Phospholipase D Enzymes Research Articles

Postharvest hexanal application delays senescence and maintains quality in persimmon fruit during low temperature storage.

Persimmon (Diospyros kaki L.) fruit is characterized by rapid metabolic changes following ripening, and softening occurs due to the gradual breakdown of the cell membrane by the direct catabolic cascades of the phospholipase-D enzyme on the phospholipid bilayer. The cell membrane weakening is also enhanced by the generation of ROS during stress conditions such as cold storage and postharvest handling. This research evaluated the postharvest hexanal dipping application on persimmon fruit storage quality. The response of "MKÜ Harbiye" persimmon fruit to exogenous hexanal at different concentrations (0.04% and 0.08%, named as HEX-I and HEX-II, respectively) on quality parameters, chilling injury (CI), microbial growth, antioxidant compounds, and free radical scavenging capacity (FRSC) during storage at 0 °C and 80-90± 5% RH for 120 d were evaluated. Both hexanal treatments retained quality and delayed senescence, as indicated by greener peel (lower a* and L* values), higher firmness, total phenol concentration (TPC), FRSC, and titratable acidity (TA), but lower weight loss, electrical conductivity (EC), rate of CO2 , ethylene production, decay, and microbial growth than the control. Total soluble solids (TSS) were lower in treated fruit than the control up to 100 days, but it was much lower in HEX-I treatment as compared to HEX-II treatment. HEX-I treatment exhibited lower CI than the other treatments during storage. Hexanal at 0.04% could be used to increase the storage period of "MKÜ Harbiye" persimmons up to 120 d at 0 °C and 80-90± 5% RH by retaining quality and delaying senescence. This article is protected by copyright. All rights reserved.

Protein salvage and repurposing in evolution: Phospholipase D toxins are stabilized by a remodeled scrap of a membrane association domain.

The glycerophosphodiester phosphodiesterase (GDPD)-like SMaseD/PLD domain family, which includes phospholipase D (PLD) toxins in recluse spiders and actinobacteria, evolved anciently in bacteria from the GDPD. The PLD enzymes retained the core (β/α)8 barrel fold of GDPD, while gaining a signature C-terminal expansion motif and losing a small insertion domain. Using sequence alignments and phylogenetic analysis, we infer that the C-terminal motif derives from a segment of an ancient bacterial PLAT domain. Formally, part of a protein containing a PLAT domain repeat underwent fusion to the C terminus of a GDPD barrel, leading to attachment of a segment of a PLAT domain, followed by a second complete PLAT domain. The complete domain was retained only in some basal homologs, but the PLAT segment was conserved and repurposed as the expansion motif. The PLAT segment corresponds to strands β7-β8 of a β-sandwich, while the expansion motif as represented in spider PLD toxins has been remodeled as an α-helix, a β-strand, and an ordered loop. The GDPD-PLAT fusion led to two acquisitions in founding the GDPD-like SMaseD/PLD family: (1) a PLAT domain that presumably supported early lipase activity by mediating membrane association, and (2) an expansion motif that putatively stabilized the catalytic domain, possibly compensating for, or permitting, loss of the insertion domain. Of wider significance, messy domain shuffling events can leave behind scraps of domains that can be salvaged, remodeled, and repurposed.

Enzyme mediated production of asymmetrically charged liposomes.

