Differences in diacylglycerol acyltransferases expression patterns and regulation cause distinct hepatic triglyceride deposition in fish.

Triglyceride (TAG) deposition in the liver is associated with metabolic disorders. In lower vertebrate, the propensity to accumulate hepatic TAG varies widely among fish species. Diacylglycerol acyltransferases (DGAT1 and DGAT2) are major enzymes for TAG synthesis. Here we show that large yellow croaker (Larimichthys crocea) has significantly higher hepatic TAG level than that in rainbow trout (Oncorhynchus mykiss) fed with same diet. Hepatic expression of DGATs genes in croaker is markedly higher compared with trout under physiological condition. Meanwhile, DGAT1 and DGAT2 in both croaker and trout are required for TAG synthesis and lipid droplet formation in vitro. Furthermore, oleic acid treatment increases DGAT1 expression in croaker hepatocytes rather than in trout and has no significant difference in DGAT2 expression in two fish species. Finally, effects of various transcription factors on croaker and trout DGAT1 promoter are studied. We find that DGAT1 is a target gene of the transcription factor CREBH in croaker rather than in trout. Overall, hepatic expression and transcriptional regulation of DGATs display significant species differences between croaker and trout with distinct hepatic triglyceride deposition, which bring new perspectives on the use of fish models for studying hepatic TAG deposition.

The role of N-terminal phosphorylation of DGK-θ.

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.

17α-Estradiol alleviates high-fat diet-induced inflammatory and metabolic dysfunction in skeletal muscle of male and female mice.

17α-estradiol (17α-E2) is a naturally occurring nonfeminizing diastereomer of 17ß-estradiol that has life span-extending effects in rodent models. To date, studies of the systemic and tissue-specific benefits of 17α-E2 have largely focused on the liver, brain, and white adipose tissue with far less focus on skeletal muscle. Skeletal muscle has an important role in metabolic and age-related disease. Therefore, this study aimed to determine whether 17α-E2 treatment has positive, tissue-specific effects on skeletal muscle during a high-fat feeding. We hypothesized that male, but not female, mice, would benefit from 17α-E2 treatment during a high-fat diet (HFD) with changes in the mitochondrial proteome to support lipid oxidation and subsequent reductions in diacylglycerol (DAG) and ceramide content. To test this hypothesis, we used a multiomics approach to determine changes in lipotoxic lipid intermediates, metabolites, and proteins related to metabolic homeostasis. Unexpectedly, we found that 17α-E2 had marked, but different, beneficial effects within each sex. In male mice, we show that 17α-E2 alleviates HFD-induced metabolic detriments of skeletal muscle by reducing the accumulation of diacylglycerol (DAG), and inflammatory cytokine levels, and altered the abundance of most of the proteins related to lipolysis and ß-oxidation. Similar to male mice, 17α-E2 treatment reduced fat mass while protecting muscle mass in female mice but had little muscle inflammatory cytokine levels. Although female mice were resistant to HFD-induced changes in DAGs, 17α-E2 treatment induced the upregulation of six DAG species. In female mice, 17α-E2 treatment changed the relative abundance of proteins involved in lipolysis, ß-oxidation, as well as structural and contractile proteins but to a smaller extent than male mice. These data demonstrate the metabolic benefits of 17α-E2 in skeletal muscle of male and female mice and contribute to the growing literature of the use of 17α-E2 for multi tissue health span benefits.NEW & NOTEWORTHY Using a multiomics approach, we show that 17α-E2 alleviates HFD-induced metabolic detriments in skeletal muscle by altering bioactive lipid intermediates, inflammatory cytokines, and the abundance of proteins related to lipolysis and muscle contraction. The positive effects of 17α-E2 in skeletal muscle occur in both sexes but differ in their outcome.

Diacylglycerols and Lysophosphatidic Acid, Enriched on Lipoprotein(a), Contribute to Monocyte Inflammation.

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-1ß 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 CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (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.

Synthesis of azide-modified glycerophospholipid precursor analogs for detection of enzymatic reactions.

