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International Journal of Molecular... Mar 2024Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell...
Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell differentiation. There are two different enzymes that take part in the direct PI synthesis: phosphatidylinositol synthase (PIS) and inositol exchange enzyme (IE). The presence of PIS is known in the human placenta, but IE activity has not been documented before. In our study, we describe the physiological properties of the two enzymes in vitro. PIS and IE were studied in different Mn and Mg concentrations that enabled us to separate the individual enzyme activities. Enzyme activity was measured by incorporation of 3[H]inositol in human primordial placenta tissue or microsomes. Optimal PIS activity was achieved between 0.5 and 2.0 mM Mn concentration, but higher concentrations inhibit enzyme activity. In the presence of Mg, the enzyme activity increases continuously up to a concentration of 100 mM. PIS was inhibited by nucleoside di- and tri-phosphates. PI production increases between 0.1 and 10 mM Mn concentration. The incorporation of [3H]inositol into PI increased by 57% when adding stabile GTP analog. The described novel pathway of inositol synthesis may provide an additional therapeutic approach of inositol supplementation before and during pregnancy.
Topics: Female; Pregnancy; Humans; Inositol; Phosphatidylinositols; CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; Transferases (Other Substituted Phosphate Groups); Placenta
PubMed: 38542419
DOI: 10.3390/ijms25063436 -
BioRxiv : the Preprint Server For... Jul 2023Sphingomyelin (SM) is a major component of mammalian cell membranes and particularly abundant in the myelin sheath that surrounds nerve fibers. Its production is...
Sphingomyelin (SM) is a major component of mammalian cell membranes and particularly abundant in the myelin sheath that surrounds nerve fibers. Its production is catalyzed by SM synthases SMS1 and SMS2, which interconvert phosphatidylcholine and ceramide to diacylglycerol and SM in the Golgi and at the plasma membrane, respectively. As the lipids participating in this reaction fulfill both structural and signaling functions, SMS enzymes have considerable potential to influence diverse important cellular processes. The nematode is an attractive model for studying both animal development and human disease. The organism contains five SMS homologues but none of these have been characterized in any detail. Here, we carried out the first systematic analysis of SMS family members in . Using heterologous expression systems, genetic ablation, metabolic labeling and lipidome analyses, we show that harbors at least three distinct SM synthases and one ceramide phosphoethanolamine (CPE) synthase. Moreover, SMS family members have partially overlapping but also unique subcellular distributions and together occupy all principal compartments of the secretory pathway. Our findings shed light on crucial aspects of sphingolipid metabolism in a valuable animal model and opens avenues for exploring the role of SM and its metabolic intermediates in organismal development.
PubMed: 37546869
DOI: 10.1101/2023.07.25.550547 -
American Journal of Physiology.... Sep 2023The endocannabinoid system (ECS) is dysregulated in various liver diseases. Previously, we had shown that the major endocannabinoid 2-arachidonoyl glycerol (2-AG)...
The endocannabinoid system (ECS) is dysregulated in various liver diseases. Previously, we had shown that the major endocannabinoid 2-arachidonoyl glycerol (2-AG) promoted tumorigenesis of intrahepatic cholangiocarcinoma (ICC). However, biosynthesis regulation and clinical significance of 2-AG remain elusive. In the present study, we quantified 2-AG by gas chromatography/mass spectrometry (GC/MS) and showed that 2-AG was enriched in patients with ICC samples as well as in thioacetamide-induced orthotopic rat ICC model. Moreover, we found that diacylglycerol lipase β (DAGLβ) was the principal synthesizing enzyme of 2-AG that significantly upregulated in ICC. DAGLβ promoted tumorigenesis and metastasis of ICC in vitro and in vivo and positively correlated with clinical stage and poor survival in patients with ICC. Functional studies showed that activator protein-1 (AP-1; heterodimers of c-Jun and FRA1) directly bound to the promoter and regulated transcription of , which can be enhanced by lipopolysaccharide (LPS). miR-4516 was identified as the tumor-suppressing miRNA of ICC that can be significantly suppressed by LPS, 2-AG, or ectopic overexpression. and were targets of miR-4516 and overexpression of miRNA-4516 significantly suppressed expression of , , and . Expression of miRNA-4516 was negatively correlated with , , and in patients with ICC samples. Our findings identify DAGLβ as the principal synthesizing enzyme of 2-AG in ICC. DAGLβ promotes oncogenesis and metastasis of ICC and is transcriptionally regulated by a novel AP-1/DAGLβ/miR4516 feedforward circuitry. Dysregulated endocannabinoid system (ECS) had been confirmed in various liver diseases. However, regulation and function of 2-arachidonoyl glycerol (2-AG) and diacylglycerol lipase β (DAGLβ) in intrahepatic cholangiocarcinoma (ICC) remain to be elucidated. Here, we demonstrated that 2-AG was enriched in ICC, and DAGLβ was the principal synthesizing enzyme of 2-AG in ICC. DAGLβ promotes tumorigenesis and metastasis in ICC via a novel activator protein-1 (AP-1)/DAGLβ/miR4516 feedforward circuitry.
