PCYT1A deficiency disturbs fatty acid metabolism and induces ferroptosis in the mouse retina.

BACKGROUND: Inherited retinal dystrophies (IRDs) are a group of debilitating visual disorders characterized by the progressive degeneration of photoreceptors, which ultimately lead to blindness. Among the causes of this condition, mutations in the PCYT1A gene, which encodes the rate-limiting enzyme responsible for phosphatidylcholine (PC) de novo synthesis via the Kennedy pathway, have been identified. However, the precise mechanisms underlying the association between PCYT1A mutations and IRDs remain unclear. To address this knowledge gap, we focused on elucidating the functions of PCYT1A in the retina. RESULTS: We found that PCYT1A is highly expressed in Müller glial (MG) cells in the inner nuclear layer (INL) of the retina. Subsequently, we generated a retina-specific knockout mouse model in which the Pcyt1a gene was targeted (Pcyt1a-RKO or RKO mice) to investigate the molecular mechanisms underlying IRDs caused by PCYT1A mutations. Our findings revealed that the deletion of Pcyt1a resulted in retinal degenerative phenotypes, including reduced scotopic electroretinogram (ERG) responses and progressive degeneration of photoreceptor cells, accompanied by loss of cells in the INL. Furthermore, through proteomic and bioinformatic analyses, we identified dysregulated retinal fatty acid metabolism and activation of the ferroptosis signalling pathway in RKO mice. Importantly, we found that PCYT1A deficiency did not lead to an overall reduction in PC synthesis within the retina. Instead, this deficiency appeared to disrupt free fatty acid metabolism and ultimately trigger ferroptosis. CONCLUSIONS: This study reveals a novel mechanism by which mutations in PCYT1A contribute to the development of IRDs, shedding light on the interplay between fatty acid metabolism and retinal degenerative diseases, and provides new insights into the treatment of IRDs.

Helper Lipid-Enhanced mRNA Delivery for Treating Metabolic Dysfunction-Associated Fatty Liver Disease.

Lipid nanoparticles (LNPs) represent the forefront of mRNA delivery platforms, yet achieving precise delivery to specific cells remains a challenge. The current targeting strategies complicate the formulation and impede the regulatory approval process. Here, through a straightforward regulation of helper lipids within LNPs, we introduce an engineered LNP designed for targeted delivery of mRNA into hepatocytes for metabolic dysfunction-associated fatty liver disease (MAFLD) treatment. The optimized LNP, supplied with POPC as the helper lipid, exhibits a 2.49-fold increase in mRNA transfection efficiency in hepatocytes compared to that of FDA-approved LNPs. CTP:phosphocholine cytidylyltransferase α mRNA is selected for delivery to hepatocytes through the optimized LNP system for self-calibration of phosphatidylcholine levels to prevent lipid droplet expansion in MAFLD. This strategy effectively regulates lipid homeostasis, while demonstrating proven biosafety. Our results present a mRNA therapy for MAFLD and open a new avenue for discovering potent lipids enabling mRNA delivery to specific cells.

Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope.

Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.

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.

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.

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.

Genetic diseases of the Kennedy pathways for membrane synthesis.

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.

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.

FIN219/JAR1 and cryptochrome1 antagonize each other to modulate photomorphogenesis under blue light in Arabidopsis.

Plant development is affected by the integration of light and phytohormones, including jasmonates (JAs). To address the molecular mechanisms of possible interactions between blue light and JA signaling in Arabidopsis thaliana, we used molecular and transgenic approaches to understand the regulatory relationships between FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) and the blue-light photoreceptor cryptochrome1 (CRY1). FIN219 overexpression in the wild type resulted in a short-hypocotyl phenotype under blue light. However, FIN219 overexpression in cry1, cry2 and cry1cry2 double mutant backgrounds resulted in phenotypes similar to their respective mutant backgrounds, which suggests that FIN219 function may require blue light photoreceptors. Intriguingly, FIN219 overexpression in transgenic plants harboring ectopic expression of the C terminus of CRY1 (GUS-CCT1), which exhibits a hypersensitive short-hypocotyl phenotype in all light conditions including darkness, led to a rescued phenotype under all light conditions except red light. Further expression studies showed mutual suppression between FIN219 and CRY1 under blue light. Strikingly, FIN219 overexpression in GUS-CCT1 transgenic lines (FIN219-OE/GUS-CCT1) abolished GUS-CCT1 fusion protein under blue light, whereas GUS-CCT1 fusion protein was stable in the fin219-2 mutant background (fin219-2/GUS-CCT1). Moreover, FIN219 strongly interacted with COP1 under blue light, and methyl JA (MeJA) treatment enhanced the interaction between FIN219 and GUS-CCT1 under blue light. Furthermore, FIN219 level affected GUS-CCT1 seedling responses such as anthocyanin accumulation and bacterial resistance under various light conditions and MeJA treatment. Thus, FIN219/JAR1 and CRY1 antagonize each other to modulate photomorphogenic development of seedlings and stress responses in Arabidopsis.

Interdomain communication in the phosphatidylcholine regulatory enzyme, CCTα, relies on a modular αE helix.

CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis. Recent work has suggested that CCTα activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (αE) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCTα dimer to the membrane-binding (M) domains. In the soluble, inactive form, the αE helices are constrained into unbroken helices by contacts with two auto-inhibitory (AI) helices from domain M. In the active, membrane-bound form, the AI helices are displaced and engage the membrane. Molecular dynamics simulations have suggested that AI displacement is associated with hinge-like bending in the middle of the αE, positioning its C terminus closer to the active site. Here, we show that CCTα activation by membrane binding is sensitive to mutations in the αE and J segments, especially within or proximal to the αE hinge. Substituting Tyr-213 within this hinge with smaller uncharged amino acids that could destabilize interactions between the αE helices increased both constitutive and lipid-dependent activities, supporting a link between αE helix bending and stimulation of CCT activity. The solvent accessibilities of Tyr-213 and Tyr-216 suggested that these tyrosines move to new partially buried environments upon membrane binding of CCT, consistent with a folded αE/J structure. These data suggest that signal transduction through the modular αE helix pair relies on shifts in its conformational ensemble that are controlled by the AI helices and their displacement upon membrane binding.

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface.

The rate-limiting step in the biosynthesis of the major membrane phospholipid, phosphatidylcholine, is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT), which is regulated by reversible membrane binding of a long amphipathic helix (domain M). The M domain communicates with the catalytic domain via a conserved ∼20-residue linker, essential for lipid activation of CCT. Previous analysis of this region (denoted as the αEC/J) using MD simulations, cross-linking, mutagenesis, and solvent accessibility suggested that membrane binding of domain M promotes remodeling of the αEC/J into a more compact structure that is required for enzyme activation. Here, using tryptophan fluorescence quenching, we show that the allosteric linker lies superficially on the membrane surface. Analyses with truncated CCTs show that the αEC/J can interact with lipids independently of the M domain. We observed strong FRET between engineered tryptophans in the αEC/J and vesicles containing dansyl-phosphatidylethanolamine that depended on the native J sequence. These data are incompatible with the extended conformation of the αE helix observed in the previously determined crystal structure of inactive CCT but support a bent αE helix conformation stabilized by J segment interactions. Our results suggest that the membrane-adsorbed, folded allosteric linker may partially cover the active site cleft and pull it close to the membrane surface, where cytidyl transfer can occur efficiently in a relatively anhydrous environment.

Disease-linked mutations in the phosphatidylcholine regulatory enzyme CCTα impair enzymatic activity and fold stability.

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes. Mutations in the gene encoding CCTα (PCYT1A) cause three distinct pathologies in humans: lipodystrophy, spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD), and isolated retinal dystrophy. Previous analyses showed that for some disease-linked PCYT1A variants steady state levels of CCTα and PC synthesis were reduced in patient fibroblasts, but other variants impaired PC synthesis with little effect on CCT levels. To explore the impact on CCT stability and function we expressed WT and mutant CCTs in COS-1 cells, which have very low endogenous CCT. Over-expression of two missense variants in the catalytic domain (V142M and P150A) generated aggregated enzymes that could not be refolded after solubilization by denaturation. Other mutations in the catalytic core that generated CCTs with reduced solubility could be purified. Five variants destabilized the catalytic domain-fold as assessed by lower transition temperatures for unfolding, and three of these manifested defects in substrate Km values. A mutation (R223S) in a signal-transducing linker between the catalytic and membrane-binding domains also impaired enzyme kinetics. E280del, a single amino acid deletion in the autoinhibitory helix increased the constitutive (lipid-independent) enzyme activity ∼4-fold. This helix also participates in membrane binding, and surprisingly E280del enhanced the enzyme's response to anionic lipid vesicles ∼4-fold. These in vitro analyses on purified mutant CCTs will complement future measurements of their impact on PC synthesis in cultured cells and in tissues with a stringent requirement for CCTα.

Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis.

Zygotic genome activation in metazoans typically occurs several hours to a day after fertilization, and thus maternal RNAs and proteins drive early animal embryo development. In plants, despite several molecular studies of post-fertilization transcriptional activation, the timing of zygotic genome activation remains a matter of debate. For example, two recent reports that used different hybrid ecotype combinations for RNA sequence profiling of early Arabidopsis embryo transcriptomes came to divergent conclusions. One identified paternal contributions that varied by gene, but with overall maternal dominance, while the other found that the maternal and paternal genomes are transcriptionally equivalent. Here we assess paternal gene activation functionally in an isogenic background, by performing a large-scale genetic analysis of 49 EMBRYO DEFECTIVE genes and testing the ability of wild-type paternal alleles to complement phenotypes conditioned by mutant maternal alleles. Our results demonstrate that wild-type paternal alleles for nine of these genes are completely functional 2 days after pollination, with the remaining 40 genes showing partial activity beginning at 2, 3 or 5 days after pollination. Using our functional assay, we also demonstrate that different hybrid combinations exhibit significant variation in paternal allele activation, reconciling the apparently contradictory results of previous transcriptional studies. The variation in timing of gene function that we observe confirms that paternal genome activation does not occur in one early discrete step, provides large-scale functional evidence that maternal and paternal genomes make non-equivalent contributions to early plant embryogenesis, and uncovers an unexpectedly profound effect of hybrid genetic backgrounds on paternal gene activity.