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs. In this study, we report the design, synthesis, and evaluation of novel analytical tools suitable to monitor the activity of GPL biosynthetic enzymes in vitro. Our synthetic targets, namely, azide-modified PA, azide-modified DAG, and azide-modified CDP-DAG, were successfully obtained from solketal as their common starting material. Moreover, using CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT), an enzyme that catalyzed the final reaction step in synthesizing phosphatidylinositol, we demonstrated that azide-modified CDP-DAG worked as a substrate for CDIPT.

Comparative analysis of PDZ-binding motifs in the diacylglycerol kinase family.

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.

A new method for quantifying the enzyme activity of DGKs.

Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent conversion of diacylglycerol (DAG) to phosphatidic acid (PtdOH). A common approach to examine the activity of these enzymes relys on a radiometric assay (Epand and Topham, 2007; Tu-Sekine and Raben, 2017). This assay quantifies the DGK-catalyzed incorporation of 32P into DAG from AT32P to generate 32PtdOH and is perhaps been the most widely used assay. While sensitive, its drawbacks are the expense and the potential negative impacts on health and the environment. In this report, we describe a new assay which utilizes fluorescent labeled NBD-DAG (1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-Glycero-3-diacylglycerol) to quantify the DGK-θ-catalyzed conversion of NBD-DAG to NBD-PtdOH. Furthermore, we show the assay is sufficiently sensitive as the measured specific activity was similar to that previously determined with AT32P (Tu-Sekine and Raben, 2012) and was able to detect the activation of DGK-θ by synaptotagmin-1 (Barber et al., 2022). Overall, this assay is inexpensive, sensitive, and reproducible making it an attractive alternative to currently established assays.

Diacylglycerol kinases: A look into the future of immunotherapy.

Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around 10 million deaths in 2020, with lung and colon tumors accounting for the deadliest forms of cancer. As tumor cells become resistant to traditional therapeutic approaches, immunotherapy has emerged as a novel strategy for tumor control. T lymphocytes are key players in immune responses against tumors. Immunosurveillance allows identification, targeting and later killing of cancerous cells. Nevertheless, tumors evolve through different strategies to evade the immune response and spread in a process called metastasis. The ineffectiveness of traditional strategies to control tumor growth and expansion has led to novel approaches considering modulation of T cell activation and effector functions. Program death receptor 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) showed promising results in the early 90s and nowadays are still being exploited together with other drugs for several cancer types. Other negative regulators of T cell activation are diacylglycerol kinases (DGKs) a family of enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). In T cells, DGKα and DGKζ limit the PLCγ/Ras/ERK axis thus attenuating DAG mediated signaling and T cell effector functions. Upregulation of either of both isoforms results in impaired Ras activation and anergy induction, whereas germline knockdown mice showed enhanced antitumor properties and more effective immune responses against pathogens. Here we review the mechanisms used by DGKs to ameliorate T cell activation and how inhibition could be used to reinvigorate T cell functions in cancer context. A better knowledge of the molecular mechanisms involved upon T cell activation will help to improve current therapies with DAG promoting agents.

DGKα/ζ inhibition lowers the TCR affinity threshold and potentiates antitumor immunity.

Diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG) signaling by converting DAG to phosphatidic acid, thereby suppressing pathways downstream of T cell receptor signaling. Using a dual DGKα/ζ inhibitor (DGKi), tumor-specific CD8 T cells with different affinities (TRP1high and TRP1low), and altered peptide ligands, we demonstrate that inhibition of DGKα/ζ can lower the signaling threshold for T cell priming. TRP1high and TRP1low CD8 T cells produced more effector cytokines in the presence of cognate antigen and DGKi. Effector TRP1high- and TRP1low-mediated cytolysis of tumor cells with low antigen load required antigen recognition, was mediated by interferon-γ, and augmented by DGKi. Adoptive T cell transfer into mice bearing pancreatic or melanoma tumors synergized with single-agent DGKi or DGKi and antiprogrammed cell death protein 1 (PD-1), with increased expansion of low-affinity T cells and increased cytokine production observed in tumors of treated mice. Collectively, our findings highlight DGKα/ζ as therapeutic targets for augmenting tumor-specific CD8 T cell function.