Topics: Animals; Rats; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinogenesis; Cell Line, Tumor; Cholangiocarcinoma; Endocannabinoids; Glycerol; Lipopolysaccharides; Lipoprotein Lipase; MicroRNAs; Transcription Factor AP-1
PubMed: 37366545
DOI: 10.1152/ajpgi.00243.2022 -
The Biochemical Journal Dec 2023Atypical protein kinase Cs (aPKCs) are part of the PKC family of protein kinases and are atypical because they don't respond to the canonical PKC activators...
Atypical protein kinase Cs (aPKCs) are part of the PKC family of protein kinases and are atypical because they don't respond to the canonical PKC activators diacylglycerol (DAG) and Ca2+. They are central to the organization of polarized cells and are deregulated in several cancers. aPKC recruitment to the plasma membrane compartment is crucial to their encounter with substrates associated with polarizing functions. However, in contrast with other PKCs, the mechanism by which atypical PKCs are recruited there has remained elusive until recently. Here, we bring aPKC into the fold, summarizing recent reports on the direct recruitment of aPKC to membranes, providing insight into seemingly discrepant findings and integrating them with existing literature.
Topics: Protein Kinase C; Cell Membrane
PubMed: 38100320
DOI: 10.1042/BCJ20230390 -
BioRxiv : the Preprint Server For... Oct 2023Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid...
Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid modification is MprF. We recently identified that MprF synthesizes lysyl-phosphatidylglycerol (Lys-PG) from anionic PG, and a novel cationic lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), from neutral glycolipid Glc-DAG. This unexpected result prompted us to investigate whether Lys-Glc-DAG occurs in other MprF-containing bacteria, and whether other novel MprF products exist. Here, we studied protein sequence features determining MprF substrate specificity. First, pairwise analyses identified several streptococcal MprFs synthesizing Lys-Glc-DAG. Second, a restricted Boltzmann machine-guided approach led us to discover an entirely new substrate for MprF in , diglucosyl-diacylglycerol (Glc2-DAG), and an expanded set of organisms that modify glycolipid substrates using MprF. Overall, we combined the wealth of available sequence data with machine learning to model evolutionary constraints on MprF sequences across the bacterial domain, thereby identifying a novel cationic lipid.
PubMed: 37873101
DOI: 10.1101/2023.10.12.562061 -
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 -
Journal of Leukocyte Biology Jul 2023The current dogma is that chemoattractants G protein-coupled receptors activate β phospholipase C while receptor tyrosine kinases activate γ phospholipase C. Here, we...
The current dogma is that chemoattractants G protein-coupled receptors activate β phospholipase C while receptor tyrosine kinases activate γ phospholipase C. Here, we show that chemoattractant/G protein-coupled receptor-mediated membrane recruitment of γ2 phospholipase C constitutes G protein-coupled receptor-mediated phospholipase C signaling and is essential for neutrophil polarization and migration during chemotaxis. In response to a chemoattractant stimulation, cells lacking γ2 phospholipase C (plcg2kd) displayed altered dynamics of diacylglycerol production and calcium response, increased Ras/PI3K/Akt activation, elevated GSK3 phosphorylation and cofilin activation, impaired dynamics of actin polymerization, and, consequently, defects in cell polarization and migration during chemotaxis. The study reveals a molecular mechanism of membrane targeting of γ2 phospholipase C and the signaling pathways by which γ2 phospholipase C plays an essential role in neutrophil chemotaxis.
Topics: Chemotaxis; Neutrophils; Phosphatidylinositol 3-Kinases; Phospholipase C gamma; Glycogen Synthase Kinase 3; Receptors, G-Protein-Coupled; Chemotactic Factors; Type C Phospholipases
PubMed: 37040618
DOI: 10.1093/jleuko/qiad043 -
Chembiochem : a European Journal of... May 2024The process of protein transport across membranes involves a variety of factors and has been extensively investigated. Traditionally, proteinaceous translocons and... (Review)
Review
The process of protein transport across membranes involves a variety of factors and has been extensively investigated. Traditionally, proteinaceous translocons and chaperones have been recognized as crucial factors in this process. However, recent studies have highlighted the significant roles played by lipids and a glycolipid present in biological membranes in membrane protein transport. Membrane lipids can influence transport efficiency by altering the physicochemical properties of membranes. Notably, our studies have revealed that diacylglycerol (DAG) attenuates mobility in the membrane core region, leading to a dramatic suppression of membrane protein integration. Conversely, a glycolipid in Escherichia coli inner membranes, named membrane protein integrase (MPIase), enhances integration not only through the alteration of membrane properties but also via direct interactions with membrane proteins. This review explores the mechanisms of membrane protein integration mediated by membrane lipids, specifically DAG, and MPIase. Our results, along with the employed physicochemical analysis methods such as fluorescence measurements, nuclear magnetic resonance, surface plasmon resonance, and docking simulation, are presented to elucidate these mechanisms.
Topics: Glycolipids; Escherichia coli; Cell Membrane; Protein Transport; Membrane Proteins; Escherichia coli Proteins; Diglycerides
PubMed: 38400776
DOI: 10.1002/cbic.202300808 -
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