Accumulation of saturated intramyocellular lipid is associated with insulin resistance.

Intramyocellular lipid (IMCL) accumulation has been linked to both insulin-resistant and insulin-sensitive (athletes) states. Biochemical analysis of intramuscular triglyceride composition is confounded by extramyocellular triglycerides in biopsy samples, and hence the specific composition of IMCLs is unknown in these states. 1H magnetic resonance spectroscopy (MRS) can be used to overcome this problem. Thus, we used a recently validated 1H MRS method to compare the compositional saturation index (CH2:CH3) and concentration independent of the composition (CH3) of IMCLs in the soleus and tibialis anterior muscles of 16 female insulin-resistant lipodystrophic subjects with that of age- and gender-matched athletes (n = 14) and healthy controls (n = 41). The IMCL CH2:CH3 ratio was significantly higher in both muscles of the lipodystrophic subjects compared with controls but was similar in athletes and controls. IMCL CH2:CH3 was dependent on the IMCL concentration in the controls and, after adjusting the compositional index for quantity (CH2:CH3adj), could distinguish lipodystrophics from athletes. This CH2:CH3adj marker had a stronger relationship with insulin resistance than IMCL concentration alone and was inversely related to VO2max The association of insulin resistance with the accumulation of saturated IMCLs is consistent with a potential pathogenic role for saturated fat and the reported benefits of exercise and diet in insulin-resistant states.

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.

From masochistic enzymology to mechanistic physiology and disease.

The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt-/- mice are protected from weight gain and insulin resistance.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE Regulates Phosphatidylcholine Biosynthesis in Arabidopsis by Phosphatidic Acid-Mediated Activation of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE Activity.

Regulation of membrane lipid biosynthesis is critical for cell function. We previously reported that disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 (PAH1) and PAH2 stimulates net phosphatidylcholine (PC) biosynthesis and proliferation of the endoplasmic reticulum (ER) in Arabidopsis thaliana. Here, we show that this response is caused specifically by a reduction in the catalytic activity of the protein and positively correlates with an accumulation of its substrate, phosphatidic acid (PA). The accumulation of PC in pah1 pah2 is suppressed by disruption of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE1 (CCT1), which encodes a key enzyme in the nucleotide pathway for PC biosynthesis. The activity of recombinant CCT1 is stimulated by lipid vesicles containing PA. Truncation of CCT1, to remove the predicted C-terminal amphipathic lipid binding domain, produced a constitutively active enzyme. Overexpression of native CCT1 in Arabidopsis has no significant effect on PC biosynthesis or ER morphology, but overexpression of the truncated constitutively active version largely replicates the pah1 pah2 phenotype. Our data establish that membrane homeostasis is regulated by lipid composition in Arabidopsis and reveal a mechanism through which the abundance of PA, mediated by PAH activity, modulates CCT activity to govern PC content.

Mutations in PCYT1A cause spondylometaphyseal dysplasia with cone-rod dystrophy.

Spondylometaphyseal dysplasia with cone-rod dystrophy is a rare autosomal-recessive disorder characterized by severe short stature, progressive lower-limb bowing, flattened vertebral bodies, metaphyseal involvement, and visual impairment caused by cone-rod dystrophy. Whole-exome sequencing of four individuals affected by this disorder from two Brazilian families identified two previously unreported homozygous mutations in PCYT1A. This gene encodes the alpha isoform of the phosphate cytidylyltransferase 1 choline enzyme, which is responsible for converting phosphocholine into cytidine diphosphate-choline, a key intermediate step in the phosphatidylcholine biosynthesis pathway. A different enzymatic defect in this pathway has been previously associated with a muscular dystrophy with mitochondrial structural abnormalities that does not have cartilage and/or bone or retinal involvement. Thus, the deregulation of the phosphatidylcholine pathway may play a role in multiple genetic diseases in humans, and further studies are necessary to uncover its precise pathogenic mechanisms and the entirety of its phenotypic spectrum.

Mutations disrupting the Kennedy phosphatidylcholine pathway in humans with congenital lipodystrophy and fatty liver disease.

Phosphatidylcholine (PC) is the major glycerophospholipid in eukaryotic cells and is an essential component in all cellular membranes. The biochemistry of de novo PC synthesis by the Kennedy pathway is well established, but less is known about the physiological functions of PC. We identified two unrelated patients with defects in the Kennedy pathway due to biallellic loss-of-function mutations in phosphate cytidylyltransferase 1 alpha (PCYT1A), the rate-limiting enzyme in this pathway. The mutations lead to a marked reduction in PCYT1A expression and PC synthesis. The phenotypic consequences include some features, such as severe fatty liver and low HDL cholesterol levels, that are predicted by the results of previously reported liver-specific deletion of murine Pcyt1a. Both patients also had lipodystrophy, severe insulin resistance, and diabetes, providing evidence for an additional and essential role for PCYT1A-generated PC in the normal function of white adipose tissue and insulin action.