RESUMO
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
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
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
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
Choline phosphate-based delivery systems can target the acidic tumor microenvironment. In this study, we set out to evaluate the diagnostic value of Choline phosphate cytidylyltransferase-α (CCTα) in laryngeal squamous cell cancer (LSCC). The expression of CCTα was detected using immunohistochemistry in 50 LSCC patients' tissues and 16 vocal polyps as control group. Then, clinical data was collected and we used receiver operating characteristic curve (ROC) to estimate the potential of CCTα as diagnostic biomarker. We found CCTα levels to be significantly high in the tissues derived from LSCC patients, (p < 0.001). Further, we observed a positive correlation of CCTα with tumor size (p < 0.001), TNM stage (p < 0.001), lymph node metastasis (p < 0.001) as well as the grade of LSCC malignancy (p < 0.001). Furthermore, AUC was determined to be 0.939 by ROC, and the optimal cutoff value 3.100, with 76.0% sensitivity and 100% specificity. We also found an epigenetic basis of CCTα over-expression in LSCC tissues with significantly reduced methylation of CCTα in LSCC tissues, compared to vocal polyps (p < 0.001). These results support epigenetically-induced over-expression of CCTα as a potential diagnostic marker for LSCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Neoplasias Laríngeas/diagnóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Microambiente TumoralRESUMO
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.
Assuntos
Tecido Adiposo/patologia , Inflamação/patologia , Macrófagos/metabolismo , Obesidade/patologia , Fosfatidilcolinas/metabolismo , Animais , Colina-Fosfato Citidililtransferase/deficiência , Colina-Fosfato Citidililtransferase/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Humanos , Resistência à Insulina , Camundongos ObesosRESUMO
The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CDP-choline diacylglycerol phosphotransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.
Assuntos
Núcleo Celular/metabolismo , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Fosfatidilcolinas/biossíntese , Precursores de Proteínas/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Colina-Fosfato Citidililtransferase/metabolismo , Células HEK293 , Células HeLa , Hepatócitos/metabolismo , Humanos , Ácido Oleico/metabolismo , Perilipina-3/metabolismo , RatosRESUMO
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
Objective: To examine the expression and potential clinical significance of CCT (cytidine triphosphate: phosphocholine cytidylyltransferase)-α in oral squamous cell carcinoma (OSCC). Methods: Fifty-eight OSCC and paired adjacent non-malignant epithelia samples (between May 2016 and July 2016) were obtained from dental center, Second Xiangya Hospital, Central South University. CCT-α expression was examined by immunohistochemistry. The relationship between CCT-α and clinicopathological features of OSCC patients was analyzed. Quantitative real-time PCR and Western blot were performed to measure the expression of CCT-α mRNA and protein level in several OSCC cell line and two normal oral epithelial cell line. Results: Immunohistochemistry showed that CCT-α positive staining was found in cell nuclear of OSCC cells and adjacent epithelial cells. CCT-α was positively expressed in OSCC, which was significantly higher than that adjacent to carcinoma tissues (P=0.000). The expression of CCT-α in oral squamous cell carcinoma was correlated with smoking, alcohol consumption, tumor size, differentiation degree and lymph node metastasis. The expression level of CCT-α protein was significantly increased in patients with a history of smoking and alcohol consumption (P=0.001, P=0.004). With the increase of tumor diameter, the expression of CCT-α protein was significantly increased (P=0.005). According to histopathological grade, the lower the degree of tumor differentiation, the higher the expression level of CCT-α protein (P=0.000). The expression of CCT-α protein was significantly higher in patients with lymph node metastasis compared with no lymph node metastasis (P=0.000). Quantitative real-time PCR results showed the CCT-α mRNA expression level was significantly higher in OSCC cells than that in normal oral epithelial cells (P=0.016). The protein expression level of CCT-α was significantly higher in OSCC cells than that in normal oral epithelial cells. Conclusions: CCT-α may play a critical role in the carcinogenesis and development of OSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Citidina Trifosfato/metabolismo , Neoplasias Bucais/metabolismo , Western Blotting , Células Epiteliais/metabolismo , Epitélio/metabolismo , Humanos , Imuno-Histoquímica , Metástase Linfática , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVES: The aim of this study was to test choline-phosphate cytidylyltransferase-α (CCT-α) protein as a biomarker for neoadjuvant cisplatin chemotherapy response in a bladder tumor setting. MATERIALS AND METHODS: A total of 238 patients with T2-T4 bladder cancer enrolled into two prior randomized trials comparing neoadjuvant cisplatin-based chemotherapy (NAC) plus cystectomy with cystectomy only (no-NAC) were used as discovery and validation cohorts. Protein expression was determined with immunohistochemistry and assessed with Histo (H)-scoring. RESULTS: In the discovery cohort, comprising 61 patients, the survival ratio after NAC treatment for CCT-α-negative patients was significantly increased (p = 0.001) while there was no survival advantage in the CCT-α-positive patient group. Similarly, in the validation cohort with 177 patients, NAC treatment improved survival only in the CCT-α-negative group (p = 0.006). Although there was a tendency for a good NAC response with negative CCT-α status, the interaction variable between biomarker and treatment was not significant (p = 0.24). In the cystectomy-only group, patients with positive CCT-α expression had a better survival than CCT-α-negative patients. This prognostic effect of CCT-α expression remained significant after adjusting for well-known prognostic factors in a multivariate analysis. In a pooled database of both patient data sets, multivariate analyses showed CCT-α status as an independent factor for overall survival (p = 0.018; hazard ratio = 1.80, 95% confidence interval 1.11-2.93). CONCLUSION: CCT-α status was not predictive of outcome of NAC response; however, in the control group with cystectomy only it was found to have prognostic value.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/terapia , Colina-Fosfato Citidililtransferase/metabolismo , Cisplatino/uso terapêutico , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/terapia , Idoso , Quimioterapia Adjuvante , Cistectomia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Taxa de SobrevidaRESUMO
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
The activation of oncogenes can reprogram tumor cell metabolism. Here, in diffuse large B-cell lymphoma (DLBCL), serum metabolomic analysis revealed that oncogenic MYC could induce aberrant choline metabolism by transcriptionally activating the key enzyme phosphate cytidylyltransferase 1 choline-α (PCYT1A). In B-lymphoma cells, as a consequence of PCYT1A upregulation, MYC impeded lymphoma cells undergo a mitophagy-dependent necroptosis. In DLBCL patients, overexpression of PCYT1A was in parallel with an increase in tumor MYC, as well as a decrease in serum choline metabolite phosphatidylcholine levels and an International Prognostic Index, indicating intermediate-high or high risk. Both in vitro and in vivo, lipid-lowering alkaloid berberine (BBR) exhibited an anti-lymphoma activity through inhibiting MYC-driven downstream PCYT1A expression and inducing mitophagy-dependent necroptosis. Collectively, PCYT1A was upregulated by MYC, which resulted in the induction of aberrant choline metabolism and the inhibition of B-lymphoma cell necroptosis. Referred as a biomarker for DLBCL progression, PCYT1A can be targeted by BBR, providing a potential lipid-modifying strategy in treating MYC-High lymphoma.
Assuntos
Colina/biossíntese , Linfoma Difuso de Grandes Células B/metabolismo , Mitofagia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Berberina/farmacologia , Linhagem Celular Tumoral , Colina/genética , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
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 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
The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs.