Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes ...Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.展开更多
Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understan...Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg<sup>2+</sup>-dependent soluble PAP and Mg<sup>2+</sup>-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg<sup>2+</sup>-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the V<sub>max</sub> and K<sub>m</sub> values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.展开更多
Phosphatidate phosphatase (EC 3.1.3.4), PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycero...Phosphatidate phosphatase (EC 3.1.3.4), PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol. A Mg2+ independent PAP activity was identified in the soluble extract of Momordica charantia cotyledons undergoing maturation. While the microsomal fraction of the extract gave only 10% of the PAP activity, the remaining 90% of the activity was associated with the soluble fraction. At pH 3.0, the soluble PAP was bound to S column and eluted with glycine-HCl buffer containing high salt. The pH and temperature optima of the PAP activity were 6.0 and 53℃, respectively. Under optimum assay condition, the Vmax and Km for dioleoyl phosphatidic acid were 1.89 ηkat/mg of protein and 142 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ- NPP, the Vmax and Km were 10.4 ηkat/mg of protein and 107 μM, respectively. The inclusion of Mg2+ and β-mercaptoethanol into the reaction mix did not change the enzyme activity nor did the addition of N-ethylmaleimide and phenylglyoxal, which indicates that cysteine and arginine are not involved in catalysis of PtdOH. The addition of Mg2+ up to 10 mM also did not change the level of PAP activity. Triton X-100, however, inhibited the activity. This is the first documented case of an in vitro PAP activity in the developing cotyledons of Momordica charantia. The PAP described here could serve as a model for lipin-1 or lipin-2 in humans. Mutations in these genes lead to acute myoglobinuria in human infants.展开更多
Cancer Stem Cells(CSCs)are cancer cells with self-renewal and tumorigenesis abilities.CSCs in tumor tissues are the leading cause of tumor progression,recurrence,and drug resistance.CSCs have distinct metabolic featur...Cancer Stem Cells(CSCs)are cancer cells with self-renewal and tumorigenesis abilities.CSCs in tumor tissues are the leading cause of tumor progression,recurrence,and drug resistance.CSCs have distinct metabolic features that contribute to maintaining their self-renewal and stemness.Phospholipids are essential components of cell membranes and play fundamental roles in cellular activities.CSCs have abnormal phospholipid metabolism,which affects their self-renewal,differentiation,invasion,and drug resistance.Compared with non-CSCs,the phospholipid metabolism of CSCs mainly focused on significantly increased fatty acid(FAs)and phospholipids synthesis,phospho-lipid unsaturation,and lipolysis-fatty acid oxidation(FAO).In brief,FA and phospholipid metabolism in the anabolic and catabolic pathways are strictly regulated in CSCs to maintain self-renewal and stemness activity.In this review,we summarize the alterations in phospholipid metabolism in CSCs and their impacts on the stemness of CSCs,and we put forward the potential applications of targeting phospholipid metabolism for CSCs,to provide directions for the development of drugs targeting the phospholipid metabolism.展开更多
Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in...Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5?C and 45?C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 ηkat/mg of protein and 200 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ-NPP, the Vmax and Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of tryp-sinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.展开更多
Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthopho...Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthophosphate. In eukaryotes, the PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG). Existing methods for measuring PAP activity rely on the use of radioactive PtdOH. These methods are costly and cumbersome. In this report, we describe a simple assay procedure to measure released inorganic orthophosphate, which is a coproduct of the PAP reaction. Each molecule of PtdOH would release one molecule of DAG and one molecule of inorganic orthophosphate (Pi) when subjected to enzymatic breakdown under optimal conditions. Given the published rates of in vitro PAP enzymatic activity from various sources, we proposed that colorimetric determination of released Pi is possible. With this view, we performed in vitro PAP activity assays using freshly isolated enzyme from bitter gourd, Momordica charantia, and measured the released Pi using two spectrophotometric methods. Both methods gave about 2.0 to 2.25 ηkat per mg of protein. Thus, it is now possible to perform PAP activity using a simple procedure that uses nonradioactive substrates, provided the sample is dialyzed extensively to lower the intrinsic concentration of free phosphate. The kinetics data presented in this study is comparable to that of other PAP enzymes reported elsewhere, which gives credence to the notion that non-radioactive methods can be used to perform PAP activity.展开更多
CIN85 （Cbl-interacting protein of 85 kDa） is an important molecule involved in receptor tyrosine kinase endocytosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic l...CIN85 （Cbl-interacting protein of 85 kDa） is an important molecule involved in receptor tyrosine kinase endocytosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic lipid - phosphatidic acid. Its coiled-coil domain plays an important role in mediating this protein-lipid interaction. Deletion of the coiled-coil domain results in loss of membrane association, and reduced interaction with c-cbl, finally causing the blockage of epidermal growth factor receptor downregulation. In addition, a significant portion of CIN85 is located on the endosomal compartment and is related to endocytic cargo sorting, characterized by CIN85＇s localization on the ＂E class＂ compartment and EGF degradation blockage in CIN85 knockdown cells. Taken together, our results suggest that CIN85 may function as a scaffold molecule in both the internalization and endocytic cargo sorting processes through its association with the endosomal membrane.展开更多