In asymmetric liposomes, the inner and outer leaflets of the lipid bilayer have different compositions. As such, these liposomes are appealing platforms for modeling and studying membrane asymmetry in biological membranes. In addition, asymmetric liposomes can serve as innovative drug delivery vehicles in which, the inner leaflet can be designed for maximal cargo encapsulation while the outer leaflet can be optimized for minimal toxicity. This study aims to explore the use of a membrane-active enzyme, phospholipase D (PLD), to produce liposomes with asymmetrically-charged membranes. PLD is an interfacial enzyme that catalyzes the hydrolysis of zwitterionic phosphatidylcholine (PC) lipids to produce choline and negatively-charged phosphatidic acid (PA) lipids. For this work, we introduced PLD to pre-formed PC liposomes and monitored the zeta potential of these liposomes at different time points. Zeta potential of these liposomes showed a time-dependent drop, presumably due to PLD activity and production of negatively-charged PA lipids in the membrane. Using a calibration curve, the zeta potential values were converted to the amount of PA formed in the outer leaflet of liposomes. Notably, the use of heat-deactivated PLD enzyme did not have an impact on liposomal zeta values. In addition, the results of a choline assay that was used to quantify the produced choline and thus PA in the system were comparable to those from zeta measurements. Furthermore, performing these studies with different PLD concentrations showed that the rate of PA production varied with the concentration of enzyme. Lastly, experiments of dye leakage from liposomes suggested that this enzymatic activity did not have a significant impact on liposomal membrane integrity. Together, these findings indicate that PLD enzymatic activity can be utilized to generate liposomes with asymmetrically charged leaflets, providing exciting opportunities for membrane biophysical studies as well as drug delivery applications.

Chemical Tools for Understanding Phosphatidic Acid Signaling

Phosphatidic acid (PA) is an important phospholipid biosynthetic intermediate and a lipid second messenger. Both the metabolism of PA and the interactions of PA with its effector proteins can occur on numerous organelle membranes, raising important questions about how cells direct specificity into the outcomes of PA signaling. We have endeavored to ‘uncouple the pleiotropy’ of PA signaling through the development of two complementary types of chemical biology toolsets for, respectively, visualizing and manipulating PA metabolism and signaling. The first of these, IMPACT, harnesses bioorthogonal chemistry to generate fluorescent lipids whose localization and abundance report on the activity of phospholipase D (PLD) enzymes that generate ‘signaling pools’ of PA. The second tool, optoPLD, involves the use of light‐controlled, or optogenetic, PLD enzymes capable of generating pools of PA at precise organelle locations upon blue light illumination. We will describe the development of these tools, including newer, second‐generation IMPACT probes and optimized ‘superPLD’ enzymes with much better catalytic properties than our original optoPLD systems. Further, we will describe select applications of IMPACT and optoPLD. These include the use of IMPACT as a readout in genome‐wide CRISPR screening to discover new regulators of PLD signaling and the use of these tools to examine the spatiotemporal dynamics of important pathways that intersect with PLD activation, such as GPCR signaling and the Hippo pathway. Collectively, this work highlights the power of applying diverse chemical biology approaches to shed light on cellular lipid signaling pathways.

Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking. Because PA metabolism and trafficking are rapid, approaches to accurately visualize and manipulate its levels require high spatiotemporal precision. Here, we describe recent efforts to create a suite of chemical tools that enable imaging and perturbation of PA signaling. First, we describe techniques to visualize PA production by phospholipase D (PLD) enzymes, which are major producers of PA, called Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). IMPACT harnesses the ability of endogenous PLD enzymes to accept bioorthogonally tagged alcohols in transphosphatidylation reactions to generate functionalized reporter lipids that are subsequently fluorescently tagged via click chemistry. Second, we describe two light-controlled approaches for precisely manipulating PA signaling. Optogenetic PLDs use light-mediated heterodimerization to recruit a bacterial PLD to desired organelle membranes, and photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. We highlight select applications of these tools for studying GPCR–Gq signaling, discovering regulators of PLD signaling, tracking intracellular lipid transport pathways, and elucidating new oncogenic signaling roles for PA. We envision that these chemical tools hold promise for revealing many new insights into lipid signaling pathways.

Aldolase A and Phospholipase D1 Synergistically Resist Alkylating Agents and Radiation in Lung Cancer.

