A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation.

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of kinase IP6K1 and repression of myo-inositol-3-P synthase.

Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-inositol-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. Here, we elucidate the role of PA in IP6K1 repression. Our results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of the MIPS protein. We found that this effect was specific to PA synthesized in the plasma membrane, as endoplasmic reticulum-derived PA did not induce IP6K1 translocation. Furthermore, we determined that PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5' AMP-activated protein kinase (AMPK). We show that activation of AMPK by glucose deprivation or by treatment with the mood-stabilizing drugs valproate or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Phospholipids (PLs) know-how: exploring and exploiting phospholipase D for its industrial dissemination.

Owing to their numerous nutritional and bioactive functions, phospholipids (PLs), which are major components of biological membranes in all living organisms, have been widely applied as nutraceuticals, food supplements, and cosmetic ingredients. To date, PLs are extracted solely from soybean or egg yolk, despite the diverse market demands and high cost, owing to a tedious and inefficient manufacturing process. A microbial-based manufacturing process, specifically phospholipase D (PLD)-based biocatalysis and biotransformation process for PLs, has the potential to address several challenges associated with the soybean- or egg yolk-based supply chain. However, poor enzyme properties and inefficient microbial expression systems for PLD limit their wide industrial dissemination. Therefore, sourcing new enzyme variants with improved properties and developing advanced PLD expression systems are important. In the present review, we systematically summarize recent achievements and trends in the discovery, their structural properties, catalytic mechanisms, expression strategies for enhancing PLD production, and its multiple applications in the context of PLs. This review is expected to assist researchers to understand current advances in this field and provide insights for further molecular engineering efforts toward PLD-mediated bioprocessing.

Key Disease Mechanisms Linked to Alzheimer's Disease in the Entorhinal Cortex.

Alzheimer's disease (AD) is a chronic, neurodegenerative brain disorder affecting millions of Americans that is expected to increase in incidence with the expanding aging population. Symptomatic AD patients show cognitive decline and often develop neuropsychiatric symptoms due to the accumulation of insoluble proteins that produce plaques and tangles seen in the brain at autopsy. Unexpectedly, some clinically normal individuals also show AD pathology in the brain at autopsy (asymptomatic AD, AsymAD). In this study, SWItchMiner software was used to identify key switch genes in the brain's entorhinal cortex that lead to the development of AD or disease resilience. Seventy-two switch genes were identified that are differentially expressed in AD patients compared to healthy controls. These genes are involved in inflammation, platelet activation, and phospholipase D and estrogen signaling. Peroxisome proliferator-activated receptor γ (PPARG), zinc-finger transcription factor (YY1), sterol regulatory element-binding transcription factor 2 (SREBF2), and early growth response 1 (EGR1) were identified as transcription factors that potentially regulate switch genes in AD. Comparing AD patients to AsymAD individuals revealed 51 switch genes; PPARG as a potential regulator of these genes, and platelet activation and phospholipase D as critical signaling pathways. Chemical-protein interaction analysis revealed that valproic acid is a therapeutic agent that could prevent AD from progressing.

Arabidopsis NRT1.2 interacts with the PHOSPHOLIPASE Dα1 (PLDα1) to positively regulate seed germination and seedling development in response to ABA treatment.

NRT1.2 has been characterized as a low-affinity nitrate transporter and an abscisic acid (ABA) transporter in Arabidopsis. In this study, we demonstrate that NRT1.2 positively regulated the ABA response during germination and seedling development. The transgenic Arabidopsis NRT1.2-over-expressionors showed increased sensitivity to ABA during these processes. qRT-PCR assays indicated that NRT1.2 over-production in 7-days-old seedlings up-regulated the expression of ABA-responsive genes: ABI1, ABI2, ABI3, ABI4, ABI5, RAB18, RD29A, and RD29B and PHOSPHOLIPASE Dα1 (PLDα1). The expression of these genes was suppressed in the nrt1.2 mutant in comparison with the wild type following ABA treatment. Importantly, bimolecular fluorescence complementation assays indicated that NRT1.2 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by using yeast two hybrid (Y2H) experiments with the mating-based split ubiquitin system (MbSUS). Moreover, genetic assays indicated that PLDα1 acts epistatically on NRT1.2 to affect ABA signaling. Taken together, our results provide detailed mechanisms of NRT1.2 in ABA-mediated seed germination and seedling development.

Intestinal NAPE-PLD contributes to short-term regulation of food intake via gut-to-brain axis.

