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Genetics Jul 2022Activated Gαq signals through phospholipase-Cβ and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to...
Activated Gαq signals through phospholipase-Cβ and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cβ and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cβ functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cβ, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cβ mutants. Phospholipase-Cβ mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cβ mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cβ, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.
Topics: Animals; Caenorhabditis elegans; Calcium; Diglycerides; GTP-Binding Proteins; Myristates; Neurotransmitter Agents; Phorbols; Phospholipases; Rho Guanine Nucleotide Exchange Factors; Serotonin; rho GTP-Binding Proteins
PubMed: 35579369
DOI: 10.1093/genetics/iyac084 -
Frontiers in Immunology 2023Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR...
BACKGROUND
Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.
RESULTS
Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.
CONCLUSION
We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.
Topics: Humans; Diacylglycerol Kinase; Diglycerides; Interleukin-2; Receptors, Antigen, T-Cell; Wiskott-Aldrich Syndrome Protein
PubMed: 37138877
DOI: 10.3389/fimmu.2023.1043603 -
International Journal of Molecular... Feb 2021Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role...
Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22()-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography-mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.
Topics: Animals; Base Sequence; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Diglycerides; Gene Deletion; Leydig Cells; Ligands; Lipid Droplets; Male; Mice, Inbred C57BL; Models, Biological; Phosphoproteins; Progesterone; Rats, Sprague-Dawley; Receptors, GABA; Signal Transduction; Steroids; Mice; Rats
PubMed: 33670702
DOI: 10.3390/ijms22042021 -
Molecules (Basel, Switzerland) Jul 2021Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid ubiquitously found in photosynthetically active organisms. It has attracted much attention in recent years due to...
Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid ubiquitously found in photosynthetically active organisms. It has attracted much attention in recent years due to its biological activities. Similarly, the increasing demand for vegan and functional foods has led to a growing interest in micronutrients such as sulfolipids and their physiological influence on human health. To study this influence, reference materials are needed for developing new analytical methods and providing enough material for model studies on the biological activity. However, the availability of these materials is limited by the difficulty to isolate and purify sulfolipids from natural sources and the unavailability of chemical standards on the market. Consequently, an alternative synthetic route for the comprehensive preparation of sulfolipids was established. Here, the synthesis of a sulfolipid with two identical saturated fatty acids is described exemplarily. The method opens possibilities for the preparation of a diverse range of interesting derivatives with different saturated and unsaturated fatty acids.
Topics: Diglycerides; Fatty Acids; Glycolipids; Lipids
PubMed: 34299550
DOI: 10.3390/molecules26144275 -
Cellular and Molecular Life Sciences :... Feb 2021The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the...
The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.
Topics: Cell Line, Tumor; Diacylglycerol Kinase; Diglycerides; Endocytosis; Endosomes; Enzyme Inhibitors; Golgi Apparatus; Humans; Lipidomics; Lipids; Phospholipase D; Protein Isoforms; Protein Transport; Proteolysis; Pyrimidinones; RNA Interference; RNA, Small Interfering; Ricin; Thiazoles
PubMed: 32447426
DOI: 10.1007/s00018-020-03551-6 -
International Journal of Molecular... Oct 2022Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available...
Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover () plants were pretreated with 70 μM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic overexpressing a encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the overexpression in could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the -transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.
Topics: Spermidine; Lipidomics; Phosphatidylserines; Cardiolipins; Sulfoglycosphingolipids; Diglycerides; Temperature; Trifolium; Ceramides; Phosphatidylcholines; Lysophospholipids; Phosphatidylinositols; Amino Acids; Vitamins
PubMed: 36293104
DOI: 10.3390/ijms232012247 -
Journal of Lipid Research Mar 2024Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To...
Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.
Topics: Animals; Mice; Phosphorylation; Diglycerides; Diacylglycerol Kinase
PubMed: 38272356
DOI: 10.1016/j.jlr.2024.100506 -
PeerJ 2023Olive ( L.) oil accumulate more diacylglycerols (DAG) than mostly vegetable oils. Unsaturated fatty acids-enriched DAG consumption enhanced wellness in subjects....
BACKGROUND
Olive ( L.) oil accumulate more diacylglycerols (DAG) than mostly vegetable oils. Unsaturated fatty acids-enriched DAG consumption enhanced wellness in subjects. However, the mechanism of DAG accumulation is not yet fully understood.
METHODS
In this study, gene network of DAG accumulation and fatty acid composition in the two olive mesocarps ("Chenggu 32" (CG) and "Koroneiki" (QJ)) were investigated by integrating lipidome and transcriptome techniques.
