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Proceedings of the National Academy of... Aug 2023Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic...
Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.
Topics: Humans; Diglycerides; Synaptic Vesicles; Exocytosis; Synaptic Transmission; Synaptotagmins; Blister
PubMed: 37590407
DOI: 10.1073/pnas.2309516120 -
Scientific Reports Oct 2023The coronavirus disease 2019 (COVID-19), which affects multiple organs, is causing an unprecedented global public health crisis. Most COVID-19 patients recover gradually...
The coronavirus disease 2019 (COVID-19), which affects multiple organs, is causing an unprecedented global public health crisis. Most COVID-19 patients recover gradually upon appropriate interventions. Viruses were reported to utilize the small extracellular vesicles (sEVs), containing a cell-specific cargo of proteins, lipids, and nucleic acids, to escape the attack from the host's immune system. This study aimed to examine the sEVs lipid profile of plasma of recovered COVID-19 patients (RCs). Plasma sEVs were separated from 83 RCs 3 months after discharge without underlying diseases, including 18 recovered asymptomatic patients (RAs), 32 recovered moderate patients (RMs), and 33 recovered severe and critical patients (RSs), and 19 healthy controls (HCs) by Total Exosome Isolation Kit. Lipids were extracted from sEVs and then subjected to targeted liquid chromatography-mass spectrometry. The size, concentration, and distribution of sEVs did not differ in RCs and HCs as validated by transmission electron microscopy, nanoparticle tracking analysis, and immunoblot analysis. Fifteen subclasses of 508 lipids were detected in plasma sEVs from HCs, RAs, RMs, and RSs, such as phosphatidylcholines (PCs) and diacylglycerols (DAGs), etc. Total lipid intensity displayed downregulation in RCs compared with HCs. The relative abundance of DAGs gradually dropped, whereas PCs, lysophosphatidylcholines, and sphingomyelins were higher in RCs relative to HCs, especially in RSs. 88 lipids out of 241 in sEVs of RCs were significantly different and a conspicuous increase was revealed with disease status. The sEVs lipids alternations were found to be significantly correlated with the clinical indices in RCs and HCs, suggesting that the impact of COVID-19 on lipid metabolism lingered for a long time. The lipid abnormalities bore an intimate link with glycerophospholipid metabolism and glycosylphosphatidylinositol anchor biosynthesis. Furthermore, the lipidomic analysis showed that RCs were at higher risk of developing diabetes and sustaining hepatic impairment. The abnormality of immunomodulation in RCs might still exist. The study may offer new insights into the mechanism of organ dysfunction and help identify novel therapeutic targets in the RCs.
Topics: Humans; Lipid Metabolism; COVID-19; Extracellular Vesicles; Exosomes; Diglycerides
PubMed: 37789017
DOI: 10.1038/s41598-023-43189-5 -
International Journal of Molecular... Apr 2020Phosphoinositides (PI) form just a minor portion of the total phospholipid content in cells but are significantly involved in cancer development and progression. In... (Review)
Review
Phosphoinositides (PI) form just a minor portion of the total phospholipid content in cells but are significantly involved in cancer development and progression. In several cancer types, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P] play significant roles in regulating survival, proliferation, invasion, and growth of cancer cells. Phosphoinositide-specific phospholipase C (PLC) catalyze the generation of the essential second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (InsP) by hydrolyzing PtdIns(4,5)P. DAG and InsP regulate Protein Kinase C (PKC) activation and the release of calcium ions (Ca) into the cytosol, respectively. This event leads to the control of several important biological processes implicated in cancer. PLCs have been extensively studied in cancer but their regulatory roles in the oncogenic process are not fully understood. This review aims to provide up-to-date knowledge on the involvement of PLCs in cancer. We focus specifically on PLCβ, PLCγ, PLCδ, and PLCε isoforms due to the numerous evidence of their involvement in various cancer types.
