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Phosphatidic acid (PA) is involved in biotic and abiotic stress responses in plants. Here, we summarize quantitative lipidomics and real-time imaging used in PA studies and highlight recent studies of diacylglycerol (DAG) kinase (DGK) 5, an enzyme involved in PA biosynthesis, facilitating fine-tuning PA production for optimal stress responses in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In plant models such as Arabidopsis thaliana, phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development and nutrition. In this article, we highlight lipid signaling existing in crop species. Based on open access databases, we update the list of sequences encoding phospholipases D, phosphoinositide-dependent phospholipases C, and diacylglycerol-kinases, enzymes that lead to the production of PA. We show that structural features of these enzymes from model plants are conserved in equivalent proteins from selected crop species. We then present an in-depth discussion of the structural characteristics of these proteins before focusing on PA binding proteins. For the purpose of this article, we consider RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), the most documented PA target proteins. Finally, we present pioneering experiments that show, by different approaches such as monitoring of gene expression, use of pharmacological agents, ectopic over-expression of genes, and the creation of silenced mutants, that lipid signaling plays major roles in crop species. Finally, we present major open questions that require attention since we have only a perception of the peak of the iceberg when it comes to the exciting field of phospholipid signaling in plants. [ABSTRACT FROM AUTHOR]

It is generally recognized that the main function of α-tocopherol (αToc), which is the most active form of vitamin E, is its antioxidant effect, while non-antioxidant effects have also been reported. We previously found that αToc ameliorates diabetic nephropathy via diacylglycerol kinase alpha (DGKα) activation in vivo, and the activation was not related to the antioxidant effect. However, the underlying mechanism of how αToc activates DGKα have been enigmatic. We report that the membrane-bound 67 kDa laminin receptor (67LR), which has previously been shown to serve as a receptor for epigallocatechin gallate (EGCG), also contains a novel binding site for vitamin E, and its association with Vitamin E mediates DGKα activation by αToc. We employed hydrogen-deuterium exchange mass spectrometry (HDX/MS) and molecular dynamics (MD) simulations to identify the specific binding site of αToc on the 67LR and discovered the conformation of the specific hydrophobic pocket that accommodates αToc. Also, HDX/MS and MD simulations demonstrated the detailed binding of EGCG to a water-exposed hydrophilic site on 67LR, while in contrast αToc binds to a distinct hydrophobic site. We demonstrated that 67LR triggers an important signaling pathway mediating non-antioxidant effects of αToc, such as DGKα activation. This is the first evidence demonstrating a membrane receptor for αToc and one of the underlying mechanisms of a non-antioxidant function for αToc.

Background: Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. Methods: Immunohistochemistry (IHC) with anti‐DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (−, 1+, 2+, 3+) and divided into two groups (DGKα− and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki‐67 expression was evaluated as a cell proliferation marker. The number of Ki‐67‐positive cells was counted, and the relationship with DGKα expression was examined. Results: DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα− group (−: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence‐free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki‐67‐positive cells increased in association with the staining levels of DGKα. Conclusion: Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC. [ABSTRACT FROM AUTHOR]

Diacylglycerol kinase (DGK) is a lipid kinase that phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Based on converting one important signaling molecule (DAG) to another (PA), DGK plays an important role in plant responses to abiotic stress, including waterlogging stress. However, no studies have been reported on the characterization of the DGK gene family in the waterlogging-tolerant kiwifruit germplasm Actinidia valvata Dunn. In this study, we identified 18 AvDGK genes in the A. valvata genome. The phylogenetic analysis showed that AvDGKs can be classified into three clusters, and members within the same cluster have similar domain distributions, exon-intron structures, and conserved motif compositions. The chromosome localization analysis revealed that all AvDGK genes are located across 18 different chromosomes. There were 29 duplicated gene pairs in A. valvata and all had undergone purifying selection during evolution. The promoter cis-element analysis revealed that the cis-elements within AvDGK genes are associated with multiple functions, including phytohormone signal transduction, stress responses, and plant growth and development. The expression pattern analyses indicated that AvDGKs play important roles in fruit development and plant responses to waterlogging stress. The AvDGK gene family in the tetraploid A. valvata genome might promote PA synthesis and subsequent signal transduction both under short- and long-term waterlogging stresses. These results provide information regarding the structural characteristics and potential function of AvDGK genes within A. valvata and lay a fundamental basis for further research into breeding to enhance the kiwifruit's tolerance to waterlogging stress. [ABSTRACT FROM AUTHOR]

This review conteins updated information on the structure, localization and regulation of phosphatidic acid (PA)-producing enzymes phospholipase D, phosphoinositide-specific and non-specific phospholipases C and diacylglycerol kinases is analyzed. The specific role of PA and PA-producing enzymes in plant stress signaling is discussed.

Expression of α‐smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFβ‐induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second‐messenger DG, thereby controlling components of DG‐mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFβ stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFβ stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFβ stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα‐knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively.

