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.

Lipid- and phospho-regulation of CTP:Phosphocholine Cytidylyltransferase α association with nuclear lipid droplets.

Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTα translocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1ß, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesis.

BACKGROUND: Studies have shown that the release of endogenous glutamate (Glu) participates in lung injury by activating N-methyl-D-aspartate receptor (NMDAR), but the mechanism is still unclear. This study was to investigate the effects and related mechanisms of Glu on the lipid synthesis of pulmonary surfactant (PS) in isolated rat lung tissues. METHODS: The cultured lung tissues of adult SD rats were treated with Glu. The amount of [3H]-choline incorporation into phosphatidylcholine (PC) was detected. RT-PCR and Western blot were used to detect the changes of mRNA and protein expression of cytidine triphosphate: phosphocholine cytidylyltransferase alpha (CCTα), a key regulatory enzyme in PC biosynthesis. Western blot was used to detect the expression of NMDAR1, which is a functional subunit of NMDAR. Specific protein 1 (Sp1) expression plasmids were used. After transfected with Sp1 expression plasmids, the mRNA and protein levels of CCTα were detected by RT-PCR and Western blot in A549 cells. After treated with NMDA and MK-801, the mRNA and protein levels of Sp1 were detected by RT-PCR and Western blot in A549 cells. RESULTS: Glu decreased the incorporation of [3H]-choline into PC in a concentration- and time- dependent manner. Glu treatment significantly reduced the mRNA and protein levels of CCTα in lungs. Glu treatment up-regulated NMDAR1 protein expression, and the NMDAR blocker MK-801 could partially reverse the reduction of [3H]-choline incorporation induced by Glu (10-4 mol/L) in lungs. After transfected with Sp1 plasmid for 30 h, the mRNA and protein expression levels of CCTα were increased and the protein expression of Sp1 was also up-regulated. After A549 cells were treated with NMDA, the level of Sp1 mRNA did not change significantly, but the expression of nucleus protein in Sp1 was significantly decreased, while the expression of cytoplasmic protein was significantly increased. However, MK-801could reverse these changes. CONCLUSIONS: Glu reduced the biosynthesis of the main lipid PC in PS and inhibited CCTα expression by activating NMDAR, which were mediated by the inhibition of the nuclear translocation of Sp1 and the promoter activity of CCTα. In conclusion, NMDAR-mediated Glu toxicity leading to impaired PS synthesis may be a potential pathogenesis of lung injury.

Spatiotemporal Variation of Residual Cortisol Obstructs Nutrient Acquisition in Animal-Derived Foods by Perturbing Glycerophospholipid Metabolism.

Cortisol is inevitably secreted by pigs due to the physical and psychological stressors produced by mixed group transportation and preslaughter handling. Accumulated cortisol in animal tissues enters the human body through the food chain and entails potential risks to human health. An integrated lipidome and proteome analysis was conducted to investigate the effect of the spatiotemporal variation of residual cortisol on nutrient acquisition in pork. A total of 55 crucial lipid molecules associated with cortisol residue were identified based on debiased sparse partial correlation analysis. Label-free proteomics was applied to screen 58 differentially abundant proteins (including phospholipase A2, lysophosphatidylcholine acyltransferase, and CTP:phosphocholine cytidylyltransferase), indicating that cortisol residue perturbed the glycerophospholipid biosynthetic process and glycerophospholipid metabolic process. Cortisol induced downregulations of cPLA2 encoding genes and decreased phospholipase A2 activity, resulting in the bioaccumulation of phosphatidylethanolamine (from 36.86 to 43.18 mg kg-1). Cortisol increased the activity of CTP:phosphocholine cytidylyltransferase by improving the availability of fatty acids and aggregating the inactive L-form (lipid-independent form) to the active H-form (lipid-associated form). The metabolic pathways perturbed by cortisol resulted in phosphatidylcholine degradation (from 93.73 to 58.28 mg kg-1) and lysophosphatidylcholine accumulation (from 3.39 to 5.16 mg kg-1). These findings indicated that cortisol residue deteriorated meat quality and obstructed nutrient acquisition in animal-origin foods.

Clinal variation in PHY (PAS domain) and CRY (CCT domain)-Signs of local adaptation to light quality in Norway spruce.

