Membrane phospholipids activate the inflammatory response in macrophages by various mechanisms.

Exosomes, which are small membrane-encapsulated particles derived from all cell types, are emerging as important mechanisms for intercellular communication. In addition, exosomes are currently envisioned as potential carriers for the delivery of drugs to target tissues. The natural population of exosomes is very variable due to the limited amount of cargo components present in these small vesicles. Consequently, common components of exosomes may play a role in their function. We have proposed that membrane phospholipids could be a common denominator in the effect of exosomes on cellular functions. In this regard, we have previously shown that liposomes made of phosphatidylcholine (PC) or phosphatidylserine (PS) induced a robust alteration of macrophage (Mϕ) gene expression. We herewith report that these two phospholipids modulate gene expression in Mϕs by different mechanisms. PS alters cellular responses by the interaction with surface receptors, particularly CD36. In contrast, PC is captured by a receptor-independent process and likely triggers an activity within endocytic vesicles. Despite this difference in the capture mechanisms, both lipids mounted similar gene expression responses. This investigation suggests that multiple mechanisms mediated by membrane phospholipids could be participating in the alteration of cellular functions by exosomes.

S-Adenosyl-l-methionine restores brain mitochondrial membrane fluidity and GSH content improving Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

Germ-free status but not subacute polychlorinated biphenyl (PCB) exposure altered hepatic phosphatidylcholine and ether-phosphatidylcholine levels in mice.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that pose a current ecosystem and human health concern. PCB exposure impacts the gut microbiome in animal models, suggesting a mechanistic link between PCB exposure and adverse health outcomes. The presence and absence of the microbiome and exposure to PCBs independently affect the lipid composition in the liver, which in turn affects the PCB disposition in target tissues, such as the liver. Here, we investigated microbiome × subacute PCB effects on the hepatic lipid composition of conventional and germ-free female mice exposed to 0, 6, or 30 mg/kg body weight of an environmental PCB mixture in sterile corn oil once daily for 3 consecutive days. Hepatic triacylglyceride and polar lipid levels were quantified using mass spectrometric methods following the subacute PCB exposure. The lipidomic analysis revealed no PCB effect on the hepatic levels. No microbiome effect was observed on levels of triacylglyceride and most polar lipid classes. The total hepatic levels of phosphatidylcholine (PC) and ether-phosphatidylcholine (ePC) lipids were lower in germ-free mice than the conventional mice from the same exposure group. Moreover, levels of several unsaturated PCs, such as PC(36:5) and PC(42:10), and ePCs, such as ePC(36:2) and ePC(4:2), were lower in germ-free than conventional female mice. Based on a KEGG pathway meta-analysis of RNA sequencing data, the ether lipid metabolism pathway is altered in the germ-free mouse liver. In contrast to the liver, extractable lipid levels, determined gravimetrically, differed in several tissues from naïve conventional vs. germ-free mice. Overall, microbiome × subacute PCB exposure effects on hepatic lipid composition are unlikely to affect PCB distribution into the mouse liver. Further studies are needed to assess how the different extractable lipid levels in other tissues alter PCB distribution in conventional vs. germ-free mice.

Spatial MS multiomics on clinical prostate cancer tissues.

Mass spectrometry (MS) and MS imaging (MSI) are used extensively for both the spatial and bulk characterization of samples in lipidomics and proteomics workflows. These datasets are typically generated independently due to different requirements for sample preparation. However, modern omics technologies now provide higher sample throughput and deeper molecular coverage, which, in combination with more sophisticated bioinformatic and statistical pipelines, make generating multiomics data from a single sample a reality. In this workflow, we use spatial lipidomics data generated by matrix-assisted laser desorption/ionization MSI (MALDI-MSI) on prostate cancer (PCa) radical prostatectomy cores to guide the definition of tumor and benign tissue regions for laser capture microdissection (LCM) and bottom-up proteomics all on the same sample and using the same mass spectrometer. Accurate region of interest (ROI) mapping was facilitated by the SCiLS region mapper software and dissected regions were analyzed using a dia-PASEF workflow. A total of 5525 unique protein groups were identified from all dissected regions. Lysophosphatidylcholine acyltransferase 1 (LPCAT1), a lipid remodelling enzyme, was significantly enriched in the dissected regions of cancerous epithelium (CE) compared to benign epithelium (BE). The increased abundance of this protein was reflected in the lipidomics data with an increased ion intensity ratio for pairs of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) in CE compared to BE.