Diacylglycerol kinases (DGKs) control local and temporal amounts of diacylglycerol (DAG) and phosphatidic acid (PA) by converting DAG to PA through phosphorylation in cells. Certain DGK enzymes possess C‐terminal sequences that encode potential PDZ‐binding motifs (PBMs), which could be involved in their recruitment into supramolecular signaling complexes. In this study, we used two different interactomic approaches, quantitative native holdup (nHU) and qualitative affinity purification (AP), both coupled to mass spectrometry (MS) to investigate the PDZ partners associated with the potential PBMs of DGKs. Complementing these results with site‐specific affinity interactomic data measured on isolated PDZ domain fragments and PBM motifs, as well as evolutionary conservation analysis of the PBMs of DGKs, we explored functional differences within different DGK groups. All our results indicate that putative PBM sequences of type II enzymes, namely DGKδ, DGKη, and DGKκ, are likely to be nonfunctional. In contrast, type IV enzymes, namely DGKζ and DGKι, possess highly promiscuous PBMs that interact with a set of PDZ proteins with very similar affinity interactomes. The combination of various interactomic assays and evolutionary analyses provides a useful strategy for identifying functional domains and motifs within diverse enzyme families. [ABSTRACT FROM AUTHOR]

Lipid phosphorylation by diacylglycerol kinase (DGK) that produces phosphatidic acid (PA) plays important roles in various biological processes, including stress responses, but the underlying mechanisms remain elusive. Here, we show that DGK5 and its lipid product PA suppress ABA biosynthesis by interacting with ABA-DEFICIENT 2 (ABA2), a key ABA biosynthesis enzyme, to negatively modulate plant response to abiotic stress tested in Arabidopsis thaliana. Loss of DGK5 function rendered plants less damaged, whereas overexpression (OE) of DGK5 enhanced plant damage to water and salt stress. The dgk5 mutant plants exhibited decreased total cellular and nuclear levels of PA with increased levels of diacylglycerol, whereas DGK5 -OE plants displayed the opposite effect. Interestingly, we found that both DGK5 and PA bind to the ABA-synthesizing enzyme ABA2 and suppress its enzymatic activity. Consistently, the dgk5 mutant plants exhibited increased levels of ABA, while DGK5 -OE plants showed reduced ABA levels. In addition, we showed that both DGK5 and ABA2 are detected in and outside the nuclei, and loss of DGK5 function decreased the nuclear association of ABA2. We found that both DGK5 activity and PA promote nuclear association of ABA2. Taken together, these results indicate that both DGK5 and PA interact with ABA2 to inhibit its enzymatic activity and promote its nuclear sequestration, thereby suppressing ABA production in response to abiotic stress. Our study reveals a sophisticated mechanism by which DGK5 and PA regulate plant stress responses. Diacylglycerol kinase (DGK) produces phosphatidic acid (PA), and both DGK and PA play important roles in stress responses. This study shows that DGK5 and PA bind to ABA-DEFICIENT 2 and suppress abscisic acid synthesis by inhibiting its enzymatic activity and promoting its nuclear sequestration. The results indicate that lipid phosphorylation interacts with abscisic acid production to mediate plant response to abiotic stress. [ABSTRACT FROM AUTHOR]

Expression of α‐smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFβ‐induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second‐messenger DG, thereby controlling components of DG‐mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFβ stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFβ stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFβ stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα‐knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively. [ABSTRACT FROM AUTHOR]

Abstract Background Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. Methods Immunohistochemistry (IHC) with anti‐DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (−, 1+, 2+, 3+) and divided into two groups (DGKα− and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki‐67 expression was evaluated as a cell proliferation marker. The number of Ki‐67‐positive cells was counted, and the relationship with DGKα expression was examined. Results DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα− group (−: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence‐free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki‐67‐positive cells increased in association with the staining levels of DGKα. Conclusion Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC.

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG‐generating sphingomyelin synthase (SMS)‐related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N‐terminal cytosolic region of SMSr binds to the N‐terminal half catalytic domain of DGKζ. However, the C‐terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.

Abstract Background Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). Methods We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. Results DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. Conclusions Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

The sleep state is widely observed in animals. The molecular mechanisms underlying sleep regulation, however, remain largely unclear. In the nematode Caenorhabditis elegans, developmentally timed sleep (DTS) and stress-induced sleep (SIS) are 2 types of quiescent behaviors that fulfill the definition of sleep and share conserved sleep-regulating molecules with mammals. To identify novel sleep-regulating molecules, we conducted an unbiased forward genetic screen based on DTS phenotypes. We isolated 2 mutants, rem8 and rem10, that exhibited significantly disrupted DTS and SIS. The causal gene of the abnormal sleep phenotypes in both mutants was mapped to dgk-1, which encodes diacylglycerol kinase. Perhaps due to the diminished SIS, dgk-1 mutant worms exhibited decreased survival following exposure to a noxious stimulus. Pan-neuronal and/or cholinergic expression of dgk-1 partly rescued the dgk-1 mutant defects in DTS, SIS, and post-stress survival. Moreover, we revealed that pkc-1/nPKC participates in sleep regulation and counteracts the effect of dgk-1; the reduced DTS, SIS, and post-stress survival rate were partly suppressed in the pkc-1; dgk-1 double mutant compared with the dgk-1 single mutant. Excessive sleep observed in the pkc-1 mutant was also suppressed in the pkc-1; dgk-1 double mutant, implying that dgk-1 has a complicated mode of action. Our findings indicate that neuronal DGK-1 is essential for normal sleep and that the counterbalance between DGK-1 and PKC-1 is crucial for regulating sleep and mitigating post-stress damage. [ABSTRACT FROM AUTHOR]

