Genome-wide identification of CK gene family suggests functional expression pattern against Cd2+ stress in Gossypium hirsutum L.

Choline kinase (CK) gene plays an important role in plants growth, development and resistance to stress. It mainly regulates the biosynthesis of phosphatidylcholine. This study aims to explore the structure-function relationship, and to provide a framework for functional validation and biochemical characterization of various CK genes. Our analysis showed that 87 CK genes were identified in cotton and 7 diploid plants, of which 43 genes encode CK proteins in 4 cotton species, and 13 genes were identified in Gossypium hirsutum. Most of GhCK genes are affected by the abiotic stress conditions, indicating the importance of CK proteins for plant development and response to abiotic stress. RT-qPCR analysis showed the tissue specificity of GhCK genes in response to Cd2+ and other abiotic stresses. Under Cd2+ stress, the expression level of GhCK gene family members has undergone different changes. The expression level of GhCK5 was enhanced, indicating that Cd2+ stress caused the increase of phosphatidylcholine content, which in turn reacted on the plant cell membrane, finally reached the absorption of Cd2+ into plant cells to repair Cd2+ the purpose of contaminated soil. This study will further broaden our understanding of the association between evolution and function of the GhCK gene family.

Evidence of multiple hepatic mechanisms to mobilize docosahexaenoic acid into dam plasma during pregnancy in chow-fed sprague dawley rats.

Fetal brain growth requires considerable amounts of docosahexaenoic acid (DHA) during late pregnancy that is associated with increased maternal/dam plasma levels of PC 16:0_22:6 (palmitoyl docosahexaenoyl phosphatidylcholine). While biosynthesis of DHA during pregnancy is upregulated, the mechanisms responsible for the incorporation of dam DHA into PC 16:0_22:6 are not understood. The present study used a discovery approach combining untargeted lipidomics of plasma and liver (n = 3/group) with semi-targeted qPCR of hepatic gene products (n = 6/group) to identify metabolic pathways related to DHA metabolism, with a hypothesis that an upregulated acyltransferase involved in PC remodeling would be identified. Sprague Dawley rats were fed a commercial rodent chow throughout the study and samples were collected before pregnancy (baseline), at 15 and 20 days of pregnancy, and 7 days postpartum. Plasma and hepatic PC 16:0_22:6 was significantly increased (by 79% and 194%, respectively) at day 20 of pregnancy. An increase in hepatic DG (diacylglycerol) 16:0_22:6 (by 243%) and significant decreases in Pla2G15 (0.4-fold) and Pla2G16 (0.6-fold) at day 20 of pregnancy, no changes in Lpcat1-4, and an abundant pool of hepatic pool PE (phosphatidylethanolamine) 16:0_22:6 suggest that plasma PC 16:0_22:6 is not being produced by fatty acyl remodeling during pregnancy. The increase in plasma PC 16:0_22:6 during pregnancy appears to be due to an increase in de novo synthesis of PC and both the CDP-choline and phosphatidylcholine methyltransferase pathways are implicated. There was also evidence suggesting channeling of DHA into PC and lipoprotein assembly may be occurring. Targeted research is necessary to confirm these findings, but the results of this study indicate metabolic adaptions to enable maternal/dam resiliency towards meeting the fetal/pup demand for DHA during pregnancy.

GlmS mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum.

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights the presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.

Role of Choline in Ocular Diseases.

Choline is essential for maintaining the structure and function of cells in humans. Choline plays an important role in eye health and disease. It is a precursor of acetylcholine, a neurotransmitter of the parasympathetic nervous system, and it is involved in the production and secretion of tears by the lacrimal glands. It also contributes to the stability of the cells and tears on the ocular surface and is involved in retinal development and differentiation. Choline deficiency is associated with retinal hemorrhage, glaucoma, and dry eye syndrome. Choline supplementation may be effective for treating these diseases.

Genes involved in phosphatidylcholine biosynthesis correlate with nuclear factor-κB in biliary tract cancer patients: Evidence from 1H NMR and computational analyses.