Binding of perilipin 3 to membranes containing diacylglycerol is mediated by conserved residues within its PAT domain.

Perilipins (PLINs) constitute an evolutionarily conserved family of proteins that specifically associate with the surface of lipid droplets (LDs). These proteins function in LD biogenesis and lipolysis and help to stabilize the surface of LDs. PLINs are typically composed of three different protein domains. They share an N-terminal PAT domain of unknown structure and function, a central region containing 11-mer repeats that form amphipathic helices, and a C-terminal domain that adopts a 4-helix bundle structure. How exactly these three distinct domains contribute to PLIN function remains to be determined. Here, we show that the N-terminal PAT domain of PLIN3 binds diacylglycerol (DAG), the precursor to triacylglycerol, a major storage lipid of LDs. PLIN3 and its PAT domain alone bind liposomes with micromolar affinity and PLIN3 binds artificial LDs containing low concentrations of DAG with nanomolar affinity. The PAT domain of PLIN3 is predicted to adopt an amphipathic triangular shaped structure. In silico ligand docking indicates that DAG binds to one of the highly curved regions within this domain. A conserved aspartic acid residue in the PAT domain, E86, is predicted to interact with DAG, and we found that its substitution abrogates high affinity binding of DAG as well as DAG-stimulated association with liposome and artificial LDs. These results indicate that the PAT domain of PLINs harbor specific lipid-binding properties that are important for targeting these proteins to the surface of LDs and to ER membrane domains enriched in DAG to promote LD formation.

Cadmium facilitates the formation of large lipid droplets via PLCß2-DAG-DGKε-PA signal pathway in Leydig cells.

Cadmium (Cd) exposure damages the reproductive system. Lipid droplets (LDs) play an important role in steroid-producing cells to provide raw material for steroid hormone. We have found that the LDs of Leydig cells exposed to Cd are bigger than those of normal cells, but the effects on steroidogenesis and its underlying mechanism remains unclear. Using Isobaric tag for relative and absolute quantitation (iTARQ) proteomics, phosphodiesterase beta-2 (PLCß2) was identified as the most significantly up-regulated protein in immature Leydig cells (ILCs) and adult Leydig cells (ALCs) derived from male rats exposed to maternal Cd. Consistent with high expression of PLCß2, the size of LDs was increased in Leydig cells exposed to Cd, accompanied by reduction in cholesterol and progesterone (P4) levels. However, the high PLCß2 did not result in high diacylglycerol (DAG) level, because Cd exposure up-regulated diacylglycerol kinases ε (DGKε) to promote the conversion from DAG to phosphatidic acid (PA). Exogenous PA, which was consistent with the intracellular PA concentration induced by Cd, facilitated the formation of large LDs in R2C cells, followed by reduced P4 level in the culture medium. When PLCß2 expression was knocked down, the increased DGKε caused by Cd was reversed, and then the PA level was decreased to normal. As results, large LDs returned to normal size, and the level of total cholesterol was improved to restore steroidogenesis. The accumulation of PA regulated by PLCß2-DAG-DGKε signal pathway is responsible for the formation of large LDs and insufficient steroid hormone synthesis in Leydig cells exposed to Cd. These data highlight that LD is an important target organelle for Cd-induced steroid hormone deficiency in males.

Diacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles.