Exposure to alkylating agents and radiation may cause damage and apoptosis in cancer cells. Meanwhile, this exposure involves resistance and leads to metabolic reprogramming to benefit cancer cells. At present, the detailed mechanism is still unclear. Based on the profiles of several transcriptomes, we found that the activity of phospholipase D (PLD) and the production of specific metabolites are related to these events. Comparing several particular inhibitors, we determined that phospholipase D1 (PLD1) plays a dominant role over other PLD members. Using the existing metabolomics platform, we demonstrated that lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC) are the most critical metabolites, and are highly dependent on aldolase A (ALDOA). We further demonstrated that ALDOA could modulate total PLD enzyme activity and phosphatidic acid products. Particularly after exposure to alkylating agents and radiation, the proliferation of lung cancer cells, autophagy, and DNA repair capabilities are enhanced. The above phenotypes are closely related to the performance of the ALDOA/PLD1 axis. Moreover, we found that ALDOA inhibited PLD2 activity and enzyme function through direct protein–protein interaction (PPI) with PLD2 to enhance PLD1 and additional carcinogenic features. Most importantly, the combination of ALDOA and PLD1 can be used as an independent prognostic factor and is correlated with several clinical parameters in lung cancer. These findings indicate that, based on the PPI status between ALDOA and PLD2, a combination of radiation and/or alkylating agents with regulating ALDOA-PLD1 may be considered as a new lung cancer treatment option.

Acyl Chain Specificity of Marine Streptomyces klenkii PhosPholipase D and Its Application in Enzymatic Preparation of Phosphatidylserine.

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S−1 mM−1), but lower catalytic efficiency toward PCs with long acyl chain (>16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties.

Specificity of Loxosceles α Clade Phospholipase D Enzymes for Choline-Containing Lipids: Role of a Conserved Aromatic Cage

Phospholipase D (PLD) enzymes are one of the major toxins in the recluse spider (genus Loxosceles) venom. Loxosceles PLD enzymes are classified in either the α clade or the β clade, and some correlation exists between α or β clade membership and high or low catalytic activity against sphingomyelin (SM), respectively. Lajoie et al., recently showed that a β clade enzyme from Sicarius terrosus (St_βID1) had a strong preference for substrates containing ethanolamine headgroups, and suggested that this substrate specificity originated from the interactions between the PLD interfacial face and lipids. To further understand the substrate specificity of these enzymes, we performed simulations of two PLDs from the α clade, Loxosceles intermedia αIA1 (Li_αIA1) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1i. Simulations were performed in the presence of two types of lipid bilayers, choline-containing and ethanolamine-containing bilayer. We observed that the two α clade PLDs bound to bilayers with choline-containing lipids (PC or SM) using the catalytic loop. By contrast, St_ βIB1i, did not bind to those bilayers. Analyses of the trajectories also reveal the importance, for the two α clade PLDs, of three aromatic residues located on the catalytic loop and forming a π-cage interacting with a choline group. A multiple sequence alignment of 18 PLDs of both clades reveal that the aromatic cage is conserved in α clade PLDs and absent from at least one major subgroup of the β clade enzymes. We suggest that this cage controls the affinity for choline headgroups, which is in agreement with earlier reports of PC lipid headgroups interacting with aromatic amino acids.

An alkali-tolerant phospholipase D from <i>Sphingobacterium thalpophilum</i> 2015: Gene cloning, overproduction and characterization

The phospholipase pl-S.t gene of Sphingobacterium thalpophilum 2015 was cloned and the gene sequence was submitted to NCBI with Accession Number KX674735.1. The phylogenetic analysis showed that this PL-S.t was clustered to phospholipase D (PLD). As far as we know, the PL-S.t with a molecular mass of 22.5 kDa is the lowest of the currently purified bacterial PLDs, which belongs to a non-HKD PLD enzyme. This PL-S.t was resistant to a wide range of alkali pHs (7.5-9.0) after 1 h incubation, retaining more than 90% of its maximum activity. The PL-S.t activity can be enhanced by Ni2+, Co2+ and Mn2+. This PL-S.t has only one cysteine residue and fewer negatively-charged amino acids (AAs). The hydrogen bonds network was found around the cystein108, which may be beneficial to the stability and activity of PL-S.t in Ni2+ solution. This study has laid the foundation for further research on the molecular mechanism of the catalytic characteristics of low molecular weight alkalic PLD from S. thalpophilum 2015.

Pre-harvest hexanal spray reduces bitter pit and enhances post-harvest quality in ‘Honeycrisp’ apples (Malus domestica Borkh.)