Gallbladder cancer (GBC) is an aggressive malignancy of gastrointestinal tract. Due to uncontrolled growth, GBC cells rapidly synthesize biomolecules including lipids. The lipids are integral component of cell membrane with a wide range of cellular functions. In this study, we measured the clinicopathological features in 40 cases of histologically confirmed GBC and 16 cases of chronic cholecystitis (CC). The female to male ratio in the GBC and CC groups were 3.44:1 and 2.2:1, respectively. The GBC patients exhibited well to poorly differentiated tumor. In the CC group, all patients showed cholecystitis with no evidence of dysplasia or malignancy. The majority of GBC and CC patients reported pain. Using 1H NMR spectroscopy, we observed 4-folds increase in the level of choline containing phospholipids (CCPLs) in the gallbladder of GBC patients as compared to CC patients. Other lipid metabolites such as cholesterol ester, C18-cholesterol and saturated fatty acids were insignificantly changed between GBC and CC patients. Moreover, the level of CCPLs in the GBC patients with BMI <25 kg/m2 was significantly higher as compared to CC patients. Further, a significant increase in the CCPLs level was observed in GBC female patients in comparison to CC patients. From the computational analyses, we observed that the genes involved in the biosynthesis of phosphatidylcholine (PtdCho) indirectly interact with the RELA, which encodes the NF-κB p65 subunit. The genes involved in the PtdCho biosynthesis were also correlated with the overall and disease-free survival of cholangiocarcinoma patients. The study opens new window for exploring the diagnostic and therapeutic potential of CCPLs in GBC patients.

Hypothyroidism Increases Cholesterol Gallstone Prevalence in Mice by Elevated Hydrophobicity of Primary Bile Acids.

Background: Thyroid hormone (TH) deficiency has been associated with increased cholesterol gallstone prevalence. Hypothyroidism impacts hepatic lipid homeostasis, biliary secretion, gallbladder motility, and gallstone (LITH) gene expression, all potential factors contributing to cholesterol gallstone disease (CGD). However, how TH deficiency may lead to gallstone formation is still poorly understood. Therefore, we performed molecular studies in a CGD mouse model under lithogenic conditions and modulation of TH status. Methods: Male, three-month-old C57BL/6 mice were randomly divided into a control (euthyroid) group, a hypothyroid (hypo) group, a gallstone (litho) group, and a gallstone+hypothyroid (litho+hypo) group and were treated for 2, 4, and 6 weeks (n = 8/treatment period). Gallstone prevalence, biliary composition and cholesterol crystals, hepatic expression of genes participating in cholesterol, bile acid (BA), and phosphatidylcholine synthesis (Hmgcr, Cyp7a1, Pcyt1a), and canalicular transport (Abcg5, Bsep, Abcb4) were investigated. Results: Increased cholesterol gallstone prevalence was observed in hypothyroid mice under lithogenic diet after 4 and 6 weeks of treatment (4 weeks: 25% vs. 0%; 6 weeks: 75% vs. 37.5%). Interestingly, neither the composition of the three main biliary components, cholesterol, BAs, and phosphatidylcholine, nor the hepatic expression of genes involved in synthesis and transport could explain the differences in cholesterol gallstone formation in the mice. However, TH deficiency resulted in significantly increased hydrophobicity of primary BAs in bile. Furthermore, downregulation of hepatic sulfonation enzymes Papss2 and Sult2a8 as well as diminished biliary BA sulfate concentrations in mice were observed under hypothyroid conditions all contributing to a lithogenic biliary milieu as evidenced by microscopic cholesterol crystals and macroscopic gallstone formation. Conclusions: We describe a novel pathogenic link between TH deficiency and CGD and suggest that the increased hydrophobic character of biliary BAs due to the diminished expression of hepatic detoxification enzymes promotes cholesterol crystal precipitation and enhances cholesterol gallstone formation in the bile of hypothyroid mice.

Aberrant activation of super enhancer and choline metabolism drive antiandrogen therapy resistance in prostate cancer.