Cells regulate phospholipase D (PLD) activity in response to numerous extracellular signals. Here, we investigated the involvement of PLD activity in transforming growth factor-β(TGF-β1)-mediated growth inhibition o...Cells regulate phospholipase D (PLD) activity in response to numerous extracellular signals. Here, we investigated the involvement of PLD activity in transforming growth factor-β(TGF-β1)-mediated growth inhibition of epithelial cells. TGFβ1 inhibits the growth of MDCK, Mv1Lu, and A-549 cells. In the presence of 0.4 % butanol, TGF-β1 induces an increase in the formation of phosp hat idylbutanol, a unique product catalyzed by PLD. TGF-β1 also induces an increase in phosphatidic acid (PA) level in A-549 and MDCK cells. TGF-β1 induces an increase in the levels of DAG labeled with [~3H]-myristic acid in A-549 and MDCK cells but not in Mv1Lu cells- No increase of DAG was observed in cells prelabeled with [~3H]-arachidonic acid.The data presented suggest that PLD activation is involved in the TGF-β1-induced cell growth inhibition.展开更多
The Heilongjiang Soybean Research Institute not only focuses on improving yield and quality of soybean, but also exploring the development of various high quality soybean byproducts. Currently, the institute uses adva...The Heilongjiang Soybean Research Institute not only focuses on improving yield and quality of soybean, but also exploring the development of various high quality soybean byproducts. Currently, the institute uses advanced extraction technology, the institute also produces new soybean chips and vitamin E from soybean pulp, soybean peptides, phosphatides and oligosaccharides. 120 broilers were randomly divided into 4 groups with 6 replicates in each group and 5 chicken in each replicate. The authors studied influence on performance and body quality of broiler chicken by using soybean phosphatides to take the place of 0.5%, 1% and 1.5% corn of basal daily grain. Conclusion indicated that rates of broiler chicken weight gain were 2.1%, 4.4% and 8.7%, feed utilization rates raised 3.5%, 5.2% and 8.1%, costs reduced 2.3%, 3.5% and 5.8%, chest muscle rates improved 14.7%, 0.9% and -0.49%, belly fat rates improved 11.06%, 20.28% and 44.75% by using soybean phosphatides to take place of corn in daily grain after 42 days. More recently, the study is also involved in the research on improving the meat quality of chicken by adding extracted soybean peptide and phosphatides into feed. Furthermore, nearly 98% post-consumer waste oil with high acid value can be converted into biodiesel by using an effective supercritical methanol method.展开更多
Phosphatidic acid(PA)is an important class of signaling lipids involved in various biological processes in plants.Functional characterization of mutants of PA-metabolizing enzymes,combined with lipidomics and protein...Phosphatidic acid(PA)is an important class of signaling lipids involved in various biological processes in plants.Functional characterization of mutants of PA-metabolizing enzymes,combined with lipidomics and protein–lipid interaction analyses,has revealed the key role of PA signaling in plant responses to biotic and abiotic stresses.Moreover,PA and its metabolizing enzymes influence several reproductive processes,including gametogenesis,pollen tube growth,self-incompatibility,haploid embryo formation,embryogen-esis,and seed development.They also play a significant role in shaping plant reproductive and root archi-tecture.Recent studies have shed light on the diverse mechanisms of PA’s action,though much remains to be elucidated.Here,we summarize recent advances in the study of PA and its metabolizing enzymes,emphasizing their roles in plant sexual reproduction and architecture.We also explore potential mecha-nisms underlying PA’s functions and discuss future research directions.展开更多
Acute liver failure(ALF)is a life-threatening condition associated with macrophagemediated inflammatory responses.Effective therapies and drugs are still lacking to date.Here,we reveal that a derivative of xanthohumol...Acute liver failure(ALF)is a life-threatening condition associated with macrophagemediated inflammatory responses.Effective therapies and drugs are still lacking to date.Here,we reveal that a derivative of xanthohumol,CAM12203,alleviates lipopolysaccharide(LPS)+D-galactosamine(D-GalN)-induced ALF through limiting macrophage-mediated inflammation,with the most significant impact on interleukin-1β(IL-1β)transcription.Through biotin labeling-mediated pull-down and LC-MS/MS analysis,diacylglycerol kinase ζ(DGKζ),a lipid-metabolizing kinase,is identified as the direct target of CAM12203.Mechanistically,DGKζ is induced in macrophages upon inflammatory stimuli and is upregulated observed on clinical liver failure samples.Its product phosphatidic acid(PA)boosts phospholipase C(PLC)-inositol 1,4,5-trisphosphate(IP_(3))-Ca^(2+)signaling and subsequent janus kinase 2(JAK2)-signal transducer and activator of transcription 3(STAT3)cascade,ultimately promoting IL1β production and liver failure.DGKζ knockdown/ablation or inhibition significantly impairs the DGKζ-STAT3-IL-1β pathway along with ALF progression.Finally,CAM12203 is confirmed to be a new DGKζ inhibitor and acts against inflammation in a DGKζ-reliant manner.Taken together,CAM12203 inhibits IL-1β transcription in macrophages by binding to DGKζ and blocking the DGKζ-STAT3 axis,thereby exerting an ameliorative effect on ALF.These results not only highlight CAM12203 as a promising lead compound for ALF treatment,but also define DGKζ as a novel therapeutic target.展开更多
