Diacylglycerol Kinase Activity Research Articles

Exogenous Methyl Jasmonate Alleviates Mechanical Damage in Banana Fruit by Regulating Membrane Lipid Metabolism

Bananas are economically important fruits, but they are vulnerable to mechanical damage during harvesting and transport. This study examined the effects of methyl jasmonate (MeJA) on the cell membrane integrity and membrane lipid metabolism of wounded banana fruits after harvest. The results showed that 10 and 50 μM MeJA treatments on mechanically wounded bananas significantly delayed ripening and senescence in comparison with the control. At the end of storage, MeJA-treated groups showed a significant reduction in electrolyte leakage and malondialdehyde content, indicating that MeJA protected cell membrane integrity. MeJA also led to a significant decrease in the activity of antioxidant enzymes, including lipoxygenase, diacylglycerol kinase, and lipid phosphate phosphatase. Furthermore, MeJA reduced phospholipase (C and D), phosphatidic acid, and diacylglycerol levels, as well as slowed down the decrease in phosphatidylcholine and phosphatidylinositol contents. Compared to the control, MeJA significantly downregulated the expression of MaPLDγ, MaPLDα, and MaPLDζ. Therefore, MeJA treatment could be a reliable method to delay the senescence of harvested banana fruits subjected to mechanical wounding.

Diacylglycerol kinase is a keystone regulator of signaling relevant to the pathophysiology of asthma.

Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGKs) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA, through a series of biochemical reactions, is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of α and ζ isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma, such as contraction and proliferation of airway smooth muscle (ASM) cells and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.

Glycoursodeoxycholic Acid Alleviates Arterial Thrombosis via Suppressing Diacylglycerol Kinases Activity in Platelet.

Glycoursodeoxycholic acid (GUDCA) has been acknowledged for its ability to regulate lipid homeostasis and provide benefits for various metabolic disorders. However, the impact of GUDCA on arterial thrombotic events remains unexplored. The objective of this study is to examine the effects of GUDCA on thrombogenesis and elucidate its underlying mechanisms. Plasma samples from patients with arterial thrombotic events and diet-induced obese mice were collected to determine the GUDCA concentrations using mass spectrometry. Multiple in vivo murine thrombosis models and in vitro platelet functional assays were conducted to comprehensively evaluate the antithrombotic effects of GUDCA. Moreover, lipidomic analysis was performed to identify the alterations of intraplatelet lipid components following GUDCA treatment. Plasma GUDCA level was significantly decreased in patients with arterial thrombotic events and negatively correlated with thrombotic propensity in diet-induced obese mice. GUDCA exhibited prominent suppressing effects on platelet reactivity as evidenced by the attenuation of platelet activation, secretion, aggregation, spreading, and retraction (P<0.05). In vivo, GUDCA administration robustly alleviated thrombogenesis (P<0.05) without affecting hemostasis. Mechanistically, GUDCA inhibited DGK (diacylglycerol kinase) activity, leading to the downregulation of the phosphatidic acid-mediated signaling pathway. Conversely, phosphatidic acid supplementation was sufficient to abolish the antithrombotic effects of GUDCA. More importantly, long-term oral administration of GUDCA normalized the enhanced DGK activity, thereby remarkably alleviating the platelet hyperreactivity as well as the heightened thrombotic tendency in diet-induced obese mice (P<0.05). Our study implicated that GUDCA reduces platelet hyperreactivity and improves thrombotic propensity by inhibiting DGKs activity, which is a potentially effective prophylactic approach and promising therapeutic agent for arterial thrombotic events.

Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis

Tumor-associated macrophages (TAMs) are often associated with chemoresistance and resultant poor clinical outcome in solid tumors. Here, we demonstrated that TAMs-released chemokine-C-C motif chemokine 22 (CCL22) in esophageal squamous cell carcinoma (ESCC) stroma was tightly correlated with the chemoresistance of ESCC patients. TAMs-secreted CCL22 was able to block the growth inhibitory and apoptosis-promoting effects of cisplatin on ESCC cells. Mechanistically, CCL22 stimulated intratumoral diacylglycerol kinase α (DGKα) to produce phosphatidic acid (PA), which suppressed the activity of NADPH oxidase 4 (NOX4) and then blocked the overproduction of intratumoral reactive species oxygen (ROS) induced by cisplatin. CCL22 activated DGKα/nuclear factor-κB (NF-κB) axis to upregulate the level of several members of ATP binding cassette (ABC) transporter superfamily, including ABC sub-family G member 4 (ABCG4), ABC sub-family A member 3 (ABCA3), and ABC sub-family A member 5 (ABCA5), to lower the intratumoral concentration of cisplatin. Consequently, these processes induced the cisplatin resistance in ESCC cells. In xenografted models, targeting DGKα with 5’-cholesterol-conjugated small-interfering (si) RNA enhanced the chemosensitivity of cisplatin in ESCC treatment, especially in the context of TAMs. Our data establish the correlation between the TAMs-induced intratumoral metabolic product/ROS axis and chemotherapy efficacy in ESCC treatment and reveal relevant molecular mechanisms.