Sleep is a fundamental state of behavioral quiescence and physiological restoration. Sleep is controlled by environmental conditions, indicating a complex regulation of sleep by multiple processes. Our knowledge of the genes and mechanisms that control sleep during various conditions is, however, still incomplete. In Caenorhabditis elegans, sleep is increased when development is arrested upon starvation. Here, we performed a reverse genetic sleep screen in arrested L1 larvae for genes that are associated with metabolism. We found over 100 genes that are associated with a reduced sleep phenotype. Enrichment analysis revealed sphingolipid metabolism as a key pathway that controls sleep. A strong sleep loss was caused by the loss of function of the diacylglycerol kinase 1 gene, dgk-1, a negative regulator of synaptic transmission. Rescue experiments indicated that dgk-1 is required for sleep in cholinergic and tyraminergic neurons. The Ring Interneuron S (RIS) neuron is crucial for sleep in C. elegans and activates to induce sleep. RIS activation transients were abolished in dgk-1 mutant animals. Calcium transients were partially rescued by a reduction-of-function mutation of unc-13, suggesting that dgk-1 might be required for RIS activation by limiting synaptic vesicle release. dgk-1 mutant animals had impaired L1 arrest survival and dampened expression of the protective heat shock factor gene hsp-12.6. These data suggest that dgk-1 impairment causes broad physiological deficits. Microcalorimetry and metabolomic analyses of larvae with impaired RIS showed that RIS is broadly required for energy conservation and metabolic control, including for the presence of sphingolipids. Our data support the notion that metabolism broadly influences sleep and that sleep is associated with profound metabolic changes. We thus provide novel insights into the interplay of lipids and sleep and provide a rich resource of mutants and metabolic pathways for future sleep studies. [ABSTRACT FROM AUTHOR]

In plant models such as Arabidopsis thaliana, phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development and nutrition. In this article, we highlight lipid signaling existing in crop species. Based on open access databases, we update the list of sequences encoding phospholipases D, phosphoinositide-dependent phospholipases C, and diacylglycerol-kinases, enzymes that lead to the production of PA. We show that structural features of these enzymes from model plants are conserved in equivalent proteins from selected crop species. We then present an in-depth discussion of the structural characteristics of these proteins before focusing on PA binding proteins. For the purpose of this article, we consider RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), the most documented PA target proteins. Finally, we present pioneering experiments that show, by different approaches such as monitoring of gene expression, use of pharmacological agents, ectopic over-expression of genes, and the creation of silenced mutants, that lipid signaling plays major roles in crop species. Finally, we present major open questions that require attention since we have only a perception of the peak of the iceberg when it comes to the exciting field of phospholipid signaling in plants.

Diacylglycerol kinase (DGK) is a lipid kinase that phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Based on converting one important signaling molecule (DAG) to another (PA), DGK plays an important role in plant responses to abiotic stress, including waterlogging stress. However, no studies have been reported on the characterization of the DGK gene family in the waterlogging-tolerant kiwifruit germplasm Actinidia valvata Dunn. In this study, we identified 18 AvDGK genes in the A. valvata genome. The phylogenetic analysis showed that AvDGKs can be classified into three clusters, and members within the same cluster have similar domain distributions, exon-intron structures, and conserved motif compositions. The chromosome localization analysis revealed that all AvDGK genes are located across 18 different chromosomes. There were 29 duplicated gene pairs in A. valvata and all had undergone purifying selection during evolution. The promoter cis-element analysis revealed that the cis-elements within AvDGK genes are associated with multiple functions, including phytohormone signal transduction, stress responses, and plant growth and development. The expression pattern analyses indicated that AvDGKs play important roles in fruit development and plant responses to waterlogging stress. The AvDGK gene family in the tetraploid A. valvata genome might promote PA synthesis and subsequent signal transduction both under short- and long-term waterlogging stresses. These results provide information regarding the structural characteristics and potential function of AvDGK genes within A. valvata and lay a fundamental basis for further research into breeding to enhance the kiwifruit’s tolerance to waterlogging stress.

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG‐generating sphingomyelin synthase (SMS)‐related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N‐terminal cytosolic region of SMSr binds to the N‐terminal half catalytic domain of DGKζ. However, the C‐terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways. [ABSTRACT FROM AUTHOR]

Background: Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). Methods: We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. Results: DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. Conclusions: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae is widely used for producing various triterpenes by exploiting its native mevalonate/ergosterol pathway. Yeasts that accumulate phospholipids can produce more triterpenes. Our recent study demonstrated that a high phospholipid-accumulating yeast phenotype, as in spt10Δ yeast, results in increased endoplasmic reticulum (ER) biogenesis, resulting in ER expansion. However, the spt10Δ strain also exhibits high reticulophagy. Dgk1 (diacylglycerol kinase) is an important enzyme in lipid metabolism, which synthesizes phosphatidic acid (PA) by phosphorylating diacylglycerol (DG). We demonstrate that spt10Δ yeast with increased Dgk1 activity offer two desired results, (i) a highly expanded ER, due to redirection of the lipid pathway away from triglycerides towards phospholipid synthesis, increasing ER biogenesis; and (ii) decreased reticulophagy, by increasing the PA pool that activates TOR complex-mediated autophagy suppression. It was speculated that more ER-bound pathway enzymes can fit in the expanded ER, and the mevalonate-ergosterol pathway, being ER bound, might have higher activity. This was demonstrated by the co-expression of Dgk1 and plant triterpene synthase in spt10Δ yeast, which shows a high accumulation of plant triterpene.