Lipidomics studies reveal dynamic changes in polar lipids of developing endosperm of oat and wheat varieties with differing oil contents.

Polar lipids have biosynthetic pathways which intersect and overlap with triacylglycerol biosynthesis; however, polar lipids have not been well characterized in the developing endosperms of oat with high oil accumulation. The polar lipids in endosperms of oat and wheat varieties having different oil contents were analyzed and compared at different developmental stages. Our study shows that the relative contents of polar lipid by mass were decreased more slowly in wheat than in oat. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, which showed similar abundance and gradual decreases during endosperm development in oat and wheat, while lysophospholipids were noticeably higher in oat. Monogalactosyldiacylglycerol showed a gradual increase in wheat and a decrease in oat during endosperm development. The relative contents of some polar lipid species and their unsaturation index were significantly different in their endosperms. These characteristics of polar lipids might indicate an adaption of oat to accommodate oil accumulation.

Association between pre-diagnostic circulating lipid metabolites and colorectal cancer risk: a nested case-control study in the European Prospective Investigation into Cancer and Nutrition (EPIC).

BACKGROUND: Altered lipid metabolism is a hallmark of cancer development. However, the role of specific lipid metabolites in colorectal cancer development is uncertain. METHODS: In a case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC), we examined associations between pre-diagnostic circulating concentrations of 97 lipid metabolites (acylcarnitines, glycerophospholipids and sphingolipids) and colorectal cancer risk. Circulating lipids were measured using targeted mass spectrometry in 1591 incident colorectal cancer cases (55% women) and 1591 matched controls. Multivariable conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for associations between concentrations of individual lipid metabolites and metabolite patterns with colorectal cancer risk. FINDINGS: Of the 97 assayed lipids, 24 were inversely associated (nominally p < 0.05) with colorectal cancer risk. Hydroxysphingomyelin (SM (OH)) C22:2 (ORper doubling 0.60, 95% CI 0.47-0.77) and acylakyl-phosphatidylcholine (PC ae) C34:3 (ORper doubling 0.71, 95% CI 0.59-0.87) remained associated after multiple comparisons correction. These associations were unaltered after excluding the first 5 years of follow-up after blood collection and were consistent according to sex, age at diagnosis, BMI, and colorectal subsite. Two lipid patterns, one including 26 phosphatidylcholines and all sphingolipids, and another 30 phosphatidylcholines, were weakly inversely associated with colorectal cancer. INTERPRETATION: Elevated pre-diagnostic circulating levels of SM (OH) C22:2 and PC ae C34:3 and lipid patterns including phosphatidylcholines and sphingolipids were associated with lower colorectal cancer risk. This study may provide insight into potential links between specific lipids and colorectal cancer development. Additional prospective studies are needed to validate the observed associations. FUNDING: World Cancer Research Fund (reference: 2013/1002); European Commission (FP7: BBMRI-LPC; reference: 313010).

Inhibition of choline metabolism in an angioimmunoblastic T-cell lymphoma preclinical model reveals a new metabolic vulnerability as possible target for treatment.

BACKGROUND: Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4+ follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option. METHODS: To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data. RESULTS: Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1high neoplastic cells. CONCLUSION: Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients.

p53 suppresses lipid droplet-fueled tumorigenesis through phosphatidylcholine.

Choline deficiency causes disorders including hepatic abnormalities and is associated with an increased risk of multiple types of cancer. Here, by choline-free diet-associated RNA-Seq analyses, we found that the tumor suppressor p53 drives the Kennedy pathway via PCYT1B to control the growth of lipid droplets (LDs) and their fueling role in tumorigenesis. Mechanistically, through upregulation of PCYT1B, p53 channeled depleted choline stores to phosphatidylcholine (PC) biosynthesis during choline starvation, thus preventing LD coalescence. Cells lacking p53 failed to complete this response to choline depletion, leading to hepatic steatosis and tumorigenesis, and these effects could be reversed by enforcement of PCYT1B expression or restoration of PC abundance. Furthermore, loss of p53 or defects in the Kennedy pathway increased surface localization of hormone-sensitive lipase on LDs to release specific fatty acids that fueled tumor cells in vivo and in vitro. Thus, p53 loss leads to dysregulation of choline metabolism and LD growth and couples perturbed LD homeostasis to tumorigenesis.

