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Proceedings of the National Academy of... Mar 2021Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated...
Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (L) and liquid-disordered, cholesterol-poor (L) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich L phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor L phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank L domains and enlarged L domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged L and shrank L Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)] and phosphatidylinositol 4-phosphate (PtdIns4) are present in both L and L domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5) and PtdIns4 more rapidly and produced diacylglycerol (DAG) more rapidly in L than in L Further, PtdIns(4,5) was restored more rapidly in L than in L Thus destruction and restoration of PtdIns(4,5) are faster in L than in L This suggests that L is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4 pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.
Topics: Bacterial Proteins; Cell Line, Transformed; Cholesterol; Diffusion; Diglycerides; Fluorescence Resonance Energy Transfer; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; Humans; Kinetics; Lipoylation; Luminescent Proteins; Membrane Lipids; Membrane Microdomains; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Protein Processing, Post-Translational; Receptors, Muscarinic; Type C Phospholipases; Unilamellar Liposomes
PubMed: 33619111
DOI: 10.1073/pnas.2025343118 -
Microbial Cell (Graz, Austria) Jun 2020Lipid droplets (LDs) are cellular compartments dedicated to the storage of metabolic energy in the form of neutral lipids, commonly known as "fat". The biogenesis of LDs...
Lipid droplets (LDs) are cellular compartments dedicated to the storage of metabolic energy in the form of neutral lipids, commonly known as "fat". The biogenesis of LDs takes place in the endoplasmic reticulum (ER), but its spatial and temporal organization is poorly understood. How exactly sites of LD formation are selected and the succession of proteins and lipids needed to mediate this process remains to be defined. In our current study we show that the yeast triacylglycerol (TAG)-synthases, Lro1 and Dga1 get recruited to discrete ER subdomains where they initiate TAG synthesis and hence LD formation (Choudhary (2020), J Cell Biol). These ER subdomains are defined by yeast seipin, Fld1, and a regulator of diacylglycerol (DAG) production, Nem1. Both Fld1 and Nem1 are ER proteins which localize at contact sites between the ER and LDs. Interestingly, even in cells lacking LDs, Fld1 and Nem1 show punctate localization at ER subdomains independently of each other, but they are required together to recruit the TAG-synthases and hence create functional sites of LD biogenesis. Fld1/Nem1-containing ER subdomains recruit additional LD biogenesis factors, such as Yft2, Pex30, Pet10 and Erg6, and these membrane domains become enriched in DAG. In conclusion, Fld1 and Nem1 play a crucial role in defining ER subdomains for the recruitment of proteins and lipids needed to initiate LD biogenesis.
PubMed: 32743002
DOI: 10.15698/mic2020.08.727 -
Proceedings of the National Academy of... Dec 2020Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains...
Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2-associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane -1,2-DAG-induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.
Topics: AMP-Activated Protein Kinases; Adiponectin; Adipose Tissue, White; Animals; Diet, High-Fat; Diglycerides; Insulin; Insulin Resistance; Lipid Metabolism; Lipoprotein Lipase; Liver; Male; Mice, Inbred C57BL; Muscle, Skeletal; Nitric Oxide Synthase Type III; Protein Kinase C; Recombinant Proteins
PubMed: 33293421
DOI: 10.1073/pnas.1922169117 -
Biochimica Et Biophysica Acta.... Apr 2021Diacylglycerol kinase (DGK) constitutes a family of enzymes that phosphorylate diacylglycerol to phosphatidic acid (PA). These lipids serve as second messengers, thereby... (Review)
Review
Diacylglycerol kinase (DGK) constitutes a family of enzymes that phosphorylate diacylglycerol to phosphatidic acid (PA). These lipids serve as second messengers, thereby activating distinct downstream cascades and different cellular responses. Therefore, DG-to-PA conversion activity induces a phase transition of signaling pathways. One member of the family, DGKζ, is involved closely with stress responses. Morphological data showing that DGKζ localizes predominantly to the nucleus and that it shuttles between the nucleus and the cytoplasm implicate DGKζ in the regulation of transcription factors during stress responses. Tumor suppressor p53 and NF-κB are major stress-responsive transcription factors. They exert opposing effects on cellular pathophysiology. Herein, we summarize DGKζ catalytic activity-dependent and -independent regulatory mechanisms of p53 and NF-κB transactivation activities, including p53 degradation and NF-κB nuclear translocation. We also discuss how each component of DGKζ-interacting protein complex modulates the specificity and selectivity of target gene expression.
Topics: Animals; Cell Nucleus; Diacylglycerol Kinase; Diglycerides; Humans; NF-kappa B; Phosphatidic Acids; Protein Transport; Proteolysis; Second Messenger Systems; Tumor Suppressor Protein p53
PubMed: 33450306
DOI: 10.1016/j.bbamcr.2021.118953 -
Molecules (Basel, Switzerland) Apr 2023Sea urchins () are among the most highly prized seafood products in Vietnam because of their nutritional value and medicinal properties. In this research, lipid classes...