‘Honeycrisp’ is a popular apple variety among consumers due to its desirable flavour and characteristic texture. However, this variety is highly prone to pre-harvest fruit drop, bitter pit and decline in quality during long-term storage. This research investigated the effects of hexanal as a formulation (named by us as Enhanced Freshness Formulation -EFF) on fruit retention and post-harvest shelf-life in ‘Honeycrisp’ apples. Hexanal is a known inhibitor of phospholipase D (PLD). Our results indicated that hexanal application reduced PLD enzyme activity from 30 days post-harvest to the end of the storage period compared to the other treatments. Ethylene production was reduced with hexanal treatment from 0 to 90 days post-harvest. An increase in total soluble solids and a decrease in physiological loss in weight was observed with hexanal application. A significantly lower occurrence of progression and severity of bitter pit was observed, while incidence of bitter was reduced by 70 %. Further electron micrographs revealed that hexanal treated apples had higher structural integrity overall. Our results reveal that application of hexanal can reduce bitter pit incidence in Honeycrisp apples, while increasing the post-harvets storage.

Effect of Pre-harvest and Post-harvest Hexanal Treatments on Fruits and Vegetables: A Review

The membrane degradation process associated with ripening and senescence of fruit is accelerated by the enzyme phospholipase D (PLD). Hexanal is a compound with high potential to inhibit phospholipase-D enzyme, which is naturally secretes from plants and also promotes the shelf-life extension of fruits during its storage. Pre-harvest hexanal application as enhanced freshness formulation helps to reduce fruit decay and other post-harvest disease by preventing the microbial growth especially mold growth. Post-harvest application of hexanal in the forms of vapour or liquid and nano emulsion for dip treatment, etc. results in improved fruit quality with better colour firmness along with other improved biochemical qualities. The cell wall degrading enzymes such as PLD, PME (Pectin methyl esterase) suppressed after hexanal treatment, whereas accelerated the antioxidant enzymes activities. The hexanal can be used as a potential compound for the preservation and post-harvest storage of perishables with better quality and safety.

Coenzyme Q10 attenuates rat hepatocarcinogenesis via the reduction of CD59 expression and phospholipase D activity.

The current study aimed to test the profile of serum lipids, phospholipase D (PLD) activity, and CD59 expression pattern in rat hepatocellular carcinoma (HCC) after therapeutic treatment with Coenzyme Q10 (CoQ10). Three rat groups were allocated as normal control, untreated HCC, and treated HCC (HCC + CoQ10). The levels of serum α-fetoprotein (AFP) and tumour necrosis factor (TNF)-α were assessed using enzyme-linked immunosorbent assay (ELISA), while proliferating cell nuclear antigen (PCNA) was detected using immunohistochemistry (IHC). Serum lipids, classical (CH50), and alternative (APH50) pathways of complement activation, the liver cell HMG-CoA reductase (HMGCR), and PLD activities were assayed colorimetrically. The protein expression of CD59, scavenger receptor class B type 1 (SRB1), B cell lymphoma-2 (Bcl2), and cleaved Caspase-3 (Casp-3) were detected using western blotting, while the level of serum CD59 (sCD59) was assessed using dot-blot. CoQ10 reduced the cell proliferation, histological alterations, and the levels of AFP and TNF-α but increased lipids, CH50, and sCD59 in serum. In the liver cell, CoQ10 decreased and increased PLD and HMGCR enzyme activities, respectively. In addition, reduction of liver CD59, Bcl2, and SRB1 vs increased cleaved Casp-3 expressions was observed. Statistical correlation indicated an inverse relationship between CH50 and each of CD59 expression and PLD activity after treatment with CoQ10. In conclusion, CoQ10 could protect against rat HCC through increased lipids and the reduction of CD59 expression and PLD activity. SIGNIFICANCE OF THE STUDY: To our knowledge, this study is the first to describe the attenuating effect of antitumour natural product like Coenzyme Q10 (CoQ10) via the reduction of CD59 expression and phospholipase D (PLD) activity. This illustrates the important role of CD59 and PLD in relation to lipids in cancer prevention.

Targeting Phospholipase D Genetically and Pharmacologically for Studying Leukocyte Function.