Topics: Animals; Diglycerides; Humans; Neoplasms; Phosphatidylinositols; Phosphoinositide Phospholipase C; Protein Kinase C; Signal Transduction
PubMed: 32276377
DOI: 10.3390/ijms21072581 -
Journal of Lipid Research Feb 2024HDL particles vary in lipidome and proteome, which dictate their individual physicochemical properties, metabolism, and biological activities. HDL dysmetabolism in...
HDL particles vary in lipidome and proteome, which dictate their individual physicochemical properties, metabolism, and biological activities. HDL dysmetabolism in nondiabetic hypertriglyceridemia (HTG) involves subnormal HDL-cholesterol and apoAI levels. Metabolic anomalies may impact the qualitative features of both the HDL lipidome and proteome. Whether particle content of bioactive lipids and proteins may differentiate HDL subclasses (HDL2b, 2a, 3a, 3b, and 3c) in HTG is unknown. Moreover, little is known of the effect of statin treatment on the proteolipidome of hypertriglyceridemic HDL and its subclasses. Nondiabetic, obese, HTG males (n = 12) received pitavastatin calcium (4 mg/day) for 180 days in a single-phase, unblinded study. ApoB-containing lipoproteins were normalized poststatin. Individual proteolipidomes of density-defined HDL subclasses were characterized prestatin and poststatin. At baseline, dense HDL3c was distinguished by marked protein diversity and peak abundance of surface lysophospholipids, amphipathic diacylglycerol and dihydroceramide, and core cholesteryl ester and triacylglycerol, (normalized to mol phosphatidylcholine), whereas light HDL2b showed peak abundance of free cholesterol, sphingomyelin, glycosphingolipids (monohexosylceramide, dihexosylceramide, trihexosylceramide, and anionic GM3), thereby arguing for differential lipid transport and metabolism between subclasses. Poststatin, bioactive lysophospholipid (lysophosphatidylcholine, lysoalkylphosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol) cargo was preferentially depleted in HDL3c. By contrast, baseline lipidomic profiles of ceramide, dihydroceramide and related glycosphingolipids, and GM3/phosphatidylcholine were maintained across particle subclasses. All subclasses were depleted in triacylglycerol and diacylglycerol/phosphatidylcholine. The abundance of apolipoproteins CI, CII, CIV, and M diminished in the HDL proteome. Statin treatment principally impacts metabolic remodeling of the abnormal lipidome of HDL particle subclasses in nondiabetic HTG, with lesser effects on the proteome.
Topics: Male; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Proteome; Diglycerides; Lipidomics; Ceramides; Cholesterol; Hypertriglyceridemia; Hyperlipidemias; Cholesterol, HDL; Triglycerides; Phosphatidylcholines; Quinolines
PubMed: 38160756
DOI: 10.1016/j.jlr.2023.100494 -
Molecular Carcinogenesis Jun 2017Few kinases have been studied as extensively as protein kinase C (PKC), particularly in the context of cancer. As major cellular targets for the phorbol ester tumor... (Review)
Review
Few kinases have been studied as extensively as protein kinase C (PKC), particularly in the context of cancer. As major cellular targets for the phorbol ester tumor promoters and diacylglycerol (DAG), a second messenger generated by stimulation of membrane receptors, PKC isozymes play major roles in the control of signaling pathways associated with proliferation, migration, invasion, tumorigenesis, and metastasis. However, despite decades of research, fundamental questions remain to be answered or are the subject of intense controversy. Primary among these unresolved issues are the role of PKC isozymes as either tumor promoter or tumor suppressor kinases and the incomplete understanding on isozyme-specific substrates and effectors. The involvement of PKC isozymes in cancer progression needs to be reassessed in the context of specific oncogenic and tumor suppressing alterations. In addition, there are still major hurdles in addressing isozyme-specific function due to the limited specificity of most pharmacological PKC modulators and the lack of validated predictive biomarkers for response, which impacts the translation of these agents to the clinic. In this review we focus on key controversial issues and upcoming challenges, with the expectation that understanding the intricacies of PKC function will help fulfill the yet unsuccessful promise of targeting PKCs for cancer therapeutics.