Phospholipase Dα1 （PLDα1） has been shown to mediate the abscisic acid regulation of stomatal movements. Arabidopsis plants deficient in PLDα1 increased, whereas PLDα1-overexpressing tobacco decreased, transpirat...Phospholipase Dα1 （PLDα1） has been shown to mediate the abscisic acid regulation of stomatal movements. Arabidopsis plants deficient in PLDα1 increased, whereas PLDα1-overexpressing tobacco decreased, transpirational water loss. In the early stage of drought, the decrease in water loss was associated with a rapid stomatal closure caused by a high level of PLD in PLDα1-overexpressing plants. However, in the late stage of drought, the overexpressing plants displayed more susceptibility to drought than control plants. PLDα1 activity in the overexpressing plants was much higher than that of control plants in which drought also induced an increase in PLDα1 activity. The high level of PLDα1 activity was correlated to membrane degradation in late stages of drought, as demonstrated by ionic leakage and lipid peroxidation. These findings indicate that a high level of PLDα1 expression has different effects on plant response to water deficits. It promotes stomatal closure at earlier stages, but disrupts membranes in prolonged drought stress. These findings are discussed in relation to the understanding of PLD functions and potential applications.展开更多
Lipid catabolism in germinating seeds provides energy and substrates for initial seedling growth,but how this process is regulated is not well understood.Here,we show that an AT-hook motif-containing nuclear localized...Lipid catabolism in germinating seeds provides energy and substrates for initial seedling growth,but how this process is regulated is not well understood.Here,we show that an AT-hook motif-containing nuclear localized(AHL)protein regulates lipid mobilization and fatty acid p-oxidation during seed germination and seedling establishment.AHL4 was identified to directly interact with the lipid mediator phosphatidic acid(PA).Knockout(KO)of AHL4 enhanced,but overexpression(OE)of AHL4 attenuated,triacylglycerol(TAG)degradation and seedling growth.Normal seedling growth of the OE lines was restored by sucrose supplementation to the growth medium.AHL4-OE seedlings displayed decreased expression of genes involved in TAG hydrolysis and fatty acid oxidation,whereas the opposite was observed in AHL4-KOs.These genes contained AHL4-binding cis elements,and AHL4 was shown to bind to the promoter regions of genes encoding the TAG lipases SDP1 and DALL5 and acyl-thioesterase KAT5.These AHL4-DNA interactions were suppressed by PA species that bound to AHL4.These results indicate that AHL4 suppresses lipid catabolism by repressing the expression of specific genes involved in TAG hydrolysis and fatty acid oxidation,and that PA relieves AHL4-mediated suppression and promotes TAG degradation.Thus,AHL4 and PA together regulate lipid degradation during seed germination and seedling establishment.展开更多
Phospholipase D （PLD） exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid （PA）. Protein kinases and phosphatases, such as mammalian target of r...Phospholipase D （PLD） exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid （PA）. Protein kinases and phosphatases, such as mammalian target of rapa- mycin （mTOR）, Protein Phosphatase 1 （PP1） and Protein Phosphatase 2C （PP2C）, are PA-binding proteins that execute crucial regulatory functions in both animals and plants. PA participates in many signaling pathways by modulating the enzymatic activity and/or subcellular localization of bound proteins. In this study, we demonstrated that PLD-derived PA interacts with the scaffolding A1 subunit of Protein Phosphatase 2A （PP2A） and regulates PP2A-mediated PIN1 dephos- phorylation in Arabidopsis. Genetic and pharmacological studies showed that both PA and PP2A participate in the regu- lation of auxin distribution. In addition, both the phosphorylation status and polar localization of PIN1 protein were affected by PLD inhibitors, Exogenous PA triggered the membrane accumulation of PP2AA1 and enhanced the PP2A activity at membrane, while PLD inhibition resulted in the reduced endosomal localization and perinuclear aggregation of PP2AA1. These results demonstrate the important role of PLD-derived PA in normal PP2A-mediated PIN dephosphoryl- ation and reveal a novel mechanism, in which PA recruits PP2AA1 to the membrane system and regulates PP2A function on membrane-targeted proteins. As PA and PP2A are conserved among eukaryotes, other organisms might use similar mechanisms to mediate multiple biological processes.展开更多
The development of molecular medicine has greatly promoted the research and development (R&D) of innovative drugs. However,drug design and development for those novel targets remains a big challenge with low succe...The development of molecular medicine has greatly promoted the research and development (R&D) of innovative drugs. However,drug design and development for those novel targets remains a big challenge with low success rates and high attrition of drug candidates1. The current methodology of new drug R&D is deeply influenced by the idea of allopathic medicine, which directly inhibits biological targets.展开更多
Phospholipids, including phosphatidic acid （PA）, phosphatidylcholine （PC）, phosphatidylethanolamine （PE）, phosphatidylglycerol （PC）, phosphatidylserine （PS） and phosphoinositides, have emerged as an importan...Phospholipids, including phosphatidic acid （PA）, phosphatidylcholine （PC）, phosphatidylethanolamine （PE）, phosphatidylglycerol （PC）, phosphatidylserine （PS） and phosphoinositides, have emerged as an important class of cellular messenger molecules in various cellular and physiological processes, of which PA attracts much attention of researchers. In addition to its effect on stimulating vesicle trafficking, many studies have demonstrated that PA plays a crucial role in various signaling pathways by binding target proteins and regulating their activity and subcellular localization. Here, we summarize the functional mechanisms and target proteins underlying PA-mediated regulation of cellular signaling, development, hormonal responses, and stress responses in plants.展开更多
Membrane fluidity,permeability,and surface charges are controlled by phospholipid metabolism and transport.Despite the importance of phosphatidic acid(PA)as a bioactive molecule,the mechanical properties of PA translo...Membrane fluidity,permeability,and surface charges are controlled by phospholipid metabolism and transport.Despite the importance of phosphatidic acid(PA)as a bioactive molecule,the mechanical properties of PA translocation and subcellular accumulation are unknown.Here,we used a mobilizable,highly responsive genetically encoded fluorescent indicator,green fluorescent protein(GFP)-N160RbohD,to monitor PA dynamics in living cells.The majority of GFP-N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels.Cellular,pharmacological,and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160RbohD fluorescence at the plasma membrane,which mainly depended on hydrolysis of phospholipase D.By contrast,heat stress induced nuclear translocation of PA indicated by GFP-N160RbohD through a process that required diacylglycerol kinase activity,as well as secretory and endocytic trafficking.Strikingly,we showed that gravity triggers asymmetric PA distribution at the root apex,a response that is suppressed by PLDz2 knockout.The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that the synthesis,transport,and metabolism of PA.展开更多
Increasing evidence suggests that mitogen-activated protein kinase(MAPK)cascades play a crucial role in plant defense against viruses.However,the mechanisms that underlie the activation of MAPK cascades in response to...Increasing evidence suggests that mitogen-activated protein kinase(MAPK)cascades play a crucial role in plant defense against viruses.However,the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear.In this study,we discovered that phosphatidic acid(PA)repre-sents a major class of lipids that respond to Potato virus Y(PVY)at an early stage of infection.We identified NbPLDa1(Nicotiana benthamiana phospholipase Da1)as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role.6K2 of PVY interacts with NbPLDa1,lead-ing to elevated PA levels.In addition,NbPLDa1 and PA are recruited by 6K2 to membrane-bound viral repli-cation complexes.On the other hand,6K2 also induces activation of the MAPK pathway,dependent on its interaction with NbPLDa1 and the derived PA.PA binds to WIPK/SIPK/NTF4,prompting their phosphoryla-tion of WRKY8.Notably,spraying with exogenous PA is sufficient to activate the MAPK pathway.Knock-down of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA.6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDa1 and induced the activation of MAPK-mediated immunity.Loss of function of NbPLDa1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation.Thus,activation of MAPK-mediated immunity by NbPLDa1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.展开更多
Background:Intrahepatic cholangiocarcinoma(iCCA)is a highly heteroge-neous and lethal hepatobiliary tumor with few therapeutic strategies.The metabolic reprogramming of tumor cells plays an essential role in the devel...Background:Intrahepatic cholangiocarcinoma(iCCA)is a highly heteroge-neous and lethal hepatobiliary tumor with few therapeutic strategies.The metabolic reprogramming of tumor cells plays an essential role in the develop-ment of tumors,while the metabolic molecular classification of iCCA is largely unknown.Here,we performed an integrated multiomics analysis and metabolic classification to depict differences in metabolic characteristics of iCCA patients,hoping to provide a novel perspective to understand and treat iCCA.Methods:We performed integrated multiomics analysis in 116 iCCA samples,including whole-exome sequencing,bulk RNA-sequencing and proteome anal-ysis.Based on the non-negative matrix factorization method and the protein abundance of metabolic genes in human genome-scale metabolic models,the metabolic subtype of iCCA was determined.Survival and prognostic gene analy-ses were used to compare overall survival(OS)differences between metabolic subtypes.Cell proliferation analysis,5-ethynyl-2’-deoxyuridine(EdU)assay,colony formation assay,RNA-sequencing and Western blotting were performed to investigate the molecular mechanisms of diacylglycerol kinaseα(DGKA)in iCCA cells.Results:Three metabolic subtypes(S1-S3)with subtype-specific biomarkers of iCCA were identified.These metabolic subtypes presented with distinct prog-noses,metabolic features,immune microenvironments,and genetic alterations.The S2 subtype with the worst survival showed the activation of some special metabolic processes,immune-suppressed microenvironment and Kirsten ratsar-coma viral oncogene homolog(KRAS)/AT-rich interactive domain 1A(ARID1A)mutations.Among the S2 subtype-specific upregulated proteins,DGKA was further identified as a potential drug target for iCCA,which promoted cell proliferation by enhancing phosphatidic acid(PA)metabolism and activating mitogen-activated protein kinase(MAPK)signaling.Conclusion:Viamultiomics analyses,we identified three metabolic subtypes of iCCA,revealing that the S2 subtype exhibited the poorest survival outcomes.We further identified DGKA as a potential target for the S2 subtype.展开更多
基金supported by the National Natural Science Foundation of China(Nos.30960032 and 31000117)the Major Technology Project of Hainan(No.ZDZX2013023-1)+2 种基金the National Nonprofit Institute Research Grants(Nos.CATAS-ITBB 110507 and CATAS-ITBB130305)the Fundamental Scientific Research Funds for Chinese Academy of Tropical Agricultural Sciences(No.1630052013009)the Natural Science Foundation of Hainan Province(No.313077),China
文摘Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.