Next generation antiandrogens such as enzalutamide (Enz) are effective initially for the treatment of castration-resistant prostate cancer (CRPC). However, the disease often relapses and the underlying mechanisms remain elusive. By performing H3-lysine-27 acetylation (H3K27ac) ChIP-seq in Enz-resistant CRPC cells, we identified a group of super enhancers (SEs) that are abnormally activated in Enz-resistant CRPC cells and associated with enhanced transcription of a subset of tumor promoting genes such as CHPT1, which catalyzes phosphatidylcholine (PtdCho) synthesis and regulates choline metabolism. Increased CHPT1 conferred CRPC resistance to Enz in vitro and in mice. While androgen receptor (AR) primarily binds to a putative CHPT1 enhancer and mediates androgen-dependent expression of CHPT1 gene in Enz-sensitive prostate cancer cells, AR binds to a different enhancer within the CHPT1 SE locus and facilities androgen-independent expression of CHPT1 in Enz-resistant cells. We further identified a long-non coding RNA transcribed at CHPT1 enhancer (also known as enhancer RNA) that binds to the H3K27ac reader BRD4 and participates in regulating CHPT1 SE activity and CHPT1 gene expression. Our findings demonstrate that aberrantly activated SE upregulates CHPT1 expression and confers Enz resistance in CRPC, suggesting that SE-mediated expression of downstream effectors such as CHPT1 can be viable targets to overcome Enz resistance in PCa.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

Phosphatidylcholine Biosynthesis in Mitis Group Streptococci via Host Metabolite Scavenging.

The mitis group streptococci include the major human pathogen Streptococcus pneumoniae and the opportunistic pathogens Streptococcus mitis and Streptococcus oralis, which are human oral cavity colonizers and agents of bacteremia and infective endocarditis in immunocompromised patients. Bacterial membrane lipids play crucial roles in microbe-host interactions; for many pathogens, however, the composition of the membrane is poorly understood. In this study, we characterized the lipidomes of selected species of mitis group streptococci and investigated the mechanistic basis for biosynthesis of the phospholipid phosphatidylcholine (PC). PC is a major lipid in eukaryotic cellular membranes, but it is considered to be comparatively rare in bacterial taxa. Using liquid chromatography-mass spectrometry in conjunction with stable isotope tracing, we determined that mitis group streptococci synthesize PC via a rare host-metabolite-scavenging pathway, the glycerophosphocholine (GPC) pathway, which is largely uncharacterized in bacteria. Our work demonstrates that mitis group streptococci, including S. pneumoniae, remodel their membranes in response to the major human metabolites GPC and lysophosphatidylcholine.IMPORTANCE We lack fundamental information about the composition of the cellular membrane even for the best-studied pathogens of critical significance for human health. The mitis group streptococci are closely linked to humans in health and disease, but their membrane biology is poorly understood. Here, we demonstrate that these streptococci scavenge major human metabolites and use them to synthesize the membrane phospholipid PC. Our work is significant because it identifies a mechanism by which the major human pathogen S. pneumoniae and the primary human oral colonizers S. mitis and S. oralis remodel their membranes in response to host metabolites.

Computational and experimental elucidation of Plasmodium falciparum phosphoethanolamine methyltransferase inhibitors: Pivotal drug target.

INTRODUCTION: Plasmodium falciparum synthesizes phosphatidylcholine for the membrane development through serine decarboxylase-phosphoethanolamine methyltransferase pathway for growth in human host. Phosphoethanolamine-methyltransferase (PfPMT) is a crucial enzyme for the synthesis of phosphocholine which is a precursor for phosphatidylcholine synthesis and is considered as a pivotal drug target as it is absent in the host. The inhibition of PfPMT may kill malaria parasite and hence is being considered as potential target for rational antimalarial drug designing. METHODS: In this study, we have used computer aided drug designing (CADD) approaches to establish potential PfPMT inhibitors from Asinex compound library virtually screened for ADMET and the docking affinity. The selected compounds were tested for in-vitro schizonticidal, gametocidal and cytotoxicity activity. Nontoxic compounds were further studied for PfPMT enzyme specificity and antimalarial efficacy for P. berghei in albino mice model. RESULTS: Our results have identified two nontoxic PfPMT competitive inhibitors ASN.1 and ASN.3 with better schizonticidal and gametocidal activity which were found to inhibit PfPMT at IC50 1.49µM and 2.31µM respectively. The promising reduction in parasitaemia was found both in orally (50 & 10 mg/kg) and intravenous (IV) (5& 1 mg/kg) however, the better growth inhibition was found in intravenous groups. CONCLUSION: We report that the compounds containing Pyridinyl-Pyrimidine and Phenyl-Furan scaffolds as the potential inhibitors of PfPMT and thus may act as promising antimalarial inhibitor candidates which can be further optimized and used as leads for template based antimalarial drug development.

