Metabolic engineering for glycoglycerolipids production in E. coli: Tuning phosphatidic acid and UDP-glucose pathways.

Glycolipids are target molecules in biotechnology and biomedicine as biosurfactants, biomaterials and bioactive molecules. An engineered E. coli strain for the production of glycoglycerolipids (GGL) used the MG517 glycolipid synthase from M. genitalium for glucosyl transfer from UDPGlc to diacylglycerol acceptor (Mora-Buyé et al., 2012). The intracellular diacylglycerol pool proved to be the limiting factor for GGL production. Here we designed different metabolic engineering strategies to enhance the availability of precursor substrates for the glycolipid synthase by modulating fatty acids, acyl donor and phosphatidic acid biosynthesis. Knockouts of tesA, fadE and fabR genes involved in fatty acids degradation, overexpression of the transcriptional regulator FadR, the acyltransferases PlsB and C, and the pyrophosphatase Cdh for phosphatidic acid biosynthesis, as well as the phosphatase PgpB for conversion to diacylglycerol were explored with the aim of improving GGL titers. Among the different engineered strains, the ΔtesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Attempts to boost UDPGlc availability by overexpressing the uridyltransferase GalU or knocking out the UDP-sugar diphosphatase encoding gene ushA did not further improve GGL titers. Most of the strains produced GGL containing a variable number of glucosyl units from mono-to tetra-saccharides. Interestingly, the strains co-expressing Cdh showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs) whereas the strains with a fadR knockout presented a higher amount of unsaturated acyl chains. In all cases, GGL production altered the lipidic composition of the E. coli membrane, observing that GGL replace phosphatidylethanolamine to maintain the overall membrane charge balance.

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis.

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

Roles of DGKs in neurons: Postsynaptic functions?

Diacylglycerol kinases (DGKs) contribute to an important part of intracellular signaling because, in addition to reducing diacylglycerol levels, they generate phosphatidic acid (PtdOH) Recent research has led to the discovery of ten mammalian DGK isoforms, all of which are found in the mammalian brain. Many of these isoforms have studied functions within the brain, while others lack such understanding in regards to neuronal roles, regulation, and structural dynamics. However, while previously a neuronal function for DGKθ was unknown, it was recently found that DGKθ is required for the regulation of synaptic vesicle endocytosis and work is currently being conducted to elucidate the mechanism behind this regulation. Here we will review some of the roles of all mammalian DGKs and hypothesize additional roles. We will address the topic of redundancy among the ten DGK isoforms and discuss the possibility that DGKθ, among other DGKs, may have unstudied postsynaptic functions. We also hypothesize that in addition to DGKθ's presynaptic endocytic role, DGKθ might also regulate the endocytosis of AMPA receptors and other postsynaptic membrane proteins.

Characterization of α-synuclein N-terminal domain as a novel cellular phosphatidic acid sensor.

Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 µm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) ß at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.

miR-543 promoted the cell proliferation and invasion of nasopharyngeal carcinoma by targeting the JAM-A.

MicroRNAs (miRNAs) are defined as small, non-coding RNAs that act as post-transcriptional regulators of gene expression. Dysfunction of miRNAs was involved in the initiation and progression of nasopharyngeal carcinoma (NPC). Here, we found that miR-543 was markedly overexpressed in NPC tissues and cell lines. Overexpression of miR-543 promoted the proliferation, cell cycle progression and invasion of NPC cells. Down-regulation of miR-543 inhibited the proliferation and induced apoptosis of NPC cells. Bioinformatics analysis suggested the junctional adhesion molecule A (JAM-A) as a potential target of miR-543. Furthermore, molecular study showed that the miR-543 bound the 3'-untranslated region (UTR) of JAM-A and decreased the expression of JAM-A in NPC cells. The expression of JAM-A in NPC tissues was decreased and negatively correlated with that of miR-543. Overexpression of JAM-A attenuated miR-543-induced proliferation of NPC cells. Collectively, these evidence indicated the important roles of miR-543/JAM-A signaling in the progression of NPC, highlighting the potential of miR-543 as a target in the treatment of NPC.

Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.