文摘Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg<sup>2+</sup>-dependent soluble PAP and Mg<sup>2+</sup>-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg<sup>2+</sup>-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the V<sub>max</sub> and K<sub>m</sub> values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.
文摘Phosphatidate phosphatase (EC 3.1.3.4), PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol. A Mg2+ independent PAP activity was identified in the soluble extract of Momordica charantia cotyledons undergoing maturation. While the microsomal fraction of the extract gave only 10% of the PAP activity, the remaining 90% of the activity was associated with the soluble fraction. At pH 3.0, the soluble PAP was bound to S column and eluted with glycine-HCl buffer containing high salt. The pH and temperature optima of the PAP activity were 6.0 and 53℃, respectively. Under optimum assay condition, the Vmax and Km for dioleoyl phosphatidic acid were 1.89 ηkat/mg of protein and 142 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ- NPP, the Vmax and Km were 10.4 ηkat/mg of protein and 107 μM, respectively. The inclusion of Mg2+ and β-mercaptoethanol into the reaction mix did not change the enzyme activity nor did the addition of N-ethylmaleimide and phenylglyoxal, which indicates that cysteine and arginine are not involved in catalysis of PtdOH. The addition of Mg2+ up to 10 mM also did not change the level of PAP activity. Triton X-100, however, inhibited the activity. This is the first documented case of an in vitro PAP activity in the developing cotyledons of Momordica charantia. The PAP described here could serve as a model for lipin-1 or lipin-2 in humans. Mutations in these genes lead to acute myoglobinuria in human infants.
基金supported by grants from National Natural Science Foundation of China(81902784,81771086)by the Fund of Sichuan Provincial Department of science and technology(2024YFFK0393,2022YFS0039)+1 种基金the Research and Develop Program,West China Hospital of Stomatology,Sichuan University(LCYJ2023-DL-2,RD-02-202002)the CAMS Innovation Fund for Medical Sciences(CIFMS,2019-I2M-5-004)。
文摘Cancer Stem Cells(CSCs)are cancer cells with self-renewal and tumorigenesis abilities.CSCs in tumor tissues are the leading cause of tumor progression,recurrence,and drug resistance.CSCs have distinct metabolic features that contribute to maintaining their self-renewal and stemness.Phospholipids are essential components of cell membranes and play fundamental roles in cellular activities.CSCs have abnormal phospholipid metabolism,which affects their self-renewal,differentiation,invasion,and drug resistance.Compared with non-CSCs,the phospholipid metabolism of CSCs mainly focused on significantly increased fatty acid(FAs)and phospholipids synthesis,phospho-lipid unsaturation,and lipolysis-fatty acid oxidation(FAO).In brief,FA and phospholipid metabolism in the anabolic and catabolic pathways are strictly regulated in CSCs to maintain self-renewal and stemness activity.In this review,we summarize the alterations in phospholipid metabolism in CSCs and their impacts on the stemness of CSCs,and we put forward the potential applications of targeting phospholipid metabolism for CSCs,to provide directions for the development of drugs targeting the phospholipid metabolism.
文摘Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5?C and 45?C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 ηkat/mg of protein and 200 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ-NPP, the Vmax and Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of tryp-sinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.
文摘Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthophosphate. In eukaryotes, the PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG). Existing methods for measuring PAP activity rely on the use of radioactive PtdOH. These methods are costly and cumbersome. In this report, we describe a simple assay procedure to measure released inorganic orthophosphate, which is a coproduct of the PAP reaction. Each molecule of PtdOH would release one molecule of DAG and one molecule of inorganic orthophosphate (Pi) when subjected to enzymatic breakdown under optimal conditions. Given the published rates of in vitro PAP enzymatic activity from various sources, we proposed that colorimetric determination of released Pi is possible. With this view, we performed in vitro PAP activity assays using freshly isolated enzyme from bitter gourd, Momordica charantia, and measured the released Pi using two spectrophotometric methods. Both methods gave about 2.0 to 2.25 ηkat per mg of protein. Thus, it is now possible to perform PAP activity using a simple procedure that uses nonradioactive substrates, provided the sample is dialyzed extensively to lower the intrinsic concentration of free phosphate. The kinetics data presented in this study is comparable to that of other PAP enzymes reported elsewhere, which gives credence to the notion that non-radioactive methods can be used to perform PAP activity.
文摘CIN85 （Cbl-interacting protein of 85 kDa） is an important molecule involved in receptor tyrosine kinase endocytosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic lipid - phosphatidic acid. Its coiled-coil domain plays an important role in mediating this protein-lipid interaction. Deletion of the coiled-coil domain results in loss of membrane association, and reduced interaction with c-cbl, finally causing the blockage of epidermal growth factor receptor downregulation. In addition, a significant portion of CIN85 is located on the endosomal compartment and is related to endocytic cargo sorting, characterized by CIN85＇s localization on the ＂E class＂ compartment and EGF degradation blockage in CIN85 knockdown cells. Taken together, our results suggest that CIN85 may function as a scaffold molecule in both the internalization and endocytic cargo sorting processes through its association with the endosomal membrane.