Designing and fabrication of colloidal nano-phytosomes with gamma-oryzanol and phosphatidylcholine for encapsulation and delivery of polyphenol-rich extract from pomegranate peel.

Nano-carriers are well-known delivery systems to encapsulate different bioactive compounds and extracts. Such nano-systems are used in various food and drug areas to protect active ingredients, increase bioavailability, control the release, and deliver bioactive substances. This study aimed to design and fabricate a stable colloidal nano-delivery system to better preserve the antioxidant properties of pomegranate peel extract (PPE) and protect its sustained release in a gastrointestinal model. To achieve this goal, a nano-phytosomal system was fabricated with plant-based, cost-effective, and food-grade compounds, i.e., phosphatidylcholine (PC) and gamma-oryzanol (GO) for encapsulation of PPE. To fabricate the nano-phytosomes, thin film hydration/sonication method was used. The parameters of particle size, zeta potential, polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) were investigated to evaluate the efficiency of the produced nano-system. In summary, the size, zeta potential, PDI, LC, and EE of homogenous spherical PC-GO-PPE nano-phytosomes (NPs) in the ratio of 8:2:2 % w/w were achieved as 60.61 ± 0.81 nm, -32.24 ± 0.84 mV, 0.19 ± 0.01, 19.13 ± 0.30 %, and 95.66 ± 1.52 %, respectively. Also, the structure of NPs was approved by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). The optimized NPs were stable during one month of storage at 4 °C, and changes in the size of particles and PPE retention rate were insignificant (p > 0.05). The nano-encapsulation of PPE significantly decreased the loss of its antioxidant activity during one month of storage at 4 °C. The optimized NPs exhibited prolonged and sustained release of PPE in a gastrointestinal model, so that after 2 h in simulated gastric fluid (SGF) and 4 h in simulated intestinal fluid (SIF), 22.66 ± 2.51 % and 69.33 ± 4.50 % of initially loaded PPE was released, respectively. Optimized NPs had considerable cytotoxicity against the Michigan Cancer Foundation-7 cell line (MCF7) (IC50 = 103 µg/ml), but not against Human Foreskin Fibroblast cell line (HFF-2) (IC50 = 453 µg/ml). In conclusion, spherical PC-GO-PPE NPs were identified as a promising delivery system to efficiently encapsulate PPE, as well as protect and preserve its bioactivity, including antioxidant and cytotoxicity against cancer cell line.

Intestinal Atp8b1 dysfunction causes hepatic choline deficiency and steatohepatitis.

Choline is an essential nutrient, and its deficiency causes steatohepatitis. Dietary phosphatidylcholine (PC) is digested into lysoPC (LPC), glycerophosphocholine, and choline in the intestinal lumen and is the primary source of systemic choline. However, the major PC metabolites absorbed in the intestinal tract remain unidentified. ATP8B1 is a P4-ATPase phospholipid flippase expressed in the apical membrane of the epithelium. Here, we use intestinal epithelial cell (IEC)-specific Atp8b1-knockout (Atp8b1IEC-KO) mice. These mice progress to steatohepatitis by 4 weeks. Metabolomic analysis and cell-based assays show that loss of Atp8b1 in IEC causes LPC malabsorption and thereby hepatic choline deficiency. Feeding choline-supplemented diets to lactating mice achieves complete recovery from steatohepatitis in Atp8b1IEC-KO mice. Analysis of samples from pediatric patients with ATP8B1 deficiency suggests its translational potential. This study indicates that Atp8b1 regulates hepatic choline levels through intestinal LPC absorption, encouraging the evaluation of choline supplementation therapy for steatohepatitis caused by ATP8B1 dysfunction.

Fetal sex differences in placental LCPUFA ether and plasmalogen phosphatidylethanolamine and phosphatidylcholine contents in pregnancies complicated by obesity.