Although recent studies have indicated that mutations in the gene encoding diacylglycerol kinase epsilon (DGKE) result in some proteinuria related hereditary kidney diseases, the DGKE expression pattern in the kidney and its contribution to acute kidney injury (AKI) remain unknown. Therefore, the present study was designed to detect the role of DGKE in mice with AKI. DGKE expression was time-dependently altered in the kidneys of mice with renal ischemia/reperfusion injury (IRI). Compared with wild-type (WT) mice, DGKE- overexpressing mice (Rosa26-Dgke+/+) exhibited protective effects against renal IRI, including reduced serum creatinine, blood urea concentration, tubular cell death and inflammatory responses as well as improved morphological injuries. Consistently, in vitro, DGKE overexpression in human renal proximal tubule (HK-2) cells also protected against oxygen-glucose deprivation (OGD)/reoxygenation-induced cell death. Mechanistically, DGKE regulated Klotho expression, at least partly via the transcription factor Krüppel-like factor (KLF) 15. Moreover, a significant reduction in DGKE was also found in kidneys from patients with ischemia-associated acute tubular necrosis (ATN). Collectively, our studies demonstrate that DGKE protects against AKI in mice at least partly through KLF15/Klotho signaling pathway, indicating that DGKE may present an innovative therapeutic strategy for treating patients with AKI.

Effective amelioration of type II diabetes requires therapies that increase both glucose uptake activity per cell and skeletal muscle mass. Myristic acid (14:0) increases diacylglycerol kinase (DGK) δ protein levels and enhances glucose uptake in myotubes in a DGKδ‐dependent manner. However, it is still unclear whether myristic acid treatment affects skeletal muscle mass. In this study, we found that myristic acid treatment increased the protein level of β‐tubulin, which constitutes microtubules and is closely related to muscle mass, in C2C12 myotubes but not in the proliferation stage in C2C12 myoblasts. However, lauric (12:0), palmitic (16:0) and oleic (18:1) acids failed to affect DGKδ and β‐tubulin protein levels in C2C12 myotubes. Moreover, knockdown of DGKδ by siRNA significantly inhibited the increased protein level of β‐tubulin in the presence of myristic acid, suggesting that the increase in β‐tubulin protein by myristic acid depends on DGKδ. These results indicate that myristic acid selectively affects β‐tubulin protein levels in C2C12 myotubes via DGKδ, suggesting that this fatty acid improves skeletal muscle mass in addition to increasing glucose uptake activity per cell.

Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (TH2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks TH2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell-specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.

Although recent studies have indicated that mutations in the gene encoding diacylglycerol kinase epsilon (DGKE) result in some proteinuria related hereditary kidney diseases, the DGKE expression pattern in the kidney and its contribution to acute kidney injury (AKI) remain unknown. Therefore, the present study was designed to detect the role of DGKE in mice with AKI. DGKE expression was time-dependently altered in the kidneys of mice with renal ischemia/reperfusion injury (IRI). Compared with wild-type (WT) mice, DGKE- overexpressing mice (Rosa26-Dgke+/+) exhibited protective effects against renal IRI, including reduced serum creatinine, blood urea concentration, tubular cell death and inflammatory responses as well as improved morphological injuries. Consistently, in vitro, DGKE overexpression in human renal proximal tubule (HK-2) cells also protected against oxygen-glucose deprivation (OGD)/reoxygenation-induced cell death. Mechanistically, DGKE regulated Klotho expression, at least partly via the transcription factor Krüppel-like factor (KLF) 15. Moreover, a significant reduction in DGKE was also found in kidneys from patients with ischemia-associated acute tubular necrosis (ATN). Collectively, our studies demonstrate that DGKE protects against AKI in mice at least partly through KLF15/Klotho signaling pathway, indicating that DGKE may present an innovative therapeutic strategy for treating patients with AKI. [ABSTRACT FROM AUTHOR]

Abstract Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA‐binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1‐KO mouse model. Here we show that adeno‐associated viral vector delivery of a modified and FMRP‐independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS‐relevant behavioral phenotypes in the Fmr1‐KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS.