Detection of the genomic basis of local adaptation to environmental conditions is challenging in forest trees. Phytochromes (PHY) and cryptochromes (CRY) perceive the red (R)/far-red (FR) and blue light respectively, thus playing a fundamental role in regulating plant growth and development. PHYO and PHYP from conifers are the equivalents of PHYA/PHYC and PHYB in angiosperms, respectively. Norway spruce shows an adaptive latitudinal cline for shade (low R:FR or FR-enriched light) tolerance and requirement of FR light for its growth. We analyzed the exome capture data that included a uniquely large data set of 1654 Norway spruce trees sampled across many latitudes in Sweden to capture the natural clines for photoperiod and FR light exposure during the growth season. Statistically significant clinal variation was detected in allele and genotype frequencies of missense mutations in coding regions belonging to well-defined functional domains of PHYO (PAS-B), PHYP2 (PAS fold-2), CRY1 (CCT1) and CRY2 (CCT2) that strongly correlates with the latitudinal gradient in response to variable light quality in Norway spruce. The missense SNP in PHYO resulting in Asn835Ser, displayed the steepest cline among all other polymorphisms. We propose that these variations in the photoreceptors represent signs of local adaptation to light quality.

Genome-wide characterization of plant CTP:phosphocholine cytidylyltransferases through evolutionary, biochemical, and structural analyses.

Phosphatidylcholine has essential functions in many eukaryotic cells, and its de novo biosynthesis is rate-limited by cytidine triphosphate:phosphocholine cytidylyltransferase (CCT). Although the biological and biochemical functions of CCT have been reported in mammals and several plants, this key enzyme has yet to be examined at a genome-wide level. As such, certain fundamental questions remain unanswered, including the evolutionary history, genetic and functional relationships, and structural variations among CCTs in the green lineage. In the current study, in-depth phylogenetic analysis, as well as the conservation and diversification in CCT gene structure and motif patterns, indicated that CCTs exist broadly in chlorophytes, bryophytes, lycophytes, monilophytes, gymnosperms, early-diverging angiosperms, monocots, and eudicots, and form eight relatively conserved clades. To further explore the potential function of selection pressure, we conducted extensive selection pressure analysis with a representative CCT gene, CCT1 from the model plant Arabidopsis thaliana (AthCCT1), and identified two positive selection sites, L59 and Q156. Site-directed mutagenesis and in vitro enzyme assays demonstrated that these positively selected sites were indeed important for the activity and substrate affinity of AthCCT1, and subsequent 3D structure analyses explained the potential biochemical mechanism. Taken together, our results unraveled the evolution and diversity of CCTs in the green lineage, as well as their association with the enzyme's biochemical and structural properties, and expanded our understanding of this important enzyme at the genome-wide level.

Differential contributions of phosphotransferases CEPT1 and CHPT1 to phosphatidylcholine homeostasis and lipid droplet biogenesis.

The cytidine diphosphate-choline (Kennedy) pathway culminates with the synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by choline/ethanolamine phosphotransferase 1 (CEPT1) in the endoplasmic reticulum (ER), and PC synthesis by choline phosphotransferase 1 (CHPT1) in the Golgi apparatus. Whether the PC and PE synthesized by CEPT1 and CHPT1 in the ER and Golgi apparatus has different cellular functions has not been formally addressed. Here, we used CRISPR editing to generate CEPT1-and CHPT1-KO U2OS cells to assess the differential contribution of the enzymes to feedback regulation of nuclear CTP:phosphocholine cytidylyltransferase (CCT)α, the rate-limiting enzyme in PC synthesis, and lipid droplet (LD) biogenesis. We found that CEPT1-KO cells had a 50 and 80% reduction in PC and PE synthesis, respectively, while PC synthesis in CHPT1-KO cells was also reduced by 50%. CEPT1 KO caused the posttranscriptional induction of CCTα protein expression as well as its dephosphorylation and constitutive localization on the inner nuclear membrane and nucleoplasmic reticulum. This activated CCTα phenotype was prevented by incubating CEPT1-KO cells with PC liposomes to restore end-product inhibition. Additionally, we determined that CEPT1 was in close proximity to cytoplasmic LDs and CEPT1 KO resulted in the accumulation of small cytoplasmic LDs, as well as increased nuclear LDs enriched in CCTα. In contrast, CHPT1 KO had no effect on CCTα regulation or LD biogenesis. Thus, CEPT1 and CHPT1 contribute equally to PC synthesis; however, only PC synthesized by CEPT1 in the ER regulates CCTα and the biogenesis of cytoplasmic and nuclear LDs.

