Showing papers on "Diacylglycerol kinase published in 2022"
TL;DR: In this paper, a comparison between standard and magnetic fermentation was performed using high-throughput quantitative lipidomics, and the results indicated that magnetic fermentation is an effective approach to potentially improve the nutritional of goat milk.
45 citations
TL;DR: In this article , a comparison between standard and magnetic fermentation was performed using high-throughput quantitative lipidomics, and the results indicated that magnetic fermentation is an effective approach to potentially improve the nutritional of goat milk.
41 citations
TL;DR: In this paper , a review on the structure, substrate specificities, reaction requirements, and acting mechanism of several phospholipase families is presented, highlighting their functional significance in plant growth, development, and stress responses.
27 citations
TL;DR: In this article , the authors reviewed the data on diabetic alteration of the cardiovascular system and found that the combination of these pathophysiological processes, ATP deficiency, and the development of myocardial fatty degeneration induce calcium stress, as well as dysfunction of mitochondria and endoplasmic reticulum.
23 citations
TL;DR: A review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications as mentioned in this paper .
17 citations
TL;DR: In this article, diacylglycerol (DAG) was applied to fabricate water-in-oil (W/O) HIPEs, which showed good retaining ability for anthocyanin and β-carotene.
14 citations
TL;DR: In this article , diacylglycerol (DAG) was applied to fabricate water-in-oil (W/O) HIPEs, which showed good retaining ability for anthocyanin and β-carotene.
14 citations
TL;DR: This article showed that 4 °C storage induced the occurrence of chilling injury (CI) in peach fruit by promoting the expression of membrane lipid metabolism genes and phosphatidic acid (PA) accumulation, and stimulated a protective mechanism for peach fruit against LT by increasing diacylglycerol (DAG), triacyl glycerol(TAG), and several PC components in the later storage stage.
11 citations
TL;DR: In this article , the authors used ultra-high liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) to dynamically monitor the lipid composition of hazelnut oil during accelerated storage for 24 d.
11 citations
TL;DR: Recent progress on understanding of lipid metabolism regulation in GBM to promote tumor growth and discuss novel strategies to specifically induce lipotoxicity to tumor cells through disrupting lipid storage, a promising new avenue for treating GBM are summarized.
Abstract: Glioblastoma (GBM) is the most lethal primary brain tumor. With limited therapeutic options, novel therapies are desperately needed. Recent studies have shown that GBM acquires large amounts of lipids for rapid growth through activation of sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor that regulates fatty acid and cholesterol synthesis, and cholesterol uptake. Interestingly, GBM cells divert substantial quantities of lipids into lipid droplets (LDs), a specific storage organelle for neutral lipids, to prevent lipotoxicity by increasing the expression of diacylglycerol acyltransferase 1 (DGAT1) and sterol-O-acyltransferase 1 (SOAT1), which convert excess fatty acids and cholesterol to triacylglycerol and cholesteryl esters, respectively. In this review, we will summarize recent progress on our understanding of lipid metabolism regulation in GBM to promote tumor growth and discuss novel strategies to specifically induce lipotoxicity to tumor cells through disrupting lipid storage, a promising new avenue for treating GBM.
9 citations
TL;DR: In this paper, fluorescence quenching data and thermodynamic parameters were analyzed to investigate the interaction mechanism between l -theanine (L-Th) and β-lactoglobulin (β-LG).
TL;DR: In this article , the interaction mechanism between l -theanine and β-lactoglobulin (β-LG) was investigated, and the results showed that the hydrophobic interaction played an important role on the conjugate of β-LG and L-Th due to the negative values for ΔG, positive values of ΔH and ΔS at pH 4.0, pH 6.0 and pH 8.0.
TL;DR: In this article , high-resolution crystal structures of a C1 domain (C1B) from protein kinase C (PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts were reported.
Abstract: Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.
