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

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.

Accelerated phosphatidylcholine turnover in macrophages promotes adipose tissue inflammation in obesity

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.

Metabolomic Study on Nude Mice Models of Gastric Cancer Treated with Modified Si Jun Zi Tang via HILIC UHPLC-Q-TOF/MS Analysis

Recently, metabolomic methods have been used to explore the complex pathogenesis of cancer and the mechanism of action of traditional Chinese medicine (TCM) formulae. In this study, first, modified Si Jun Zi Tang (MSJZT) was prepared with strict quality control using the instrument method of ultra performance liquid chromatography and photodiode array detector (UPLC-PDA). Subsequently,in vivoexperiments with tumour-bearing nude mice demonstrated that MSJZT exerted good antitumour effects. MSJZT not only significantly increased mouse body weight but also shrank the tumour volume. Then, the HILIC UHPLC-Q-TOF/MS-based metabolomics approach was used for exploring the pathogenesis of gastric cancer and the molecular mechanism of MSJZT. A total of 59 potential biomarkers in plasma were identified, and 6 pathways were found to be disturbed in gastric cancer. In contrast, after 3 weeks of MSJZT intervention, 32 potential biomarkers were identified, and 4 altered pathways were detected. The changes in glycolytic, amino acid, and lipid metabolisms could be partially regulated by MSJZT through decreasing the content of lactic dehydrogenase (LDH), glutamine synthetase (GS), phosphocholine cytidylyltransferase (PCYT2) mRNA, and protein level. In conclusion, we established a HILIC UHPLC-Q-TOF/MS metabolomic analysis method to demonstrate a complex metabolic profile of gastric cancer. The disordered metabolism could be partially regulated by MSJZT. These findings not only establish a solid foundation for TCM to treat gastric cancer but also provide a basis for further exploration of the precise mechanism of MSJZT activity.

Accelerated phosphatidylcholine turnover in macrophages promotes adipose tissue inflammation in obesity

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 from obese mice and humans exhibit markers of increased de novo phosphatidylcholine (PC) biosynthesis rate. 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 PC turnover, leading to elevated membrane polyunsaturated fatty acids that negated the pro-inflammatory effects of palmitate. Our results reveal a link between obesity-associated increase in PC synthesis, accelerated PC turnover and pro-inflammatory activation of ATMs.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTβ), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Impaired phosphatidylcholine biosynthesis does not attenuate liver regeneration after 70% partial hepatectomy in hepatic CTP:phosphocholine cytidylyltransferase-α deficient mice

Phosphatidylcholine (PC) is the major component of mammalian membranes, and the induction of PC biosynthesis has been shown to be an essential step in cell proliferation in various cell lines. Cytidine triphosphate (CTP):phosphocholine cytidylyltransferase α (CTα) regulates the primary pathway of PC biosynthesis in the liver. The targeted disruption of CTα in murine liver (LCTα−/− mice) decreases hepatic PC mass and the number of cells in the liver, suggesting CTα as an important factor for hepatocyte proliferation. To elucidate the role of CTα in hepatic cell division in vivo, we monitored liver regeneration after 70% partial hepatectomy in LCTα−/− and loxP flanked (floxed) LCTα (control) mice. To our surprise, liver re-growth, DNA synthesis, and PC mass after surgery were not impaired in LCTα−/− mice, despite reduced total PC synthesis. Furthermore, PC synthesis in the control mice was not induced after 70% partial hepatectomy. We conclude that CTα is not essential for proliferation of hepatocytes in vivo, and that basal hepatic PC biosynthesis is sufficient to sustain regeneration after 70% partial hepatectomy.