Munc13-1 is essential for vesicle docking and fusion at the active zone of synapses. Here, we report that Munc13-1 self-assembles into molecular clusters within diacylglycerol-rich microdomains present in phospholipid bilayers. Although the copy number of Munc13-1 molecules in these clusters has a broad distribution, a systematic Poisson analysis shows that this is most likely the result of two molecular species: monomers and mainly hexameric oligomers. Each oligomer is able to capture one vesicle independently. Hexamers have also been observed in crystals of Munc13-1 that form between opposed phospholipid bilayers [K. Grushin, R. V. Kalyana Sundaram, C. V. Sindelar, J. E. Rothman, Proc. Natl. Acad. Sci. U.S.A. 119, e2121259119 (2022)]. Mutations targeting the contacts stabilizing the crystallographic hexagons also disrupt the isolated hexamers, suggesting they are identical. Additionally, these mutations also convert vesicle binding from a cooperative to progressive mode. Our study provides an independent approach showing that Munc13-1 can form mainly hexamers on lipid bilayers each capable of vesicle capture.

Cerebral Gray and White Matter Monogalactosyl Diglyceride Levels Rise with the Progression of Alzheimer's Disease.

BACKGROUND: Multiple studies have reported brain lipidomic abnormalities in Alzheimer's disease (AD) that affect glycerophospholipids, sphingolipids, and fatty acids. However, there is no consensus regarding the nature of these abnormalities, and it is unclear if they relate to disease progression. OBJECTIVE: Monogalactosyl diglycerides (MGDGs) are a class of lipids which have been recently detected in the human brain. We sought to measure their levels in postmortem human brain and determine if these levels correlate with the progression of the AD-related traits. METHODS: We measured MGDGs by ultrahigh performance liquid chromatography tandem mass spectrometry in postmortem dorsolateral prefrontal cortex gray matter and subcortical corona radiata white matter samples derived from three cohorts of participants: the Framingham Heart Study, the Boston University Alzheimer's Disease Research Center, and the Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program (total n = 288). RESULTS: We detected 40 molecular species of MGDGs (including diacyl and alkyl/acyl compounds) and found that the levels of 29 of them, as well as total MGDG levels, are positively associated with AD-related traits including pathologically confirmed AD diagnosis, clinical dementia rating, Braak and Braak stage, neuritic plaque score, phospho-Tau AT8 immunostaining density, levels of phospho-Tau396 and levels of Aß40. Increased MGDG levels were present in both gray and white matter, indicating that they are widespread and likely associated with myelin-producing oligodendrocytes-the principal cell type of white matter. CONCLUSIONS: Our data implicate the MGDG metabolic defect as a central correlate of clinical and pathological progression in AD.

Integrated lipidomic and transcriptomic analysis reveals diacylglycerol accumulation in olive of Longnan (China).

Background: Olive (Olea europaea 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.

Phospholipid:diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants.

BACKGROUND: Extensive population growth and climate change accelerate the search for alternative ways of plant-based biomass, biofuel and feed production. Here, we focus on hitherto unknow, new promising cold-stimulated function of phospholipid:diacylglycerol acyltransferase1 (PDAT1) - an enzyme catalyzing the last step of triacylglycerol (TAG) biosynthesis. RESULT: Overexpression of AtPDAT1 boosted seed yield by 160% in Arabidopsis plants exposed to long-term cold compared to standard conditions. Such seeds increased both their weight and acyl-lipids content. This work also elucidates PDAT1's role in leaves, which was previously unclear. Aerial parts of AtPDAT1-overexpressing plants were characterized by accelerated growth at early and vegetative stages of development and by biomass weighing three times more than control. Overexpression of PDAT1 increased the expression of SUGAR-DEPENDENT1 (SDP1) TAG lipase and enhanced lipid remodeling, driving lipid turnover and influencing biomass increment. This effect was especially pronounced in cold conditions, where the elevated synergistic expression of PDAT1 and SDP1 resulted in double biomass increase compared to standard conditions. Elevated phospholipid remodeling also enhanced autophagy flux in AtPDAT1-overexpresing lines subjected to cold, despite the overall diminished autophagy intensity in cold conditions. CONCLUSIONS: Our data suggest that PDAT1 promotes greater vitality in cold-exposed plants, stimulates their longevity and boosts oilseed oil production at low temperature.

Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation.

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

Ectocytosis renders T cell receptor signaling self-limiting at the immune synapse.