Phospholipase D (PLD), is a protein that breaks down phospholipids, maintaining structural integrity and remodeling of cellular or intracellular membranes, as well as mediating protein trafficking and cytoskeletal dynamics during cell motility. One of the reaction products of PLD action is phosphatidic acid (PA). PA is a mitogen involved in a large variety of physiological cellular functions, such as cell growth, cell cycle progression, and cell motility. We have chosen as cell models the leukocyte polymorphonuclear neutrophil and the macrophage as examples of cell motility. We provide a three-part method for targeting PLD genetically and pharmacologically to study its role in cell migration. In the first part, we begin with genetically deficient mice PLD1-KO and PLD2-KO. We describe bone marrow neutrophil (BMN) isolation; BMN is labeled fluorescently and can be used for studying tissue-damaging neutrophilia in ischemia-reperfusion injury (IRI). In the second part, we begin also with PLD1-KO and PLD2-KO and prepare bone marrow-derived macrophages (BMDM), first from monocytes and then inducing macrophage differentiation in culture with continuous incubation of cytokines. We use BMDM to find experimentally if PLD woul play a role in cholesterol phagocytosis, which is the first step in atherosclerosis progression. In the third part, we study PLD function in BMN and BMDM with PLD enzyme pharmacological inhibitors instead of genetically deficient mice, to ascertain the particular contributions of isoforms PLD1 and PLD2 on leukocyte function. By using the three-step thorough approach, we could understand the molecular underpinning of PLD in the pathological conditions indicated above, IRI-neutrophilia and atherosclerosis.

Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity.

Chemical imaging techniques have played instrumental roles in dissecting the spatiotemporal regulation of signal transduction pathways. Phospholipase D (PLD) enzymes affect cell signaling by producing the pleiotropic lipid second messenger phosphatidic acid via hydrolysis of phosphatidylcholine. It remains a mystery how this one lipid signal can cause such diverse physiological and pathological signaling outcomes, due in large part to a lack of suitable tools for visualizing the spatial and temporal dynamics of its production within cells. Here, we report a chemical method for imaging phosphatidic acid synthesis by PLD enzymes in live cells. Our approach capitalizes upon the enzymatic promiscuity of PLDs, which we show can accept azidoalcohols as reporters in a transphosphatidylation reaction. The resultant azidolipids are then fluorescently tagged using the strain-promoted azide–alkyne cycloaddition, enabling visualization of cellular membranes bearing active PLD enzymes. Our method, termed IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation), reveals pools of basal and stimulated PLD activities in expected and unexpected locations. As well, we reveal a striking heterogeneity in PLD activities at both the cellular and subcellular levels. Collectively, our studies highlight the importance of using chemical tools to directly visualize, with high spatial and temporal resolution, the subset of signaling enzymes that are active.

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

ABSTRACTPhosphatidylserine (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.

Identification of a novel phospholipase D with high transphosphatidylation activity and its application in synthesis of phosphatidylserine and DHA-phosphatidylserine

Phosphatidylserine (PS) and docosahexaenoic acid-phosphatidylserine (DHA-PS) have significant nutritional and biological functions, which are extensively used in functional food industries. Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) or DHA-PC with l-serine, is an effective method for PS and DHA-PS preparation. However, because of the hydrolysis activity of PLD, PC and DHA-PC would be converted to the undesirable byproduct, phosphatidic acid (PA) and DHA-PA. In this study, a novel phospholipase D (PLDa2) was firstly cloned from Acinetobacter radioresistens a2 with high transphosphatidylation activity and no hydrolysis activity. In the PLD-catalyzed synthesis process (12h), both the transphosphatidylation conversion rate and selectivity of PS and DHA-PS were about 100%, which is the only PLD enzyme reported with this superiority up till now. In comparison with the majority of other known PLDs, PLDa2 exerted the highest activity at neutral pH, and it was stable from pH 4.0 to pH 9.0. In addition, PLDa2 had excellent thermal stability, with an optimum reaction temperature of 40°C and keeping more than 80% activity from 20°C to 60°C. The high catalytic selectivity mechanism of PLDa2 was explained by utilizing homology modeling, two-step docking, and binding energy and conformation analysis. PLDa2 ensured a stable supply of the biocatalyst with its most preponderant transphosphatidylation activity and PS selectivity, and had great potential in phospholipids industrial production.