文摘Cells regulate phospholipase D (PLD) activity in response to numerous extracellular signals. Here, we investigated the involvement of PLD activity in transforming growth factor-β(TGF-β1)-mediated growth inhibition of epithelial cells. TGFβ1 inhibits the growth of MDCK, Mv1Lu, and A-549 cells. In the presence of 0.4 % butanol, TGF-β1 induces an increase in the formation of phosp hat idylbutanol, a unique product catalyzed by PLD. TGF-β1 also induces an increase in phosphatidic acid (PA) level in A-549 and MDCK cells. TGF-β1 induces an increase in the levels of DAG labeled with [~3H]-myristic acid in A-549 and MDCK cells but not in Mv1Lu cells- No increase of DAG was observed in cells prelabeled with [~3H]-arachidonic acid.The data presented suggest that PLD activation is involved in the TGF-β1-induced cell growth inhibition.
文摘The Heilongjiang Soybean Research Institute not only focuses on improving yield and quality of soybean, but also exploring the development of various high quality soybean byproducts. Currently, the institute uses advanced extraction technology, the institute also produces new soybean chips and vitamin E from soybean pulp, soybean peptides, phosphatides and oligosaccharides. 120 broilers were randomly divided into 4 groups with 6 replicates in each group and 5 chicken in each replicate. The authors studied influence on performance and body quality of broiler chicken by using soybean phosphatides to take the place of 0.5%, 1% and 1.5% corn of basal daily grain. Conclusion indicated that rates of broiler chicken weight gain were 2.1%, 4.4% and 8.7%, feed utilization rates raised 3.5%, 5.2% and 8.1%, costs reduced 2.3%, 3.5% and 5.8%, chest muscle rates improved 14.7%, 0.9% and -0.49%, belly fat rates improved 11.06%, 20.28% and 44.75% by using soybean phosphatides to take place of corn in daily grain after 42 days. More recently, the study is also involved in the research on improving the meat quality of chicken by adding extracted soybean peptide and phosphatides into feed. Furthermore, nearly 98% post-consumer waste oil with high acid value can be converted into biodiesel by using an effective supercritical methanol method.
基金supported by grants from the National Institute of General Medical Sciences of the National Institutes of Health under award no.R01GM141374the National Science Foundation under grant nos.2222157 and 2302424the USDA National Institute of Food and Agriculture 2020-67013-30908/project accession no.1022148.
文摘Phosphatidic acid(PA)is an important class of signaling lipids involved in various biological processes in plants.Functional characterization of mutants of PA-metabolizing enzymes,combined with lipidomics and protein–lipid interaction analyses,has revealed the key role of PA signaling in plant responses to biotic and abiotic stresses.Moreover,PA and its metabolizing enzymes influence several reproductive processes,including gametogenesis,pollen tube growth,self-incompatibility,haploid embryo formation,embryogen-esis,and seed development.They also play a significant role in shaping plant reproductive and root archi-tecture.Recent studies have shed light on the diverse mechanisms of PA’s action,though much remains to be elucidated.Here,we summarize recent advances in the study of PA and its metabolizing enzymes,emphasizing their roles in plant sexual reproduction and architecture.We also explore potential mecha-nisms underlying PA’s functions and discuss future research directions.
基金supported by the National Science and Technology Major Project(2024ZD0523103,China)the National Natural Science Foundation of China(92457302,82425058,82304500,and 22494693)+5 种基金the National Key R&D Program of China(2020YFE0205600)the Yangfan Project of Shanghai Science and Technology Commission(23YF1456500,China)the Shanghai“Super Postdoctoral”Incentive Plan(2022692,China)the fellowship of China Postdoctoral Science Foundation(2023M733630)the Tian-Shan Talent Program(2022TSYCLJ0064,China)the Youth Innovation Promotion Association of Chinese Academy of Sciences(E32R4001,China).
文摘Acute liver failure(ALF)is a life-threatening condition associated with macrophagemediated inflammatory responses.Effective therapies and drugs are still lacking to date.Here,we reveal that a derivative of xanthohumol,CAM12203,alleviates lipopolysaccharide(LPS)+D-galactosamine(D-GalN)-induced ALF through limiting macrophage-mediated inflammation,with the most significant impact on interleukin-1β(IL-1β)transcription.Through biotin labeling-mediated pull-down and LC-MS/MS analysis,diacylglycerol kinase ζ(DGKζ),a lipid-metabolizing kinase,is identified as the direct target of CAM12203.Mechanistically,DGKζ is induced in macrophages upon inflammatory stimuli and is upregulated observed on clinical liver failure samples.Its product phosphatidic acid(PA)boosts phospholipase C(PLC)-inositol 1,4,5-trisphosphate(IP_(3))-Ca^(2+)signaling and subsequent janus kinase 2(JAK2)-signal transducer and activator of transcription 3(STAT3)cascade,ultimately promoting IL1β production and liver failure.DGKζ knockdown/ablation or inhibition significantly impairs the DGKζ-STAT3-IL-1β pathway along with ALF progression.Finally,CAM12203 is confirmed to be a new DGKζ inhibitor and acts against inflammation in a DGKζ-reliant manner.Taken together,CAM12203 inhibits IL-1β transcription in macrophages by binding to DGKζ and blocking the DGKζ-STAT3 axis,thereby exerting an ameliorative effect on ALF.These results not only highlight CAM12203 as a promising lead compound for ALF treatment,but also define DGKζ as a novel therapeutic target.