Dynamic responses of PA to environmental stimuli imaged by a genetically encoded mobilizable fluorescent sensor

Membrane fluidity, permeability, and surface charges are controlled by phospholipid metabolism and transport. Despite the importance of phosphatidic acid (PA) as a bioactive molecule, the mechanical properties of PA translocation and subcellular accumulation are unknown. Here, we used a mobilizable, highly responsive genetically encoded fluorescent indicator, green fluorescent protein (GFP)–N160RbohD, to monitor PA dynamics in living cells. The majority of GFP–N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels. Cellular, pharmacological, and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP–N160RbohD fluorescence at the plasma membrane, which mainly depended on hydrolysis of phospholipase D. By contrast, heat stress induced nuclear translocation of PA indicated by GFP–N160RbohD through a process that required diacylglycerol kinase activity, as well as secretory and endocytic trafficking. Strikingly, we showed that gravity triggers asymmetric PA distribution at the root apex, a response that is suppressed by PLDζ2 knockout. The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that affect the synthesis, transport, and metabolism of PA.

The Role of Diacylglycerol Kinase in the Amelioration of Diabetic Nephropathy.

The drastic increase in the number of patients with diabetes and its complications is a global issue. Diabetic nephropathy, the leading cause of chronic kidney disease, significantly affects patients’ quality of life and medical expenses. Furthermore, there are limited drugs for treating diabetic nephropathy patients. Impaired lipid signaling, especially abnormal protein kinase C (PKC) activation by de novo-synthesized diacylglycerol (DG) under high blood glucose, is one of the causes of diabetic nephropathy. DG kinase (DGK) is an enzyme that phosphorylates DG and generates phosphatidic acid, i.e., DGK can inhibit PKC activation under diabetic conditions. Indeed, it has been proven that DGK activation ameliorates diabetic nephropathy. In this review, we summarize the involvement of PKC and DGK in diabetic nephropathy as therapeutic targets, and its mechanisms, by referring to our recent study.

Turnover of diacylglycerol kinase 4 by cytoplasmic acidification induces vacuole morphological change and nuclear DNA degradation in the early stage of pear self-incompatibility response.

Pear has an S-RNase-based gametophytic self-incompatibility (SI) system. Nuclear DNA degradation is a typical feature of incompatible pollen tube death, and is among the many physiological functions of vacuoles. However, the specific changes that occur in vacuoles, as well as the associated regulatory mechanism in pear SI, are currently unclear. Although research in tobacco has shown that decreased activity of diacylglycerol kinase (DGK) results in the morphological change of pollen tube vacuole, whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response, is currently unclear. We found that DGK activity is essential for pear pollen tube growth, and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear. Specifically, incompatible S-RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V-ATPase V0 domain a1 subunit gene expression as early as 30 min after treatment, when the pollen tube is still alive. Cytoplasmic acidification induced by incompatible S-RNase results in reduced DGK4 abundance and deposition, leading to morphological change of the vacuole and fragmentation of nuclear DNA, which indicates that DGK4 is a key factor in pear SI response.

Using Dictyostelium to Advance Our Understanding of the Role of Medium Chain Fatty Acids in Health and Disease.

Ketogenic diets have been utilized for many years to improve health, and as a dietary approach for the treatment of a range of diseases, where the mechanism of these low carbohydrate and high fat diets is widely considered to be through the production of metabolic products of fat breakdown, called ketones. One of these diets, the medium chain triglyceride ketogenic diet, involves high fat dietary intake in the form of medium chain fatty acids (MCFAs), decanoic and octanoic acid, and is commonly used in endurance and high intensity exercises but has also demonstrated beneficial effects in the treatment of numerous pathologies including drug resistant epilepsy, cancer, and diabetes. Recent advances, using Dictyostelium discoideum as a model, have controversially proposed several direct molecular mechanisms for decanoic acid in this diet, independent of ketone generation. Studies in this model have identified that decanoic acid reduces phosphoinositide turnover, diacylglycerol kinase (DGK) activity, and also inhibits the mechanistic target of rapamycin complex 1 (mTORC1). These discoveries could potentially impact the treatment of a range of disorders including epilepsy, cancer and bipolar disorder. In this review, we summarize the newly proposed mechanisms for decanoic acid, identified using D. discoideum, and highlight potential roles in health and disease treatment.