PCYT1A Missense Variant in Vizslas with Disproportionate Dwarfism.

Disproportionate dwarfism phenotypes represent a heterogeneous subset of skeletal dysplasias and have been described in many species including humans and dogs. In this study, we investigated Vizsla dogs that were affected by disproportionate dwarfism that we propose to designate as skeletal dysplasia 3 (SD3). The most striking skeletal changes comprised a marked shortening and deformation of the humerus and femur. An extended pedigree with six affected dogs suggested autosomal recessive inheritance. Combined linkage and homozygosity mapping localized a potential genetic defect to a ~4 Mb interval on chromosome 33. We sequenced the genome of an affected dog, and comparison with 926 control genomes revealed a single, private protein-changing variant in the critical interval, PCYT1A:XM_038583131.1:c.673T>C, predicted to cause an exchange of a highly conserved amino acid, XP_038439059.1:p.(Y225H). We observed perfect co-segregation of the genotypes with the phenotype in the studied family. When genotyping additional Vizslas, we encountered a single dog with disproportionate dwarfism that did not carry the mutant PCYT1A allele, which we hypothesize was due to heterogeneity. In the remaining 130 dogs, we observed perfect genotype-phenotype association, and none of the unaffected dogs were homozygous for the mutant PCYT1A allele. PCYT1A loss-of-function variants cause spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) in humans. The skeletal changes in Vizslas were comparable to human patients. So far, no ocular phenotype has been recognized in dwarf Vizslas. We propose the PCYT1A missense variant as a candidate causative variant for SD3. Our data facilitate genetic testing of Vizslas to prevent the unintentional breeding of further affected puppies.

Molecular diagnosis in a cohort of 114 patients with rare skeletal dysplasias.

Molecular diagnosis is important to provide accurate genetic counseling of skeletal dysplasias (SD). Although next-generation sequencing (NGS) techniques are currently the preferred methods for analyzing these conditions, some of the published results have not shown a detection rate as high as it would be expected. The present study aimed to assess the diagnostic yield of targeted NGS combined with Sanger sequencing (SS) for low-coverage exons of genes of interest and exome sequencing (ES) in a series of patients with rare SD and use two patients as an example of our strategy. This study used two different in-house panels. Of 93 variants found in 88/114 (77%) patients, 57 are novel. The pathogenic variants found in the following genes: B3GALT6, PCYT1A, INPPL1, LIFR, of four patients were only detected by SS. In conclusion, the high diagnostic yield reached in the present study can be attributed to both a good selection of patients and the utilization of the SS for the insufficiently covered regions. Additionally, the two case reports-a patient with acrodysostosis related to PRKAR1A and another with ciliopathy associated with KIAA0753, add new and relevant clinical information to the current knowledge.

Combination of biomarkers for neoadjuvant systemic chemotherapy before cystectomy in patients with urinary bladder cancer.

Clinical utility of cisplatin based neoadjuvant chemotherapy (NAC) prior to radical cystectomy is limited because of lack of tools that can guide for a better patient selection. We aim to explore if a combination of biomarkers is superior to a single marker. Pretreatment tumor specimens and clinical data from two randomized trials including 250 patients with T2-T4 urothelial bladder cancer, were used. The information on the expressions on tumor tissue of four biomarkers; CCTα, emmprin, survivin, and BCL-2, detected by immunohistochemistry in our previous studies, was used. Cox proportional hazard models, including treatment-by-biomarker interaction terms, were used to assess the predictive value of the biomarkers for efficacy of NAC on overall survival. CCTα provided predictive information about the efficacy of NAC (interaction P=0.009). None of the other biomarkers provided statistically significant information additional to CCTα. The adjusted hazard ratio for NAC treated versus no-NAC was 0.42 (95% CI: 0.27-0.64) for patients with negative CCTα expression, when adding information about emmprin it decreased to 0.33 (95% CI: 0.19-0.56) for patients with both negative CCTα and emmprin. This corresponds to a decrease in number needed to treat from 4 to 3 patients. The combination of CCTα with survivin or BCL-2 yielded similar results. In a group of patients with muscle invasive bladder cancer a combination of two biomarkers might improve the possibility to identify patients most likely to benefit from the use of NAC. Further studies designed to have sufficient power to detect an interaction effect are needed.

