The Roles And Mechanisms Of MGAT2/DGAT1,LPGAT1/ALCAT1 In TAG Synthesis,Mitochondrial Damage And Viral Replication

Acyltransferases play crucial roles in living cell activities. First of all, they regulate intracellular energy production, storage and release; Secondly, they modulate the structure of phospholipids which affected the structure integrities and functions of organelles membranes; moreover,the substrates of acyltransferases served as the second messenger to take part in the physiological and pathological process of various diseases. The monoacylglycerol acyltransferases(MGAT) and diacylglycerol acyltransferases(DGAT) catalyze a key process for triacylglycerols(TAG) metabolism. Lysocardiolipin acyltransferase(ALCAT1) and Lysophosphatidylglycerol Acyltransferase1(LPGAT1) are the key enzymes regulating phospholipid metabolism and mitochondrial function. However, the interaction sites of MGAT/DGAT in promoting TAG synthesis was not clear, plus the mechanism of ALCAT1/LPGAT1 expression on the morphology and function of mitochondria was not elaborated, as well as the role of MGAT2/DGAT1, LPGAT1/ALCAT1 on viral replication, all of them require to be further in-depth studied.The role of MGAT2/DGAT1, LPGAT1/ALCAT1 on TAG synthesis, mitochondrial dysfunction and viral infection were analyzed by overexpression and siRNA knockdown assay. The results are listed as follows:1. MGATs and DGATs catalyze a key process for triacylglycerol synthesis in small intestine. By overexpress the MGAT/DGAT on cos-7 to investigate the roles on TAG synthesis. Tris-glycine-PAGE electrophoresis demonstrated that MGAT2 enzyme functions as a homotetrameric protein and as a tetrameric protein. Likewise, MGAT2 specifically heterodimerized with DGAT1, however, MGAT2 did not heterodimerize with DGAT2. Furthermore, systematic deletion analysis of MGAT2 and DGAT2 demonstrated that N-terminal amino acids 35~80 of DGAT1, but not a signal peptide at the N terminus of MGAT2, is required for the heterodimerization. Finally, co-expression of MGAT2 with DGAT1 significantly increased lipogenesis in COS-7 cells, indicating the functional importance of the dimerization.2. Generated of Stable expression of LPGAT1, ALCAT1 H9C2 Cell Lines. Firstly, confocal laser experiments showed that LPGAT1 significantly reduced the ALCAT1 induced mitochondrial fragmentation, take it into a mix status of tubular and dot fragment. In response to H2O2 resulted Oxidative Stress Damage, the LPGAT1 relieved ALCAT1 caused H2O2 leakage, loss of mtDNA, high intracellular lipid peroxidation level as well as the up-regulated oxidative stress-related genes. Mitochondrial respiratory function tests showed LPGAT1 exhibited the highest mitochondrial oxygen consumption rate, which diminished the ALCAT1 resulted mitochondrial respiratory dysfunction. LPGAT1 also can alleviate ALCAT1 caused increased autophagosome. The data indicated that, under oxidative stress, the interactcion of LPGAT1 and ALCAT1 work together to modulate the mitochondria structure and function and to further regulate the cell metabolism and function.3. Oil red O staining showed that PK 15 cells infected with TGEV showed the neutral lipid content was increased combined with viral-DNA qPCR detection demonstrated a MOI related increase manner in infected cells. ELISA studies further confirmed that the increase of neutral lipid caused by DAG content promotion rather than TAG content. qPCR displayed TGEV infection up-regulated the expression of MGAT2, DGAT1, as well as ATGL, while siRNA respectively knockdown the DAG synthetase enzymes found that the variation of DAG content effected the virus replication and virus titer, but had no significant effect of virus entry. Western blotting showed that TGEV infection modulated the DAG content to activate the PKC signaling pathways. In addition, even though TGEV infections can cause mitochondrial damage, the ALCAT1/LPGAT1 had a relatively steady expression during TGEV infection.In conclusion, this study confirmed the MGAT2 heterodimerized with DGAT1 to promote the synthesis of the TAG; and LPGAT1 alleviate the ALCAT1 induced mitochondria dysfunction; In addition, TGEV infection in PK 15 cells, up-regulated the expression of MGAT2, DGAT2, ATGL to increase the DAG content, which may act as the second messenger to activate the PKC signal pathway, and affected the virus replication and virus titer. However, TGEV infection did not affect the expression of ALCAT1 and LPGAT1. The Results illustrated the role of MGAT2/DGAT1 LPGAT1/ALCAT1 in TAG synthesis, mitochondrial function and viral replication and, which may expend the understanding of the lipid metabolism key enzyme in cellular physiological process.