New Era of Diacylglycerol Kinase, Phosphatidic Acid and Phosphatidic Acid-Binding Protein.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α–κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK–PA–PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG–DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.

The mechanisms of ameliorating effect of a green tea polyphenol on diabetic nephropathy based on diacylglycerol kinase\xa0\u03b1

Significant efforts have been made to ameliorate diabetic nephropathy (DN) by inhibiting protein kinase C. However, these efforts have not been successful in human trials, suggesting that novel therapeutic strategies are required. Thus far, it has been reported that green tea polyphenol epigallocatechin gallate (EGCg) improved albuminuria in DN in a human trial. Our previous study revealed that activation of diacylglycerol kinase α (DGKα) plays a crucial role in the amelioration of DN and that EGCg activates DGKα. Here, we investigated whether and how DGKα contributes to the amelioration of DN upon stimulation by EGCg by using streptozotocin-induced type 1 diabetic model mice. Our results revealed that EGCg ameliorated albuminuria in DN through DGKα in vivo, and methylated EGCg, which has higher absorption in the plasma improved albuminuria in DN effectively. Additionally, we showed that c-Src mediated EGCg-induced DGKα translocation and colocalized with the 67 kDa laminin receptor, which is an EGCg receptor. Furthermore, EGCg attenuated the loss of podocytes in DN by preventing a decrease in focal adhesion under high glucose conditions. Our results indicate that the DGKα pathway is an attractive therapeutic target and that activating this pathway is a novel strategy for treating DN.

A Pair of Arabidopsis Diacylglycerol Kinases Essential for Gametogenesis and Endoplasmic Reticulum Phospholipid Metabolism in Leaves and Flowers.

Phosphatidic acid (PA) is a key phospholipid in glycerolipid metabolism and signaling. Diacylglycerol kinase (DGK) produces PA by phosphorylating diacylglycerol, a crucial step in PA metabolism. Although DGK activity is known to be involved in plant development and stress response, how specific DGK isoforms function in development and phospholipid metabolism remains elusive. Here, we showed that Arabidopsis (Arabidopsis thaliana) DGK2 and DGK4 are crucial for gametogenesis and biosynthesis of phosphatidylglycerol and phosphatidylinositol in the endoplasmic reticulum (ER). With comprehensive transcriptomic data of seven DGKs and genetic crossing, we found that dgk2-1/- dgk4-1/- plants were gametophyte lethal, although parental single homozygous plants were viable. The dgk2-1/+ dgk4-1/+ double heterozygote showed defective pollen tube growth and seed development because of nonviable mutant gametes. DGK2 and DGK4 were localized to the ER and were involved in PA production for pollen tube growth. Transgenic knockdown lines of DGK2 and DGK4 confirmed the gametophyte defect and also revealed defective leaf and root growth. Glycerolipid analysis in the knockdown lines showed that phosphatidylglycerol and phosphatidylinositol metabolism was affected differently in floral buds and leaves. These results suggest that DGK2 and DGK4 are essential during gametogenesis and are required for ER-localized phospholipid metabolism in vegetative and reproductive growth.

Mechanistic models of PLC/PKC signaling implicate phosphatidic acid as a key amplifier of chemotactic gradient sensing.

Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.

Graded diacylglycerol kinases α and ζ activities ensure mucosal-associated invariant T-cell development in mice.

Mucosal-associated invariant T (MAIT) cells participate in both protective immunity and pathogenesis of diseases. Most murine MAIT cells express an invariant TCRVα19-Jα33 (iVα19) TCR, which triggers signals crucial for their development. However, signal pathways downstream of the iVα19TCR and their regulation in MAIT cells are unknown. Diacylglycerol (DAG) is a critical second messenger that relays the TCR signal to multiple downstream signaling cascades. DAG is terminated by DAG kinase (DGK)-mediated phosphorylation and conversion to phosphatidic acid. We have demonstrated here that downregulation of DAG caused by enhanced DGK activity impairs late-stage MAIT cell maturation in both thymus and spleen. Moreover, deficiency of DGKζ but not DGKα by itself causes modest decreases in MAIT cells, and deficiency of both DGKα and ζ results in severe reductions of MAIT cells in an autonomous manner. Our studies have revealed that DAG signaling is not only critical but also must be tightly regulated by DGKs for MAIT cell development and that both DGKα and, more prominently, DGKζ contribute to the overall DGK activity for MAIT cell development.