BACKGROUND: We have previously reported that maternal obesity reduces placental transport capacity for lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA), a preferred form for transfer of DHA (omega 3) to the fetal brain, but only in male fetuses. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC), have either sn-1 ester, ether or vinyl ether (plasmalogen) linkages to primarily unsaturated and monounsaturated fatty acids and DHA or arachidonic acid (ARA, omega 6) in the sn-2 position. Whether ether and plasmalogen PC and PE metabolism in placenta impacts transfer to the fetus is unexplored. We hypothesized that ether and plasmalogen PC and PE containing DHA and ARA are reduced in maternal-fetal unit in pregnancies complicated by obesity and these differences are dependent on fetal sex. METHODS: In maternal, umbilical cord plasma and placentas from obese women (11 female/5 male infants) and normal weight women (9 female/7 male infants), all PC and PE species containing DHA and ARA were analyzed by LC-MS/MS. Placental protein expression of enzymes involved in phospholipid synthesis, were determined by immunoblotting. All variables were compared between control vs obese groups and separated by fetal sex, in each sample using the Benjamini-Hochberg false discovery rate adjustment to account for multiple testing. RESULTS: Levels of ester PC containing DHA and ARA were profoundly reduced by 60-92% in male placentas of obese mothers, while levels of ether and plasmalogen PE containing DHA and ARA were decreased by 51-84% in female placentas. PLA2G4C abundance was lower in male placentas and LPCAT4 abundance was lower solely in females in obesity. In umbilical cord, levels of ester, ether and plasmalogen PC and PE with DHA were reduced by 43-61% in male, but not female, fetuses of obese mothers. CONCLUSIONS: We found a fetal sex effect in placental PE and PC ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Docosahexaenoic acid (DHA) is a critical omega 3 long chain polyunsaturated fatty acid (LCPUFA) for fetal brain development. We have recently reported that maternal obesity reduces placental transport capacity for LysophosPhatidylCholine-DHA (LPC-DHA), a preferred form for transfer of DHA to the fetal brain, but only in male fetuses. Other important lipids, the plasmalogen phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are considered DHA reservoirs, but its roles in the maternal­fetal unit are largely unexplored. We examined these lipid species in maternal and fetal circulation and in placental tissue to uncover potential novel roles for ether and plasmalogen lipids in the regulation of placenta delivery of these vital nutrients in pregnancies complicated by obesity depending of fetal sex. We demonstrated for the first time, that female fetuses of obese mothers decrease placental ether and plasmalogen PE containing DHA and arachidonic acid (ARA, omega 6), and show a high fetal­placental adaptability and placental reserve capacity that can maintain the PC-LCPUFA synthesis and the transfer of these crucial species to the fetus to preserve brain development. Our study also demonstrated that male fetuses, in response to maternal obesity, reduce the placental ester PC species containing DHA and ARA and reduce the ether and plasmalogen PE reservoir of DHA and ARA in fetal circulation. Our findings support a fetal sex effect in placental ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Phosphatidylcholine-Specific Phospholipase C as a Promising Drug Target.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is an enzyme that catalyzes the formation of the important secondary messengers phosphocholine and diacylglycerol (DAG) from phosphatidylcholine. Although PC-PLC has been linked to the progression of many pathological conditions, including cancer, atherosclerosis, inflammation and neuronal cell death, studies of PC-PLC on the protein level have been somewhat neglected with relatively scarce data. To date, the human gene expressing PC-PLC has not yet been found, and the only protein structure of PC-PLC that has been solved was from Bacillus cereus (PC-PLCBc). Nonetheless, there is evidence for PC-PLC activity as a human functional equivalent of its prokaryotic counterpart. Additionally, inhibitors of PC-PLCBc have been developed as potential therapeutic agents. The most notable classes include 2-aminohydroxamic acids, xanthates, N,N'-hydroxyureas, phospholipid analogues, 1,4-oxazepines, pyrido[3,4-b]indoles, morpholinobenzoic acids and univalent ions. However, many medicinal chemistry studies lack evidence for their cellular and in vivo effects, which hampers the progression of the inhibitors towards the clinic. This review outlines the pathological implications of PC-PLC and highlights current progress and future challenges in the development of PC-PLC inhibitors from the literature.

Lipidome Profiling in Childhood Obesity Compared to Adults: A Pilot Study.

The objective is to assess the circulating lipidome of children with obesity before and after lifestyle intervention and to compare the data to the circulating lipidome of adults with obesity before and after bariatric surgery. Ten pediatric (PE) and thirty adult (AD) patients with obesity were prospectively recruited at a referral single center. The PE cohort received lifestyle recommendations. The AD cohort underwent bariatric surgery. Clinical parameters and lipidome were analyzed in serum before and after six months of metabolic intervention. The abundance of phosphatidylinositols in the PE cohort and phosphatidylcholines in the AD significantly increased, while O-phosphatidylserines in the PE cohort and diacyl/triacylglycerols in the AD decreased. Fifteen lipid species were coincident in both groups after lifestyle intervention and bariatric surgery. Five species of phosphatidylinositols, sphingomyelins, and cholesteryl esters were upregulated. Eight species of diacylglycerols, glycerophosphoglycerols, glycerophosphoethanolamines, and phosphatidylcholines were downregulated. Most matching species were regulated in the same direction except for two phosphatidylinositols: PI(O-36:2) and PI(O-34:0). A specific set of lipid species regulated after bariatric surgery in adult individuals was also modulated in children undergoing lifestyle intervention, suggesting they may constitute a core circulating lipid profile signature indicative of early development of obesity and improvement after clinical interventions regardless of individual age.

