RESUMO
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
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
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
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.
Assuntos
Gotículas Lipídicas , Fosfatidilcolinas , Humanos , Gotículas Lipídicas/metabolismo , Fosfatidilcolinas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Colina/metabolismo , Metabolismo dos Lipídeos , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismoRESUMO
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.
Assuntos
Lesão Pulmonar , Surfactantes Pulmonares , Fator de Transcrição Sp1 , Animais , Ratos , Colina/metabolismo , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Maleato de Dizocilpina , Ácido Glutâmico , N-Metilaspartato , Fosfatidilcolinas , Surfactantes Pulmonares/metabolismo , Ratos Sprague-Dawley , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/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
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.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Craterostigma/enzimologia , Proteínas de Plantas/metabolismo , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/fisiologia , Clonagem Molecular , Craterostigma/genética , Desidratação , Regulação da Expressão Gênica de Plantas , Fosfatidilcolinas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Alinhamento de SequênciaRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Sinais de Localização Nuclear , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Núcleo Celular/genética , Colina-Fosfato Citidililtransferase/genética , Cricetinae , Cricetulus , Citidina Trifosfato/metabolismo , Fosforilcolina/metabolismo , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
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
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.
Assuntos
Autofagia/fisiologia , Colina-Fosfato Citidililtransferase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/genética , Meios de Cultura , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Gotículas Lipídicas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfatidilcolinas/metabolismoRESUMO
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
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.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Gotículas Lipídicas/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Células CHO , Núcleo Celular/metabolismo , Colina-Fosfato Citidililtransferase/fisiologia , Cricetulus , Ácidos Graxos/metabolismo , Humanos , Gotículas Lipídicas/fisiologia , Membrana Nuclear/metabolismo , Ácido Oleico/metabolismo , Fosfatidato Fosfatase/fisiologia , Fosfatidilcolinas/química , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/fisiologiaRESUMO
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.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Citidina Trifosfato/metabolismo , Lipídeos de Membrana/metabolismo , Catálise , Domínio Catalítico , Membrana Celular/enzimologia , Colina-Fosfato Citidililtransferase/química , Modelos MolecularesRESUMO
CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTß catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Gotículas Lipídicas/metabolismo , Membrana Nuclear/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Colina/metabolismo , Colina-Fosfato Citidililtransferase/química , Células HeLa , Humanos , Modelos Biológicos , Ácido Oleico/metabolismo , Fosforilação , Transporte Proteico , RatosRESUMO
Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
Assuntos
Antineoplásicos/farmacologia , Autofagossomos/enzimologia , Autofagossomos/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Furanos/farmacologia , Macroautofagia , Fosfatidilcolinas/biossíntese , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Células CHO , Linhagem Celular Tumoral , Colina/metabolismo , Colina-Fosfato Citidililtransferase/genética , Cricetulus , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Macroautofagia/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metabolômica , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Chronic hepatitis B Virus (HBV) infection has high morbidity, high pathogenicity and unclear pathogenesis. To elucidate the relationship between HBV replication and host phospholipid metabolites, we measured 10 classes of phospholipids in serum of HBV infected patients and cells using ultra performance liquid chromatograph-triple quadruple mass spectrometry. We found that the levels of phosphatidylcholine (PC), phosphatidylethanolamine, and lyso-phosphatidic acid were increased in HBsAg (+) serum of infected patients compared with HBsAg (-), while phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin were decreased, which were confirmed in an HBV infected HepG2.2.15 cell line. We further evaluated the enzyme levels of PC pathways and found that PCYT1A and LPP1 for PC synthesis were up-regulated after HBV infection. Moreover, HBV replication was inhibited when PCYT1A and LPP1 were inhibited. These results indicated that the PC synthesis in HBV infected host are regulated by PCYT1A and LPP1, which suggests that PCYT1A, LPP1 could be new potential targets for HBV treatment.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/virologia , Lipogênese , Fosfatidato Fosfatase/metabolismo , Replicação Viral , Colina-Fosfato Citidililtransferase/genética , Feminino , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/patologia , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidato Fosfatase/genéticaRESUMO
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
White adipose tissue (WAT) inflammation contributes to the development of insulin resistance in obesity. While the role of adipose tissue macrophage (ATM) pro-inflammatory signalling in the development of insulin resistance has been established, it is less clear how WAT inflammation is initiated. Here, we show that ATMs isolated from obese mice and humans exhibit markers of increased rate of de novo phosphatidylcholine (PC) biosynthesis. Macrophage-specific knockout of phosphocholine cytidylyltransferase A (CCTα), the rate-limiting enzyme of de novo PC biosynthesis pathway, alleviated obesity-induced WAT inflammation and insulin resistance. Mechanistically, CCTα-deficient macrophages showed reduced ER stress and inflammation in response to palmitate. Surprisingly, this was not due to lower exogenous palmitate incorporation into cellular PCs. Instead, CCTα-null macrophages had lower membrane PC turnover, leading to elevated membrane polyunsaturated fatty acid levels that negated the pro-inflammatory effects of palmitate. Our results reveal a causal link between obesity-associated increase in de novo PC synthesis, accelerated PC turnover and pro-inflammatory activation of ATMs.