Of local translation control and lipid signaling in neurons.

Fine-tuned regulation of new proteins synthesis is key to the fast adaptation of cells to their changing environment and their response to external cues. Protein synthesis regulation is particularly refined and important in the case of highly polarized cells like neurons where translation occurs in the subcellular dendritic compartment to produce long-lasting changes that enable the formation, strengthening and weakening of inter-neuronal connection, constituting synaptic plasticity. The changes in local synaptic proteome of neurons underlie several aspects of synaptic plasticity and new protein synthesis is necessary for long-term memory formation. Details of how neuronal translation is locally controlled only start to be unraveled. A generally accepted view is that mRNAs are transported in a repressed state and are translated locally upon externally cued triggering signaling cascades that derepress or activate translation machinery at specific sites. Some important yet poorly considered intermediates in these cascades of events are signaling lipids such as diacylglycerol and its balancing partner phosphatidic acid. A link between these signaling lipids and the most common inherited cause of intellectual disability, Fragile X syndrome, is emphasizing the important role of these secondary messages in synaptically controlled translation.

Diacylglycerol kinases: Relationship to other lipid kinases.

Lipid kinases regulate a wide variety of cellular functions and have emerged as one the most promising targets for drug design. Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PtdOH). Despite the critical role in lipid biosynthesis, both DAG and PtdOH have been shown as bioactive lipids mediating a number of signaling pathways. Although there is increasing recognition of their role in signaling systems, our understanding of the key enzyme which regulate the balance of these two lipid messages remain limited. Solved structures provide a wealth of information for understanding the function and regulation of these enzymes. Solving the structures of mammalian DGKs by traditional NMR and X-ray crystallography approaches have been challenging and so far, there are still no three-dimensional structures of these DGKs. Despite this, some insights may be gained by examining the similarities and differences between prokaryotic DGKs and other mammalian lipid kinases. This review focuses on summarizing and comparing the structure of prokaryotic and mammalian DGKs as well as two other lipid kinases: sphingosine kinase and phosphatidylinositol-3-kinase. How these known lipid kinases structures relate to mammalian DGKs will also be discussed.

Cucumber Phospholipase D alpha gene overexpression in tobacco enhanced drought stress tolerance by regulating stomatal closure and lipid peroxidation.

BACKGROUND: Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. Both PLD and PA play key roles in plant growth, development, and cellular processes. PLD was suggested to mediate the regulation of stomatal movements by abscisic acid (ABA) as a response to water deficit. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (CsPLDα, GenBank accession number EF363796) in the growth and tolerance of transgenic tobacco (Nicotiana tabacum) to drought stress. RESULTS: The CsPLDα overexpression in tobacco lines correlated with the ABA synthesis and metabolism, regulated the rapid stomatal closure in drought stress, and reduced the water loss. The NtNCED1 expression levels in the transgenic lines and wild type (WT) were sharply up-regulated after 16 days of drought stress compared with those before treatment, and the expression level in the transgenic lines was significantly higher than that in the WT. The NtAOG expression level evidently improved after 8 and 16 days compared with that at 0 day of treatment and was significantly lower in the transgenic lines than in the WT. The ABA content in the transgenic lines was significantly higher than that in the WT. The CsPLDα overexpression could increase the osmolyte content and reduce the ion leakage. The proline, soluble sugar, and soluble protein contents significantly increased. By contrast, the electrolytic leakage and malondialdehyde accumulation in leaves significantly decreased. The shoot and root fresh and dry weights of the overexpression lines significantly increased. These results indicated that a significant correlation between CsPLDα overexpression and improved resistance to water deficit. CONCLUSIONS: The plants with overexpressed CsPLDα exhibited lower water loss, higher leaf relative water content, and heavier fresh and dry matter accumulation than the WT. We proposed that CsPLDα was involved in the ABA-dependent pathway in mediating the stomatal closure and preventing the elevation of intracellular solute potential.

FLOURY SHRUNKEN ENDOSPERM1 Connects Phospholipid Metabolism and Amyloplast Development in Rice.

Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during endosperm development.

Identification of novel phosphatidic acid binding domain on sphingosine kinase 1 of Arabidopsis thaliana.