TL;DR: It is reported that PI(4,5)P2 regulates TRPC3 in three independent modes, and an FFAT site at the TR PC3 N-terminal loop within the linker helices that envelope the C-terminus pole helix is identified.
Abstract: PI(4,5)P2, a key lipid at ER/PM junctions, has multiple roles in regulating TRPC channels, which includes recruitment of the channel to the junctions to facilitate activation by receptor stimulation, channel pore opening, and channel ionic selectivity.
TL;DR: Wang et al. as mentioned in this paper used mass spectrometry-based metabolomic and lipidomic analyses to characterize the endogenous metabolite differences among black wolfberry fruits grown in different geographical regions, from which 204 primary and specialized metabolites, and 267 lipids were identified in their fruits.
TL;DR: In this article , high-resolution crystal structures of a C1 domain (C1B) from protein kinase C (PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts were reported.
Abstract: Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.
TL;DR: Wang et al. as mentioned in this paper used mass spectrometry-based metabolomic and lipidomic analyses in black wolfberry grown in nine locations throughout five provinces in China, from which 204 primary and specialized metabolites and 267 lipids were identified in their fruits.
TL;DR: The fatty acid side‐chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.
Abstract: Phosphoinositide lipids (PPIn) are enriched in stearic‐ and arachidonic acids (38:4) but how this enrichment is established and maintained during phospholipase C (PLC) activation is unknown. Here we show that the metabolic fate of newly synthesized phosphatidic acid (PA), the lipid precursor of phosphatidylinositol (PI), is influenced by the fatty acyl‐CoA used with preferential routing of the arachidonoyl‐enriched species toward PI synthesis. Furthermore, during agonist stimulation the unsaturated forms of PI(4,5P)2 are replenished significantly faster than the more saturated ones, suggesting a favored recycling of the unsaturated forms of the PLC‐generated hydrolytic products. Cytidine diphosphate diacylglycerol synthase 2 (CDS2) but not CDS1 was found to contribute to increased PI resynthesis during PLC activation. Lastly, while the lipid transfer protein, Nir2 is found to contribute to rapid PPIn resynthesis during PLC activation, the faster re‐synthesis of the 38:4 species does not depend on Nir2. Therefore, the fatty acid side‐chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.
TL;DR: In this paper , the effect of PDAT overexpression on lipid metabolism in oilseed rape (Brassica napus) seeds was investigated and it was shown that despite severalfold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG.
Abstract: The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.
TL;DR: In this article , a wide spectrum of DAG signals from an integrated perspective could underscore meaningful advances in targeted cancer therapy and enable integrated therapy of tumors and immune cells, thus representing powerful modifiers of immune checkpoint and adoptive T cell-directed immunotherapy.
Abstract: Diacylglycerol (DAG) is a lipid second messenger that is generated in response to extracellular stimuli and channels intracellular signals that affect mammalian cell proliferation, survival, and motility. DAG exerts a myriad of biological functions through protein kinase C (PKC) and other effectors, such as protein kinase D (PKD) isozymes and small GTPase-regulating proteins (such as RasGRPs). Imbalances in the fine-tuned homeostasis between DAG generation by phospholipase C (PLC) enzymes and termination by DAG kinases (DGKs), as well as dysregulation in the activity or abundance of DAG effectors, have been widely associated with tumor initiation, progression, and metastasis. DAG is also a key orchestrator of T cell function and thus plays a major role in tumor immunosurveillance. In addition, DAG pathways shape the tumor ecosystem by arbitrating the complex, dynamic interaction between cancer cells and the immune landscape, hence representing powerful modifiers of immune checkpoint and adoptive T cell–directed immunotherapy. Exploiting the wide spectrum of DAG signals from an integrated perspective could underscore meaningful advances in targeted cancer therapy. Description Targeting the lipid DAG may enable integrated therapy of tumors and immune cells.
TL;DR: In this article , two chloroplast-localized FA exporters (FAX1 and FAX2) and one endoplasmic reticulum (ER) localized FA transporter (ABCA2) were co-overexpressed in Chlamydomonas.
