RESUMO
Phosphatidylserine (PS) is an important anionic phospholipid that is synthesized within the endoplasmic reticulum (ER). While PS shows the highest enrichment and serves important functional roles in the plasma membrane (PM) but its role in the nucleus is poorly explored. Using three orthogonal approaches, we found that PS is also uniquely enriched in the inner nuclear membrane (INM) and the nuclear reticulum (NR). Nuclear PS is critical for supporting the translocation of CCTα and Lipin1α, two key enzymes important for phosphatidylcholine (PC) biosynthesis, from the nuclear matrix to the INM and NR in response to oleic acid treatment. We identified the PS-interacting regions within the M-domain of CCTα and M-Lip domain of Lipin1α, and show that lipid droplet formation is altered by manipulations of nuclear PS availability. Our studies reveal an unrecognized regulatory role of nuclear PS levels in the regulation of key PC synthesizing enzymes within the nucleus.
Assuntos
Colina-Fosfato Citidililtransferase , Membrana Nuclear , Fosfatidilcolinas , Fosfatidilserinas , Membrana Nuclear/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilcolinas/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Colina-Fosfato Citidililtransferase/genética , Humanos , Animais , Camundongos , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismoRESUMO
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.
Assuntos
Ácidos Graxos , Ferroptose , Camundongos Knockout , Retina , Animais , Camundongos , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Ácidos Graxos/metabolismo , Ferroptose/fisiologia , Ferroptose/genética , Retina/metabolismo , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismoRESUMO
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.
Assuntos
Hepatócitos , Nanopartículas , Fosfatidilcolinas , RNA Mensageiro , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Humanos , Hepatócitos/metabolismo , Fosfatidilcolinas/química , Nanopartículas/química , Animais , Transfecção/métodos , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Fígado Gorduroso/terapia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Lipídeos/química , Técnicas de Transferência de Genes , Camundongos , LipossomosRESUMO
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.
Assuntos
Gotículas Lipídicas , Membrana Nuclear , Humanos , Células CACO-2 , Membrana Nuclear/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Camundongos , Diferenciação Celular/efeitos dos fármacos , Colina-Fosfato Citidililtransferase/metabolismo , Colina-Fosfato Citidililtransferase/genética , Ácido Oleico/farmacologia , Ácido Oleico/metabolismo , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Gotículas Lipídicas , Fosfatidilcolinas , Fosfatidilcolinas/metabolismo , Gotículas Lipídicas/metabolismo , Fosfotransferases/metabolismo , Homeostase , Colina/metabolismo , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismoRESUMO
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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Bactérias Gram-Positivas/imunologia , Imunidade Inata , Metabolismo dos Lipídeos/imunologia , Animais , Animais Geneticamente Modificados , Peptídeos Catiônicos Antimicrobianos/genética , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Enterococcus faecalis/imunologia , Corpo Adiposo/enzimologia , Corpo Adiposo/imunologia , Feminino , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Larva/enzimologia , Larva/imunologia , Metabolismo dos Lipídeos/genética , Masculino , Fosfolipídeos/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/metabolismo , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/genética , Colina-Fosfato Citidililtransferase/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Fosfatidilcolinas/metabolismo , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Alinhamento de SequênciaRESUMO
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 â¼20-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.
Assuntos
Membrana Celular/enzimologia , Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Sítio Alostérico , Biocatálise , Domínio Catalítico , Membrana Celular/química , Membrana Celular/genética , Colina-Fosfato Citidililtransferase/genética , Ativação Enzimática , Humanos , Lipídeos/química , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Arabidopsis/química , Domínio Catalítico , Colina-Fosfato Citidililtransferase/química , Sequência Conservada , Evolução Molecular , Cinética , Modelos Biológicos , Fosforilação , Fosforilcolina/metabolismo , Fosfosserina/metabolismo , Folhas de Planta/genética , Domínios Proteicos , Ratos , Homologia Estrutural de Proteína , Nicotiana/genéticaRESUMO
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 â¼4-fold. This helix also participates in membrane binding, and surprisingly E280del enhanced the enzyme's response to anionic lipid vesicles â¼4-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.
Assuntos
Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Lipodistrofia/genética , Mutação , Osteocondrodisplasias/genética , Dobramento de Proteína , Distrofias Retinianas/genética , Retinose Pigmentar/genética , Animais , Células COS , Catálise , Domínio Catalítico , Chlorocebus aethiops , Colina-Fosfato Citidililtransferase/genética , Cristalografia por Raios X , Humanos , Lipodistrofia/patologia , Osteocondrodisplasias/patologia , Fosfatidilcolinas/metabolismo , Ligação Proteica , Estabilidade Proteica , Distrofias Retinianas/patologia , Retinose Pigmentar/patologiaRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Adenocarcinoma de Pulmão/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologiaRESUMO
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 â¼30% 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.
Assuntos
Gorduras na Dieta/metabolismo , Homeostase/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fosfatidilcolinas/biossíntese , Animais , Transporte Biológico/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Colesterol/metabolismo , Colina-Fosfato Citidililtransferase/deficiência , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/efeitos adversos , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Aterosclerose , Colina-Fosfato Citidililtransferase , Resistência à Insulina , Obesidade , Fosfatidilcolinas , Fosfatidiletanolamina N-Metiltransferase , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Dieta/efeitos adversos , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Marcação de Genes , História do Século XX , História do Século XXI , Humanos , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/enzimologia , Obesidade/genética , Obesidade/patologia , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/genética , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Fosfatidato Fosfatase/metabolismo , Fosforilcolina/metabolismo , Proteínas de Arabidopsis/genética , Colina-Fosfato Citidililtransferase/genética , Fosfatidato Fosfatase/genéticaRESUMO
Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location.
