Disease relevance of Pcyt1a

• The 17 monoclonal antibodies were divided into a minimum of seven different specificity groups based on their abilities to bind to the following purified test antigens in solid-phase radioimmunoassays: CT, the A and B polypeptides of CT (CT-A and CT-B, respectively), and the heat-labile enterotoxins designated LTh and LTp from Escherichia coli [1].

High impact information on Pcyt1a

• In contrast, enzymatic and real-time PCR data revealed that CCTbeta (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTalpha expression in adult liver and other tissues from Pcyt1a+/- heterozygous mice [2].
• PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a+/- mice [2].
• In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTalpha transcripts are significantly more abundant than transcripts encoding CCTbeta or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho [2].
• The importance of CCTalpha in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system [2].
• Caspase processing and nuclear export of CTP:phosphocholine cytidylyltransferase alpha during farnesol-induced apoptosis [3].

Biological context of Pcyt1a

• After synchronizing the cell cycle in NIH3T3 cells, CTalpha mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA [4].
• RNA interference targeting Net caused an increase of endogenous CTalpha mRNA [4].
• Previous studies (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692) demonstrated that CT alpha mRNA accumulates during S phase in preparation for cellular mitosis [5].
• These results enhance our understanding of the regulatory mechanisms involved in the expression of CTalpha in preparation for cell division [6].
• Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CTalpha in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338-12344) [7].

Anatomical context of Pcyt1a

• 9% to 29.5% in CTalpha-deficient macrophages [8].
• In this study, we investigated whether or not histone deacetylation is involved in repression of CTalpha expression in quiescent C3H10T1/2 mouse embryo fibroblasts [9].
• CTP:phosphocholine cytidylyltransferase alpha is a cytosolic protein in pulmonary epithelial cells and tissues [10].
• To identify the most important transcription factor involved in basal CTalpha transcription, we made CTalpha promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells [7].
• Surprisingly, the number of peritoneal macrophages harvested from CTalpha-deficient mice and their overall health under normal culture conditions appeared normal [8].

Associations of Pcyt1a with chemical compounds

• Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT) encoded by the Pcyt1a and Pcyt1b genes [4].
• Furthermore, CTalpha expression is decreased in cells overexpressing a dominant-negative form of CDK2 and in cells treated with the CDK2 kinase inhibitors roscovitine and olomoucine [6].
• We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine [11].
• The plasma phosphatidylcholine concentration was reduced in the CTalpha knockout (independent of gender), as were levels of high density lipoproteins (cholesterol and apoAI) and very low density lipoproteins (triacylglycerols and apoB100) [12].
• Experiments in which mice were injected with Triton WR1339 indicated that apoB secretion was decreased in hepatic-specific CTalpha knockout mice compared with controls [12].
• When knock-out hepatocytes were infected with an adenovirus expressing CTalpha, apoAI-dependent PC efflux returned partially, whereas cholesterol efflux and ABCA1 levels were not restored to normal levels [13].

Physical interactions of Pcyt1a

• Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CTalpha transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722) [4].
• We conclude that Sp1 binding to the CT alpha proximal promoter is necessary to enhance transcription during the S phase [5].

Enzymatic interactions of Pcyt1a

• Sp1 binds in a phosphorylated state to the CTalpha promoter [6].

Regulatory relationships of Pcyt1a

• Inhibition of Sp1 expression by RNA interference abolished the enhanced expression of CT alpha [5].
• Sp1 and Sp3 activate CTalpha gene transcription through sequence specific binding within three promoter domains [14].

Other interactions of Pcyt1a

• Multiple CCT isoforms, CCTalpha, CCTbeta2, and CCTbeta3, are encoded by two genes, Pcyt1a and Pcyt1b [2].
• Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-alpha [15].
• Activation of CTP:phosphocholine cytidylyltransferase alpha expression during the S phase of the cell cycle is mediated by the transcription factor Sp1 [5].
• In CTalpha knockout mice, hepatic CT activity (due to residual CTbeta2 activity as well as activity in nonhepatic cells) was 15% of normal, whereas phosphatidylethanolamine N-methyltransferase activity was elevated 2-fold compared with controls [12].
• These data place the Ctpct gene on mouse chromosome 16 between the Smst and Stf-1 genes [16].

Analytical, diagnostic and therapeutic context of Pcyt1a

• A genomic clone was isolated and the chromosomal location of the Ctpct locus determined by Southern blot hybridization of DNAs from a panel of interspecific backcross progeny derived from matings of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice [16].
• Indirect immunofluorescence of these cells with anti-CCT alpha antibodies resulted in a similar distribution [10].
• The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests [17].
• By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity [17].
• The binding of these antibodies to the following subunits and fragments of CT was also determined in Western blots: pentameric CT-B, monomeric CT-B, intact CT-A, and the A1 fragment of CT-A [1].

References