文摘Phospholipase Dα1 （PLDα1） has been shown to mediate the abscisic acid regulation of stomatal movements. Arabidopsis plants deficient in PLDα1 increased, whereas PLDα1-overexpressing tobacco decreased, transpirational water loss. In the early stage of drought, the decrease in water loss was associated with a rapid stomatal closure caused by a high level of PLD in PLDα1-overexpressing plants. However, in the late stage of drought, the overexpressing plants displayed more susceptibility to drought than control plants. PLDα1 activity in the overexpressing plants was much higher than that of control plants in which drought also induced an increase in PLDα1 activity. The high level of PLDα1 activity was correlated to membrane degradation in late stages of drought, as demonstrated by ionic leakage and lipid peroxidation. These findings indicate that a high level of PLDα1 expression has different effects on plant response to water deficits. It promotes stomatal closure at earlier stages, but disrupts membranes in prolonged drought stress. These findings are discussed in relation to the understanding of PLD functions and potential applications.
基金The work is supported by grants from the National Key Research and Development Program of China(2017YFE0104800)National Natural Science Foundation of China(31801029)+1 种基金the U.S.Department of Energy(DESC0001295)the Agriculture and Food Research Initiative(AFRI)(award no.2016-67013-24429,project accession number 1007600)from the USDA National Institute of Food and Agriculture,and the International Postdoctoral Exchange Fellowship Program 2016 of the Office of China Postdoctoral Council(20160034).
文摘Lipid catabolism in germinating seeds provides energy and substrates for initial seedling growth,but how this process is regulated is not well understood.Here,we show that an AT-hook motif-containing nuclear localized(AHL)protein regulates lipid mobilization and fatty acid p-oxidation during seed germination and seedling establishment.AHL4 was identified to directly interact with the lipid mediator phosphatidic acid(PA).Knockout(KO)of AHL4 enhanced,but overexpression(OE)of AHL4 attenuated,triacylglycerol(TAG)degradation and seedling growth.Normal seedling growth of the OE lines was restored by sucrose supplementation to the growth medium.AHL4-OE seedlings displayed decreased expression of genes involved in TAG hydrolysis and fatty acid oxidation,whereas the opposite was observed in AHL4-KOs.These genes contained AHL4-binding cis elements,and AHL4 was shown to bind to the promoter regions of genes encoding the TAG lipases SDP1 and DALL5 and acyl-thioesterase KAT5.These AHL4-DNA interactions were suppressed by PA species that bound to AHL4.These results indicate that AHL4 suppresses lipid catabolism by repressing the expression of specific genes involved in TAG hydrolysis and fatty acid oxidation,and that PA relieves AHL4-mediated suppression and promotes TAG degradation.Thus,AHL4 and PA together regulate lipid degradation during seed germination and seedling establishment.
文摘Phospholipase D （PLD） exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid （PA）. Protein kinases and phosphatases, such as mammalian target of rapa- mycin （mTOR）, Protein Phosphatase 1 （PP1） and Protein Phosphatase 2C （PP2C）, are PA-binding proteins that execute crucial regulatory functions in both animals and plants. PA participates in many signaling pathways by modulating the enzymatic activity and/or subcellular localization of bound proteins. In this study, we demonstrated that PLD-derived PA interacts with the scaffolding A1 subunit of Protein Phosphatase 2A （PP2A） and regulates PP2A-mediated PIN1 dephos- phorylation in Arabidopsis. Genetic and pharmacological studies showed that both PA and PP2A participate in the regu- lation of auxin distribution. In addition, both the phosphorylation status and polar localization of PIN1 protein were affected by PLD inhibitors, Exogenous PA triggered the membrane accumulation of PP2AA1 and enhanced the PP2A activity at membrane, while PLD inhibition resulted in the reduced endosomal localization and perinuclear aggregation of PP2AA1. These results demonstrate the important role of PLD-derived PA in normal PP2A-mediated PIN dephosphoryl- ation and reveal a novel mechanism, in which PA recruits PP2AA1 to the membrane system and regulates PP2A function on membrane-targeted proteins. As PA and PP2A are conserved among eukaryotes, other organisms might use similar mechanisms to mediate multiple biological processes.
文摘The development of molecular medicine has greatly promoted the research and development (R&D) of innovative drugs. However,drug design and development for those novel targets remains a big challenge with low success rates and high attrition of drug candidates1. The current methodology of new drug R&D is deeply influenced by the idea of allopathic medicine, which directly inhibits biological targets.
基金supported by the National Natural Science Foundation of China(31721001 and 31400261)the“Ten Thousand Talent Program”Collaborative Innovation Center of Crop Stress Biology,Henan Province
文摘Phospholipids, including phosphatidic acid （PA）, phosphatidylcholine （PC）, phosphatidylethanolamine （PE）, phosphatidylglycerol （PC）, phosphatidylserine （PS） and phosphoinositides, have emerged as an important class of cellular messenger molecules in various cellular and physiological processes, of which PA attracts much attention of researchers. In addition to its effect on stimulating vesicle trafficking, many studies have demonstrated that PA plays a crucial role in various signaling pathways by binding target proteins and regulating their activity and subcellular localization. Here, we summarize the functional mechanisms and target proteins underlying PA-mediated regulation of cellular signaling, development, hormonal responses, and stress responses in plants.