Phosphatidic acid (PA) is an important lipid signaling molecule which interacts with Arabidopsis thaliana Sphingosine kinase1 (AtSPHK1) during several abiotic stresses particularly drought stress as a result of Abscisic acid (ABA) signaling in guard cells. PA molecules respond by generating lipid signal and/or by binding and translocating target proteins to membrane. However, site of interaction and role of PA binding to AtSPHK1 is not clear yet. Owing to the importance of AtSPHK1 during stress signaling it is imperative to decipher the site of PA interaction with AtSPHK1. To identify the PA binding region of AtSPHK1, various deletion fragments from N-terminal and C-terminal region were prepared. Results from protein lipid overlay assay using various truncated proteins of AtSPHK1 suggested the involvement of N-terminal region, between 110 and 205 amino acids, in binding with PA. In-silico analyses performed to build homologous structure of AtSPHK1 revealed that PA docking occurs in the hydrophobic cavity of DAG-Kinase domain. Deletion of amino acids 182VSGDGI187 perturbed PA-AtSPHK1 binding, indicating an essential role of these six amino acids in PA-AtSPHK1 binding.

Where do substrates of diacylglycerol kinases come from? Diacylglycerol kinases utilize diacylglycerol species supplied from phosphatidylinositol turnover-independent pathways.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprises ten isozymes (α-κ) and regulates a wide variety of physiological and pathological events, such as cancer, type II diabetes, neuronal disorders and immune responses. DG and PA consist of various molecular species that have different acyl chains at the sn-1 and sn-2 positions, and consequently, mammalian cells contain at least 50 structurally distinct DG/PA species. Because DGK is one of the components of phosphatidylinositol (PI) turnover, the generally accepted dogma is that all DGK isozymes utilize 18:0/20:4-DG derived from PI turnover. We recently established a specific liquid chromatography-mass spectrometry method to analyze which PA species were generated by DGK isozymes in a cell stimulation-dependent manner. Interestingly, we determined that DGKδ, which is closely related to the pathogenesis of type II diabetes, preferentially utilized 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG species (X:Y = the total number of carbon atoms: the total number of double bonds) supplied from the phosphatidylcholine-specific phospholipase C pathway, but not 18:0/20:4-DG, in high glucose-stimulated C2C12 myoblasts. Moreover, DGKα mainly consumed 14:0/16:0-, 16:0/18:1-, 18:0/18:1- and 18:1/18:1-DG species during cell proliferation in AKI melanoma cells. Furthermore, we found that 16:0/16:0-PA was specifically produced by DGKζ in Neuro-2a cells during retinoic acid- and serum starvation-induced neuronal differentiation. These results indicate that DGK isozymes utilize a variety of DG molecular species derived from PI turnover-independent pathways as substrates in different stimuli and cells. DGK isozymes phosphorylate various DG species to generate various PA species. It was revealed that the modes of activation of conventional and novel protein kinase isoforms by DG molecular species varied considerably. However, PA species-selective binding proteins have not been found to date. Therefore, we next attempted to identify PA species-selective binding proteins from the mouse brain and identified α-synuclein, which has causal links to Parkinson's disease. Intriguingly, we determined that among phospholipids, including several PA species (16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PA and 18:0/20:4-PA); 18:1/18:1-PA was the most strongly bound PA to α-synuclein. Moreover, 18:1/18:1-PA strongly enhanced secondary structural changes from the random coil form to the α-helix form and generated a multimeric and proteinase K-resistant α-synuclein protein. In contrast with the dogma described above, our recent studies strongly suggest that PI turnover-derived DG species and also various DG species derived from PI turnover-independent pathways are utilized by DGK isozymes. DG species supplied from distinct pathways may be utilized by DGK isozymes based on different stimuli present in different types of cells, and individual PA molecular species would have specific targets and exert their own physiological functions.

Phosphatidic acid-producing enzymes regulating the synaptic vesicle cycle: Role for PLD?