Inhibitors of DAG metabolism suppress CCR2 signalling in human monocytes.

CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. Freshly isolated human monocytes and THP-1 cells were used. Fura-2 loaded cells were used to measure intracellular Ca2+ responses. Transwell migration to measure chemotaxis. siRNA-mediated gene knock-down was used to support pharmacological approaches. CCL2 evoked intracellular Ca2+ signals and stimulated migration in THP-1 monocytic cells and human CD14+ monocytes in a CCR2-dependent fashion. Attenuation of DAG catabolism in monocytes by inhibiting DAG kinase (R59949) or DAG lipase (RHC80267) activity suppressed CCL2-evoked Ca2+ signalling and transwell migration in monocytes. These effects were not due to a reduction in the number of cell surface CCR2. The effect of inhibiting DAG kinase or DAG lipase could be mimicked by addition of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) but was not rescued by application of exogenous phosphatidylinositol 4,5-bisphosphate. Suppressive effects of R59949, RHC80267, and OAG were partially or fully reversed by Gö6983 (pan PKC isoenzyme inhibitor) but not by Gö6976 (PKCα and PKCβ inhibitor). RNAi-mediated knock-down of DAG kinase α isoenzyme modulated CCL2-evoked Ca2+ responses in THP-1 cells. Taken together, these data suggest that DAG production resulting from CCR2 activation is metabolised by both DAG kinase and DAG lipase pathways in monocytes and that pharmacological inhibition of DAG catabolism or application suppresses signalling on the CCL2-CCR2 axis via a mechanism dependent upon a PKC isoenzyme that is sensitive to Gö6983 but not Gö6976.

Dual effect of reduced type I diacylglycerol kinase activity on insulin secretion from MIN6 β-cells

The role of type I diacylglycerol kinases (DGKs) in the regulation of insulin secretion was investigated in MIN6 β-cells. In intracellular Ca2+ concentration ([Ca2+]i) measurement experiments, 1 μM R59949, a type I DGK inhibitor, and 10 μM DiC8, a diacylglycerol (DAG) analog, amplified 22.2 mM glucose-induced [Ca2+]i oscillations in a protein kinase C (PKC)-dependent manner, whereas 10 μM R59949 and 100 μM DiC8 decreased [Ca2+]i independent of PKC. High concentrations of R59949 and DiC8 attenuated voltage-dependent Ca2+ channel currents. According to these results, 22.2 mM glucose-stimulated insulin secretion (GSIS) was potentiated by 1 μM R59949 but suppressed by 10 μM of the same. The DGKα inhibitor R59022 showed a similar dual effect. Conversely, DiC8 at 10 and 100 μM potentiated GSIS, although 100 μM DiC8 decreased [Ca2+]i. These results suggest that DAG accumulated through declined type I DGK activity shows a dual effect on insulin secretion depending on the degree of accumulation; a mild DAG accumulation induces a PKC-dependent stimulatory effect on insulin secretion, whereas an excessive DAG accumulation suppresses it in a PKC-independent manner, possibly via attenuation of VDCC activity.

Chemoproteomic discovery of ligand binding hotspots in the lipid kinome

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through phosphorylation of the ubiquitous secondary messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to develop chemical probes for studying isoform‐specific functions. Here, I will discuss efforts from my group to utilize ATP acyl phosphate activity‐based probes and quantitative mass spectrometry to map previously undefined ATP‐ and small molecule‐binding sites of representative members from all subtypes of the DGK family. We use our chemoproteomics strategy to discover an unusual binding mode for the DGK‐alpha (DGKA) inhibitor ritanserin, including interactions at a novel binding site remote from the ATP binding pocket. I will also describe our efforts towards discovery of fragment leads for future development of highly potent and selective DGKA inhibitors. Collectively, our studies illustrate the power of chemoproteomics to site‐specifically profile protein‐small molecule interactions and reveal key ligand binding sites for selective inactivation of the DGK family of lipid kinases.Support or Funding InformationThis work was supported by the University of Virginia, the LaunchPad for Diabetes Program funded by the Manning Family Foundation at the University of Virginia, National Institutes of Health Grants (DA035864 and DA043571) and U.S. Department of Defense (W81XWH‐17‐1‐0487 to K.‐L.H.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Ligand Binding Landscape of Diacylglycerol Kinases