基金supported by the National Key R&D Program of China(2022YFA1303400 and 2022YFD1201700)the National Natural Science Foundation of China(32270301 and 31970300)+1 种基金the Jiangsu Agriculture Science and Technology Innovation Fund(CX-20-2007)the Fundamental Research Funds for the Central Universities(XUEKEN2022002).
文摘Membrane fluidity,permeability,and surface charges are controlled by phospholipid metabolism and transport.Despite the importance of phosphatidic acid(PA)as a bioactive molecule,the mechanical properties of PA translocation and subcellular accumulation are unknown.Here,we used a mobilizable,highly responsive genetically encoded fluorescent indicator,green fluorescent protein(GFP)-N160RbohD,to monitor PA dynamics in living cells.The majority of GFP-N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels.Cellular,pharmacological,and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160RbohD fluorescence at the plasma membrane,which mainly depended on hydrolysis of phospholipase D.By contrast,heat stress induced nuclear translocation of PA indicated by GFP-N160RbohD through a process that required diacylglycerol kinase activity,as well as secretory and endocytic trafficking.Strikingly,we showed that gravity triggers asymmetric PA distribution at the root apex,a response that is suppressed by PLDz2 knockout.The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that the synthesis,transport,and metabolism of PA.
基金supported by the National Natural Science Foundation of China (31901855)the Youth Talent Support Program of Henan Province (2020HYTP042)the Special Fund for Young Talents of Henan Agricultural University。
文摘Increasing evidence suggests that mitogen-activated protein kinase(MAPK)cascades play a crucial role in plant defense against viruses.However,the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear.In this study,we discovered that phosphatidic acid(PA)repre-sents a major class of lipids that respond to Potato virus Y(PVY)at an early stage of infection.We identified NbPLDa1(Nicotiana benthamiana phospholipase Da1)as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role.6K2 of PVY interacts with NbPLDa1,lead-ing to elevated PA levels.In addition,NbPLDa1 and PA are recruited by 6K2 to membrane-bound viral repli-cation complexes.On the other hand,6K2 also induces activation of the MAPK pathway,dependent on its interaction with NbPLDa1 and the derived PA.PA binds to WIPK/SIPK/NTF4,prompting their phosphoryla-tion of WRKY8.Notably,spraying with exogenous PA is sufficient to activate the MAPK pathway.Knock-down of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA.6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDa1 and induced the activation of MAPK-mediated immunity.Loss of function of NbPLDa1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation.Thus,activation of MAPK-mediated immunity by NbPLDa1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.
基金This project was supported by grants from the National Natural Science Foundation of China(82273387,82273386,82073217,32270711,82073218 and 82003084)the National Key Research and Develop-ment Program of China(2018YFC1312100)+3 种基金Beijing Nova Program(20220484230)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)Shanghai Municipal Key Clinical Specialty,CAMS Innovation Fund for Medical Sciences(CIFMS)(2019-I2M-5-058)the State Key Laboratory of Proteomics(SKLP-K202004).
文摘Background:Intrahepatic cholangiocarcinoma(iCCA)is a highly heteroge-neous and lethal hepatobiliary tumor with few therapeutic strategies.The metabolic reprogramming of tumor cells plays an essential role in the develop-ment of tumors,while the metabolic molecular classification of iCCA is largely unknown.Here,we performed an integrated multiomics analysis and metabolic classification to depict differences in metabolic characteristics of iCCA patients,hoping to provide a novel perspective to understand and treat iCCA.Methods:We performed integrated multiomics analysis in 116 iCCA samples,including whole-exome sequencing,bulk RNA-sequencing and proteome anal-ysis.Based on the non-negative matrix factorization method and the protein abundance of metabolic genes in human genome-scale metabolic models,the metabolic subtype of iCCA was determined.Survival and prognostic gene analy-ses were used to compare overall survival(OS)differences between metabolic subtypes.Cell proliferation analysis,5-ethynyl-2’-deoxyuridine(EdU)assay,colony formation assay,RNA-sequencing and Western blotting were performed to investigate the molecular mechanisms of diacylglycerol kinaseα(DGKA)in iCCA cells.Results:Three metabolic subtypes(S1-S3)with subtype-specific biomarkers of iCCA were identified.These metabolic subtypes presented with distinct prog-noses,metabolic features,immune microenvironments,and genetic alterations.The S2 subtype with the worst survival showed the activation of some special metabolic processes,immune-suppressed microenvironment and Kirsten ratsar-coma viral oncogene homolog(KRAS)/AT-rich interactive domain 1A(ARID1A)mutations.Among the S2 subtype-specific upregulated proteins,DGKA was further identified as a potential drug target for iCCA,which promoted cell proliferation by enhancing phosphatidic acid(PA)metabolism and activating mitogen-activated protein kinase(MAPK)signaling.Conclusion:Viamultiomics analyses,we identified three metabolic subtypes of iCCA,revealing that the S2 subtype exhibited the poorest survival outcomes.We further identified DGKA as a potential target for the S2 subtype.