De novo lipogenesis at the mitotic exit is used for nuclear envelope reassembly/expansion. Implications for combined chemotherapy.

Mitosis has been traditionally considered a metabolically inactive phase. We have previously shown, however, that extensive alterations in lipids occur as the cells traverse mitosis, including increased de novo fatty acid (FA) and phosphatidylcholine (PtdCho) synthesis and decreased lysophospholipid content. Given the diverse structural and functional properties of these lipids, we sought to study their metabolic fate and their importance for cell cycle completion. Here we show that FA and PtdCho synthesized at the mitotic exit are destined to the nuclear envelope. Importantly, FA and PtdCho synthesis, but not the decrease in lysophospholipid content, are necessary for cell cycle completion beyond G2/M. Moreover, the presence of alternative pathways for PtdCho synthesis renders the cells less sensitive to its inhibition than to the impairment of FA synthesis. FA synthesis, thus, represents a cell cycle-related metabolic vulnerability that could be exploited for combined chemotherapy. We explored the combination of fatty acid synthase (FASN) inhibition with agents that act at different phases of the cell cycle. Our results show that the effect of FASN inhibition may be enhanced under some drug combinations.

Nuclear lipid droplets derive from a lipoprotein precursor and regulate phosphatidylcholine synthesis.

The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CDP-choline diacylglycerol phosphotransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.

A Methyltransferase Trio Essential for Phosphatidylcholine Biosynthesis and Growth.

Phosphatidylcholine (PC) is a primary class of membrane lipids in most eukaryotes. In plants, the primary PC biosynthetic pathway and its role in plant growth and development remain elusive due to lack of a mutant model with substantially decreased PC content. Recently, a double mutant of Arabidopsis (Arabidopsis thaliana) PHOSPHO-BASE N-METHYLTRANSFERASE 1 (PMT1) and PMT3 was reported with reduced PC content and defective plant growth. However, residual PC content as well as the nonlethal phenotype of the mutant suggests an additional enzyme contributes to PC biosynthesis. In this article, we report on the role of three PMTs in PC biosynthesis and plant development, with a focus on PMT2. PMT2 had the highest expression level among the three PMTs, and it was highly expressed in roots. The pmt1 pmt2 double mutant enhanced the defects in root growth, cell viability, and PC content of pmt1, suggesting that PMT2 functions together with PMT1 in roots. Chemical inhibition of PMT activity in wild-type roots reproduced the short root phenotype observed in pmt1 pmt2, suggesting that PMT1 and PMT2 are the major PMT isoforms in roots. In shoots, pmt1 pmt2 pmt3 enhanced the phenotype of pmt1 pmt3, showing seedling lethality and further reduced PC content without detectable de novo PC biosynthesis. These results suggest that PMTs catalyze an essential reaction step in PC biosynthesis and that the three PMTs have differential tissue-specific functions in PC biosynthesis and plant growth.

Choline Rescues Behavioural Deficits in a Mouse Model of Rett Syndrome by Modulating Neuronal Plasticity.