Sea urchins () are among the most highly prized seafood products in Vietnam because of their nutritional value and medicinal properties. In this research, lipid classes and the phospholipid (PL) molecular species compositions from the body and eggs of collected in Hon Tam, Nha Trang, Khanh Hoa, Vietnam, were investigated. Hydrocarbon and wax (HW), triacylglycerol (TG), mono- and diacylglycerol (MDAG), free fatty acid (FFA), sterol (ST), polar lipid (PoL), and monoalkyl-diacylglycerol are the major lipid classes. In PL, five main glycerophospholipid classes have been identified, in which 137 PL molecular species were detected in the body and eggs of , including 20 inositol glycerophospholipids (PI), 11 serine glycerophospholipids (PS), 22 ethanolamine glycerophospholipids (PE), 11 phosphatidic acids (PA), and 73 choline glycerophospholipids (PC). PI 18:0/20:4, PS 20:1/20:1, PE 18:1e/20:4, PA 20:1/20:1, and PC 18:0e/20:4 are the most abundant species with the highest content values of 38.65-48.19%, 42.48-44.41%, 41.21-40.03%, 52.42-52.60%, and 7.77-7.18% in each class of the body-eggs, respectively. Interestingly, PL molecules predominant in the body sample were also found in the egg sample. The molecular species with the highest content account for more than 40% of the total species in each molecular class. However, in the PC class containing 73 molecular species, the highest content species amounted to only 7.77%. For both the body and egg TL samples of the sea urchin , a substantial portion of C20:4n polyunsaturated fatty acid was found in PI, PE, and PC, but C16, C18, C20, and C22 saturated fatty acids were reported at low levels. The most dominant polyunsaturated fatty acid in PI, PE, and PC was tetracosapolyenoic C20, while unsaturated fatty acid C20:1 was the most dominant in PS and PA. To our knowledge, this is the first time that the chemical properties of TL and phospholipid molecular species of the PoL of Vietnamese sea urchin () have been studied.
Topics: Animals; Diglycerides; Fatty Acids; Fatty Acids, Unsaturated; Glycerophospholipids; Phospholipids; Sea Urchins; Seafood; Vietnam
PubMed: 37175131
DOI: 10.3390/molecules28093721 -
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 -
JHEP Reports : Innovation in Hepatology Jun 2023Lipid metabolism plays an important role in liver pathophysiology. The liver lobule asymmetrically distributes oxygen and nutrition, resulting in heterogeneous metabolic...
BACKGROUND & AIMS
Lipid metabolism plays an important role in liver pathophysiology. The liver lobule asymmetrically distributes oxygen and nutrition, resulting in heterogeneous metabolic functions. Periportal and pericentral hepatocytes have different metabolic functions, which lead to generating liver zonation. We developed spatial metabolic imaging using desorption electrospray ionisation mass spectrometry to investigate lipid distribution across liver zonation with high reproducibility and accuracy.
METHODS
Fresh frozen livers from healthy mice with control diet were analysed using desorption electrospray ionisation mass spectrometry imaging. Imaging was performed at 50 μm × 50 μm pixel size. Regions of interest (ROIs) were manually created by co-registering with histological data to determine the spatial hepatic lipids across liver zonation. The ROIs were confirmed by double immunofluorescence. The mass list of specific ROIs was automatically created, and univariate and multivariate statistical analysis were performed to identify statistically significant lipids across liver zonation.
RESULTS
A wide range of lipid species was identified, including fatty acids, phospholipids, triacylglycerols, diacylglycerols, ceramides, and sphingolipids. We characterised hepatic lipid signatures in three different liver zones (periportal zone, midzone, and pericentral zone) and validated the reproducibility of our method for measuring a wide range of lipids. Fatty acids were predominantly detected in the periportal region, whereas phospholipids were distributed in both the periportal and pericentral zones. Interestingly, phosphatidylinositols, PI(36:2), PI(36:3), PI(36:4), PI(38:5), and PI(40:6) were located predominantly in the midzone (zone 2). Triacylglycerols and diacylglycerols were detected mainly in the pericentral region. triacylglycerol biosynthesis appeared to be the most influenced pathway across the three zones.
CONCLUSIONS
The ability to accurately assess zone-specific hepatic lipid distribution in the liver could lead to a better understanding of lipid metabolism during the progression of liver disease.
IMPACT AND IMPLICATIONS
Zone-specific hepatic lipid metabolism could play an important role in lipid homoeostasis during disease progression. Herein, we defined the zone-specific references of hepatic lipid species in the three liver zones using molecular imaging. The triacylglycerol biosynthesis was highlighted as the most influenced pathway across the three zones.
PubMed: 37284141
DOI: 10.1016/j.jhepr.2023.100725 -
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 -
IScience Jul 2023A sustainable and green approach was developed for the scalable synthesis of uncommon naturally occurring phospholipid species, Hemi-bis(monoacylglycero)phosphates...
A sustainable and green approach was developed for the scalable synthesis of uncommon naturally occurring phospholipid species, Hemi-bis(monoacylglycero)phosphates (Hemi-BMPs) and bis(diacylglycero)phosphates (BDPs) via the phospholipase D (PLD) mediated transphosphatidylation. PLD from . showed great substrate promiscuity for both phospholipids from different biological sources, and alcohol donors with diverse regiochemistry; monoacylglycerols with diverse fatty acyl structures (C12-C22), affording 74-92 wt% yields in 2 h. Experimental results demonstrated that the reaction rate is rather independent of phosphatidyls but to a large extent governed by the size, shape and regiolocation of fatty acyls incorporated on the glycerol backbone, particularly for the regio-isomers of bulky diacylglycerols (-1,3 or -1,2), which displays great diversity. In addition, a plausible mechanism is proposed based on molecular simulations for an elaborated explanation of the reaction thermodynamic and kinetic favorability toward the synthesis of Hemi-BMPs and BDPs.
PubMed: 37448559
DOI: 10.1016/j.isci.2023.107075