PEMT Mediates Hepatitis C Virus-Induced Steatosis, Explains Genotype-Specific Phenotypes and Supports Virus Replication.

The hepatitis C virus (HCV) relies on cellular lipid pathways for virus replication and also induces liver steatosis, but the mechanisms involved are not clear. We performed a quantitative lipidomics analysis of virus-infected cells by combining high-performance thin-layer chromatography (HPTLC) and mass spectrometry, using an established HCV cell culture model and subcellular fractionation. Neutral lipid and phospholipids were increased in the HCV-infected cells; in the endoplasmic reticulum there was an ~four-fold increase in free cholesterol and an ~three-fold increase in phosphatidyl choline (p < 0.05). The increase in phosphatidyl choline was due to the induction of a non-canonical synthesis pathway involving phosphatidyl ethanolamine transferase (PEMT). An HCV infection induced expression of PEMT while knocking down PEMT with siRNA inhibited virus replication. As well as supporting virus replication, PEMT mediates steatosis. Consistently, HCV induced the expression of the pro-lipogenic genes SREBP 1c and DGAT1 while inhibiting the expression of MTP, promoting lipid accumulation. Knocking down PEMT reversed these changes and reduced the lipid content in virus-infected cells. Interestingly, PEMT expression was over 50% higher in liver biopsies from people infected with the HCV genotype 3 than 1, and three times higher than in people with chronic hepatitis B, suggesting that this may account for genotype-dependent differences in the prevalence of hepatic steatosis. PEMT is a key enzyme for promoting the accumulation of lipids in HCV-infected cells and supports virus replication. The induction of PEMT may account for virus genotype specific differences in hepatic steatosis.

An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

FACS-assisted single-cell lipidome analysis of phosphatidylcholines and sphingomyelins in cells of different lineages.

Recent advances in single-cell genomics and transcriptomics technologies have transformed our understanding of cellular heterogeneity in growth, development, ageing, and disease; however, methods for single-cell lipidomics have comparatively lagged behind in development. We have developed a method for the detection and quantification of a wide range of phosphatidylcholine and sphingomyelin species from single cells that combines fluorescence-assisted cell sorting with automated chip-based nanoESI and shotgun lipidomics. We show herein that our method is capable of quantifying more than 50 different phosphatidylcholine and sphingomyelin species from single cells and can easily distinguish between cells of different lineages or cells treated with exogenous fatty acids. Moreover, our method can detect more subtle differences in the lipidome between cell lines of the same cancer type. Our approach can be run in parallel with other single-cell technologies to deliver near-complete, high-throughput multi-omics data on cells with a similar phenotype and has the capacity to significantly advance our current knowledge on cellular heterogeneity.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

sn-1 Specificity of Lysophosphatidylcholine Acyltransferase-1 Revealed by a Mass Spectrometry-Based Assay.

Lysophosphatidylcholine acyltransferase-1 (LPCAT1) plays a critical role in the remodeling of phosphatidylcholines (PCs) in cellular lipidome. However, evidence is scarce regarding its sn-selectivity, viz. the preference of assembling acyl-Coenzyme A (CoA) at the C1 or C2-hydroxyl on a glycerol backbone because of difficulty to quantify the thus-formed PC sn-isomers. We have established a multiplexed assay to measure both sn- and acyl-chain selectivity of LPCAT1 toward a mixture of acyl-CoAs by integrating isomer-resolving tandem mass spectrometry. Our findings reveal that LPCAT1 shows exclusive sn-1 specificity regardless of the identity of acyl-CoAs. We further confirm that elevated PC 18 : 1/16:0 relative to its sn-isomer results from an increased expression of LPCAT1 in human hepatocellular carcinoma (HCC) tissue as compared to normal liver tissue. MS imaging via desorption electrospray ionization of PC 18 : 1/16:0 thus enables visualization of HCC margins in human liver tissue at a molecular level.