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls, with 95% of RTT cases resulting from mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Choline, a dietary micronutrient found in most foods, has been shown to be important for brain development and function. However, the exact effects and mechanisms are still unknown. We found that 13 mg/day (1.7 × required daily intake) of postnatal choline treatment to Mecp2-conditional knockout mice rescued not only deficits in motor coordination, but also their anxiety-like behaviour and reduced social preference. Cortical neurons in the brains of Mecp2-conditional knockout mice supplemented with choline showed enhanced neuronal morphology and increased density of dendritic spines. Modelling RTT in vitro by knocking down the expression of the MeCP2 protein with shRNA, we found that choline supplementation to MeCP2-knockdown neurons increased their soma sizes and the complexity of their dendritic arbors. Rescue of the morphological defects could lead to enhanced neurotransmission, as suggested by an observed trend of increased expression of synaptic proteins and restored miniature excitatory postsynaptic current frequency in choline-supplemented MeCP2-knockdown neurons. Through the use of specific inhibitors targeting each of the known physiological pathways of choline, synthesis of phosphatidylcholine from choline was found to be essential in bringing about the changes seen in the choline-supplemented MeCP2-knockdown neurons. Taken together, these data reveal a role of choline in modulating neuronal plasticity, possibly leading to behavioural changes, and hence, a potential for using choline to treat RTT.

Loss of Phosphoethanolamine N-Methyltransferases Abolishes Phosphatidylcholine Synthesis and Is Lethal.

Plants use several pathways to synthesize phosphatidylcholine (PC), the major phospholipid of eukaryotic cells. PC has important structural and signaling roles. One pathway plants use for synthesis is the phospho-base methylation pathway, which forms the head-group phosphocholine through the triple methylation of phosphoethanolamine (PEA) catalyzed by phosphoethanolamine N-methyltransferases (PEAMTs). Our understanding of that pathway and its physiological importance remains limited. We recently reported that disruption of Arabidopsis thaliana PEAMT1/NMT1 and PEAMT3/NMT3 induces severe PC deficiency leading to dwarfism and impaired development. However, the double nmt1 nmt3 knock-out mutant is viable. Here, we show that this is enabled by residual PEAMT activity through a third family member, NMT2. The triple nmt1 nmt2 nmt3 knock-out mutant cannot synthesize PC from PEA and is lethal. This shows that, unlike mammals and yeast, Arabidopsis cannot form PC from phosphatidyl ethanolamine (PE), and demonstrates that methylation of PEA is the sole, and vital, entry point to PC synthesis. We further show that Arabidopsis has evolved an expanded family of four nonredundant PEAMTs through gene duplication and alternate use of the NMT2 promoter. NMT2 encodes two PEAMT variants, which greatly differ in their ability to perform the initial phospho-base methylation of PEA. Five amino acids at the N terminus of PEAMTs are shown to each be critical for the catalysis of that step committing to PC synthesis. As a whole, these findings open new avenues for enzymatic engineering and the exploration of ways to better tune phosphocholine and PC synthesis to environmental conditions for improved plant performance.

Lipid bilayer stress-activated IRE-1 modulates autophagy during endoplasmic reticulum stress.

Metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD), are emerging as epidemics that affect the global population. One facet of these disorders is attributed to the disturbance of membrane lipid composition. Perturbation of endoplasmic reticulum (ER) homeostasis through alteration in membrane phospholipids activates the unfolded protein response (UPR) and causes dramatic transcriptional and translational changes in the cell. To restore cellular homeostasis, the three highly conserved UPR transducers ATF6, IRE1 (also known as ERN1 in mammals) and PERK (also known as EIF2AK3 in mammals) mediate adaptive responses upon ER stress. The homeostatic UPR cascade is well characterised under conditions of proteotoxic stress, but much less so under lipid bilayer stress-induced UPR. Here, we show that disrupted phosphatidylcholine (PC) synthesis in Caenorhabditiselegans causes lipid bilayer stress, lipid droplet accumulation and ER stress induction. Transcriptional profiling of PC-deficient worms revealed a unique subset of genes regulated in a UPR-dependent manner that is independent from proteotoxic stress. Among these, we show that autophagy is modulated through the conserved IRE-1-XBP-1 axis, strongly suggesting of the importance of autophagy in maintaining cellular homeostasis during the lipid bilayer stress-induced UPR.