Phospholipase C (PLC) β1 hydrolyzes 1‐stearoyl‐2‐arachidonoyl (18:0/20:4)‐phosphatidylinositol (PtdIns) 4,5‐bisphosphate to produce diacylglycerol, which is converted to phosphatidic acid (PtdOH), in the PtdIns cycle and plays pivotal roles in intracellular signal transduction. The present study identified PLCβ1 as a PtdOH‐binding protein using PtdOH‐containing liposomes. Moreover, the comparison of the binding of PLCβ1 to various PtdOH species, including 14:0/14:0‐PtdOH, 16:0/16:0‐PtdOH, 16:0/18:1‐PtdOH, 18:0/18:1‐PtdOH, 18:0/18:0‐PtdOH, 18:1/18:1‐PtdOH, 18:0/20:4‐PtdOH, and 18:0/22:6‐PtdOH, indicated that the interaction of PLCβ1 with 16:0/16:0‐PtdOH was the strongest. The PLCβ1‐binding activity of 18:0/18:0‐PtdOH was almost the same as the binding activity of 16:0/16:0‐PtdOH. Furthermore, the binding of PLCβ1 to 16:0/16:0‐PtdOH was substantially stronger than 16:0/16:0‐phosphatidylserine, 16:0/16:0/16:0/16:0‐cardiolipin, 16:0/16:0‐PtdIns, and 18:0/20:4‐PtdIns. We revealed that a PLCβ1 mutant whose Lys946 and Lys951 residues were replaced with Glu (PLCβ1‐KE) did not interact with 16:0/16:0‐PtdOH and failed to localize to the plasma membrane in Neuro‐2a cells. Retinoic acid‐dependent increase in neurite length and numbers was significantly inhibited in PLCβ1‐expressing cells; however, this considerable attenuation was not detected in the cells expressing PLCβ1‐KE. Overall, these results strongly suggest that PtdOHs containing only saturated fatty acids, including 16:0/16:0‐PtdOH, which are not derived from the PtdIns cycle, selectively bind to PLCβ1 and regulate its function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Airway remodeling in asthma involves the hyperproliferation of airway smooth muscle (ASM) cells. However, the molecular signals that regulate ASM growth are not completely understood. Gq-coupled G protein-coupled receptor and receptor tyrosine kinase signaling regulate ASM cell proliferation via activation of phospholipase C, generation of inositol triphosphate (IP3) and diacylglycerol (DAG). Diacylglycerol kinase (DGK) converts DAG into phosphatidic acid (PA) and terminates DAG signaling while promoting PA-mediated signaling and function. Herein, we hypothesized that PA is a pro-mitogenic second messenger in ASM, and DGK inhibition reduces the conversion of DAG into PA resulting in inhibition of ASM cell proliferation. We assessed the effect of pharmacological inhibition of DGK on pro-mitogenic signaling and proliferation in primary human ASM cells. Pretreatment with DGK inhibitor I (DGKI) significantly inhibited platelet-derived growth factor-stimulated ASM cell proliferation. Anti-mitogenic effect of DGKI was associated with decreased mTOR signaling and expression of cyclin D1. Exogenous PA promoted pro-mitogenic signaling and rescued DGKI-induced attenuation of ASM cell proliferation. Finally, house dust mite (HDM) challenge in wild type mice promoted airway remodeling features, which were attenuated in DGKζ-/- mice. We propose that DGK serves as a potential drug target for mitigating airway remodeling in asthma. [ABSTRACT FROM AUTHOR]

Effective amelioration of type II diabetes requires therapies that increase both glucose uptake activity per cell and skeletal muscle mass. Myristic acid (14:0) increases diacylglycerol kinase (DGK) δ protein levels and enhances glucose uptake in myotubes in a DGKδ‐dependent manner. However, it is still unclear whether myristic acid treatment affects skeletal muscle mass. In this study, we found that myristic acid treatment increased the protein level of β‐tubulin, which constitutes microtubules and is closely related to muscle mass, in C2C12 myotubes but not in the proliferation stage in C2C12 myoblasts. However, lauric (12:0), palmitic (16:0) and oleic (18:1) acids failed to affect DGKδ and β‐tubulin protein levels in C2C12 myotubes. Moreover, knockdown of DGKδ by siRNA significantly inhibited the increased protein level of β‐tubulin in the presence of myristic acid, suggesting that the increase in β‐tubulin protein by myristic acid depends on DGKδ. These results indicate that myristic acid selectively affects β‐tubulin protein levels in C2C12 myotubes via DGKδ, suggesting that this fatty acid improves skeletal muscle mass in addition to increasing glucose uptake activity per cell. [ABSTRACT FROM AUTHOR]

