Phosphatidic acid-enabled MKL1 contributes to liver regeneration:Translational implication in liver failure

Liver regeneration following injury aids the restoration of liver mass and the recovery of liver function.In the present study we investigated the contribution of megakaryocytic leukemia 1(MKL1),a transcriptional modulator,to liver regeneration.We report that both MKL1 expression and its nuclear translocation correlated with hepatocyte proliferation in cell and animal models of liver regeneration and in liver failure patients.Mice with MKL1 deletion exhibited defective regenerative response in the liver.Transcriptomic analysis revealed that MKL1 interacted with E2F1 to program pro-regenerative transcription.MAPKAPK2 mediated phosphorylation primed MKL1 for its interaction with E2F1.Of interest,phospholipase d2 promoted MKL1 nuclear accumulation and liver regeneration by catalyzing production of phosphatidic acid(PA).PA administration stimulated hepatocyte proliferation and enhanced survival in a MKL1-dependent manner in a pre-clinical model of liver failure.Finally,PA levels was detected to be positively correlated with expression of pro-regenerative genes and inversely correlated with liver injury in liver failure patients.In conclusion,our data reveal a novel mechanism whereby MKL1 contributes to liver regeneration.Screening for small-molecule compounds boosting MKL1 activity may be considered as a reasonable approach to treat acute liver failure.

Identification of an Mg2+-Independent Soluble Phosphatidate Phosphatase in Cottonseed (Gossypium hirsutum L.)

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 Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg2+-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 Vmax and Km 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.

Lipid phosphorylation by a diacylglycerol kinase suppresses ABA biosynthesis to regulate plant stress responses

Lipid phosphorylation by diacylglycerol kinase(DGK)that produces phosphatidic acid(PA)plays important roles in various biological processes,including stress responses,but the underlying mechanisms remain elusive.Here,we show that DGK5 and its lipid product PA suppress ABA biosynthesis by interacting withABA-DEFICIENT2(ABA2),a key ABA biosynthesis enzyme,to negatively modulate plant responseto abiotic stress tested in Arabidopsis thaliana.Loss of DGK5 function rendered plants less damaged,whereas overexpression(OE)of DGK5 enhanced plant damage to water and salt stress.The dgk5 mutant plants exhibited decreased total cellular and nuclear levels of PA with increased levels of diacylglycerol,whereas DGK5-OE plants displayed the opposite effect.Interestingly,we found that both DGK5 and PA bind to the ABA-synthesizing enzyme ABA2 and suppress its enzymatic activity.Consistently,the dgk5 mutant plants exhibited increased levels of ABA,while DGK5-OE plants showed reduced ABA levels.In addition,we showed that both DGK5 and ABA2 are detected in and outside the nuclei,and loss of DGK5 function decreased the nuclear association of ABA2.We found that both DGK5 activity and PA promote nuclear association of ABA2.Taken together,these results indicate that both DGK5 and PA interact with ABA2 to inhibit its enzymatic activity and promote its nuclear sequestration,thereby sup-pressing ABA production in response to abiotic stress.Our study reveals a sophisticated mechanism by which DGK5 and PA regulate plant stress responses.

Multiomics analysis reveals metabolic subtypes and identifies diacylglycerol kinase α (DGKA) as a potential therapeutic target for intrahepatic cholangiocarcinoma

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.

Phosphatidic Acid （PA） PP2A Activity and PIN1 Binds PP2AA1 to Regulate Polar Localization

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.

Phosphatidic acid plays key roles regulating plant development and stress responses

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.

The discovery of the fat-regulating phosphatidic acid phosphatase gene

Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid(substrate)and diacylglycerol(product),which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol.Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression,membrane expansion,secretion,and endocytosis.While this important enzyme has been known for several decades,its gene was only identified recently from yeast.This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans.

Dynamic responses ofPAto environmental stimuli imaged by a genetically encoded mobilizable fluorescent sensor

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.

Dual Functions of Phospholipase Dα1 in Plant Response to Drought

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.

Transcriptional Regulation of Lipid Catabolism during Seedling Establishment

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.

Commentary:Indirect action pattern:A remote and cross-organ pharmacological mechanism for drug innovation

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.