Topics: Animals; Antineoplastic Agents; Diglycerides; Disease Progression; Humans; Isoenzymes; Molecular Targeted Therapy; Neoplasms; Phorbol Esters; Protein Kinase C; Substrate Specificity
PubMed: 28112438
DOI: 10.1002/mc.22617 -
Advances in Biological Regulation Jan 2015The synaptic vesicle (SV) cycle includes exocytosis of vesicles loaded with a neurotransmitter such as glutamate, coordinated recovery of SVs by endocytosis, refilling... (Review)
Review
The synaptic vesicle (SV) cycle includes exocytosis of vesicles loaded with a neurotransmitter such as glutamate, coordinated recovery of SVs by endocytosis, refilling of vesicles, and subsequent release of the refilled vesicles from the presynaptic bouton. SV exocytosis is tightly linked with endocytosis, and variations in the number of vesicles, and/or defects in the refilling of SVs, will affect the amount of neurotransmitter available for release (Sudhof, 2004). There is increasing interest in the roles synaptic vesicle lipids and lipid metabolizing enzymes play in this recycling. Initial emphasis was placed on the role of polyphosphoinositides in SV cycling as outlined in a number of reviews (Lim and Wenk, 2009; Martin, 2012; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Other lipids are now recognized to also play critical roles. For example, PLD1 (Humeau et al., 2001; Rohrbough and Broadie, 2005) and some DGKs (Miller et al., 1999; Nurrish et al., 1999) play roles in neurotransmission which is consistent with the critical roles for phosphatidic acid (PtdOH) and diacylglycerol (DAG) in the regulation of SV exo/endocytosis (Cremona et al., 1999; Exton, 1994; Huttner and Schmidt, 2000; Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). PLD generates phosphatidic acid by catalyzing the hydrolysis of phosphatidylcholine (PtdCho) and in some systems this PtdOH is de-phosphorylated to generate DAG. In contrast, DGK catalyzes the phosphorylation of DAG thereby converting it into PtdOH. While both enzymes are poised to regulate the levels of DAG and PtdOH, therefore, they both lead to the generation of PtdOH and could have opposite effects on DAG levels. This is particularly important for SV cycling as PtdOH and DAG are both needed for evoked exocytosis (Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Two lipids and their involved metabolic enzymes, two sphingolipids have also been implicated in exocytosis: sphingosine (Camoletto et al., 2009; Chan et al., 2012; Chan and Sieburth, 2012; Darios et al., 2009; Kanno et al., 2010; Rohrbough et al., 2004) and sphingosine-1-phosphate (Chan, Hu, 2012; Chan and Sieburth, 2012; Kanno et al., 2010). Finally a number of reports have focused on the somewhat less well studies roles of sphingolipids and cholesterol in SV cycling. In this report, we review the recent understanding of the roles PLDs, DGKs, and DAG lipases, as well as sphingolipids and cholesterol play in synaptic vesicle cycling.
Topics: Animals; Cholesterol; Diacylglycerol Kinase; Diglycerides; Endocytosis; Humans; Lipoprotein Lipase; Phosphatidic Acids; Phospholipase D; Sphingolipids; Synaptic Vesicles
PubMed: 25446883
DOI: 10.1016/j.jbior.2014.09.010 -
International Journal of Molecular... Jun 2020Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized... (Review)
Review
Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell-cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell-cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell-cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell-APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.
Topics: Antigen-Presenting Cells; Cell Communication; Diacylglycerol Kinase; Diglycerides; Humans; Lymphocyte Activation; Sorting Nexins; T-Lymphocytes
PubMed: 32549284
DOI: 10.3390/ijms21124254 -
Frontiers in Immunology 2022Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen...
INTRODUCTION
Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen exposure is less understood. Here, we investigated the role of diacylglycerol (DAG)-mediated signaling downstream of the TCR during chronic lymphocytic choriomeningitis virus clone 13 (LCMV CL13) infection by blocking DAG kinase zeta (DGKζ), a negative regulator of DAG.