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprise ten isozymes (α-κ) that regulate a wide variety of physiological and pathological events. Recently, we revealed that DGK isozymes use saturated fatty acid (SFA)/monosaturated fatty acid (MUFA)-containing and docosahexaenoic acid (22:6)-containing DG species, but not phosphatidylinositol (PI) turnover-derived 18:0/20:4-DG. For example, DGKδ, which is involved in the pathogenesis of type 2 diabetes, preferentially uses SFA/MUFA-containing DG species, such as 16:0/16:0- and 16:0/18:1-DG species, in high glucose-stimulated skeletal muscle cells. Moreover, DGKδ, which destabilizes the serotonin transporter (SERT) and regulates the serotonergic system in the brain, primarily generates 18:0/22:6-PA. Furthermore, 16:0/16:0-PA is produced by DGKζ in Neuro-2a cells during neuronal differentiation. We searched for SFA/MUFA-PA- and 18:0/22:6-PA-selective binding proteins (candidate downstream targets of DGKδ) and found that SFA/MUFA-PA binds to and activates the creatine kinase muscle type, an energy-metabolizing enzyme, and that 18:0/22:6-PA interacts with and activates Praja-1, an E3 ubiquitin ligase acting on SERT, and synaptojanin-1, a key player in the synaptic vesicle cycle. Next, we searched for SFA/MUFA-DG-generating enzymes upstream of DGKδ. We found that sphingomyelin synthase (SMS)1, SMS2, and SMS-related protein (SMSr) commonly act as phosphatidylcholine (PC)-phospholipase C (PLC) and phosphatidylethanolamine (PE)-PLC, generating SFA/MUFA-DG species, in addition to SMS and ceramide phosphoethanolamine synthase. Moreover, the orphan phosphatase PHOSPHO1 showed PC- and PE-PLC activities that produced SFA/MUFA-DG. Although PC- and PE-PLC activities were first described 70-35 years ago, their proteins and genes were not identified for a long time. We found that DGKδ interacts with SMSr and PHOSPHO1, and that DGKζ binds to SMS1 and SMSr. Taken together, these results strongly suggest that there are previously unrecognized signal transduction pathways that include DGK isozymes and generate and utilize SFA/MUFA-DG/PA or 18:0/22:6-DG/PA but not PI-turnover-derived 18:0/20:4-DG/PA., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Atypical hemolytic uremic syndrome (aHUS) is a rare, mostly complement-mediated thrombotic microangiopathy. The majority of patients are infants. In contrast to infantile-onset aHUS, the clinical and genetic characteristics of adolescence-onset aHUS have not been sufficiently addressed to date., Methods: A total of 28 patients (21 girls, 7 boys) who were diagnosed as aHUS between the ages of ≥10 years and <18 years were included in this study. All available data in the Turkish Pediatric aHUS registry were collected and analyzed., Results: The mean age at diagnosis was 12.8±2.3 years. Extra-renal involvement was noted in 13 patients (46.4%); neurological involvement was the most common (32%). A total of 21 patients (75%) required kidney replacement therapy. Five patients (17.8%) received only plasma therapy and 23 (82%) of the patients received eculizumab. Hematologic remission and renal remission were achieved in 25 (89.3%) and 17 (60.7%) of the patients, respectively. Compared with the infantile-onset aHUS patients, adolescent patients had a lower complete remission rate during the first episode (p = 0.002). Genetic analyses were performed in all and a genetic variant was detected in 39.3% of the patients. The mean follow-up duration was 4.9±2.6 years. At the last visit, adolescent patients had lower eGFR levels (p = 0.03) and higher rates of chronic kidney disease stage 5 when compared to infantile-onset aHUS patients (p = 0.04)., Conclusions: Adolescence-onset aHUS is a rare disease but tends to cause more permanent renal dysfunction than infantile-onset aHUS. These results may modify the management approaches in these patients., (© 2024. The Author(s), under exclusive licence to Japanese Society of Nephrology.)

Simple Summary: Store-operated Ca2+ entry (SOCE) has long been known to regulate the differentiation and effector functions of T cells as well as to be instrumental to the ability of cytotoxic T lymphocytes to target cancer cells. Currently, no information is available regarding the expression and function of SOCE in tumour-infiltrating lymphocytes (TILs) that have been expanded in vitro for adoptive cell therapy (ACT). This study provides the first evidence that SOCE is up-regulated in ex vivo-expanded TILs from metastatic colorectal cancer (mCRC) patients. The up-regulation of SOCE mainly depends on diacylglycerol kinase (DGK), which prevents the protein kinase C-dependent inhibition of Ca2+ entry in normal T cells. Of note, the pharmacological blockade of SOCE with the selective inhibitor, BTP-2, during target cell killing significantly increases cytotoxic activity at low TIL density, i.e., when TILs-mediated cancer cell death is rarer. This study, albeit preliminary, could lay the foundation to propose an alternative strategy to effect ACT. It has been shown that ex vivo-expanded TILs did not improve the disease-free survival rate in mCRC patients. Our results strongly suggest that pre-treating autologous TILs with a SOCE or DGK inhibitor before being infused into the patient could improve their cytotoxic activity against cancer cells. (1) Background: Store-operated Ca2+ entry (SOCE) drives the cytotoxic activity of cytotoxic T lymphocytes (CTLs) against cancer cells. However, SOCE can be enhanced in cancer cells due to an increase in the expression and/or function of its underlying molecular components, i.e., STIM1 and Orai1. Herein, we evaluated the SOCE expression and function in tumour-infiltrating lymphocytes (TILs) from metastatic colorectal cancer (mCRC) patients. (2) Methods: Functional studies were conducted in TILs expanded ex vivo from CRC liver metastases. Peripheral blood T cells from healthy donors (hPBTs) and mCRC patients (cPBTs) were used as controls. (3) Results: SOCE amplitude is enhanced in TILs compared to hPBTs and cPBTs, but the STIM1 protein is only up-regulated in TILs. Pharmacological manipulation showed that the increase in SOCE mainly depends on tonic modulation by diacylglycerol kinase, which prevents the protein kinase C-dependent inhibition of SOCE activity. The larger SOCE caused a stronger Ca2+ response to T-cell receptor stimulation by autologous mCRC cells. Reducing Ca2+ influx with BTP-2 during target cell killing significantly increases cytotoxic activity at low target:effector ratios. (4) Conclusions: SOCE is enhanced in ex vivo-expanded TILs deriving from mCRC patients but decreasing Ca2+ influx with BTP-2 increases cytotoxic activity at a low TIL density. [ABSTRACT FROM AUTHOR]

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.

Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA‐binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1‐KO mouse model. Here we show that adeno‐associated viral vector delivery of a modified and FMRP‐independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS‐relevant behavioral phenotypes in the Fmr1‐KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS. Synopsis: Brain neuron‐restricted expression of an FMRP‐independent DGKk enzyme via intra‐cerebral adeno‐associated virus (AAV) gene therapy corrects phosphatidic acid deficiency and rescues disease relevant behavioral phenotypic alterations in a mouse model of Fragile X syndrome (FXS). The expression of diacylglycerol kinase kappa (DGKk), a favored mRNA target of FMRP, is reduced in the FXS brain in both humans and mice.∆N‐DGKk is a truncated active enzyme whose synthesis bypasses the need for FMRP.AAV‐Rh10‐∆N‐DGKk enables the synthesis of active DGKk enzyme in neurons lacking FMRP.∆N‐DGKk neuronal expression has no adverse effect in wild type control mice and is associated with normal behavioral functions of the rescued Fmr1‐null mice. [ABSTRACT FROM AUTHOR]

Knockout mice of diacylglycerol kinase (DGK) η, which has been repeatedly suggested to be associated with bipolar disorder (BPD) by genome-wide association studies, exhibited abnormal behaviors similar to the manic phase of BPD. Chronic stress is also linked to changes in mood symptoms, including BPD. In the present study, we analyzed the effects of the glucocorticoid stress hormones, triamcinolone acetonide (TAA) and dexamethasone (DEX), on DGKη protein levels in neuroblastoma cell lines, Neuro-2a and SH-SY5Y. The protein levels of DGKη were significantly increased in the undifferentiated Neuro-2a and SH-SY5Y cells by TAA and DEX, but not in the differentiated neuroblastoma cells. To assess the functions of DGKη in undifferentiated neuroblastoma cells, we established DGKη-deficient SH-SY5Y cells using the clustered regularly interspaced palindromic repeat/caspase 9 system. Notably, proliferation of DGKη-deficient SH-SY5Y cells was markedly attenuated, concomitant with the decrease in levels of phosphorylated extracellular signal-regulated kinase. Taken together, these results suggest that DGKη levels are increased in undifferentiated neuroblastoma cells by glucocorticoid stress hormones and regulate cell proliferation. • Diacylglycerol kinase (DGK) η is a key enzyme in bipolar disorder (BPD) pathogenesis • BPD is associated with abnormalities in the stress hormone-generating HPA axis • DGKη levels were increased by stress hormones in undifferentiated neuroblastoma cells • DGKη-KO attenuated the proliferation of undifferentiated neuroblastoma cells • DGKη-KO decreased the levels of phosphorylated ERK. [ABSTRACT FROM AUTHOR]

Cells sense a variety of extracellular signals balancing their metabolism and physiology according to changing growth conditions. Plasma membranes are the outermost informational barriers that render cells sensitive to regulatory inputs. Membranes are composed of different types of lipids that play not only structural but also informational roles. Hormones and other regulators are sensed by specific receptors leading to the activation of lipid metabolizing enzymes. These enzymes generate lipid second messengers. Among them, phosphatidic acid (PA) is a well-known intracellular messenger that regulates various cellular processes. This lipid affects the functional properties of cell membranes and binds to specific target proteins leading to either genomic (affecting transcriptome) or non-genomic responses. The subsequent biochemical, cellular and physiological reactions regulate plant growth, development and stress tolerance. In the present review, we focus on primary (genome-independent) signaling events triggered by rapid PA accumulation in plant cells and describe the functional role of PA in mediating response to hormones and hormone-like regulators. The contributions of individual lipid signaling enzymes to the formation of PA by specific stimuli are also discussed. We provide an overview of the current state of knowledge and future perspectives needed to decipher the mode of action of PA in the regulation of cell functions. [ABSTRACT FROM AUTHOR]

Summary: Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear.We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)‐localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant.Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase‐dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5‐formed PA regulates phosphatidylinositol 4‐phosphate 5‐kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5‐bisphosphate levels and suppressed related growth phenotypes.We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5‐kinases. [ABSTRACT FROM AUTHOR]

Objectives: Our study aimed to better understand the different thermal adaptation in Mucor irregularis (M. irregularis) strains under high temperature and the involved virulence‐related genes, and to offer more appropriate explanation for the diverse pathogenicity of M. irregularis in human infections. Methods: M. irregularis isolates were incubated at 30 and 35°C for Illumina HiSeq technology (RNA‐seq), as well as the virulence difference detected through Galleria mellonella infection models. We verified their transcriptional profile with RT‐PCR and analysed differentially expressed genes with GO and KEGG annotations. Results: All 25 isolates formed the biggest colonies at 28°C and did not grow at 37°C, while were differently inhibited at 22 and 35°C. Six selected M. irregularis displayed virulence in sync with their growth condition at high temperature. From the outcomes of RNA‐seq, a total of 1559 differentially expressed genes (FC ≥ 2, FDR < 0.05) were obtained, of which 1021 genes were upregulated, and 538 genes were downregulated. Cell wall structure genes related to Ras‐like and GH16 proteins, influx‐efflux pumps consist of transmembrane proteins as ABC and MFS proteins, and metabolic genes as DGKɛ and Hsfs, seem to be essential in thermal adaptation and virulence of M. irregularis. Conclusion: We found some common genes expressed at high temperature, while some others specifically related to M. irregularis isolates with different virulence and thermal adaptation. Further research of genes involved in the pathogenic process is needed for the development of potential targeted antifungal. [ABSTRACT FROM AUTHOR]