Our objective was to explore the physiological role of the intestinal endocannabinoids in the regulation of appetite upon short-term exposure to high-fat-diet (HFD) and understand the mechanisms responsible for aberrant gut-brain signaling leading to hyperphagia in mice lacking Napepld in the intestinal epithelial cells (IECs). We generated a murine model harboring an inducible NAPE-PLD deletion in IECs (NapepldΔIEC). After an overnight fast, we exposed wild-type (WT) and NapepldΔIEC mice to different forms of lipid challenge (HFD or gavage), and we compared the modification occurring in the hypothalamus, in the vagus nerve, and at endocrine level 30 and 60 min after the stimulation. NapepldΔIEC mice displayed lower hypothalamic levels of N-oleoylethanolamine (OEA) in response to HFD. Lower mRNA expression of anorexigenic Pomc occurred in the hypothalamus of NapepldΔIEC mice after lipid challenge. This early hypothalamic alteration was not the consequence of impaired vagal signaling in NapepldΔIEC mice. Following lipid administration, WT and NapepldΔIEC mice had similar portal levels of glucagon-like peptide-1 (GLP-1) and similar rates of GLP-1 inactivation. Administration of exendin-4, a full agonist of GLP-1 receptor (GLP-1R), prevented the hyperphagia of NapepldΔIEC mice upon HFD. We conclude that in response to lipid, NapepldΔIEC mice displayed reduced OEA in brain and intestine, suggesting an impairment of the gut-brain axis in this model. We speculated that decreased levels of OEA likely contributes to reduce GLP-1R activation, explaining the observed hyperphagia in this model. Altogether, we elucidated novel physiological mechanisms regarding the gut-brain axis by which intestinal NAPE-PLD regulates appetite rapidly after lipid exposure.

Integrated Analyses Identify Immune-Related Signature Associated with Qingyihuaji Formula for Treatment of Pancreatic Ductal Adenocarcinoma Using Network Pharmacology and Weighted Gene Co-Expression Network.

The study aimed to clarify the potential immune-related targets and mechanisms of Qingyihuaji Formula (QYHJ) against pancreatic cancer (PC) through network pharmacology and weighted gene co-expression network analysis (WGCNA). Active ingredients of herbs in QYHJ were identified by the TCMSP database. Then, the putative targets of active ingredients were predicted with SwissTargetPrediction and the STITCH databases. The expression profiles of GSE32676 were downloaded from the GEO database. WGCNA was used to identify the co-expression modules. Besides, the putative targets, immune-related targets, and the critical module genes were mapped with the specific disease to select the overlapped genes (OGEs). Functional enrichment analysis of putative targets and OGEs was conducted. The overall survival (OS) analysis of OGEs was investigated using the Kaplan-Meier plotter. The relative expression and methylation levels of OGEs were detected in UALCAN, human protein atlas (HPA), Oncomine, DiseaseMeth version 2.0 and, MEXPRESS database, respectively. Gene set enrichment analysis (GSEA) was conducted to elucidate the key pathways of highly-expressed OGEs further. OS analyses found that 12 up-regulated OGEs, including CDK1, PLD1, MET, F2RL1, XDH, NEK2, TOP2A, NQO1, CCND1, PTK6, CTSE, and ERBB2 that could be utilized as potential diagnostic indicators for PC. Further, methylation analyses suggested that the abnormal up-regulation of these OGEs probably resulted from hypomethylation, and GSEA revealed the genes markedly related to cell cycle and proliferation of PC. This study identified CDK1, PLD1, MET, F2RL1, XDH, NEK2, TOP2A, NQO1, CCND1, PTK6, CTSE, and ERBB2 might be used as reliable immune-related biomarkers for prognosis of PC, which may be essential immunotherapies targets of QYHJ.

Targeting of Phospholipase D1 Ameliorates Collagen-Induced Arthritis via Modulation of Treg and Th17 Cell Imbalance and Suppression of Osteoclastogenesis.

Phospholipase D1 (PLD1) plays a crucial role in various inflammatory and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease. However, the role of PLD1 in the pathogenesis of RA remains unknown. Here, we first investigated the role and effects of PLD1 in collagen-induced arthritis (CIA) and found that genetic and pharmacological inhibition of PLD1 in DBA1/J mice with CIA reduced the incidence of CIA, decreased the clinical score, and abrogated disease symptoms including infiltration of leukocytes, synovial inflammation, bone erosion, and cartilage destruction. Moreover, ablation and inhibition of PLD1 suppressed the production of type II collagen-specific IgG2a autoantibody and proinflammatory cytokines, accompanied by an increase in the regulatory T (Treg) cell population and a decrease in the Th17 cell population in CIA mice. The PLD1 inhibitor also promoted differentiation of Treg cells and suppressed differentiation of Th17 cells in vitro. Furthermore, the PLD1 inhibitor attenuated pathologic bone destruction in CIA mice by suppressing osteoclastogenesis and bone resorption. Thus, our findings indicate that the targeting of PLD1 can ameliorate CIA by modulating the imbalance of Treg and Th17 cells and suppressing osteoclastogenesis, which might be a novel strategy to treat autoimmune diseases, such as RA.