Methylome and transcriptome profiling revealed epigenetic silencing of LPCAT1 and PCYT1A associated with lipidome alterations in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine diseases of fertile women and a major cause of infertility. The regulatory effects of DNA methylation on gene transcription and downstream lipid metabolism have not been explored in PCOS. In this study, MBD-seq and RNA-seq were performed on ovarian granulosa cells of PCOS patients and controls, and methylation specific PCR and quantitative polymerase chain reaction were used to validate the results. Then lipidomic profiling was conducted on serum of PCOS patients and controls using UPLC-MS. We identified 73 genes with differently methylated promoters and 830 differently expressed genes. The promoter regions of LPCAT1 and PCYT1A were hypermethylated, accompanied by downregulation of their messenger RNA expression, which may be involved in the regulation of PCOS through downstream glycerophospholipid metabolism and phosphatidylcholine synthesis. The lipid profiling results showed significant changes in 21 lipids, which demonstrated the disturbance in glycerophospholipid metabolism and glycerolipid metabolism pathways. Furthermore, the metabolites-genes interaction network was constructed to illustrate the association of aberrant methylome and transcriptome with lipidome alterations in glycerolipid and glycerophospholipid metabolism pathways. Our study suggested that the methylation silencing of LPCAT1 and PCYT1A may promote glycerophospholipids metabolism dysregulation, which provided a novel genetic and lipometabolic basis for the pathogenesis of PCOS.

Intestinal Phospholipid Disequilibrium Initiates an ER Stress Response That Drives Goblet Cell Necroptosis and Spontaneous Colitis in Mice.

BACKGROUND & AIMS: Patients with ulcerative colitis have low concentrations of the major membrane lipid phosphatidylcholine (PC) in gastrointestinal mucus, suggesting that defects in colonic PC metabolism might be involved in the development of colitis. To determine the precise role that PC plays in colonic barrier function, we examined mice with intestinal epithelial cell (IEC)-specific deletion of the rate-limiting enzyme in the major pathway for PC synthesis: cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTαIKO mice). METHODS: Colonic tissue of CTαIKO mice and control mice was analyzed by histology, immunofluorescence, electron microscopy, quantitative polymerase chain reaction, Western blot, and thin-layer chromatography. Histopathologic colitis scores were assigned by a pathologist blinded to the experimental groupings. Intestinal permeability was assessed by fluorescein isothiocyanate-dextran gavage and fecal microbial composition was analyzed by sequencing 16s ribosomal RNA amplicons. Subsets of CTαIKO mice and control mice were treated with dietary PC supplementation, antibiotics, or 4-phenylbutyrate. RESULTS: Inducible loss of CTα in the intestinal epithelium reduced colonic PC concentrations and resulted in rapid and spontaneous colitis with 100% penetrance in adult mice. Colitis development in CTαIKO mice was traced to a severe and unresolving endoplasmic reticulum stress response in IECs with altered membrane phospholipid composition. This endoplasmic reticulum stress response was linked to the necroptotic death of IECs, leading to excessive loss of goblet cells, formation of a thin mucus barrier, increased intestinal permeability, and infiltration of the epithelium by microbes. CONCLUSIONS: Maintaining the PC content of IEC membranes protects against colitis development in mice, showing a crucial role for IEC phospholipid equilibrium in colonic homeostasis. SRA accession number: PRJNA562603.

Identification and characterization of CTP:phosphocholine cytidylyltransferase CpCCT1 in the resurrection plant Craterostigma plantagineum.