PurposeImpaired glucose metabolism-related genetic variants likely interact with obesity-modifiable factors in response to glucose intolerance, yet their interconnected pathways have not been fully characterized.MethodsWith data from 1,027 postmenopausal participants of the Genomics and Randomized Trials Network study and 15 single-nucleotide polymorphisms (SNPs) associated with glucose homeostasis, we assessed whether obesity, physical activity, and high dietary fat intake interact with the SNP-glucose variations. We used regression analysis plus stratification and graphic approaches.ResultsAcross carriers of the 15 SNPs, fasting levels of glucose, insulin, and homeostatic model assessment-insulin resistance (HOMA-IR) were higher in obese, inactive, and high fat-diet women than in their respective counterparts. Carriers within subgroups differently demonstrated the direction and/or magnitude of the variants' effect on glucose-relevant traits. Variants in GCKR, GCK, DGKB/TMEM195 (P for interactions = 0.02, 0.02, and 0.01), especially, showed interactions with obesity: obese, inactive, and high fat-diet women had greater increases in fasting glucose, insulin, and HOMA-IR levels. Obese carriers at TCF7L2 variant had greater increases in fasting glucose levels than nonobese carriers (P for interaction = 0.04), whereas active women had greater decreases in insulin and HOMA-IR levels than inactive women (P for interaction = 0.02 in both levels).ConclusionsOur data support the important role of obesity in modifying glucose homeostasis in response to glucose metabolism-relevant variants. These findings may inform research on the role of glucose homeostasis in the etiology of chronic disease and the development of intervention strategies to reduce risk in postmenopausal women.

BackgroundWe examined risks associated with joint exposure of gene variants and pesticides.MethodsAnalyses included 189 cases and 390 male controls born from 1991 to 2003 in California's San Joaquin Valley. We used logistic regression to examine risks associated with joint exposures of gene variants and pesticides that our previous work identified as associated with hypospadias. Genetic variables were based on variants in DGKK, genes involved in sex steroid synthesis/metabolism, and genes involved in genital tubercle development. Pesticide exposure was based on residential proximity to commercial agricultural pesticide applications.ResultsOdds ratios (ORs) were highest among babies with joint exposures, who had two- to fourfold increased risks; for example, the OR was 3.7 (95% confidence interval [CI], 0.8-16.5) among subjects with the risk-associated DGKK haplotype and pesticide exposure; OR, 1.5 (95% CI, 0.7-3.1) among subjects with the haplotype and no pesticide exposure; and OR, 0.9 (95% CI, 0.5-1.6) among subjects without the haplotype but with pesticide exposure, relative to subjects with neither. However, results did not provide statistical evidence that these risks were significantly greater than expected on an additive scale, relative to risks associated with one exposure at a time.ConclusionWe observed elevated risks associated with joint exposures to selected pesticides and genetic variants but no statistical evidence for interaction. Birth Defects Research (Part A) 106:653-658, 2016. © 2016 Wiley Periodicals, Inc.

Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

Diacylglycerol (DG) is unique in lipid metabolism because it serves not only as an intermediate product for triglyceride synthesis, but also as a signaling molecule that activates proteins containing DG-responsive elements, such as protein kinase C. Consequently, DG acts as a hub between energy metabolism and intracellular signaling. Of DG metabolizing pathways, DG kinase (DGK) phosphorylates DG to produce phosphatidic acid, which also serves as a second messenger. Several lines of evidence suggest that DGK is deeply involved in metabolic diseases such as obesity and insulin resistance. Of DGK isozymes, DGKε is simplest in terms of structure, but it is characterized by substrate specificity toward arachidonoyl-DG. Recently, we have reported that DGKε deficiency promotes adipose tissue remodeling in mice during the course of high fat diet (HFD) feeding regimen including obesity, insulin resistance, and beige adipogenesis. DGKε ablation engenders altered expression of other lipid metabolizing enzymes, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and diacylglycerol acyltransferase (DGAT). Subcellular localization of DGKε in the endoplasmic reticulum suggests involvement of this isozyme in lipid energy homeostasis. This review presents current findings of DGKε in lipid-orchestrated pathophysiology, especially unique phenotypes of DGKε-knockout mice in the early and late stages of obesogenic conditions. [ABSTRACT FROM AUTHOR]

The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2), which is known to associate with AP180. An AP180 N-terminal homology domain (1–289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38–K39–K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P 2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages. [Display omitted] • Clathrin coat assembly protein AP180 binds to 18:0/22:6-phosphatidic acid (PA). • The Lys-rich motif in ANTH is essential for 18:0/22:6-PA binding. • 18:0/22:6-PA inhibits AP180-clathrin assembly. • 18:0/22:6-PA is an effective regulator of the disassembly of clathrin from CME cages. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors causes airway hyperresponsiveness in asthma. Activation of Gq-coupled G protein-coupled receptors leads to phospholipase C (PLC)- mediated generation of inositol triphosphate (IP3) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKz and a isoform knockout mice are protected from the development of allergen-induced airway hyperresponsiveness. Here we aimed to determine the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKa and z, whereas pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP3, and calcium levels were assessed. Furthermore, we used precision-cut human lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholineinduced bronchoconstriction. DGKa and z knockdown or pretreatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and MLC phosphatase in ASM cells. Furthermore, DGK inhibition decreased Gq agonist-induced calcium elevation and generation of IP3 and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio, resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction. [ABSTRACT FROM AUTHOR]