METHODS
We examined the activation, survival, expansion, and phenotype of virus-specific T cell in the acute and chronic phases of LCMV CL13-infected in mice after DGKζ blockade or selective activation of ERK.
RESULTS
Upon LCMV CL13 infection, DGKζ deficiency promoted early short-lived effector cell (SLEC) differentiation of LCMV-specific CD8+ T cells, but this was followed by abrupt cell death. Short-term inhibition of DGKζ with ASP1570, a DGKζ-selective pharmacological inhibitor, augmented CD8+ T cell activation without causing cell death, which reduced virus titers both in the acute and chronic phases of LCMV CL13 infection. Unexpectedly, the selective enhancement of ERK, one key signaling pathway downstream of DAG, lowered viral titers and promoted expansion, survival, and a memory phenotype of LCMV-specific CD8+ T cells in the acute phase with fewer exhausted T cells in the chronic phase. The difference seen between DGKζ deficiency and selective ERK enhancement could be potentially explained by the activation of the AKT/mTOR pathway by DGKζ deficiency, since the mTOR inhibitor rapamycin rescued the abrupt cell death seen in virus-specific DGKζ KO CD8+ T cells.
DISCUSSION
Thus, while ERK is downstream of DAG signaling, the two pathways lead to distinct outcomes in the context of chronic CD8+ T cell activation, whereby DAG promotes SLEC differentiation and ERK promotes a memory phenotype.
Topics: Animals; Mice; CD8-Positive T-Lymphocytes; Diglycerides; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Receptors, Antigen, T-Cell; Signal Transduction; TOR Serine-Threonine Kinases; MAP Kinase Signaling System
PubMed: 36846018
DOI: 10.3389/fimmu.2022.1032113 -
Plant Physiology Jul 2018Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these...
Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these lipids to mediate cold responses remains unknown. The enzymes ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) and DIACYLGLYCEROL KINASE (DGK) catalyze the conversion of DAG to TAG and PA, respectively. Here, we show that DGAT1, DGK2, DGK3, and DGK5 contribute to the response to cold in Arabidopsis (). With or without cold acclimation, the mutants exhibited higher sensitivity upon freezing exposure compared with the wild type. Under cold conditions, the mutants showed reduced expression of and its regulons, which are essential for the acquisition of cold tolerance. Lipid profiling revealed that freezing significantly increased the levels of PA and DAG while decreasing TAG in the rosettes of mutant plants. During freezing stress, the accumulation of PA in plants stimulated NADPH oxidase activity and enhanced RbohD-dependent hydrogen peroxide production compared with the wild type. Moreover, the cold-inducible transcripts of , , and were significantly more up-regulated in the mutants than in the wild type during cold stress. Consistent with this observation, , , and knockout mutants showed improved tolerance and attenuated PA production in response to freezing temperatures. Our findings demonstrate that the conversion of DAG to TAG by is critical for plant freezing tolerance, acting by balancing TAG and PA production in Arabidopsis.
Topics: Arabidopsis; Arabidopsis Proteins; Cold-Shock Response; Diacylglycerol Kinase; Diacylglycerol O-Acyltransferase; Diglycerides; Freezing; Gene Expression Regulation, Plant; Gene Knockout Techniques; Hydrogen Peroxide; Mutation; NADPH Oxidases; Phosphatidic Acids; Salicylic Acid; Trans-Activators; Triglycerides
PubMed: 29853600
DOI: 10.1104/pp.18.00402 -
Arteriosclerosis, Thrombosis, and... Mar 2024Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into...
BACKGROUND
Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a).
METHODS
Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions.
RESULTS
Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1β after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68IL-18TLR4 (toll-like receptor) TREM2 (triggering receptor expressed on myeloid cells) resident macrophages and CD68CASP1 (caspase) IL-1BSELL (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]).
CONCLUSIONS
Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.
Topics: Humans; Diglycerides; Inflammasomes; Inflammation; Interleukin-1beta; Interleukin-6; Lipoprotein(a); Lysophospholipids; Monocytes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein
PubMed: 38269588
DOI: 10.1161/ATVBAHA.123.319937