PTDINS BIOSYNTHESIS · Regulation of mammalian phosphatidylinositol biosynthesis
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2005-01-01 → 2006-12-31
- EU contribution
- €80,000
- Participants
- 1
- Scheme
- IRG
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Results in brief
Final Activity Report Summary - PTDINS BIOSYNTHESIS (Regulation of mammalian phosphatidylinositol biosynthesis)
Phosphatidate phosphatases, PAPs, are key enzymes in lipid biosynthesis and signaling. Functionally, PAP1 enzymes participate in de-novo phospholipid biosynthesis, whereas PAP2 enzymes have an established role in lipid signalling. To identify PAP2 enzymes that were potentially involved in de-novo phospholipid synthesis we first screened the human genome for PAP2 enzymes and after identifying all family members, we screened these for exposure of their predicted active site residues to the cytosolic side of membranes. These criteria identified two related enzymes, PAP2F and PAP2G, which show differential tissue and subcellular distribution, as well as novel yet differential roles in lipid metabolism. Specifically, we found that myc/His-PAP2F, but not myc/His-PAP2G, is a potent Mg2+-independent type II PAP which exhibited surface dilution kinetics. Subcellular fractionation detection showed that both PAPs were associated with membranes, while immunofluorescent imaging revealed an exclusive ER distribution. Most importantly, PAP2F accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol while decreased phosphatidylinositol. On the contrary, myc/His-PAP2G had no effect on lipid metabolism. Coexpression of CTP:phosphocholine cytidylyltransferase-a with PAP2F enhanced the effect of PAP2F on phosphatidylcholine levels, yet attenuated its effect on triacylglycerol. Taken together, our studies provide the first evidence that, in addition to PAP1, there is a eukaryotic, ER-resident PAP2 enzyme, PAP2F, which regulates de-novo biosynthesis of phospholipids and triacylglycerols.
Data: CORDIS, © European Union
Project objective
The goal of the current proposal is to define the mechanisms that control phosphatidylinositol (PtdIns) biosynthesis. PtdIns is a structural component of the membrane and a precursor to signalling molecules. Its biosynthetic pathway utilizes phosphatidic ac id (PtdOH) and cytidylyldiphosphodiacylglycerol (CDP-DAG). There are two mammalian genes encoding CDP-DAG synthetases (CDS1 and CDS2). In the current proposal we will test whether one CDS isoform is involved in de novo PtdIns formation whereas the other participates in the turnover of PtdIns after agonist-induced stimulation of phospholipase C. There are strong computational evidence for CDS1 and CDS2 phosphorylation, and we propose to develop isoform specific antibodies to characterize the post-translation al modifications as well as the sub-cellular distribution of the two isoforms. The second goal is to identify the enzyme(s) that regulate the distribution of PtdOH between the CDP-DAG and diacylglycerol.Our alternative hypothesis suggests that PtdIns bio-synthesis is regulated by the supply of PtdOH, the substrate of CDS. Magnesium-dependent phosphatidate phosphohydrolase (PAP1) is the enzyme involved in the de novo pathway of PtdOH utilization. The gene(s) coding for PAP1 enzyme(s) have not been characterized. We used a bio-informatics approach to identify two novel genes as candidates for PAP1 and we propose to characterize their biochemical activities and cellular functions as well as their involvement in PtdIns biosynthesis. Propranolol and bromoenol lactone have been previously identified as PAP1 inhibitors and additional micro-array experiments are proposed to characterize the transcriptional response to them, which may lead to the identification of their target PAP1 gene(s). The results of these studies will identify novel players in PtdIns biosynthesis, advance our understanding of the mechanisms that govern mammalian PtdIns metabolism and may lead to the development of novel therapeutics.
Original text from CORDIS.
Participants
- FOUNDATION FOR BIOMEDICAL RESEARCH OF THE ACADEMY OF ATHENS · ATHENSCoordinatorCity levelGreece
Links
Data: CORDIS, © European Union