A pair of phospho-base methyltransferases important for phosphatidylcholine biosynthesis in Arabidopsis.

Phosphatidylcholine (PtdCho) is a predominant membrane lipid class in eukaryotes. Phospho-base N-methyltransferase (PMT) catalyzes a critical step in PtdCho biosynthesis. However, in Arabidopsis thaliana, the discovery of involvement of the specific PMT isoform in PtdCho biosynthesis remains elusive. Here, we show that PMT1 and PMT3 redundantly play an essential role in phosphocholine (PCho) biosynthesis, a prerequisite for PtdCho production. A pmt1 pmt3 double mutant was devoid of PCho, which affected PtdCho biosynthesis in vivo, showing severe growth defects in post-embryonic development. PMT1 and PMT3 were both highly expressed in the vasculature. The pmt1 pmt3 mutants had specifically affected leaf vein development and showed pale-green seedlings that were rescued by exogenous supplementation of PCho. We suggest that PMT1 and PMT3 are the primary enzymes for PCho biosynthesis and are involved in PtdCho biosynthesis and vascular development in Arabidopsis seedlings.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice.

Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

Phosphatidylcholine synthesis regulates triglyceride storage and chylomicron secretion by Caco2 cells.

Intracellular lipid droplets (LDs) supply fatty acids for energy, membrane biogenesis, and lipoprotein secretion. The surface monolayer of LDs is composed of phospholipids, primarily phosphatidylcholine (PC), that stabilize the neutral lipid core of triglyceride (TG). To determine the relationship between PC synthesis and TG storage and secretion in chylomicrons, we used a model of intestinal-derived human epithelial colorectal adenocarcinoma (Caco2) cells with knockout of PCYT1A, which encodes the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT)α in the CDP-choline pathway, that were treated with the fatty acid oleate. CRISPR/Cas9 knockout of CCTα in Caco2 cells (Caco2-KO cells) reduced PC synthesis by 50%. Compared with Caco2 cells, Caco2-KO cells exposed to oleate had fewer and larger LDs and greater TG accumulation as a result. The addition of exogenous lysophosphatidylcholine to Caco2-KO cells reversed the LD morphology defect. Caco2-KO cells, differentiated into epithelial monolayers, accumulated intracellular TG and had deficient TG and chylomicron-associated apoB48 secretion; apoB100 secretion was unaffected by CCTα knockout or oleate. Metabolic-labeling and LD imaging of Caco2-KO cells indicated preferential shuttling of de novo synthesized TG into larger LDs rather than into chylomicrons. Thus, reduced de novo PC synthesis in Caco2 cells enhances TG storage in large LDs and inhibits apoB48 chylomicron secretion.

Intestinal de novo phosphatidylcholine synthesis is required for dietary lipid absorption and metabolic homeostasis.

De novo phosphatidylcholine (PC) synthesis via CTP:phosphocholine cytidylyltransferase-α (CTα) is required for VLDL secretion. To determine the precise role of de novo PC synthesis in intestinal lipid metabolism, we deleted CTα exclusively in the intestinal epithelium of mice (CTαIKO mice). When fed a chow diet, CTαIKO mice showed normal fat absorption despite a ∼30% decrease in intestinal PC concentrations relative to control mice, suggesting that biliary PC can fully support chylomicron secretion under these conditions. However, when fed a high-fat diet, CTαIKO mice showed impaired passage of FAs and cholesterol from the intestinal lumen into enterocytes. Impaired intestinal lipid uptake in CTαIKO mice was associated with lower plasma triglyceride concentrations, higher plasma glucagon-like peptide 1 and peptide YY, and disruption of intestinal membrane lipid transporters after a high-fat meal relative to control mice. Unexpectedly, biliary bile acid and PC secretion was enhanced in CTαIKO mice due to a shift in expression of bile-acid transporters to the proximal intestine, indicative of accelerated enterohepatic cycling. These data show that intestinal de novo PC synthesis is required for dietary lipid absorption during high-fat feeding and that the reacylation of biliary lyso-PC cannot compensate for loss of CTα under these conditions.