Phosphatidylcholine is a major phospholipid which is shown to be involved in stress adaptation. Phosphatidylcholine increased during dehydration in Craterostigma plantagineum, and therefore we characterized CTP:phosphocholine cytidylyltransferase (CpCCT1), a key regulatory enzyme for phosphatidylcholine synthesis in plants. The CpCCT1 gene from the resurrection plant C. plantagineum was cloned and the amino acid sequence was compared with homologs from other species including yeast and rat. CCT proteins have conserved catalytic and membrane-binding domains while the N-terminal and C-terminal domains have diverged. The tissue specific expression analysis indicated that CpCCT1 is expressed in all tested tissues and it is induced by dehydration and in response to 0.5 M NaCl solutions. In plants exposed to low temperature in the dark, the CpCCT1 transcript increased after 4 h at 4 °C. CpCCT1 expression also increased during mannitol and sorbitol treatments in a concentration dependent manner. Phytohormones such as abscisic acid and indole-3-acetic acid also trigged transcript accumulation. Comparisons of transcript and protein accumulations for different treatments (except for dehydration) suggest transcriptional and translational control mechanisms. Analysis of promoter activity and polysome occupancy suggest that CpCCT1 gene expression is mainly under translational regulation during dehydration.

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.

Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function.

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme.

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

Long-term autophagy is sustained by activation of CCTß3 on lipid droplets.

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase ß3 (CCTß3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTß3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTß3 and is enhanced by its overexpression. This CCTß3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTß3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTß3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.

PCYT1A suppresses proliferation and migration via inhibiting mTORC1 pathway in lung adenocarcinoma.

Lung cancer is one of most common malignant cancer worldwide. It is emerging that PCYT1A, a rate-limiting enzyme required for the biosynthesis of phosphatidylcholine, is associated with cancer progression. However, the biological functions and underlying molecular mechanisms of PCYT1A in lung adenocarcinoma is still unknown. Here we found that PCYT1A suppressed lung adenocarcinoma cancer cell proliferation and migration. Mechanically, PCYT1A served as a novel negative regulator of mTORC1 signaling. PCYT1A knockdown enhanced the malignant proliferation and migration of lung adenocarcinoma cells by activating mTORC1. The promoting effects of PCYT1A silencing on cell proliferation and migration could be abolished when mTORC1 signaling was inhibited by rapamycin or RAPTOR depletion. Importantly, PCYT1A high expression predicted longer survival of lung cancer patients. The expression of PCYT1A was also negatively correlated with mTORC1 activation in the clinical lung cancer samples. We therefore reveal that PCYT1A suppresses proliferation and migration by inhibiting the mTORC1 signaling pathway in lung adenocarcinoma. PCYT1A shows as a potential promising biomarker in lung adenocarcinoma.

Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1.

Nuclear lipid droplets (nLDs) form on the inner nuclear membrane by a mechanism involving promyelocytic leukemia (PML), the protein scaffold of PML nuclear bodies. We report that PML structures on nLDs in oleate-treated U2OS cells, referred to as lipid-associated PML structures (LAPS), differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100, and DAXX. These nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1, and DAG. Translocation of CCTα onto nLDs was mediated by its α-helical M-domain but was not correlated with its activator DAG. High-resolution imaging revealed that CCTα and LAPS occupied distinct polarized regions on nLDs. PML knockout U2OS (PML KO) cells lacking LAPS had a 40-50% reduction in nLDs with associated CCTα, and residual nLDs were almost devoid of Lipin1 and DAG. As a result, phosphatidylcholine and triacylglycerol synthesis was inhibited in PML KO cells. We conclude that in response to excess exogenous fatty acids, LAPS are required to assemble nLDs that are competent to recruit CCTα and Lipin1.

Membrane Lipids Assist Catalysis by CTP: Phosphocholine Cytidylyltransferase.

While most of the articles in this issue review the workings of integral membrane enzymes, in this review, we describe the catalytic mechanism of an enzyme that contains a soluble catalytic domain but appears to catalyze its reaction on the membrane surface, anchored and assisted by a separate regulatory amphipathic helical domain and inter-domain linker. Membrane partitioning of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme of phosphatidylcholine metabolism, is regulated chiefly by changes in membrane phospholipid composition, and boosts the enzyme's catalytic efficiency >200-fold. Catalytic enhancement by membrane binding involves the displacement of an auto-inhibitory helix from the active site entrance-way and promotion of a new conformational ensemble for the inter-domain, allosteric linker that has an active role in the catalytic cycle. We describe the evidence for close contact between membrane lipid, a compact allosteric linker, and the CCT active site, and discuss potential ways that this interaction enhances catalysis.