RESULTS
A total of 1,408 lipid molecules were identified by lipidomic analysis in olive mesocarp, of which DAG (DAG36:3, DAG36:4 and DAG36:5) showed higher content, and triacylglycerols (TAG54:3, TAG54:4) exhibited opposite trend in CG. Specifically, DAG was rich in polyunsaturated fatty acids (especially C18:2) at the sn-2 position, which was inconsistent with TAG at the same positions (Primarily C18:1). Transcriptomic analysis revealed that phospholipase C (NPC, EC 3.1.4.3) were up-regulated relative to QJ, whereas diacylglycerol kinase (ATP) (DGK, EC 2.7.1.107), diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), and phospholipid: diacylglycerol acyltransferase (PDAT, EC 2.3.1.158) were down-regulated.
CONCLUSION
We speculated that the non-acyl coenzyme A pathway played a significant role in DAG biosynthesis. Additionally, fatty acyl-ACP thioesterase B (FATB, EC 3.1.2.14), stearoyl [acyl-carrier-protein] 9-desaturase (SAD, EC 1.14.19.2) and omega-6 fatty acid desaturase (FAD2, EC 1.14.19.6) were highly expressed in CG and may be involved in regulating fatty acid composition. Meanwhile, phospholipase A1 (LCAT, EC 3.1.1.32) involved in the acyl editing reaction facilitated PUFA linkage at the sn-2 position of DAG. Our findings provide novel insights to increase the DAG content, improve the fatty acid composition of olive oil, and identify candidate genes for the production of DAG-rich oils.
Topics: Humans; Olea; Lipidomics; Diacylglycerol O-Acyltransferase; Diglycerides; Transcriptome; Fatty Acids; Fatty Acids, Unsaturated
PubMed: 37583911
DOI: 10.7717/peerj.15724 -
Molecular Carcinogenesis Oct 2018Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of...
Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/βII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.
Topics: Aldo-Keto Reductase Family 1 member B10; Animals; Breast Neoplasms; Cell Line, Tumor; Diglycerides; Female; HEK293 Cells; Humans; Lipogenesis; MAP Kinase Signaling System; MCF-7 Cells; Mice, Nude; Protein Kinase C; Second Messenger Systems; Transplantation, Heterologous; Tumor Burden
PubMed: 29846015
DOI: 10.1002/mc.22844 -
Proceedings of the National Academy of... Aug 2022Archaeal membrane lipids are widely used for paleotemperature reconstructions, yet these molecular fossils also bear rich information about ecology and evolution of...
Archaeal membrane lipids are widely used for paleotemperature reconstructions, yet these molecular fossils also bear rich information about ecology and evolution of marine ammonia-oxidizing archaea (AOA). Here we identified thermal and nonthermal behaviors of archaeal glycerol dialkyl glycerol tetraethers (GDGTs) by comparing the GDGT-based temperature index (TEX) to the ratio of GDGTs with two and three cyclopentane rings (GDGT-2/GDGT-3). Thermal-dependent biosynthesis should increase TEX and decrease GDGT-2/GDGT-3 when the ambient temperature increases. This presumed temperature-dependent (PTD) trend is observed in GDGTs derived from cultures of thermophilic and mesophilic AOA. The distribution of GDGTs in suspended particulate matter (SPM) and sediments collected from above the pycnocline-shallow water samples-also follows the PTD trend. These similar GDGT distributions between AOA cultures and shallow water environmental samples reflect shallow ecotypes of marine AOA. While there are currently no cultures of deep AOA clades, GDGTs derived from deep water SPM and marine sediment samples exhibit nonthermal behavior deviating from the PTD trend. The presence of deep AOA increases the GDGT-2/GDGT-3 ratio and distorts the temperature-controlled correlation between GDGT-2/GDGT-3 and TEX. We then used Gaussian mixture models to statistically characterize these diagnostic patterns of modern AOA ecology from paleo-GDGT records to infer the evolution of marine AOA from the Mid-Mesozoic to the present. Long-term GDGT-2/GDGT-3 trends suggest a suppression of today's deep water marine AOA during the Mesozoic-early Cenozoic greenhouse climates. Our analysis provides invaluable insights into the evolutionary timeline and the expansion of AOA niches associated with major oceanographic and climate changes.
Topics: Ammonia; Archaea; Diglycerides; Evolution, Molecular; Membrane Lipids; Oxidation-Reduction; Phylogeny; Water
PubMed: 35905325
DOI: 10.1073/pnas.2123193119