Assuntos
Núcleo Celular/enzimologia , Colina-Fosfato Citidililtransferase/metabolismo , Células Epiteliais/efeitos dos fármacos , Membrana Nuclear/enzimologia , Prostaglandina D2/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Western Blotting , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/metabolismo , Indometacina/farmacologia , Rim/citologia , Masculino , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Ratos WistarRESUMO
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.
Assuntos
Colina-Fosfato Citidililtransferase/genética , Osteocondrodisplasias/genética , Retinose Pigmentar/genética , Adolescente , Brasil , Criança , Pré-Escolar , Colina-Fosfato Citidililtransferase/metabolismo , Feminino , Genes Recessivos , Homozigoto , Humanos , Lactente , Masculino , Oftalmologia/métodos , LinhagemRESUMO
Cytidine diphosphate choline (CDP-choline) has been applied for treating acute craniocerebral injury and allowing recovery of consciousness after brain surgery. In this study, an acetate kinase (ACK)/acetyl phosphate system was used to supply ATP and combined with Escherichia coli-overexpressed CMP kinase (CMK), NDP kinase (NDK), choline phosphate cytidylyltransferase (CCT), and choline kinase (CKI) to produce CDP-choline from CMP and choline chloride. Within 1 h, 49 mM CDP-choline was produced, for a molar yield of 89.9 and 68.4 % based on CMP and choline chloride, respectively; the utilization efficiency of energy (UEE) was 79.5 %. Acetyl phosphate, sodium acetate, and CTP inhibited the reaction when the concentration exceeded 18.5, 600, and 30 mM, respectively. This inhibition could be overcome by controlling the rate of acetyl phosphate, CMP addition or using KOH instead of NaOH to regulate the pH in fed-batch transformation. After 24 h, the maximum titer was 124.1 ± 2.7 mM, the productivity was 5.1 ± 0.1 mM l-1 h-1, the molar yield to CMP and choline chloride were 83.8 and 63.7 %, respectively, and the UEE was 58.2 %. This high yield and productivity of CDP-choline through biocatalysis suggest future application at the industrial scale.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Corynebacterium/enzimologia , Corynebacterium/metabolismo , Citidina Difosfato Colina/metabolismo , Trifosfato de Adenosina/metabolismo , BiocatáliseRESUMO
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.
Assuntos
Colina-Fosfato Citidililtransferase/genética , Fígado Gorduroso/genética , Lipodistrofia/congênito , Lipodistrofia/genética , Fosfatidilcolinas/química , Células 3T3-L1 , Tecido Adiposo/metabolismo , Adolescente , Alelos , Animais , Criança , HDL-Colesterol/química , Colina-Fosfato Citidililtransferase/metabolismo , Biologia Computacional , Fígado Gorduroso/metabolismo , Feminino , Glicerofosfolipídeos/química , Humanos , Insulina/química , Lipídeos/química , Lipodistrofia/metabolismo , Camundongos , Mutação , Fenótipo , Distribuição TecidualRESUMO
In activated B cells, increased production of phosphatidylcholine (PtdCho), the most abundant cellular phospholipid, is handled primarily by the CDP-choline pathway. B cell-specific deletion of CTP:phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme in the CDP-choline pathway, led to augmented IgM secretion and reduced IgG production, suggesting that PtdCho synthesis is required for germinal center reactions. To specifically assess whether PtdCho influences B cell fate during germinal center responses, we examined immune responses in mice whereby PtdCho synthesis is disrupted in B cells that have undergone class switch recombination to IgG1 (referred to as either Cγ1wt/wt, Cγ1Cre/wt or Cγ1Cre/Cre based on Cre copy number). Serum IgG1 was markedly reduced in naïve Cγ1Cre/wt and Cγ1Cre/Cre mice, while levels of IgM and other IgG subclasses were similar between Cγ1Cre/wt and Cγ1wt/wt control mice. Serum IgG2b titers were notably reduced and IgG3 titers were increased in Cγ1Cre/Cre mice compared with controls. Following immunization with T cell-dependent antigen NP-KLH, control mice generated high titer IgG anti-NP while IgG anti-NP titers were markedly reduced in both immunized Cγ1Cre/wt and Cγ1Cre/Cre mice. Correspondingly, the frequency of NP-specific IgG antibody-secreting cells was also reduced in spleens and bone marrow of Cγ1Cre/wt and Cγ. 1Cre/Cre mice compared to control mice. Interestingly, though antigen-specific IgM B cells were comparable between Cγ1Cre/wt, Cγ1Cre/Cre and control mice, the frequency and number of IgG1 NP-specific B cells was reduced only in Cγ1Cre/Cre mice. These data indicate that PtdCho is required for the generation of both germinal center-derived B cells and antibody-secreting cells. Further, the reduction in class-switched ASC but not B cells in Cγ1Cre/wt mice suggests that ASC have a greater demand for PtdCho compared to germinal center B cells.
Assuntos
Linfócitos B/imunologia , Colina-Fosfato Citidililtransferase/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Colina-Fosfato Citidililtransferase/genética , Centro Germinativo/patologia , Imunidade Humoral/genética , Imunoglobulina G/sangue , Memória Imunológica/genética , Ativação Linfocitária , Camundongos , Camundongos Knockout , Linfócitos T/imunologia , Resposta a Proteínas não DobradasRESUMO
CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves disruption of an inhibitory interaction between the αE helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-αE interaction as a cluster of four amphipathic helices (two αE and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-αE interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device.