBJECTIVES: This study aimed to identify the essential proteins and signaling pathways involved in glutamine's promotion of porcine intestinal epithelial cell proliferation. METHODS: Phosphoproteomics was applied to describe the protein phosphorylation landscape under glutamine treatment. Kinase-substrate enrichment analysis was subjected to predict kinase activity and validated by qRT-PCR and Western blotting. Cell Counting Kit-8, glutamine rescue experiment, chloroquine treatment, and 5-fluoro-2-indolyl deschlorohalopemide inhibition assay revealed the possible underlying mechanism of glutamine promoting porcine intestinal epithelial cell proliferation. RESULTS: In this study, glutamine starvation was found to significantly suppress the proliferation of intestinal epithelial cells and change phosphoproteomic profiles with 575 downregulated sites and 321 upregulated sites. Interestingly, phosphorylation of eukaryotic initiation factor 4E-binding protein 1 at position Threonine70 was decreased, which is a crucial downstream of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Further studies showed that glutamine supplementation rescued cell proliferation and mTORC1 activity, dependent on lysosomal function and phospholipase D activation. CONCLUSION: In conclusion, glutamine activates mTORC1 signaling dependent on phospholipase D and a functional lysosome to promote intestinal epithelial cell proliferation. This discovery provides new insight into regulating the homeostasis of the intestinal epithelium, particularly in pig production.

High-Yield Synthesis of Phosphatidylserine in a Well-Designed Mixed Micellar System.

A sustainable enzymatic system is essential for efficient phosphatidylserine (PS) synthesis in industrial production. Conventional biphasic systems face challenges such as excessive organic solvent usage, enzyme-intensive processes, and increased costs. This study introduces a novel approach using chitin nanofibrils (ChNFs) as an immobilization material for phospholipase D (PLD) in a mixed micellar system stabilized by the food-grade emulsifier sodium deoxycholate (SDC). The immobilized enzyme, ChNF-chiA1, was quickly prepared in a one-step process, eliminating the need for purification. By optimizing the reaction conditions, including l-Ser concentration (1.0 M), SDC concentration (10 mM), reaction time (8 h), and enzyme dosage (1.0 U), a remarkable PS yield of 96.74% was achieved in the solvent-free mixed micellar system. The catalytic efficiency of ChNF-chiA1 surpassed that of the free PLD-chiA1 biphasic system by 6.0-fold. This innovative and green biocatalytic technology offers a reusable solution for the high-value enzymatic synthesis of phospholipids, providing a promising avenue for industrial applications.

The wide world of non-mammalian phospholipase D enzymes.

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

The role and regulation of phospholipase D in metabolic disorders.

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and free choline. In mammals, PLD exists in two well-characterized isoforms, PLD1 and PLD2, and it plays pivotal roles as signaling mediators in various cellular functions, such as cell survival, differentiation, and migration. These isoforms are predominantly expressed in diverse cell types, including many immune cells, such as monocytes and macrophages, as well as non-immune cells, such as epithelial and endothelial cells. Several previous studies have revealed that the stimulation of these cells leads to an increase in PLD expression and its enzymatic products, potentially influencing the pathological responses in a wide spectrum of diseases. Metabolic diseases, exemplified by conditions, such as diabetes, obesity, hypertension, and atherosclerosis, pose significant global health challenges. Abnormal activation or dysfunction of PLD emerges as a potential contributing factor to the pathogenesis and progression of these metabolic disorders. Therefore, it is crucial to thoroughly investigate and understand the intricate relationship between PLD and metabolic diseases. In this review, we provide an in-depth overview of the functional roles and molecular mechanisms of PLD involved in metabolic diseases. By delving into the intricate interplay between PLD and metabolic disorders, this review aims to offer insights into the potential therapeutic interventions.

A Brief Overview of the Toxic Sphingomyelinase Ds of Brown Recluse Spider Venom and Other Organisms and Simple Methods To Detect Production of Its Signature Cyclic Ceramide Phosphate.