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases.Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region.Unexpectedly, deconstruction of ritanserin yielded afragment compound that blocks DGKα activitythrough a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

Diacylglycerol Kinase alpha is Involved in the Vitamin E-Induced Amelioration of Diabetic Nephropathy in Mice

Diabetic nephropathy (DN) is one of vascular complications of diabetes and is caused by abnormal protein kinase C activation as a result of increased diacylglycerol (DG) production in diabetic hyperglycaemia. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. Therefore, it is expected that the activation of DGK would ameliorate DN. Indeed, it has been reported that vitamin E (VtE) ameliorates DN in rat by activating DGK, and we recently reported that VtE specifically activates DGKα isoform in vitro. However, whether DGKα is involved in the VtE-induced amelioration of DN in vivo remains unknown. Therefore, we investigated the VtE-induced amelioration of DN in wild-type (DGKα+/+) and DGKα–deficient (DGKα−/−) mice in which diabetes was induced by streptozocin. Several symptoms of DN were ameliorated by VtE treatment in the DGKα+/+ mice but not in the DGKα−/− mice. Moreover, transmission electron microscopy of glomeruli and immunofluorescent staining of glomerular epithelial cells (podocytes) indicated that VtE ameliorates podocyte pathology and prevents podocyte loss in the DGKα+/+ mice but not in the DGKα−/− mice. We showed that VtE can ameliorate DN in mice and that DGKα is involved in the VtE-induced amelioration of DN in vivo, suggesting that DGKα is an attractive therapeutic target for DN.

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK-1-regulated functions in T cells.

Diacylglycerol kinase (DGK)-mediated consumption of the diacylglycerol (DAG) generated in response to antigen recognition is an important mechanism to limit T-cell function. Targeting DGK activity presents new opportunities for therapeutic manipulation of the immune response, but assessment of individual DGK functions is complex. T cells express two DGK isoforms, DGKα and DGKζ, and there are no isoform-specific inhibitors. Here we used short interfering RNA-mediated gene silencing in human T cells and DGKα- and DGKζ-deficient mice to define DGK isoform-specific regulation of key signaling pathways during T-cell activation. Our results identify DGKζ as the predominant brake on basal/tonic conditions as well as on downstream T-cell receptor/co-stimulatory signals. DGKζ silencing triggers basal RasGTP activation and facilitates enhanced membrane stability of protein kinase C alpha as well as increased activity of AGC kinases. Downstream of T-cell receptor/co-stimulation, DGKζ silencing results in enhanced and maintained recruitment of PKC theta to the membrane, as well as phosphoinositide-dependent protein kinase-1 activation and scaffolding functions. Our studies identify a previously unrecognized DGKζ contribution as a negative regulator of the crosstalk between phospholipase C-gamma- and phosphoinositide 3-kinase-regulated pathways. This DGKζ input helps to explain previous observations in DGK-deficient mice and suggests that the development of isoform-specific DGK inhibitors is of great interest for the manipulation of distinct aspects of T-cell responses.

Lipid metabolic changes in an early divergent fungus govern the establishment of a mutualistic symbiosis with endobacteria

The recent accumulation of newly discovered fungal-bacterial mutualisms challenges the paradigm that fungi and bacteria are natural antagonists. To understand the mechanisms that govern the establishment and maintenance over evolutionary time of mutualisms between fungi and bacteria, we studied a symbiosis of the fungus Rhizopus microsporus (Mucoromycotina) and its Burkholderia endobacteria. We found that nonhost R. microsporus, as well as other mucoralean fungi, interact antagonistically with endobacteria derived from the host and are not invaded by them. Comparison of gene expression profiles of host and nonhost fungi during interaction with endobacteria revealed dramatic changes in expression of lipid metabolic genes in the host. Analysis of the host lipidome confirmed that symbiosis establishment was accompanied by specific changes in the fungal lipid profile. Diacylglycerol kinase (DGK) activity was important for these lipid metabolic changes, as its inhibition altered the fungal lipid profile and caused a shift in the host-bacterial interaction into an antagonism. We conclude that adjustments in host lipid metabolism during symbiosis establishment, mediated by DGKs, are required for the mutualistic outcome of the Rhizopus-Burkholderia symbiosis. In addition, the neutral and phospholipid profiles of R. microsporus provide important insights into lipid metabolism in an understudied group of oleaginous Mucoromycotina. Lastly, our study revealed that the DGKs involved in the symbiosis form a previously uncharacterized clade of DGK domain proteins.