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.

EjFAD8 Enhances the Low-Temperature Tolerance of Loquat by Desaturation of Sulfoquinovosyl Diacylglycerol (SQDG).

Loquat (Eriobotrya japonica Lindl.) is an evergreen fruit tree of Chinese origin, and its autumn-winter flowering and fruiting growth habit means that its fruit development is susceptible to low-temperature stress. In a previous study, the triploid loquat (B431 × GZ23) has been identified with high photosynthetic efficiency and strong resistance under low-temperature stress. Analysis of transcriptomic and lipidomic data revealed that the fatty acid desaturase gene EjFAD8 was closely associated with low temperatures. Phenotypic observations and measurements of physiological indicators in Arabidopsis showed that overexpressing-EjFAD8 transgenic plants were significantly more tolerant to low temperatures compared to the wild-type. Heterologous overexpression of EjFAD8 enhanced some lipid metabolism genes in Arabidopsis, and the unsaturation of lipids was increased, especially for SQDG (16:0/18:1; 16:0/18:3), thereby improving the cold tolerance of transgenic lines. The expression of ICE-CBF-COR genes were further analyzed so that the relationship between fatty acid desaturase and the ICE-CBF-COR pathway can be clarified. These results revealed the important role of EjFAD8 under low-temperature stress in triploid loquat, the increase expression of FAD8 in loquat under low temperatures lead to desaturation of fatty acids. On the one hand, overexpression of EjFAD8 in Arabidopsis increased the expression of ICE-CBF-COR genes in response to low temperatures. On the other hand, upregulation of EjFAD8 at low temperatures increased fatty acid desaturation of SQDG to maintain the stability of photosynthesis under low temperatures. This study not only indicates that the EjFAD8 gene plays an important role in loquat under low temperatures, but also provides a theoretical basis for future molecular breeding of loquat for cold resistance.

Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species.

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.

A Trifolium repens flavodoxin-like quinone reductase 1 (TrFQR1) improves plant adaptability to high temperature associated with oxidative homeostasis and lipids remodeling.

Maintenance of stable mitochondrial respiratory chains could enhance adaptability to high temperature, but the potential mechanism was not elucidated clearly in plants. In this study, we identified and isolated a TrFQR1 gene encoding the flavodoxin-like quinone reductase 1 (TrFQR1) located in mitochondria of leguminous white clover (Trifolium repens). Phylogenetic analysis indicated that amino acid sequences of FQR1 in various plant species showed a high degree of similarities. Ectopic expression of TrFQR1 protected yeast (Saccharomyces cerevisiae) from heat damage and toxic levels of benzoquinone, phenanthraquinone and hydroquinone. Transgenic Arabidopsis thaliana and white clover overexpressing TrFQR1 exhibited significantly lower oxidative damage and better photosynthetic capacity and growth than wild-type in response to high-temperature stress, whereas AtFQR1-RNAi A. thaliana showed more severe oxidative damage and growth retardation under heat stress. TrFQR1-transgenic white clover also maintained better respiratory electron transport chain than wild-type plants, as manifested by significantly higher mitochondrial complex II and III activities, alternative oxidase activity, NAD(P)H content, and coenzyme Q10 content in response to heat stress. In addition, overexpression of TrFQR1 enhanced the accumulation of lipids including phosphatidylglycerol, monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and cardiolipin as important compositions of bilayers involved in dynamic membrane assembly in mitochondria or chloroplasts positively associated with heat tolerance. TrFQR1-transgenic white clover also exhibited higher lipids saturation level and phosphatidylcholine:phosphatidylethanolamine ratio, which could be beneficial to membrane stability and integrity during a prolonged period of heat stress. The current study proves that TrFQR1 is essential for heat tolerance associated with mitochondrial respiratory chain, cellular reactive oxygen species homeostasis, and lipids remodeling in plants. TrFQR1 could be selected as a key candidate marker gene to screen heat-tolerant genotypes or develop heat-tolerant crops via molecular-based breeding.