Abstract: Chloroplast and endoplasmic reticulum (ER)-localized fatty acid (FA) transporters have been reported to play important roles in oil (mainly triacylglycerols, TAG) biosynthesis. However, whether these FA transporters synergistically contribute to lipid accumulation, and their effect on lipid metabolism in microalgae are unknown.Here, we co-overexpressed two chloroplast-localized FA exporters (FAX1 and FAX2) and one ER-localized FA transporter (ABCA2) in Chlamydomonas. Under standard growth conditions, FAX1/FAX2/ABCA2 over-expression lines (OE) accumulated up to twofold more TAG than the parental strain UVM4, and the total amounts of major polyunsaturated FAs (PUFA) in TAG increased by 4.7-fold. In parallel, the total FA contents and major membrane lipids in FAX1/FAX2/ABCA2-OE also significantly increased compared with those in the control lines. Additionally, the total accumulation contribution ratio of PUFA, to total FA and TAG synthesis in FAX1/FAX2/ABCA2-OE, was 54% and 40% higher than that in UVM4, respectively. Consistently, the expression levels of genes directly involved in TAG synthesis, such as type-II diacylglycerol acyltransferases (DGTT1, DGTT3 and DGTT5), and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), significantly increased, and the expression of PGD1 (MGDG-specific lipase) was upregulated in FAX1/FAX2/ABCA2-OE compared to UVM4.These results indicate that the increased expression of FAX1/FAX2/ABCA2 has an additive effect on enhancing TAG, total FA and membrane lipid accumulation and accelerates the PUFA remobilization from membrane lipids to TAG by fine-tuning the key genes involved in lipid metabolism under standard growth conditions. Overall, FAX1/FAX2/ABCA2-OE shows better traits for lipid accumulation than the parental line and previously reported individual FA transporter-OE. Our study provides a potential useful strategy to increase the production of FA-derived energy-rich and value-added compounds in microalgae.
TL;DR: In this paper , the authors characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.
Abstract: Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.
TL;DR: It is shown that adeno‐associated viral vector delivery of a modified and FMRP‐independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS‐relevant behavioral phenotypes in the Fmr1‐KO mouse.
Abstract: Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA‐binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1‐KO mouse model. Here we show that adeno‐associated viral vector delivery of a modified and FMRP‐independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS‐relevant behavioral phenotypes in the Fmr1‐KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS.
TL;DR: It is concluded that the NoPDAT pathway is parallel to and independent of the NoDGAT pathway for oil production, and can serve as an alternative carbon sink for photosynthetically assimilated carbon in N. oceanica when PDAT-mediated TAG biosynthesis is compromised or under stress in the presence of high CO2 levels.
Abstract: Triacylglycerols (TAGs) are the main storage lipids in photosynthetic organisms under stress. In the oleaginous alga Nannochloropsis oceanica, while multiple acyl CoA:diacylglycerol acyltransferases (NoDGATs) are involved in TAG production, the role of the unique phospholipid:diacylglycerol acyltransferase (NoPDAT) remains unknown. Here, we performed a functional complementation assay in TAG-deficient yeast (Saccharomyces cerevisiae) and an in vitro assay to probe the acyltransferase activity of NoPDAT. Subcellular localization, overexpression, and knockdown experiments were also conducted to elucidate the role of NoPDAT in N. oceanica. NoPDAT, residing at the outermost plastid membrane, does not phylogenetically fall into the clades of algae or plants and uses phosphatidylethanolamine (PE) and phosphatidylglycerol with 16:0, 16:1, and 18:1 at position sn-2 as acyl-donors in vivo. NoPDAT knockdown, not triggering any compensatory mechanism via DGATs, led to an ∼30% decrease of TAG content, accompanied by a vast accumulation of PEs rich in 16:0, 16:1, and 18:1 fatty acids (referred to as "LU-PE") that was positively associated with CO2 availability. We conclude that the NoPDAT pathway is parallel to and independent of the NoDGAT pathway for oil production. LU-PE can serve as an alternative carbon sink for photosynthetically assimilated carbon in N. oceanica when PDAT-mediated TAG biosynthesis is compromised or under stress in the presence of high CO2 levels.