Inhibition of phospholipase D enzyme activity through hexanal leads to delayed mango (Mangifera indica L.) fruit ripening through changes in oxidants and antioxidant enzymes activity

Fruit ripening is a senescence process and phospholipase D (PLD) is a key enzyme causing degradation of membrane phospholipids during that process. Our earlier studies showed that hexanal, is known to inhibit the PLD activity. The objectives were (i) to quantify the effects of postharvest hexanal treatment in mango fruit on physiochemical traits, (ii) to assess the changes in oxidants, antioxidants, and antioxidant enzymes activity in mango fruit after hexanal treatment. Fully matured mango fruit, var. Neelum were harvested from the tree, dipped in 0.02% hexanal solution, and stored under ambient conditions to study the physio and biochemical changes during storage period. The results indicated that hexanal treatment significantly reduced ethylene evolution rate, oxidants content and PLD enzyme activity in the fruit compared with control, key factors to delay ripening and senescence in fruit. However, the activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase and the contents of ascorbic acid were increased in response to hexanal treatment. The decreased ethylene evolution rate, PLD enzyme activity and oxidant production caused by hexanal treatment might have led to increased shelf life. Overall, the results suggest that post-harvest dip of mango fruits in 0.02% hexanal solution extended the shelf life of mango fruit under ambient storage conditions, without the loss of quality of fruits.

Targeting phospholipase D in cancer, infection and neurodegenerative disorders.

Lipid second messengers have essential roles in cellular function and contribute to the molecular mechanisms that underlie inflammation, malignant transformation, invasiveness, neurodegenerative disorders, and infectious and other pathophysiological processes. The phospholipase D (PLD) isoenzymes PLD1 and PLD2 are one of the major sources of signal-activated phosphatidic acid (PtdOH) generation downstream of a variety of cell-surface receptors, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and integrins. Recent advances in the development of isoenzyme-selective PLD inhibitors and in molecular genetics have suggested that PLD isoenzymes in mammalian cells and pathogenic organisms may be valuable targets for the treatment of several human diseases. Isoenzyme-selective inhibitors have revealed complex inter-relationships between PtdOH biosynthetic pathways and the role of PtdOH in pathophysiology. PLD enzymes were once thought to be undruggable owing to the ubiquitous nature of PtdOH in cell signalling and concerns that inhibitors would be too toxic for use in humans. However, recent promising discoveries suggest that small-molecule isoenzyme-selective inhibitors may provide novel compounds for a unique approach to the treatment of cancers, neurodegenerative disorders and other afflictions of the central nervous system, and potentially serve as broad-spectrum antiviral and antimicrobial therapeutics.

Analysis of Phosphatidic Acid Binding and Regulation of PIPKI In Vitro and in Intact Cells.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a lipid second messenger that regulates a wide array of essential cellular events, such as signal transduction, vesicle trafficking, actin cytoskeleton dynamics, adhesion, and motility. To control the spatiotemporal production of PI(4,5)P2, the activity of type 1 phosphotidylinositol-4-phosphate-5-kinases (PIPKIs) is tightly regulated by small GTPases and another signaling lipid, phosphatidic acid (PA). It is of interest that PI(4,5)P2 is also a critical cofactor for the activation of the PA-generating enzyme, phospholipase D (PLD). It has been proposed that the reciprocal stimulation of PLD and PIPKI enzymes enables a rapid feedforward stimulation loop for the localized and acute generation of signaling lipids that are critical for the regulation of actin cytoskeletal reorganization and membrane trafficking. Here, we outline the methods for the expression and purification of PIPKIγ from bacteria, determination of direct PA binding, and activation of PIPKIγ using in vitro liposomes assays, and examination of actin cytoskeletal reorganization promoted by the PA-PIPKIγ signaling in intact cells using fluorescent microscopy.

Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity.

Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [3H]phosphatidic acid (PA) released from [3H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.