Study On Central Role Of Pap In Regulation Of Triacylglycerol Biosynthesis And Its Related Molecular Mechanism

Nearly95percent of fat in human belongs to triacylglycerol (TAG), whose synthesis and mobilization is one of the primary factors in the regulation of energy balance. When this regulation is unbalanced, it will induce hypertension, cardiopathy, subcutaneous fat loss and other related diseases in human.The fat-regulatory enzymes, which are encoded by fat mass and obesity associated gene, directly regulate the lipid metabolism. This process is highly conservative in all eukaryotic system. The biosynthesis of TAG is an important step in the lipid metabolism. Phosphatidate phosphatase (PAP), called as lipin, is involved in the biosynthesis of TAG.Recently, its related gene PAH1(Saccharomyces cerevisiae), LIPIN1, LIPIN2, LIPIN3(mammalian) are confirmed to be the key genes in regulation of TAG metabolism.By DNA sequence blastn analysis, basic information of genes can be obtained to predict structure and function of its encoded proteins. According to the encoded sequences of PAH1and LIPIN1, their homology was analyzed using bio-information tools of NCBI, HHpred,Bioedit, Then, the tertiary structure of HAD-LIKE domain, which is a conservative region of encoded protein of PAH1and LIPIN1, was successfully predicted by the analysis software Phyre2.Meanwhile, PAH1-defect type strain was constructed on the basis of wild type of yeast BY4742α.Its phenotype showed high sensitivity to temperature and fatty acid toxicity. By thin layer chromatography of lipid components and fluorescence microscopy, we found that the content of TAG and numbers of lipid droplet in PAH1-defect cells were decreased90%and70%compared to the wild type ones, respectively. Therefore, defect of PAH1suppressed the biosynthesis of TAG. When transfected the LIPINI from human into the PAH1-defect cells, the numbers of lipid droplets were increased and the sensitivity to temperature and fatty acid toxicity was reversed compared to those of PAH1-defect cells. The evidence above strongly proved that PAH1p and LIPIN1p are highly conserved in the regulation of TAG biosynthesis.Moreover, TGL3and TGL4, which are the main encoding genes of hydrolytic enzymes of TAG, were knockout combined with the overexpression of PAH1in the wild type yeast cells,. It resulted in the disruption of TAG metabolism and a very good model of fat-yeast was successfully constructed. The four usual lipid-lowering drugs including Propranolol Hydrochloride, Clenbuterol Hydrochloride, Berberine Hydrochloride, and Taurine were chosen to treat the fat model. It showed that Berberine Hydrochloride had the best effect on lipid-lowering.In summary, a platform regarding the regulation of PAP in TAG biosynthesis was successfully constructed in yeast system. Compared with the platform using animals, the advantages of yeast one include time saving, lower cost and easy operating. Since PAH1plays an important role in yeast growth and nutrient metabolism, especially in lipid metabolism, these studies help us to understand the clues of the regulation mechanism of lipid metabolism. Also, the construction of fat-yeast model and the tentative exploration in lipid lowering drugs provide the strategy for the research and development of new lipid lowering drugs.