25 Feb 2022
TL;DR: In this paper , transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation, aiming to identify the target genes that could be used in the biotechnological production of high-value oils.
Abstract: Vernonia galamensis native to Africa is an annual oleaginous plant of Asteraceae family. As a newly established industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), which is an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation, aiming to identify the target genes that could be used in the biotechnological production of high-value oils.Based on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates for each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferases, and phospholipid:diacylglycerol acyltransferases. Expression profiling revealed that various genes associated with acyl editing, fatty acid β-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which were consistent with the fast accumulation of EFA in V. galamensis developing seed, these genes were detected to play fundamental roles in EFA production. In addition, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient expression of the selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established.Our findings provide new insights into molecular mechanisms underlying the natural epoxy oil production in V. galamensis. A set of genes identified here could be used as the targets to develop other oilseeds highly accumulating valued epoxy oils for commercial production.
TL;DR: The ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C‐DAG pathway is demonstrated.
Abstract: Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca2+ signaling function. Single particle cryo‐EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure‐guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG‐probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C‐DAG pathway.
TL;DR: In this paper , the authors found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT 1 in a yeast microsome assay.
Abstract: Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1-expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1-overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content.
TL;DR: In this paper , the authors found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis DGAT 1 and soybean DG 1 in a yeast microsome assay.
Abstract: Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the DGAT activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis DGAT1 and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1 expressing lines showed a significantly higher oil content (49% mean increase compared to wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1 over-expressing lines did not show a significant increase. In addition to these four DGAT1s, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content more than 10 times than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were performed by site-directed mutagenesis and substantially increased the TAG content.
TL;DR: In this article , C. reinhardtii cells (wild-type CC124 and cyclic electron transport dependant mutants pgrl1 and pgr5) were grown photoheterotrophically in high light 500 μmol photons m-2 s-1, where Pgr5 growth was retarded due to an increase in reactive oxygen species (ROS).
Abstract: Chlamydomonas (C.) reinhardtii is a potential microalga for lipid production. Autophagy-triggered lipid metabolism in microalgae has not being studied so far from a mutant of proton gradient regulation 1 like (PGRL1) and proton gradient regulation 5 (PGR5). In this study, C. reinhardtii cells (wild-type CC124 and cyclic electron transport dependant mutants pgrl1 and pgr5) were grown photoheterotrophically in high light 500 μmol photons m-2 s-1, where pgr5 growth was retarded due to an increase in reactive oxygen species (ROS). The lipid contents were increased; however, carbohydrate content was decreased in pgr5. Further, the Nile Red (NR) fluorescence shows many lipid bodies in pgr5 cells under high light. Similarly, the electron micrographs show that large vacuoles were formed in high light stress despite the grana stacks structure. We also observed increased production of reactive oxygen species, which could be one reason the cells underwent autophagy. Further, a significant increase of autophagy ATG8 and detections of ATG8-PE protein was noticed in pgr5, a hallmark characteristic for autophagy formation. Consequently, the triacylglycerol (TAG) content was increased due to diacylglycerol acyltransferases (DGAT) and phospholipid diacylglycerol acyl-transference (PDAT) enzymes' expression, especially in pgr5. Here the TAG synthesis would have been obtained from degraded membrane lipids in pgr5. Additionally, mono, polyunsaturated, and saturated fatty acids were identified more in the high light condition. Our study shows that the increased light induces the reactive oxygen species, which leads to autophagy and TAG accumulation. Therefore, the enhanced accumulation of TAGs can be used as feedstock for biodiesel production and aqua feed.
TL;DR: The results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.
Abstract: Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ50-DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ50-DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.