Novel allelic variation in the Phospholipase D alpha1 gene (OsPLDα1) of wild Oryza species implies to its low expression in rice bran.

Rice bran, a by-product after milling, is a rich source of phytonutrients like oryzanols, tocopherols, tocotrienols, phytosterols, and dietary fibers. Moreover, exceptional properties of the rice bran oil make it unparalleled to other vegetable oils. However, a lipolytic enzyme Phospholipase D alpha1 (OsPLDα1) causes rancidity and 'stale flavor' in the oil, and thus limits the rice bran usage for human consumption. To improve the rice bran quality, sequence based allele mining at OsPLDα1 locus (3.6 Kb) was performed across 48 accessions representing 11 wild Oryza species, 8 accessions of African cultivated rice, and 7 Oryza sativa cultivars. From comparative sequence analysis, 216 SNPs and 30 InDels were detected at the OsPLDα1 locus. Phylogenetic analysis revealed 20 OsPLDα1 cDNA variants which further translated into 12 protein variants. The O. officinalis protein variant, when compared to Nipponbare, showed maximum variability comprising 22 amino acid substitutions and absence of two peptides and two ß-sheets. Further, expression profiling indicated significant differences in transcript abundance within as well as between the OsPLDα1 variants. Also, a new OsPLDα1 transcript variant having third exon missing in it, Os01t0172400-06, has been revealed. An O. officinalis accession (IRGC101152) had lowest gene expression which suggests the presence of novel allele, named as OsPLDα1-1a (GenBank accession no. MF966931). The identified novel allele could be further deployed in the breeding programs to overcome rice bran rancidity in elite cultivars.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

Characterization of a PLDζ2 Homology Gene from Developing Castor Bean Endosperm.

Castor oil contains approximately 90% ricinoleic acid (RA) which is stored mainly in the form of tri-ricinoleic acid containing triacylglycerols (TAG). Ricinoleate is synthesized from oleate (18:1n-9) esterified to the sn-2 position of phosphatidylcholine (PtdCho) catalyzed by oleoyl-12-hydroxylase. PtdCho-derived diacylglycerol (DAG) is an important substrate pool for TAG synthesis, and the interconversion between PtdCho and DAG has been shown to play a critical role in channeling hydroxy fatty acids (HFA) to TAG. Although phospholipase D (PLD) has been reported to catalyze the hydrolysis of PtdCho to produce phosphatidic acid which can then be converted to DAG, its potential functions in the channeling of RA from PtdCho to DAG and the assembly of RA on TAG is largely unknown. In the present study, 11 PLD genes were identified from the Castor Bean Genome Database. Gene expression analysis indicated that RcPLD9 is expressed at relatively high levels in developing seeds compared to other plant tissues. Sequence and phylogenetic analyses revealed that RcPLD9 is a homolog of Arabidopsis PLDζ2. Overexpression of RcPLD9 in the Arabidopsis CL7 line producing C18-HFA resulted in RA content reductions in the polar lipid fraction (mainly PtdCho) and mono-HFA-TAG, but increased RA content in di-HFA-TAG. Since part of RA in di-HFA-TAG is derived from HFA-DAG, the results indicated that RcPLD9 facilitates the channeling of RA from PtdCho to DAG for its assembly on TAG in developing seeds.

Functional analysis of phospholipase Dδ family in tobacco pollen tubes.

Phosphatidic acid (PA), an important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD), but the isoforms responsible for production of PM PA were not identified yet and their functional roles remain unknown. Following genome-wide bioinformatic analysis of the PLD family in tobacco, we focused on the pollen-overrepresented PLDδ class. Combining live-cell imaging, gene overexpression, lipid-binding and structural bioinformatics, we characterized five NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. Moreover, only PLDδ3 shows significant constitutive PLD activity in vivo and, in turn, PA promotes binding of PLDδ3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes that are crucial for plant cell tip growth.

Treatment with Gold Nanoparticles Using Cudrania tricuspidata Root Extract Induced Downregulation of MMP-2/-9 and PLD1 and Inhibited the Invasiveness of Human U87 Glioblastoma Cells.

