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Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter.

ABSTRACT: Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268-1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal approximately 1900 bp of the 5' flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between -169 and +71 bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from -1867 to +71, was induced 2-3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240 bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5' flanking region.

SUBMITTER: Mallampalli RK

PROVIDER: S-EPMC1222362 | biostudies-other | 2002 Feb

REPOSITORIES: biostudies-other

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Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis.

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Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

Project description:Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.

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Localization and expression of CTP: Phosphocholine cytidylyltransferase in rat brain following cocaine exposure.

Project description:Phosphatidylcholine (PC) is a primary phospholipid and major source of secondary lipid messengers and also serves as a biosynthetic precursor for other membrane phospholipids. Phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme responsible for catalyzing the formation of PC. Changes in CCT activity have been associated with lipid dysregulation across various neurological disorders. Additionally, intermediates in PC synthesis, such as CDP-choline, have been suggested to attenuate drug craving during cocaine addiction. Recent work from our group demonstrated that cocaine exposure and conditioning alter the level of PC in the brain, specifically in the cerebellum and hippocampus. The present study examines the role of CCT expression in the brain and determines the effect of cocaine exposure on CCT expression. Immunohistochemical analysis (IHC) was performed to assess region-specific expression of CCT, including both of its isoforms; alpha (CCT?) and beta (CCT?). IHC did not detect any staining of CCT? throughout the rat brain. In contrast, CCT? expression was detected in the Purkinje cells of the cerebellum with decreases in expression following cocaine exposure. Collectively, these data demonstrate the region- and cell-specific localization of CCT? and CCT? in the rat brain, as well as the altered expression of CCT? in the cerebellum following cocaine exposure.

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CTP:phosphocholine cytidylyltransferase alpha is required for B-cell proliferation and class switch recombination.

Project description:CTP:phosphocholine cytidylyltransferase (CCT) is a key rate-controlling enzyme in the biosynthetic pathway leading to the principle membrane phospholipid, phosphatidylcholine. CCTalpha is the predominant isoform expressed in mammalian cells. To investigate the role of CCTalpha in the development and function of B-lymphocytes, mice with B-lymphocytes that selectively lacked CCTalpha were derived using the CD19-driven Cre/loxP system. When challenged with a T-cell-dependent antigen, the animals harboring CCTalpha-deficient B-cells exhibited a hyper-IgM secretion phenotype coupled with a lack of IgG production. The inability of CCTalpha-/- B-cells to undergo class switch recombination correlated with a proliferation defect in vivo and in vitro in response to antigenic and mitogenic stimuli. Lipopolysaccharide stimulation of CCTalpha-/- B-cells resulted in an early trigger of the unfolded protein response-mediated splicing of Xbp-1 mRNA, and this was accompanied by accelerated kinetics of IgM secretion and higher incidence of IgM-secreting cells. Thus, the inability of stimulated B-cells to produce enough phosphatidylcholine prevents proliferation and class switch recombination but leads to unfolded protein response activation and a hyper-IgM secretion phenotype.

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

Project description: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.

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Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption.

Project description:A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.

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