A special category of phospholipase D (PLD) in the venom of the brown recluse spider (Loxosceles reclusa) and several other sicariid spiders accounts for the dermonecrosis and many of the other clinical symptoms of envenomation. Related proteins are produced by other organisms, including fungi and bacteria. These PLDs are often referred to as sphingomyelinase Ds (SMase Ds) because they cleave sphingomyelin (SM) to choline and "ceramide phosphate." The lipid product has actually been found to be a novel sphingolipid: ceramide 1,3-cyclic phosphate (Cer1,3P). Since there are no effective treatments for the injury induced by the bites of these spiders, SMase D/PLDs are attractive targets for therapeutic intervention, and some of their features will be described in this minireview. In addition, two simple methods are described for detecting the characteristic SMase D activity using a fluorescent SM analog, (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-SM (C12-NBD-SM), that is cleaved to C12-NBD-Cer1,3P, which is easily separated from other potential metabolites by thin-layer chromatography and visualized under UV light. Besides confirming that C12-NBD-Cer1,3P is the only product detected upon incubation of C12-NBD-SM with brown recluse spider venom, the method was also able to detect for the first time very low levels of activity in venom from another spider, Kukulcania hibernalis The simplicity of the methods makes it relatively easy to determine this signature activity of SMase D/PLD. SIGNIFICANCE STATEMENT: The sphingomyelinase D/phospholipase D that are present in the venom of the brown recluse spider and other sources cause considerable human injury, but detection of the novel sphingolipid product, ceramide 1,3-cyclic phosphate, is not easy by previously published methods. This minireview describes simple methods for detection of this activity that will be useful for studies of its occurrence in spider venoms and other biological samples, perhaps including lesions from suspected spider bites and infections.

The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Phospholipase D Immobilization on Lignin Nanoparticles for Enzymatic Transformation of Phospholipids.

Lignin nanoparticles (LNPs) are promising components for various materials, given their controllable particle size and spherical shape. However, their origin from supramolecular aggregation has limited the applicability of LNPs as recoverable templates for immobilization of enzymes. In this study, we show that stabilized LNPs are highly promising for the immobilization of phospholipase D (PLD), the enzyme involved in the biocatalytic production of high-value polar head modified phospholipids of commercial interest, phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine. Starting from hydroxymethylated lignin, LNPs were prepared and successively hydrothermally treated to obtain c-HLNPs with high resistance to organic solvents and a wide range of pH values, covering the conditions for enzymatic reactions and enzyme recovery. The immobilization of PLD on c-HLNPs (PLD-c-HLNPs) was achieved through direct adsorption. We then successfully exploited this new enzymatic preparation in the preparation of pure polar head modified phospholipids with high yields (60-90 %). Furthermore, the high stability of PLD-c-HLNPs allows recycling for a number of reactions with appreciable maintenance of its catalytic activity. Thus, PLD-c-HLNPs can be regarded as a new, chemically stable, recyclable and user-friendly biocatalyst, based on a biobased inexpensive scaffold, to be employed in sustainable chemical processes for synthesis of value-added phospholipids.

Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Phospholipase D1 activity is crucial for cytosolic phospholipase A2 -dependent prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells.

In bone, prostaglandin E2 (PGE2) is highly osteogenic and formed by osteoblasts, a key modulatory event in the regulation of bone cell activity. MC3T3-E1 cells are widely used as an in vitro model of osteoblast function. It is still not clear which pathways contribute to the release of AA in these cells. In this study we have focussed on the contribution of phospholipase D (PLD) enzymes to osteoblastic PGE2 formation after stimulation with endothelin-1 (ET-1). Using specific inhibitors of PLD1 and PLD2 we could show that PGE2 formation was strictly dependent on PLD1 but not PLD2 activity and cytosolic phospholipase A2 (cPLA2) was activated by triggering through PLD1. We have identified diacyl glycerol (DAG) as a possible effector molecule which may serve as a triggering signal for PKC activation and subsequent cPLA2 phosphorylation.

NME3 binds to phosphatidic acid and mediates PLD6-induced mitochondrial tethering.

Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

The role of GPLD1 in chronic diseases.

Glycosylphosphatidylinositol-specific phospholipase D (GPLD1) is a specific enzyme for glycosylphosphatidylinositol (GPI) anchors, thereby exerting its biological functions by cleaving membrane-associated GPI molecules. GPLD1 is abundant in serum, with a concentration of approximately 5-10 µg/mL. Previous studies have demonstrated that GPLD1 plays a crucial role in the pathogenesis of numerous chronic diseases including disorders of lipid and glucose metabolism, cancer, and neurological disorders. In the present study, we reviewed the structure, functions, and localization of GPLD1 in chronic diseases, as well as exercise-mediated regulation of GPLD1, thus providing a theoretical support to develop GPLD1 as a new therapeutic target for chronic diseases.