In this study, we aimed to elucidate the anti-invasive effects of Cudrania tricuspidata root-gold nanoparticles (CTR-GNPs) using glioblastoma cells. We demonstrated the rapid synthesis of CTR-GNPs using UV-vis spectra. The surface morphology, crystallinity, reduction, capsulation, and stabilization of CTR-GNPs were analyzed using high resolution transmission electron microscopy (HR-TEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). Furthermore, CTR-GNPs displayed excellent photocatalytic activity as shown by the photo-degradation of methylene blue and rhodamine B. Cell migration and invasion assays with human glioblastoma cells were performed to investigate the anti-invasive effect of CTR-GNPs on U87 cells that were treated with phorbol 12-myristate 13-acetate. The results show that CTR-GNPs can significantly inhibit both basal and phorbol 12-myristate 13-acetate (PMA)-induced migration and invasion ability. Importantly, treatment with CTR-GNPs significantly decreased the levels of metalloproteinase (MMP)-2/-9 and phospholipase D1 (PLD1) and protein but not PLD2, which is involved in the modulation of migration and the invasion of glioblastoma cells. These results present a novel mechanism showing that CTR-GNPs can attenuate the migration and invasion of glioblastoma cells induced by PMA through transcriptional and translational regulation of MMP-2/-9 and PLD1. Taken together, our results suggest that CTR-GNPs might be an excellent therapeutic alternative for wide range of glioblastomas.

Phospholipase D-derived phosphatidic acid promotes root hair development under phosphorus deficiency by suppressing vacuolar degradation of PIN-FORMED2.

Root hair development is crucial for phosphate absorption, but how phosphorus deficiency affects root hair initiation and elongation remains unclear. We demonstrated the roles of auxin efflux carrier PIN-FORMED2 (PIN2) and phospholipase D (PLD)-derived phosphatidic acid (PA), a key signaling molecule, in promoting root hair development in Arabidopsis thaliana under a low phosphate (LP) condition. Root hair elongation under LP conditions was greatly suppressed in pin2 mutant or under treatment with a PLDζ2-specific inhibitor, revealing that PIN2 and polar auxin transport and PLDζ2-PA are crucial in LP responses. PIN2 was accumulated and degraded in the vacuole under a normal phosphate (NP) condition, whereas its vacuolar accumulation was suppressed under the LP or NP plus PA conditions. Vacuolar accumulation of PIN2 was increased in pldζ2 mutants under LP conditions. Increased or decreased PIN2 vacuolar accumulation is not observed in sorting nexin1 (snx1) mutant, indicating that vacuolar accumulation of PIN2 is mediated by SNX1 and the relevant trafficking process. PA binds to SNX1 and promotes its accumulation at the plasma membrane, especially under LP conditions, and hence promotes root hair development by suppressing the vacuolar degradation of PIN2. We uncovered a link between PLD-derived PA and SNX1-dependent vacuolar degradation of PIN2 in regulating root hair development under phosphorus deficiency.

Overexpression of soybean GmPLDγ enhances seed oil content and modulates fatty acid composition in transgenic Arabidopsis.

Phospholipase D (PLD) hydrolyzes the phosphodiester bond of glycerophospholipids to yield phosphatidic acid (PA) and a free headgroup. PLDs are important for plant growth, development, and responses to external stresses. However, their roles in triacylglycerol (TAG) synthesis are still unclear. Here, we report that a soybean (Glycine max) PLDγ (GmPLDγ) is involved in glycerolipid turnover and seed oil production. GmPLDγ was targeted to mitochondria and exhibited PLD activity that was activated by oleate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Overexpression of GmPLDγ (abbreviated GmPLDγ-OE) in Arabidopsis thaliana resulted in enhanced seed weight, elevated levels of TAGs with 18-, 20-, and 22-carbon fatty acids (FAs), and altered oil-body morphology. Furthermore, the levels of membrane lipids in vegetative tissues decreased significantly, whereas no overt changes were found in mature seeds except for a decrease in the digalactosyldiacylglycerol (DGDG) level in the GmPLDγ-OE lines. Additionally, the expression of genes involved in glycerolipid metabolism was significantly upregulated in developing siliques in GmPLDγ-OE lines. Together, our data indicate a regulatory role for GmPLDγ in TAG synthesis and fatty-acid remodeling, highlighting the importance of mitochondria-directed glycerophospholipid homeostasis in seed oil accumulation.

Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering.

Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7-10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.

Mining of a phospholipase D and its application in enzymatic preparation of phosphatidylserine.

Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+ at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.

Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D.

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.

Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight.

Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1-/- and Pld2-/- mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.