In cortical and hippocampal neurons of the mammalian brain, the synaptic vesicle cycle is a series of steps that tightly regulate exo- and endocytosis of vesicles. Many proteins contribute to this regulation, but lipids have recently emerged as critical regulators as well. Of all the many lipid signaling molecules, phosphatidic acid is important to the physical processes of membrane fusion. Therefore, the lipid-metabolizing enzymes that produce phosphatidic acid are vital to the regulation of the cycle. Our lab is particularly interested in the potential regulatory mechanisms and neuronal roles of two phosphatidic acid-producing enzymes: diacylglycerol kinase theta (DGKθ) and phospholipase D (PLD). We recently discovered a regulatory role of DGKθ on evoked endocytosis (Goldschmidt et al., 2016). In addition to this enzyme, studies implicate PLD1 in neurotransmission, although its precise role is of some debate. Altogether, the production of phosphatidic acid by these enzymes offer an interesting and novel pathway for the regulation of the synaptic vesicle cycle.

Proliferative and metastatic roles for Phospholipase D in mouse models of cancer.

Phospholipase D (PLD) activity has been proposed to facilitate multiple steps in cancer progression including growth, metabolism, angiogenesis, and mobility. The canonical enzymes PLD1 and PLD2 enact their diverse effects through hydrolyzing the membrane lipid phosphatidylcholine to generate the second messenger and signaling lipid phosphatidic acid (PA). However, the widespread expression of PLD1 and PLD2 in normal tissues and the additional distinct enzymatic mechanisms through which PA can be generated have produced uncertainty regarding the optimal settings in which PLD inhibition might ameliorate cancer. Recent studies in mouse model systems have demonstrated that inhibition or elimination of PLD activity reduces tumor growth and metastasis. One mechanism proposed for this outcome involves proliferative signaling mediated by receptor tyrosine kinases (RTK) and G protein-coupled receptors (GPCR), which is attenuated when downstream PLD signal propagation is suppressed. The reduced proliferative signaling has been reported to be compounded by dysfunctional energetic metabolism in the tumor cells under conditions of nutrient deprivation. Moreover, cancer cells lacking PLD activity display inefficiencies across multiple steps of the metastatic cascade, limiting the tumor's lethal spread. Using PLD isoform knockout mice, recent studies have reported on the net effects of inhibition and ablation in multiple cancer models through examining the role of PLD in the non-tumor cells comprising the stroma and microenvironment. The promising results of such in vivo studies, combined with the apparent low toxicity of highly-specific and potent inhibitors, highlights PLD as an attractive target for therapeutic inhibition in cancer. We discuss here the array of anti-tumor effects produced by PLD inhibition and ablation in cancer models with a focus on animal studies.

Lipid sensing by mTOR complexes via de novo synthesis of phosphatidic acid.

mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein-12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the de novo synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for de novo synthesis of PA. Suppression of the de novo synthesis of PA resulted in G1 cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA.

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA.

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells.

Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.

Molecular Pathways: Targeting Diacylglycerol Kinase Alpha in Cancer.

Lipid kinases have largely been neglected as targets in cancer, and an increasing number of reports suggest diacylglycerol kinase alpha (DGKα) may be one with promising therapeutic potential. DGKα is one of 10 DGK family members that convert diacylglycerol (DAG) to phosphatidic acid (PA), and both DAG and PA are critical lipid second messengers in the plasma membrane. A host of important oncogenic proteins and pathways affect cancer cells in part through DGKα, including the c-Met and VEGF receptors. Others partially mediate the effects of DGKα inhibition in cancer, such as mTOR and HIF-1α. DGKα inhibition can directly impair cancer cell viability, inhibits angiogenesis, and notably may also boost T-cell activation and enhance cancer immunotherapies. Although two structurally similar inhibitors of DGKα were established decades ago, they have seen minimal in vivo usage, and it is unlikely that either of these older DGKα inhibitors will have utility for cancer. An abandoned compound that also inhibits serotonin receptors may have more translational potential as a DGKα inhibitor, but more potent and specific DGKα inhibitors are sorely needed. Other DGK family members may also provide therapeutic targets in cancer, but require further investigation.

RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction.

Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover.

RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide.

Myostatin (MSTN), a member of the transforming growth factor-ß superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.